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04 University of Plymouth Research Theses 01 Research Theses Main Collection
2014 CELLULAR AND MOLECULAR MECHANISMS OF BIOMINERALISATION IN A SILICIFYING HAPTOPHYTE PRYMNESIUM NEOLEPIS
Durak, Grazyna http://hdl.handle.net/10026.1/3098
Plymouth University
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CELLULAR AND MOLECULAR MECHANISMS OF BIOMINERALISATION IN A SILICIFYING HAPTOPHYTE PRYMNESIUM NEOLEPIS
by
GRAŻYNA MAŁGORZATA DURAK
A thesis submitted to the University of Plymouth in partial fulfilment for the degree of
DOCTOR OF PHILOSOPHY
School of Marine Science & Engineering Faculty of Science & Technology
In collaboration with The Marine Biological Association of the United Kingdom
-August 2013-
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Copyright Statement
This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the author's prior consent.
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Grażyna Małgorzata Durak
ABSTRACT
CELLULAR AND MOLECULAR MECHANISMS OF BIOMINERALISATION IN A SILICIFYING HAPTOPHYTE PRYMNESIUM NEOLEPIS
Haptophytes are renowned for the most prominent and biogeochemically important group of marine calcifiers: coccolithophores. The unexpected discovery of a unique, silicifying member of this clade - Prymnesium neolepis - prompted questions regarding mechanisms of silicification and their origin in the calcifier-dominated haptophytes. To address these questions I used cell physiology, biochemistry and molecular approaches, investigating cellular and molecular mechanisms involved in silicification in haptophytes. Comparisons of this system with calcification in coccolithophores and other silica-based systems in eukaryotes were also made. Here I report that P. neolepis is an obligate silicifier, producing silica scales in a process fundamentally different to that observed in coccolithophores. Scale deposition and secretion in P. neolepis is localized in the posterior, vacuolar part of the cell rather than in the anterior part near the flagellar roots as in calcifying coccolithophores. The organic matrix underlying silica scales in P. neolepis was found to be non-homologous with organic scales, which in coccolithophores serve as coccolith baseplates. This suggests, that silica scales and coccoliths arise from two distinct, most likely non-homologous processes, which is further supported by the comparative investigation of the role of cytoskeleton in silica scale production in P. neolepis and coccolithogenesis in a representative calcifier, Coccolithus pelagicus. Using cytoskeleton inhibitors I established, that the cytoskeleton components used for morphogenesis and secretion of biomineralised structures are different in these two systems. Analysis of P. neolepis biosilica revealed the presence of an intimately associated organic fraction consisting of a putatively chitin-containing material, potentially serving as an organic matrix underlying silica scales. Further biochemical investigation of the biosilica-associated organics confirmed the presence of long chain polyamines (LCPAs) dissimilar to those previously reported in diatoms and sponges. Additionally, a potentially novel, proline and lysine-rich protein sharing a weak homology with lipocalins was recovered, suggesting that this silicification system is unique to haptophytes. Several theories concerning acquisition of the ability to silicify in haptophytes were proposed. Overall, the findings presented in this study provide a detailed description of Si biomineralisation system in this unique, silicifying haptophyte and supply novel information on biomineralisation systems in marine haptophytes. This study contributes a basis on which the phenomenon of silicification in haptophytes can be further investigated, as well as novel information which can be further used in elucidation of origins of silicification in algae and other Eucarya.
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LIST OF CONTENTS PAGE
ABSTRACT ...... 3
LIST OF FIGURES AND TABLES ...... 11
LIST OF ABBREVIATIONS ...... 17
ACKNOWLEDGEMENTS ...... 20
AUTHOR'S DECLARATION ...... 21
CHAPTER I: METABOLISM AND PHYSIOLOGY OF SILICON. MECHANISMS AND FUNCTIONS OF BIOMINERALISATION IN DIFFERENT PHYLA ...... 23
I.1. INTRODUCTION ...... 23
I.2. ALGAE: SILICA ...... 26
I.3. DIATOMS (HETEROKONTOPHYTA: BACILLARIOPHYCEAE) ...... 27
I.3.1. Cell cycle ...... 27
I.3.2. Transport and storage ...... 28
I.3.3. Si Uptake and transport models ...... 29
I.3.4. Valve formation: SDV ...... 33
I.3.5. Components involved in silica polymerization ...... 34
I.3.6. Organic matrix of the frustule ...... 36
I.3.7. Polyanionic polysaccharides ...... 38
I.4. CHRYSOPHYTES ...... 38
I.5. HAPTOPHYTES (HAPTOHPYTA: COCCOLITHOPHYCEAE) ...... 39
I.5.1. Coccolithales and Isochrysidales: calcification...... 40
I.5.1.1. Heterococcolith formation ...... 40
I.5.I.2. Holococcolith formation ...... 43
I.5.2. Prymnesiales: Prymnesium neolepis: silicification in haptophytes ...... 44
I.6. SPONGES (PORIFERA) ...... 45
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I.6.1. Spicule formation ...... 46
I.6.2. Silicateins ...... 50
I.6.3. Silicase ...... 52
I.6.4. Silintaphins ...... 53
I.6.5. Long chain polyamines...... 54
I.7. PLANTS ...... 54
I.7.1. Si transporters ...... 55
I.8. SUMMARY AND AIMS ...... 56
CHAPTER II: PRYMNESIUM NEOLEPIS: GENERAL ORGANISM DESCRIPTION, BIOLOGY AND PHYSIOLOGY OF SILICIFICATION ...... 59
II.1. INTRODUCTION ...... 59
II.1.1. Putative capacity for silicification in Prymnesium sp...... 61
II.2. METHODS ...... 63
II.2.1. Culture conditions ...... 63
II.2.2. DNA extraction, gene amplification, sequencing and molecular analysis ...... 64
II.2.3. Confocal microscopy ...... 66
II.2.4. Differential Interference Contrast (DIC)/light microscopy imaging...... 67
II.2.5. NaOH etching of biosilica material ...... 67
II.2.6. Scanning Electron Microscopy (SEM), Transmission Electron Microscopy
(TEM) and Energy Dispersive Microanalysis (EDX) ...... 68
II.2.7. Fourier Transform Infrared Spectroscopy (FTIR) ...... 68
II.2.8. Light: dark experiment: flow cytometry assessment of silica scale production
...... 69
II.2.9. High/Low Si experiment ...... 69
II.2.10. Si concentration determination: silicomolybdate assay ...... 70
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II.2.11. Ge supplementation of P. neolepis cultures and biosilica material purification
for EDX analysis ...... 72
II.3. RESULTS ...... 73
II.3.1. Molecular identification of P. neolepis strain ...... 73
II.3.2. P. neolepis: general morphology ...... 77
II.3.3. In-vivo visualization of silicification in P. neolepis: silica scale formation and
exocytosis ...... 79
II.3.4. Morphological observations of silica scales ...... 84
II.3.5. Non-biomineralised elements of P. neolepis cell covering ...... 86
II.3.6. FT-IR characterization of chemical structure of P. neolepis biosilica ...... 89
II.3.7. Physiology of silicification in P. neolepis ...... 92
II.3.8. Dependency of silicification on light availability ...... 92
II.3.9. Dependency of silica scale production on silica availability ...... 94
II.3.11. Inhibition of silica scale production in P. neolepis with Ge: CLSM
observations ...... 98
II.3.12. Ge/Si co-feeding of P. neolepis: growth ...... 99
II.3.13. Ge/Si co-feeding of P. neolepis: Ge incorporation into silica scales ...... 101
II.4. DISCUSSION ...... 102
II.4.1. P. neolepis strain TMR5: morphological and biological characteristics and
comparisons with closely related algae ...... 102
II.4.2. Organic and biomineralised cell covering ...... 103
II.4.3. Silica scale formation...... 105
II.4.4. Physiology of silicification in P. neolepis ...... 106
II.4.5. Ge inhibition of silicification ...... 107
II.4.6. P. neolepis: the only silicifying haptophyte described to date? ...... 108
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II.5. SUMMARY ...... 109
CHAPTER III: THE ROLE OF CYTOSKELETON IN ALGAL BIOMINERALISATION ...... 111
III.1. INTRODUCTION ...... 111
III.2. METHODS ...... 115
III.2.1. Culture conditions and cytoskeleton inhibitor dosing ...... 115
III.2.2. Monitoring of culture health under experimental conditions ...... 116
III.2.3. Confocal microscopy observations of P. neolepis under cytoskeleton inhibitor
treatment ...... 116
III.2.4. Flow cytometry monitoring of silica scale production in P. neolepis ...... 117
III.2.5. C. pelagicus: Differential Interference Contrast (DIC) imaging of
cytoskeleton inhibitor treated cells ...... 118
III.2.6. C. pelagicus: Electron Microscopy imaging of cytoskeleton inhibitor treated
coccoliths ...... 119
III.2.7. Tubulin Immunofluorescence labelling ...... 120
III.3. RESULTS ...... 121
III.3.1. Assays to determine effects of cytoskeleton inhibition on culture health ..... 121
III.3.2. Effects of cytoskeleton inhibitors on P. neolepis health ...... 122
III.3.3. Effects of cytoskeleton disruptors on silica scale morphogenesis in P.
neolepis ...... 126
III.3.4. Quantification of cytoskeleton inhibitor effects on silica scale production in P.
neolepis...... 131
III.3.5. P. neolepis: immmunofluorescence imaging of the MT cytoskeleton...... 133
III.3.6. Effects of cytoskeleton inhibition on culture health of C. pelagicus ...... 137
III.4. Effects of cytoskeleton disruptors on biomineralisation in C. pelagicus: light
microscopy imaging ...... 138
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III.4.1. SEM analysis of cytoskeleton inhibitor-induced coccolith malformations in C.
pelagicus ...... 142
III.4.2. C. pelagicus: immunofluorescence imaging of the MT cytoskeleton ...... 145
III.5. DISCUSSION ...... 148
III.5.1. Cytoskeleton inhibitors and culture health ...... 148
III.5.2. The role of actin in biomineralisation ...... 150
III.5.3. The role of tubulin in biomineralisation ...... 152
III.5.4. Differential role of cytoskeleton in silicification in haptophyte and
Bacillariophyte (diatom) algae ...... 155
III.6. CONCLUSIONS ...... 156
CHAPTER IV: BIOCHEMICAL ANALYSIS OF PRYMNESIUM NEOLEPIS SILICA SCALES . 159
IV.1. INTRODUCTION ...... 159
IV.2. METHODS ...... 162
IV.2.1. Silica scale purification ...... 162
IV.2.2. Glycerol cushion separation of the purified silica scales and further
assessment of sample purity ...... 163
IV.2.3. Ammonium fluoride extraction of the organic phase...... 164
IV.2.4. Separation of the putative polyamine fraction from the NH4F flow-through on
an ion exchange column ...... 166
IV.2.5. Polyacrylamide gel analysis ...... 166
IV.2.6. Trypsin digestion of the NH4F soluble organic extract ...... 167
IV.2.7. Amino acid analysis ...... 168
IV.2.8. Mass spectrometry analysis ...... 168
IV.2.9. FESEM, TEM and EDX analysis of the NH4F insoluble extract ...... 169
IV.2.10. Confocal microscopy imaging of the NH4F insoluble extract ...... 169
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IV.3. RESULTS ...... 171
IV.3.1. Amino acid analysis of purified P. neolepis biosilica...... 171
IV.3.3. Sample purity verification: Schägger-PAGE analysis of the NH4F soluble
phase generated from glycerol cushion-separated silica scales and EDTA extraction
of protein from whole scales ...... 174
IV.3.4. Trypsin digest of the NH4F soluble extract: verification of presence of protein
...... 175
IV.3.5. Cation-exchange liquid chromatography column samples: purification of
putative LCPAs from the NH4Fextraction flow through ...... 177
IV.3.6. Mass spectrometry analysis of the protein extracts...... 178
IV.3.7. Mass spectrometry analysis of the putative polyamine phase isolated on the
cation exchanger ...... 179
IV.3.8. FESEM and EDX and confocal analysis of the NH4F insoluble extract,
detection of polysaccharides with fluorescent markers ...... 182
IV.4. DISCUSSION ...... 184
IV.4.1. NH4F soluble organic extract: silica-associated proteins ...... 185
IV.4.2. LCPAs ...... 185
IV.4.3. NH4F insoluble organic extract ...... 188
IV.5. SUMMARY ...... 189
CHAPTER V: GENERAL DISCUSSION AND CONCLUDING REMARKS ...... 191
V.1. INTRODUCTION ...... 191
V.1.1. Origins of photosynthetic Eucarya ...... 193
V.1.2. Evolution of silicification in Eucarya ...... 195
V.1.3. Evolution of other biomineralisation systems in Eucarya ...... 196
V.2. SILICIFICATION IN P. NEOLEPIS: EVOLUTIONARY CONTEXT...... 198
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V.3. SUMMARY ...... 200
LIST OF APPENDICES ...... 202
REFERENCES ...... 209
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LIST OF FIGURES AND TABLES
Fig. I. 1. A tree of life with clades including silicifying representatives underlined in red
...... 26
Fig. I. 2. A: silicon starvation induced arrest points in the cell cycle ...... 28
Fig. I. 3. Reconstruction of a diatom silicon transporter ...... 30
Fig. I. 4. Diatom Si uptake models. Gray oval represents SDV (after Thamatrakoln and
Hildebrand, 2008)...... 32
Fig. I. 5. Examples of LCPAs from different diatom species with different attachment
moieties...... 35
Fig. I. 6. Chemical representation of native Silaffin 1a structure ...... 36
Fig. I. 7. A schematic of a diatom frustule ...... 37
Fig. I. 8. Coccolithus pelagicus ssp. braarudi heterococcolith bearing diploid phase ... 41
Fig. I. 9. A diagram of a coccolithophore cell ...... 42
Fig. I. 10. Holococcoliths of Syracosphaera anthos...... 43
Fig. I. 11. SEM of a P. neolepis cell ...... 45
Fig. I. 12. SEM cross section of a Suberites domuncula ...... 47
Fig. I. 13. Immunogold TEM images of primmorph cross sections ...... 49
Fig. I. 14. Silicatein ...... 51
Fig. I. 15. A - model of silicase ...... 53
Fig. II. 1. A - P. parvum light microscopy image...... 61
Fig. II. 2. Electron micrographs of Prymnesium sp ...... 62
Table II. 1. Chemical composition of ASW...... 64
Table II. 2. Primers used for amplification of SSU, LSU and rbcL genes...... 65
Table II. 3. List of reagents used in the silicomolybdate assay and their composition .. 71
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Table II. 4. A summary of the degree of homology between rbcL and SSU gene
sequences obtained from P. neolepis strains isolated from different locations...... 74
Fig. II. 3. Unrooted maximum likelihood phylogenetic tree of selected haptophytes
based on the rbcL gene sequences ...... 75
Fig. II. 4. Unrooted maximum likelihood phylogenetic tree constructed for haptophytes
and heterokontophyte silicifiers based on SSU rRNA sequences...... 76
Fig. II. 5. P. neolepis: A - light microscopy image of a naked and silicified cells ...... 78
Fig. II. 6. DIC images of P. neolepis ...... 79
Fig. II. 7. Confocal microscopy 3D projection of a Z-stack of P. neolepis cells ...... 80
Fig. II. 8. 3D projections of Z-stacks of P. neolepis ...... 81
Fig. II. 9. Time course of a silica scale secretion event ...... 82
Fig. II. 10. Silica scales at different developmental stages ...... 83
Fig. II. 11. SEM images of a P. neolepis biosilica sample etched with 0.5 M NaOH .... 84
Fig. II. 12. SEM image of KMnO4/HCl cleaned P. neoelpis silica scales ...... 85
Fig. II. 13. P. neolepis: A - polarized light micrograph of a cell with a disturbed scale
cover ...... 86
Fig. II. 14. 3D projections of confocal microscopy Z-stacks of Concanavalin-A labelled
cells of P. neolepis: A - 3D reconstruction with labelled detached scales (arrows),
B - individual slice showing irregularities and partial detachment of the Con-A
labelled cell coating. C - decalcified C. pelagicus cells with holes in polysaccharide
covering marking spaces where coccoliths were attached, D - individual C.
pelagicus coccoliths. P. neolepis was additionally labelled with PDMPO (in blue)
to visualize cell body. Con-A is shown in green, chlorophyll autofluorescence in
red. Scale bars=5 µm...... 88
Fig. II. 15. A,B-TEM images of uranyl-acetate stained organic scales ...... 89
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Fig. II. 16. FT-IR transmission spectra of purified P. neolepis (red), Odontella sinensis
(black) biosilica ...... 91
Fig. II. 17. Box-whisker plots of the newly deposited to old scale ratios as measured
with flow cytometry ...... 94
Fig. II. 18. Confocal microscopy images of PDMPO stained P. neolepis cells ...... 95
Fig. II. 19. Validation of the method used for cell counts in the low Si experiment ...... 96
Fig. II. 20. Quantification of silica scales produced after 18 h incubation period under
low Si and control conditions ...... 97
Fig. II. 21. A - silicified cells in control, B - non-silicifying cells post 10 µM Ge
treatment with partially formed intracellular scales ...... 99
Fig. II. 22. A-Growth curve for 3 µM Ge supplemented and control P. neolepis cultures.
...... 100
Fig. II. 23. EDX spectrum of purified P. neolepis silica scales ...... 101
Fig. III. 1. Mechanism of motor protein interaction with MTs and F-actin ...... 113
Table III. 1. Cytoskeleton inhibitor and control DMSO doses...... 115
Table III. 2. Photosynthetic efficiency and cell viability counts recorded for P. neolepis
26.5 h post cytoskeleton inhibitor treatment. Measurements were taken from
triplicate samples of each treatment and averaged...... 123
Fig. III. 2. SYTOX Green labelled P. neolepis cells representative of the three cell
viability categories ...... 124
Table III. 3. Results of χ2 test between DMSO and nocodazole treatment labelled with
SYTOX Green ...... 126
Fig. III. 3. Confocal microscopy images representative of P. neolepis cells following a
24 h treatment with 1 µM latrunculin B ...... 127
Fig. III. 4. 3D projections of confocal microscopy generated Z-stacks of P. neolepis
after 24 h treatment with cytoskeleton inhibitors ...... 128
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Table III. 4. T-test comparisons of P. neolepis scales between different cytoskeleton
inhibitor treatments ...... 129
Fig. III. 5. A - frequency distribution of P. neolepis scales according to different size
classes ...... 130
Fig. III. 6. Box-whisker plots of fluorescent vs. non-fluorescent scales ratios post 24h
treatments ...... 132
Table III. 5. Results of the t-test comparisons of the fluorescent vs. non-fluorescent
scales ratio...... 133
Fig. III. 7. Confocal microscopy 3D projection of a Z-stack of P. neolepis tubulin-
immunolabelled control cells ...... 134
Fig. III. 8. P. neolepis: 3D confocal microscopy projection of a Z-stack of anti-α-tubulin
immunolabelled nocodazole treated cells ...... 135
Fig. III. 9. Confocal microscopy image of a P. neolepis immunofluorescence labelling
control ...... 136
Table III. 6. Photosynthetic efficiency recorded post 25 h cytoskeleton inhibitor
treatment of C. pelagicus...... 137
Fig. III. 10. DIC of decalcified C. pelagicus cells at the beginning of the experiment. 138
Fig. III. 11. DIC images of cytoskeleton inhibitor treated C. pelagicus ...... 140
Fig. III. 12. DIC image of C. pelagicus post 48 h recovery from latrunculin B treatment
...... 142
Fig. III. 13. FESEM images of C. pelagicus coccoliths used for morphological
aberration ranking of coccoliths produced ...... 143
Table III. 7. χ2 statistic results of DMSO and nocodazole treatments...... 144
Fig. III. 14. A-C - malformed/incomplete coccoliths representative of latrunculin B
treatment ...... 145
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Fig. III. 15. 3D projections of confocal microscopy Z-stacks of anti-α-tubulin
immunolabelled C. pelagicus control cells ...... 146
Fig. III. 16. 3D projections of confocal microscopy Z-stacks of anti-α-tubulin
immunolabelled C. pelagicus ...... 147
Fig. III. 17. 3D confocal microscopy projections of Z-stacks of C. pelagicus nonspecific
labelling control ...... 148
Table III. 8. Summary of the effects of cytoskeleton inhibitors on P. neolepis and C.
pelagicus ...... 158
Fig. IV. 1. A schematic summarising processing of the P. neolepis biosilica ...... 165
Table IV. 1. Concentrations and incubation times of fluorophores used...... 170
Fig. IV. 2. HPLC chromatogram of amino acids of the whole scales ...... 172
Fig. IV. 3. NH4F soluble extract run on a 16% Schägger gel ...... 173
Fig. IV. 4. Coomassie Blue staining of the NH4F soluble extract of the silica scale
fraction ...... 175
Fig. IV. 5. A - Coomassie stained 16% Schägger gel ...... 176
Fig. IV. 6. Coomassie stained 16% Schägger gel with samples isolated on a cation
exchanger ...... 177
Table IV. 2. Summary of putative properties of the comp1037 peptide recovered from
the transcriptome ...... 178
Fig. IV. 7. Amino acid composition of comp1037 peptide retrieved from P. neolepis
transcriptome. Abbreviations are as follows: Ala - alanine, Cys - cysteine, Asp -
aspartic acid, Glu - glutamic acid, Phe - phenylalanine, Gly - glycine, His -
histidine, Ile - isoleucine, Lys - lysine, Leu - leucine, Met - methionine, Asn -
aspargine, Pro - proline, Gln - glutamine, Arg - arginine, Ser - serine, Thr -
threonine, Val - valine, Trp - tryptophan, Tyr - tyrosine...... 179
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Fig. IV. 8. A,B - mass spectra of the ammonia eluate isolated from the NH4F flow
through on a cation exchanger ...... 180
Fig. IV. 9. Collision induced fragmentation spectrum of the m/z=574.6 peak...... 181
Fig. IV. 10. FESEM images of the NH4F insoluble biosilica-associated organic phase
...... 183
Fig. IV. 11. EDX analysis of the NH4F insoluble extract ...... 184
Table V. 1. Table summarizing differences between the formation of the silica scales
and coccoliths...... 192
Fig. V. 1. The hypothetical chain of evolutionary events leading to plastid acquisition
and subsequent lineage differentiation in eukaryotes...... 194
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LIST OF ABBREVIATIONS
ASW-artificial sea water
BLAST-Basic Local Alignment Search Tool
BSA-bovine serum albumin
BYA-billion years ago
Con-A-Concanavalin A
CV-coccolith vesicle
CW-Calcofluor White
CLSM-confocal laser scanning microscopy
DF-degrees of freedom
DIC-differential interference contrast microscopy
DMSO-dimethyl sulfoxide dNTP-deoxyribonucleotide triphosphate
DNA-deoxyribonucleic acid
EDTA-ethylene glycol tetraacetic acid
EDX-energy dispersive microanalysis
F-actin-linear polymer microfilament of actin
FESEM-field emission scanning electron microscopy
FSW-filtered sea water
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FT-IR-Fourier transform infrared spectroscopy
HCK-123- LysoTracker Yellow HCK-123
HPLC- High-performance liquid chromatography
LC-MS- Liquid chromatography–mass spectrometry
LCPAs-Long Chain Polyamines
LSU rDNA-28S Large subunit of the rRNA gene
MAFFT-Multiple Alignment using Fast Fourier Transform
MS-mass spectrometry
MT-microtubule
MYA-million years ago
NCBI-National Centre for Biotechnology Information
NH4Ac-ammonium acetate
NTP- nucleoside triphosphate
PCR- polymerase chain reaction
PDMPO-[2-(4-pyridyl)-5-[[4-(2-dimethylaminoethylaminocarbamoyl)methoxy] phenyl]-oxazole]
PITC- phenylisothiocyanate
PSII-photosystem II rbcL-large subunit of RuBisCO (Ribulose-1,5-bisphosphate carboxylase oxygenase)
RNA-ribonucleic acid
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SDS-sodium dodecyl sulfate
SDS-PAGE-sodium dodecyl sulfate polyacrylamide gel electrophoresis
SDV-silica deposition vesicle
SEM-scanning electron microscopy
SIT-silicon transporter
STVs-Silica Transport Vesicles
TEM-transmission electron microscopy
TPCK- Tosyl phenylalanyl chloromethyl ketone
WGA- Wheat Germ Agglutinin
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ACKNOWLEDGEMENTS
This study was funded by the EU Interreg IV Marinexus project and carried out at the
Marine Biological Association of the United Kingdom and the University of Plymouth.
I would like to thank Prof. Colin Brownlee for all help and support during the course of my study and for securing funding, enabling me to further broaden my expertise and form new, fruitful collaborations. I would especially like to thank Dr. Glen Wheeler, for his inexhaustible patience and his help and guidance during my Ph.D. study. I would also like to thank Prof. Jason Hall-Spencer for his supervision of the project and Dr. Ian
Probert for providing transcriptome data.
Further I would like to thank Dr. Declan Schroeder for his help and advice with the molecular work and Mr. Matt Hall for assistance with the laboratory equipment.
Thanks also go to Dr. Roy Moate, Mr. Glenn Harper and Mr. Peter Bond of the Electron
Microscopy unit at the University of Plymouth, for all their help with various electron microscopy techniques. Special thanks also go to Dr. Rowena Stern and Dr. Andrea
Highfield, for their friendship, help with phylogenetic analysis and stimulating discussions on various scientific and non-scientific subjects.
I am also grateful to Prof. Nils Kröger and Dr. Nicole Poulsen for hosting me at the B-
Cube Dresden Centre for Molecular Engineering and their kind help with biochemical analysis undertaken in this study.
I would also like to thank my siblings, especially my sister Marta for their encouragement and support during my Ph.D. study.
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AUTHOR'S DECLARATION
At no time during the registration for the degree of Doctor of Philosophy has the author been registered for any other University award without prior agreement of the Graduate
Committee.
This study was funded by the EU Interreg IV Marinexus project and carried out in collaboration with the Marine Biological Association of the UK.
Relevant presentations and conferences attended:
-Biomineralisation in Hyalolithus. neolepis. Oral presentation given at the EU
Marinexus Meeting, Roscoff, France, January 2010.
- Cellular and Molecular Mechanisms of Biomineralisation in a Silicifying haptophyte
Hyalolithus neolepis. Poster presented at the MBA Annual Council Science Meeting,
Plymouth, UK, April 2010.
-Biomineralisation in Hyalolithus. neolepis. Poster presented at the EU Marinexus
Meeting, Plymouth, UK, July 2011.
-Biomineralisation in Hyalolithus. neolepis. Oral presentation given at the MBA
Laboratory Group Meeting, Plymouth, UK, June 2012.
-Biomineralisation in haptophytes. Poster presented at the MBA Annual Council
Science Meeting, Plymouth, UK, April 2012.
-Calcification and silicification in marine haptophyte algae. Oral presentation given at the B-Cube Centre for Molecular Engineering, Dresden, Germany, June 2012.
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-Biomineralisation in marine haptophyte algae. Poster presented at Gordon Research
Conference in Biomineralisation, New London, USA, August 2012.
Word count of main body of thesis: 34,232
Signed
Date
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Durak, G.M. Chapter I: Introduction
CHAPTER I: METABOLISM AND PHYSIOLOGY OF SILICON. MECHANISMS AND
FUNCTIONS OF BIOMINERALISATION IN DIFFERENT PHYLA
I.1. INTRODUCTION
Biomineralisation is a process by which living organisms deposit minerals - a phenomenon widespread in both prokaryotic and eukaryotic taxa (Weiner and Dove
2003). Mineral biodeposition serves a variety of purposes, e.g. formation of exo- and endo-skeletal components, such as mollusc shells, algal cell walls, vertebrate bones and other functional structures, such as teeth and statoliths. Biomineralisation can occur both intracellularly, as is the case in e.g. metazoan holothurians (Märkel et al. 1986) and unicellular algae such as diatoms or coccolithophores (Schmid and Schulz 1979;
Westbroek et al. 1984) or extracellularly, e.g. in mollusc shells, bones and teeth
(Addadi et al. 2006; Veis 2003). Some organisms, such as silicifying sponges, deposit biomineralised structures (spicules) sequentially, with an intracellular and extracellular phase to the process (Müller et al. 2006; Müller et al. 2005). Calcium based biominerals
(e.g. carbonates, phosphates, oxalates) account for 50% of known types of minerals of biological origin (Weiner and Dove 2003). Iron-based minerals contribute about 40% of the total, with the remaining part comprising silicon, lead, barium, strontium, zinc, arsenic, manganese, copper and fluorine based minerals (Lowenstam and Weiner 1989;
Weiner and Dove 2003).
Silicon is a metalloid element and the second most common element in the Earth's crust
(Earnshaw and Greenwood 1997). Silicon is utilized by many organisms for a variety
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Durak, G.M. Chapter I: Introduction of reasons, mainly related to its superior structural, mechanical and optical properties.
Si can be taken up by organisms in aqueous solution as an uncharged orthosilicic acid,
3- Si(OH)4, (e.g. plants) or in an anionic form as silicates (e.g. as SiO(OH) in diatom algae) (Del Amo and Brzezinski 1999; Earnshaw and Greenwood 1997; Ma and
Yamaji 2006). As a structural component, silicon in a form of silica (SiO2) is particularly attractive due to its inherent toughness, relatively high density (~1.7-2.0 g/cm3), and low chemical reactivity (Hamm et al. 2003; Hurd et al. 1981; Pak and Yoo
2013). Silica is also characterised by a low refractive index (n≈1.43), making it a material highly transparent to visible light (Aizenberg et al. 2004).
Properties of silica are determined by its structure, from the organization on a molecular level, through nano-, micro-, meso- to macrostructure, dictating parameters such as surface area, porosity, interaction with light etc. (Perry 2003). Both mechanical and optical properties of silica can be further enhanced by the specific architecture, and interaction with e.g. organics via formation of biocomposites with highly modified mechanical and optical properties, tailored to a specific function (Aizenberg et al. 2004;
Yamanaka et al. 2008).
As a structural component, Si is deposited in a form of amorphous, hydrated silica
(SiO2*nH2O) in a process of biosilicification in organisms such as algae (e.g. diatoms), radiolarians, cercozoans, plants and sponges (summarized in Fig. I. 1.). Apart from the obvious structural benefit of the ability to silicify in these organisms, a number of other factors relating to metabolism and physiology have been identified. In higher animals for example, Si facilitates the formation of connective tissue, bone and calcification in avians and mammals (Carlisle 1981; Carlisle 1988). It is involved in aging processes, where the decline in Si is subject to hormonal regulation (Charnot 1971). In algae such as diatoms and in sponges, Si has also been shown to function as a regulator of gene
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Durak, G.M. Chapter I: Introduction expression (Schröder et al. 2004b; Schröder et al. 2006; Schröder et al. 2003;
Thamatrakoln and Hildebrand 2007).
In this chapter, the so far described silicification systems are summarized. Their respective physiological, structural and metabolic roles in different organisms, as well as homologies and analogies and their evolutionary connections in different taxa are also discussed. Particular attention is paid to silicon metabolism and biomineralisation in algae and sponges, as these two systems are so far best described. Also, a brief overview of the other main algal biomineralisation mode-calcification, utilized by haptophytes is provided, enabling comparisons with the only silicifying representative of this group described to date - Prymnesium neolepis.
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Durak, G.M. Chapter I: Introduction
Fig. I. 1. A tree of life with clades including silicifying representatives underlined in red
(modified from (Adl et al. 2012). haptophytes (mainly calcifiers, underlined in green) and heterokontophytes (Stramenopiles) are marked with a star.
I.2. ALGAE: SILICA
Silica is used for structural component formation in several algal groups belonging to
Heterokontophyta (i.e. Chrysophyceae, Dictyochophyceae, Synurophyceae,
Parmophyceae, Xanthophyceae and most importantly Bacillariophyceae) as well and one species belonging to Haptophytea, Prymnesium neolepis (Prymnesiophyceae)
(Graham et al. 2009; Van den Hoek et al. 1995; Yoshida et al. 2006).
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Durak, G.M. Chapter I: Introduction
I.3. DIATOMS (HETEROKONTOPHYTA: BACILLARIOPHYCEAE)
Evolved around 250 MYA diatoms are obligatory silicifiers, producing a silica cell wall known as the frustule (Medlin 2011). Various explanations have been put forward to elucidate the role of biomineralisation in these algae. The silica frustule has been so far suggested to serve as a defensive structure due to its rigidity, as well as to provide protection against enzymatic attack, regulate sinking rate, confer UV protection, maintain cell shape and aid osmoregulation (Hamm et al.; 2003, Raven 1982; Raven;
1983; Raven, Waite 2004; Davidson et al., 1994). Using silica as a cell wall material may also be favoured due to the relatively low energetic costs of silica deposition in comparison to e.g. organic carbon material (Raven 1983). Another potential role of silicified frustules in diatoms is facilitating the functioning of carbon concentrating
- mechanisms via buffering the proton transfer between HCO3 , CO2, and the active site of the extracellular carbonic anhydrase (Milligan and Morel 2002). Si in diatoms has also been reported to be involved in DNA synthesis, as Si is essential for thymidylate kinase and DNA polymerase synthesis (Sullivan and Volcani 1973). Si dependence of chlorophyll and protein production has also been demonstrated (Werner 1966; Werner
1967).
I.3.1. Cell cycle
The diatom cell cycle is Si dependent, as cell division requires formation of new valves
(Fig. I. 2. B), and the majority of cells will arrest in the G1/S and G2/M phase if deprived of Si (Fig I. 2. A), (Hildebrand et al. 2007; Vaulot et al. 1987). These two arrest points have been linked to active silica deposition, also suggesting that the G1/S
-27-
Durak, G.M. Chapter I: Introduction halt might serve as a check point to determine, if extracellular Si is present in sufficient quantity for cell division to proceed (Vaulot 1985). Diatoms respond to lowering of the
Si concentration by producing thinner cell walls, in order to maintain the rate of cell division (Sullivan 1977). Si in diatoms also acts as a regulator for gene expression and a metabolite of various functions (Hildebrand et al. 1993).
Fig. I. 2. A: silicon starvation induced arrest points in the cell cycle (after Martin-
Jézéquel et al., 2000). B: schematic diagram of a diatom cell cycle. SDV and daughter valves are shown in red (after Sumper and Kröger, 2004).
I.3.2. Transport and storage
Diatoms have been shown to be able to retain soluble, supersaturated Si pools intracellularly at 19-340 mM concentrations (Martin-Jézéquel et al. 2000). This suggests that the chemical form of Si is altered once transported into the cell, likely via complexation with a so far undescribed organic component/stabilising factor, as
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Durak, G.M. Chapter I: Introduction otherwise orthosilicic acid undergoes auto-polymerisation at concentrations exceeding
2 mM (at pH 7.0) (Azam et al. 1974; Iler 1979). However, evidence that only ca. 5-10% of intracellular Si forms organosilicon complexes and the remaining Si exists as silicic acid have been later supplied (Kinrade et al. 2002). Sumper and Brunner (2008) therefore suggested that Si is intracellularly maintained as pre-polymerysed silicic acid, forming silica sols that might be stabilised by long-chain polyamines, to enable its subsequent use for silica biodeposition. The alteration of the chemical form of Si is also supported by the lack of Si efflux from diatom cells with high internal Si pools in the absence of extracellular Si (Martin-Jézéquel et al. 2000).
I.3.3. Si Uptake and transport models
Diatom silicon transporters - SITs - are a unique family of membrane-bound proteins that mediate active transport of orthosilicic acid into the cell by acting as Na+/Si symporters, using inwards Na+ gradient to drive the uptake of Si (Fig. I. 3.) (Hildebrand
2000). The highest peaks of SIT protein levels have been shown to be controlled internally by the requirement for active silicification with SIT gene expression peaking at S phase of the cell cycle, preceding the period of maximum uptake of Si, and subsequent frustule deposition (Thamatrakoln and Hildebrand 2007). The so far isolated SITs have been divided into two groups: SIT1 and SIT2 have been suggested to be active transporters with potential differences in their transport capacities/affinities linked to differences in their amino acid composition (Thamatrakoln and Hildebrand
2007). SIT3 has low Si affinity, and has been suggested to play a role as Si sensor in Si- replete conditions, triggering high affinity transporter expression, or to function as a
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Durak, G.M. Chapter I: Introduction specific Si targeting component acting intracellularly (Curnow et al. 2012;
Thamatrakoln and Hildebrand 2007).
Fig. I. 3. Reconstruction of a diatom silicon transporter (after Curnow et al., 2012).
Si uptake in diatoms usually conforms to the Michaelis-Menten saturable kinetics model of nutrient uptake, however, under certain conditions, a biphasic and non- saturable modes can be observed (Thamatrakoln and Hildebrand 2008). In an attempt to explain the variability of Si uptake kinetics, Thamatrakoln and Hildebrand (2008) revised information that has been already published, and proposed two main Si(OH)4 uptake models for exponentially growing cultures:
1. Biphasic mode of Si uptake in cultures starved for a short period of time (between
5-10 min), involving a transition from unsaturable to saturable (Michaelis-Menten)
uptake.
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Durak, G.M. Chapter I: Introduction
2. Saturable, Michaelis-Menten mode of Si uptake in cultures with an extended Si
starvation period (24 h).
In both cases, Si uptake has been proposed to be controlled by three main factors: Si availability in the media, the rate of Si incorporation, and the binding capacity of internal Si pools. In the first model, the internal Si level is assumed to be low, as the Si released by intracellular binding components for incorporation into the cell wall is not replenished, increasing their Si binding capacity (Fig. I. 4. B). Therefore upon Si addition, a non-saturable surge uptake occurs (Fig. I. 4. C) until the capacity of Si binding components returns to equilibrium with the rate of Si incorporation into the cell wall (Fig. I. 4. B).
According to the second model involving a prolonged Si starvation period, the internal
Si levels drop to minimum, causing a decrease in Si binding components (Fig. I. 4. E).
Si uptake becomes saturable upon Si replenishment due to Si binding component level
(and hence internal Si pool) reduction (Fig. I. 4. F). The amount of Si binding components increases over time to re-equilibrate the internal Si pool capacity with Si incorporation into the cell wall (Fig. I. 4. D).
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Durak, G.M. Chapter I: Introduction
Fig. I. 4. Diatom Si uptake models. Gray oval represents SDV (after Thamatrakoln and
Hildebrand, 2008).
Depending on the external Si concentration, the modes of Si transportation change. At
Si(OH)4 concentrations exceeding 30 µM, Si transport occurs mainly via simple diffusion due to limitations in the amount of carriers available for facilitated diffusion.
The presence of an inward Si gradient is suggested to be a result of a change in
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Durak, G.M. Chapter I: Introduction chemical form of intracellular Si via complexation with organic compounds
(Thamatrakoln and Hildebrand 2008). Below 30 µM the inward Si transport is likely
SIT mediated (Thamatrakoln and Hildebrand 2008).
Additionally to the above, it has been proposed that the Si efflux, which only occurs if the threshold, intracellular concentration of Si is exceeded (Sullivan 1976) may play a role in equilibration of the internal Si pool capacity and the rate of Si incorporation into the frustule (Thamatrakoln and Hildebrand 2008).
I.3.4. Valve formation: SDV
The valve formation process in diatoms occurs within a specialised, membrane enclosed acidic compartment, derived from coalescence of small, putatively Golgi and endoplasmic reticulum originating vesicles and is termed the Silica Deposition Vesicle
(SDV) (Lee and Li 1992; Li et al. 1989; Schmid 1987). The new valves are formed by highly coordinated interactions between cytoskeleton and cytoplasmic organelles responsible for shaping of the SDV, as well as organic matrix forming components, also involved in subsequent Si deposition (Pickett-Heaps 1990; Schmid 1994; Tesson and Hildebrand 2010). Si transport into the SDV has been proposed to occur via Silica
Transport Vesicles (STVs), as fusion of a type of cytoplasmic vesicles during the SDV maturation has been observed (Schmid and Schulz 1979; Sumper 2004). However, the
STVs are yet to be proven to contain Si (Sumper and Brunner 2008).
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Durak, G.M. Chapter I: Introduction
I.3.5. Components involved in silica polymerization
The most important components involved in diatom biosilicification are two phosphoprotein families: silaffins and silacidins, along with long-chain polyamines
(LCPAs) (Kröger et al. 1999; Kröger and Poulsen 2008a; Sumper and Brunner 2008;
Sumper and Kröger 2004; Wenzl et al. 2008).
LCPAs are linear chains of N-methyl polypropyleneimine attached mainly to a putrescine, ornithine, propylenediamine or spermidine/spermine basis (Kröger et al.
1999; Kröger and Poulsen 2008a; Sumper et al. 2005; Sumper and Kröger 2004).
LCPAs are involved in silicic acid polycondensation and silica sol stabilisation
(Mizutani et al. 1998; Sumper and Kröger 2004). They exhibit both variability within and between diatom species (e.g. differences in chain length, attachment moieties, degree of methylation), (Fig. I. 5.), which properties might be a factor involved in producing the unique frustule ornamentation (Sumper 2004; Sumper and Lehmann
2006). Si polymerization by LCPAs proceeds in the presence of polyanions, i.e. phosphate, which act as cross-linkers, facilitating LCPA assembly into higher order structures, significantly affecting the morphology of the resulting silica precipitates
(Sumper 2004). Some diatom species (e.g. Cylindrotheca fusiformis) do not posses any free LCPAs, but only contain LCPAs attached to a peptide backbone of silaffins
(Kröger et al. 2000; Otzen 2012).
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Durak, G.M. Chapter I: Introduction
Fig. I. 5. Examples of LCPAs from different diatom species with different attachment moieties: propyldiamine - red, putrescine - green, spermidine - blue; n, m - denote propyleneimine unit repeat number (Kröger and Poulsen 2008a).
Silacidins are polyanionic phosphopeptides composed mainly of serine phosphates, aspartic and glutamic acids and have been proven capable of facilitating LCPA assembly by acting as cross-linking molecules, with the LCPA/silacidin ratios controlling the size of Si nanospheres formed (Wenzl et al. 2008). Phosphorylated silacidins are highly efficient at facilitating silica precipitation and their abundance in frustule increases relative to remaining proteins under Si-replete conditions
(Richthammer et al. 2011).
Another group of components involved in guiding the silica deposition via matrix formation due to zwitterionic properties are silaffins (Kröger et al. 1999). These highly post-translationally modified peptides are capable of ionic-interaction driven self- assembly. They consist of a phosphorylated peptide backbone, with repeated polyamine
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Durak, G.M. Chapter I: Introduction modified lysine units forming side-chains (Fig. I. 6.) (Kröger et al. 1999; Kröger et al.
2002). Their role in Si deposition is similar to that of LCPAs (Sumper, Kröger, 2004).
Additionally, a serine-specific group of endoplasmic reticulum-associated silaffin kinases with a capacity to phosphorylate silaffins, but not silacidins has been found to be partially responsible for silaffin phosphorylation (Otzen 2012; Sheppard et al. 2010).
Fig. I. 6. Chemical representation of native Silaffin 1a structure. Charges predicted for solution at pH=5. Polyamine chains circled in red (after Kröger et al., 1999).
I.3.6. Organic matrix of the frustule
Silica frustules of diatoms are underlain by an organic matrix, containing frustule- specific proteins and polysaccharides involved in frustule morphogenesis via active silica precipitation, interaction with other silica precipitating components, or by providing a scaffolding (Kröger and Poulsen 2008a; Scheffel et al. 2011). The ability to
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Durak, G.M. Chapter I: Introduction actively precipitate silica has so far only been documented for one type of organic matrix-forming proteins: cingulins (Scheffel et al. 2011). These water insoluble proteins along with chitin, form biotemplates for girdle bands (Fig. I. 7. gbs and arrows), with a species-specific nanopattern (Durkin et al. 2009; Scheffel et al. 2011).
Another group of proteins isolated from silica frustules are frustulins. These were found to contain multiple acidic and cysteine-rich domains, and to exhibit a specific affinity for Ca2+ ions (Kröger et al. 1994; Kröger et al. 1997). Frustulins create a coat on the frustule once silica deposition has been completed and are suggested to prevent cell wall dissolution (Bidle and Azam 1999; Poll et al. 1999). The last group of proteins isolated to date from diatom organic matrix are pleuralins. These proteins are tightly associated with the biosilica of pleural bands of the epitheca (Fig. I. 7. e), and are suggested to play a role in hypotheca-epitheca differentiation during diatom cell division (Kröger et al. 1997; Kröger and Wetherbee 2000).
Fig. I. 7. A schematic of a diatom frustule. Epitheca (e) and hypotheca (h) are capped
by valves with species-specific patterning. Thecae are composed of silicified,
overlapping girdle bands (gbs and arrows). In some diatoms girdle bands are not
full rings, but taper to fit the curve (the ligula-L) of the adjacent girdle band (after
Hildebrand et al., 2008).
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Durak, G.M. Chapter I: Introduction
I.3.7. Polyanionic polysaccharides
Recently, a polysaccharide containing mannose as the main component has been isolated from Stephanopyxis turris biosilica and suggested to be involved in silicification process (Hedrich et al. 2013). Polysaccharides have been proposed to be components of the silicification system, creating a polyanionic matrix for LCPAs, and thus contributing to control over silica deposition (Chiovitti et al. 2005; Gautier et al.
2008b; Hedrich et al. 2013).
I.4. CHRYSOPHYTES
Chrysophytes (Heterokontophyta: Chrysophyceae) are an algal clade that evolved in the late Precambrian (600 MYA) (Preisig et al. 1991). A large number of species within this group is able to produce siliceous scales, spines, bristles and distinct stomatocysts
(resting cysts) (Preisig 1994; Preisig et al. 1991). Very little is known about the formation of these structures, however, some similarities between diatoms, chrysophytes and synurophytes can be observed, i.e. formation of an SDV occurs during frustule deposition in diatoms, and the encystment process of the two latter classes
(Preisig 1994; Sandgren 1983). The process of stomatocyst silicification has been divided into two phases (Sandgren 1983):
1. Rapid silicification within the SDV resulting in a deposition of a thin primary wall,
a process occurring without close association between the silica structure and the
SDV membrane.
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Durak, G.M. Chapter I: Introduction
2. Thickening of the wall and addition of a collar and surface patterns. This is a much
slower process resembling deposition of highly complex diatom frustules, where
the SDV membrane adheres very closely to the structures formed.
Unlike in diatoms though, the SDV is fully formed prior to deposition of the cyst wall
(Preisig 1994; Sandgren 1983). The pore in the cyst is formed either via rupture of SDV during the early stage of encystment, or from a pre-formed aperture (Preisig 1994;
Sandgren 1983). Recently, Likhoshway et al., (2006) have been able to confirm the presence of a SIT protein in a synurophyte: Synura petersenii and a chrysophyte:
Ochromonas ovalis, concluding that the ability to transport Si into the cell and to biomineralise appeared long before the evolution of diatoms, which appeared only ca.
250 MYA (Medlin 2011).
I.5. HAPTOPHYTES (HAPTOHPYTA: COCCOLITHOPHYCEAE)
Belonging to the haptophyte clade, coccolithophores, next to diatoms, are the most prominent group of marine phytoplankters capable of depositing biomineral structures.
Unlike diatoms, coccolithophores use a calcium carbonate based biomineralisation system, producing calcitic exoskeletons surrounding the cell (Billard 2004).
Haptophytes diverged from Stramenopiles ca. 824 MYA, with the ability to calcify estimated to have evolved ca. 243 MYA or even earlier, between Carboniferous and early Triassic eras (Liu et al. 2010). Within this group, in addition to being able to form organic scales, two orders (Coccolithales and Isochrysidales) posses the ability to produce highly complex calcitic scales (coccoliths), forming a characteristic scale covering (coccosphere) around the cell. The exact function of coccoliths is still
-39-
Durak, G.M. Chapter I: Introduction unknown, however, a number of potential uses have been suggested, such as maintenance of the microenvironment around the cell, regulation of sinking through the water column, protection from pathogens, grazing and physiological implications
(Buitenhuis et al. 1999; Raven and Waite 2004; Young 1994). Coccoliths generally comprise an organic baseplate, on which calcite crystals are deposited in an intricate, species specific pattern (Young and Henriksen 2003). Coccolithophores deposit two types of coccoliths, depending on their ploidy level: heterococcoliths (diploid phase) and holococcoliths (haploid phase) (Young and Henriksen 2003).
I.5.1. Coccolithales and Isochrysidales: calcification
I.5.1.1. Heterococcolith formation
Heterococcoliths consist of an organic base plate on which calcite crystals are radially arranged to form a structure typically consisting of two shields connected by tube elements, and interlock with neighbouring coccoliths to form a mono- or multi-layered cell covering (Fig. I. 8. A,B), (Marsh 2003; Young et al. 1997; Young and Henriksen
2003).
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Durak, G.M. Chapter I: Introduction
Fig. I. 8. Coccolithus pelagicus ssp. braarudi heterococcolith bearing diploid phase: A -
SEM of a whole cell, B-Backscattered Electron (BSE) SEM image of an individual coccolith. Scale bars =5 and 1 µm respectively.
Heterococcoliths are produced intracellularly, within a specialised, Golgi derived compartment - coccolith vesicle (CV, Fig. I. 9.), sometimes associated with additional vesicular extensions termed coccolithosomes (Outka and Williams 1971; Parke and
Adams 1960; Westbroek et al. 1984). Coccolithogenesis begins with deposition of an organic base plate (Young et al. 1999). This acts as a base on which calcite crystal nucleation proceeds, giving rise to the first mineralized structure - the protococcolith ring, which is forms around the edge of the organic scale (van der Wal et al. 1983;
Young et al. 1999). The crystal growth then progresses in an upward and outward direction, with concomitant CV expansion, until the double-shielded coccolith structure is completed (Young et al. 1999). At this stage organic/polysaccharide coating is added, and the mature coccolith is secreted outside the cell and arranged in a particular manner on the plasma membrane (Taylor et al. 2007). Additionally, coccolith associated polysaccharides, putatively involved in control over the growth of individual crystal components, the cytoskeleton, actively shaping the CV and the reticular body are also
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Durak, G.M. Chapter I: Introduction involved in coccolith morphogenesis (Didymus et al. 1994; Drescher et al. 2012;
Fichtinger-Schepman et al. 1981; Langer et al. 2010; Marsh et al. 2002; van der Wal et al. 1983; Westbroek et al. 1984).
Fig. I. 9. A diagram of a coccolithophore cell combining morphological features observed in various coccolithophores. Different coccolith types are represented by silhouettes (black for heterococcoliths, dotted for holococcoliths). Coccolith vesicle
(CV) types of Pleurochrysis and Emiliania are marked at the top right and bottom of the
Golgi body respectively. P1 and 2 are pyrenoids. D - peculiar dilations of the Golgi vesicles, F -flagellum, H - haptonema, N - nucleus, Re - reticular body, CL - columnar deposit, SC - organic scales (unmineralized) - diagram reproduced from Billard (2004).
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Durak, G.M. Chapter I: Introduction
I.5.I.2. Holococcolith formation
Holococcoliths are composed of euhedral rhombohedral calcite crystallites assembled into species-specific shapes and patterns (Fig. I. 10. A,B) (Henriksen et al. 2004;
Young and Henriksen 2003).
Details of holococcolith formation to date are still lacking, however, the process is hypothesized to occur either extracellularly, within an envelope ("skin") compartment closely associated with the plasma membrane (Manton and Leedale 1963; Rowson et al.
1986), or intracellularly. In the latter scenario, the holococcolith deposition might be a rapid process localised just below the plasma membrane, succeeded by immediate exocytosis (Young and Henriksen 2003).
Fig. I. 10. Holococcoliths of Syracosphaera anthos: A - a coccosphere, B - detail of individual coccoliths. Images reproduced from Henriksen et al. (2004).
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Durak, G.M. Chapter I: Introduction
I.5.2. Prymnesiales: Prymnesium neolepis: silicification in haptophytes
P. neolepis (formerly Hyalolithus neolepis) is a widely distributed marine alga belonging to the non-biomineralizing haptophyte order: Prymnesiales. It is the only haptophyte species described to date utilising silica for scale mineralization and subsequent formation of multilayered cell covering, very similar to that produced by coccolithophores (Fig. I. 11.) (Yoshida et al. 2006). The ultrastructural studies by
Yoshida et al. (2006) revealed the intracellular arrangement typical of haptophyte algae, as well as the presence of typical haptophyte unmineralized, organic scales formed in
Golgi body. However, the mineralized scales were not found to be produced in Golgi- associated vesicles, as it is the case in the calcifying coccolithophores. The developing silica scales were found in a compartment suggested to be a silica deposition vesicle more similar to that found in diatoms and chrysophytes, localized on the posterior end of the cell instead (see section II.3.3.). The evolutionary events leading to acquisition of the ability to silicify in this haptophyte are not known. Potential scenarios leading to development of silica biomineralisation system in P. neolepis are further discussed in chapters IV and V.
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Durak, G.M. Chapter I: Introduction
Fig. I. 11. SEM of a P. neolepis cell (after Yoshida et al., 2006). Scale bar=5 µm.
I.6. SPONGES (PORIFERA)
Poriferans evolved between Neoproterozoic and Cambrian periods and are the oldest metazoans (Xiao et al. 2005). Two out of three sponge classes, Demospongiae and
Hexactinelida, utilize amorphous, hydrated silica as a structural component for formation of a skeleton, consisting of siliceous spicules, which can constitute up to 75% dry weight of a sponge (Bergquist 1978; Shimizu et al. 1998). The remaining class,
Calcarea utilizes CaCO3 for the same purpose (Bergquist 1978). Siliceous skeletal parts, spicules, apart from the inherent structural role, have many other functions, such as deterring predators, forming anchoring structures enabling soft sediment colonization and acting as light collecting fibres in an internal, light-signalling system in an Antarctic species Rosella racovitzae, suggested to possess endosymbiotic algae
(Cattaneo-Vietti R. 1996; Müller et al. 2010; Simpson 1984; Wiens et al. 2010).
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Durak, G.M. Chapter I: Introduction
I.6.1. Spicule formation
It has been postulated that all silicified sponges are able to actively accumulate Si by
+ − utilizing a putative Na / HCO3 [Si(OH)4] cotransporter (Schröder et al. 2004a), and to be able to use silicatein-mediated enzymatic processes allowing silica precipitation, using silicon alcoxides as a substrate (Cha et al. 1999; Müller et al. 2007b). Sponges are also able to intracellularly accumulate silica in specialized compartments within sclerocytes, termed silicasomes, also containing silicateins, responsible for the gel-sol silica state. Silicasomes have an ability to exocytose their contents (Schröder et al.
2007; Schröder et al. 2008).
Spiculogenesis is a process regulated by growth, differentiation and Si availability factors, as well as formation of the matrix composed of structural and enzymatic components shaping the spicules (Schröder et al. 2007). The primary structural component controlling spicule formation is galectin, localized concentrically around the spicule, and in unorganized bundles in the extracellular space (mesohyl), interacting with Ca2+ ions to provide a matrix for silicatein and silicase molecule assembly into ring-like structures around the axial filament (Schröder et al. 2007; Schröder et al.
2006; Wang et al. 2011). The secondary structural component, collagen, is involved in formation of a highly organized fibrillar network, further shaping growing spicules
(Schröder et al. 2007). Finally, a pair of anabolic/catabolic enzymes, silicatein and silicase are responsible for polymerizing and depolymerising silica respectively
(Schröder et al. 2006).
The spicule production process is initiated in vesicles of specialized cells, sclerocytes, where a proteinaceous, silicatein-containing axial filament positioned within the axial
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Durak, G.M. Chapter I: Introduction canal is deposited (Fig. I. 12. "ac") (Müller et al. 2005; Simpson 1984). The spicule formation process in demosponges consists of an initial intracellular phase (1), occurring in sclerocytes, and the final phase localized in the extracellular space (2), where the spicules increase in size (appositional growth sub-phase) and acquire their final shape (Müller et al. 2005).
Fig. I. 12. SEM cross section of a Suberites domuncula. Spicule showing axial canal
(ac) and the surrounding Si lamellae deposited during the appositional growth (image after Müller et al., 2006).
1. Intracellular phase:
Si substrate is actively transported into sclerocytes, and stored in specialised vesicles - silicasomes prior to deposition (Schröder et al. 2006; Wang et al. 2012). Silicatein in a form of a pro-enzyme is transported through endoplasmic reticulum and Golgi, undergoes post translational modification, mainly concerning phosphorylation and is subsequently transported to vesicles within sclerocytes, where the mature enzyme is
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Durak, G.M. Chapter I: Introduction deposited in a form of a rod-like, axial filament, followed by deposition of the initial Si layer (Fig. I. 13. A, C, E-G) (Müller et al. 2006; Müller et al. 2005).
2. Extracellular phase
Immature spicules are extruded into the extracellular space, where the appositional growth commences, increasing the length and the diameter of the spicule due to silicatein presence in both extracellular space and the surface of the lamellar depositions (Fig. I. 13. B, H, I, Fig. I. 5.). Silicateins have been shown to form larger, galectin and collagen associated string-like aggregations parallel to the spicule surface, where their function is to mediate lamellar and stepwise Si deposition (Müller et al.
2006; Müller et al. 2005).
3. Spicule hardening via water syneresis (removal)
Water, a by-product of silica polycondensation trapped in the galectin scaffolding surrounding the growing spicule is taken up by cells that then migrate away from the silicification site to maintain water equilibrium (Brinker and Scherer 1990; Wang et al.
2012). This process was also suggested to be implicated in shaping of spicules via localised water removal (Uriz 2006).
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Durak, G.M. Chapter I: Introduction
Fig. I. 13. Immunogold TEM images of primmorph cross sections: nanogold anti-rabbit
IgG visualization of the immune complexes between the polyclonal anti-silicatein antibodies. A-Sclerocytes with silicatein molecules, B-concentric rings (ri) surrounding the immunostained silicatein. C-first electron dense ring associated linear clods (> <) .
D-fusion of the inner rings, formation of linear clods, E,F-Si accumulation in the centre of the multi-lamellar silicatein-containing material. G-deposition of the first Si layer onto the spicule (sp), H-further growth of the silica layer and an increase in the ring (ri) number. I-silicatein axial filament (af) with Si rings. Scale bars=2 μm.
Images reproduced from Müller et al. (2006).
Exogenous Si in demosponges and hexactinellids is also an element up-regulating a number of spicule formation-associated genes, such as those coding for silicase
(Schröder et al. 2003), galectin (Schröder et al. 2006), collagen (Krasko et al. 2000), and morphogenetic protein, noggin (Schröder et al. 2004b). Overall, the spiculogenesis
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Durak, G.M. Chapter I: Introduction process in sponges is highly energetically expensive, requiring active exogenous Si transport, followed by internal transportation to specialised storage and spicule-forming cells and production of structural and enzymatic components involved in spicule morphogenesis (Schröder et al. 2004a; Schröder et al. 2008).
I.6.2. Silicateins
Belonging to the papain-like cysteine protease superfamily and most closely related to cathepsin family of proteases, silicateins exist in three isoforms: α, β and γ (Cha et al.
1999; Schröder et al. 2008; Shimizu et al. 1998). Silicateins exhibit cathepsin L-like proteolytic ability, as well as their main silica polymerase and esterase activity (Cha et al. 1999; Müller et al. 2003; Schröder et al. 2008; Shimizu et al. 1998). As an in vitro catalyst for silica polymerization, silicatein acts as an esterase, sequentially hydrolysing silicon alkoxides, allowing for condensation of the produced silanol groups (Fig. I. 14.
A) (Schröder et al. 2007). As silica polymerase, silicatein is involved in sequential addition of silica monomers (Schröder et al. 2008). The in vivo catalytic function of silicatein is interlinked with a range of other components occurring in sponges, allowing Si(OH)4 or even SiO2 to be used as substrate for the enzyme (Müller et al.
2003). The catalytic properties of silicatein have been proposed to result from presence of the imidazole nitrogen of the histidine and the hydroxyl group of serine localized in the active site of the enzyme (Fig. I. 14. A), (Zhou et al. 1999). The hydrolytic activity of silicateins has been shown to be unspecific, as other metal oxides, such as TiO2 and
ZnO2 have been precipitated using this enzyme (Bansal et al. 2004; Sumerel et al.
2003).
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Durak, G.M. Chapter I: Introduction
Fig. I. 14. Silicatein: A-Active silicatein enzyme. Cc-catalytic centre, signal sequence displayed in green. Serine (Ser) cluster amino acids marked in red (Ser26, His165
Asn185), (after Schröder et al., 2007). B-silicatein mediated hydrolysis of bis(p- aminophenoxy)-dimethyLsilane. Serine oxygen localized in the silicatein catalytic centre mediates a nucleophilic attack on the silicon, displacing the p-aminophenol. This is followed by formation of a (alkoxyl)-monosilane and facilitated by formation of a hydrogen bond between the imidazole nitrogen of Histidine (His) and Serines’ hydroxyl group. (after Müller et al., 2008).
An alternative hypothesis for the role of silicateins in spicule formation has been proposed by Ehrlich et al. (2008). In their model, silicateins are suggested to exhibit collagenolytic capacity similar to that of cathepsins. It is then proposed, that silicateins are involved in restructuring of collagen, forming templates on which silica is precipitated rather than being actively involved in silica deposition. Instead, silica precipitation is suggested to be a non-enzymatic self-assembly process, promoted by
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Durak, G.M. Chapter I: Introduction the positively charged amine groups of collagen matrix (Ehrlich et al. 2008; Heinemann et al. 2007).
I.6.3. Silicase
Located both on the surface and within the axial filament of a growing spicule, silicase is a member of carbonic anhydrase family (Fig. I. 15. A) and catalyzes silica depolymerisation (Schröder et al. 2003). The reaction of silicase-mediated silica depolymerization (Fig. I. 15. B) proposed by Schröder et al. (2007) involves a reaction between Zn ion, acting as a Lewis acid and H2O (Lewis base). Zn ion binds the hydroxide group of H2O, and then facilitates a nucleophilic attack on the oxygen-bound
Si. Subsequently, the zinc complex binds to the Si atom of the silica polymer, cleaving the oxygen bond and releasing Si(OH)4 in a reaction requiring consumption of water for regeneration of the hydroxide group to continue the catalytic cycle.
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Durak, G.M. Chapter I: Introduction
Fig. I. 15. A - model of silicase: 3 histidine residues marked in red (His181, His183
His206), cc - catalytic centre bound to a Zn2+ ion. B - the proposed mechanism of Si polymer hydrolysis (after Schröder et al., 2007).
I.6.4. Silintaphins
Silintaphins 1 and 2 do not resemble any known proteins, apart from Silintaphin 1 possesing a pleckstrin homology domain (Wiens et al. 2009). Both proteins co-localize with silicateins inside and around spicules, controlling silica deposition via interaction with silicateins (Wiens et al. 2009; Wiens et al. 2011) Silintaphin 1 is thought to interact with silicatein to promote fibre formation and assembly into organized structures (Wang et al. 2012; Wiens et al. 2009). Silintaphin 1 was also reported to significantly increase the catalytic activity of the silicatein enzyme, as well as to promote non-enzymatic silica polycondensation (Schloßmacher et al. 2011). Silintaphin
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Durak, G.M. Chapter I: Introduction
2 exhibits Ca2+ binding affinity, and is suggested to facilitate silicatein interaction with
Si substrate and signal transduction during spicule morphogenesis (Wang et al. 2012;
Wiens et al. 2011).
I.6.5. Long chain polyamines
An interesting discovery was also made by Matsunaga at al.. (2007), namely isolation of long chain polyamines (LCPAs ) similar to those controlling biosilicification in diatoms. Sponge LCPAs were identified as oligomeric, methylated chains of polypropylamines present as sulphates or phosphates, attached to a butaneamine unit. It has been suggested that LCPAs of sponges facilitate silicification in association with larger molecules, as their presence in spicules as well as in cellular extract has been verified.
I.7. PLANTS
Silicon in plants is an important quasi-nutrient accumulated in substantial amounts, ranging from 0.1-10% of dry weight (Epstein 1994). It is essential for growth of plants from the Equisetales order (Chen and Lewin 1969). Silicon has been shown to be an inductor of defensive responses via interaction with plant signalling system and increasing synthesis of defence compounds, enhancing their resistance to various pathogens (Callot 1992; Raven 1983). Si has also been implicated in alleviation of abiotic stress, such as dehydration, or uptake of toxic metals due to Si deposition
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Durak, G.M. Chapter I: Introduction beneath the cuticle, decreasing cuticular transpiration, chelation and decreased uptake of toxic metals due to Si deposition in roots and as phytoliths (Hinsinger et al. 1992;
Sangster et al. 2001). Si in plants also serves as a structural component, promoting cell elongation, enhancing the strength of the stem and maintaining leaves erect, contributing to improvement of light interception (Epstein 1994; Ma and Takahashi
2002). Si also seems to play a very important role in enhancing growth at different developmental stages in e.g. rice, where Si deficiency during the heading stage results in severe decrease in grain yield (Yamaji and Ma 2007).
I.7.1. Si transporters
Si(OH)4 transport in plants is an active, energy requiring process (Mitani and Ma 2005).
Expressed primarily in roots, plant Si transporters belong to the aquaporin gene family and are upregulated by the level of exogenous Si, diurnal pattern, and down regulated by stress caused by dehydration and abscisic acid (Hattori et al. 2003; Ma et al. 2006).
Two coupled Si transporters, influx (Lsi1) and efflux (Lsi2) exist in the root cells and are responsible for Si transport into the stele for further distribution within the plant
(Ma et al. 2006; Ma et al. 2007). Subsequently, located mainly at the leaf xylem parenchyma cells, Lsi6 (Lsi1 homologue) becomes responsible for Si distribution within the shoot and its unloading from xylem (Yamaji et al. 2008). Lsi1 belongs to a subfamily of Nod26 major intrinsic protein (NIPIII) unique to plants, and is localized within the plasma membrane of both endo- and exodermis of the root cells (Ma et al.
2006). Lsi2 is a proton gradient driven, energy dependent putative anion transporter bearing no resemblance to Lsi1 (Ma et al. 2007). Si transporters belonging to the NIPIII
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Durak, G.M. Chapter I: Introduction subfamily were previously found in e.g. rice, wheat pumpkin and barley (Chiba et al.
2009; Ma et al. 2006; Ma et al. 2007; Mitani et al. 2011; Montpetit et al. 2012).
Another group of plant Si transporters described, NIP3s, belongs to NIPII protein group responsible for transporting of small, uncharged molecules (Grégoire et al. 2012).
These transporters were found to be highly expressed in roots and shoots of a horsetail
Equisetum arvense, where Si transporters were mainly localized to roots (Chiba et al.
2009; Grégoire et al. 2012; Ma et al. 2006).
NIP selectivity for Si(OH)4 has been suggested to be linked with two highly conserved aspargine-proline-alanine motifs and four aminoacid residues of the aromatic/arginine substrate selectivity filter in the pore (Grégoire et al. 2012; Wu and Beitz 2007). All previously described NIPIII plant Si influx transporters are characterised by GSGR
(glycine, serine, glycine, and arginine ) pore, whereas the NIPII NIP3s possess STAR
(serine, threonine, alanine, arginine) pores, (Grégoire et al. 2012; Mitani-Ueno et al.
2011).
I.8. SUMMARY AND AIMS
In summary, in all taxa described in this review Si serves multiple functions, from structural component to regulation of gene expression (Thamatrakoln and Hildebrand
2007). In terms of transport, in all cases Si is taken up in a soluble form of silicic acid.
Apart from that, the mechanisms of silicification described in this chapter were quite divergent, the plant system being entirely unique to this taxon. The sponge and diatom silicification systems bear little resemblance to each other. Sponges posses the ability to silicify both intracellularly in sclerocytes and extracellularly, upon the extrusion of the
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Durak, G.M. Chapter I: Introduction young spicule into the mesohyl, whereas the entire biosilicification process in diatoms or chrysophytes is confined to an intracellular SDV (Sandgren 1983; Schröder et al.
2007; Schröder et al. 2006). Also, the principal components involved in silicification in those organisms function on a fundamentally different basis: in diatoms Si deposition proceeds via Si polycondensation by silaffins and LCPAs in the presence of polyanions, whereas in sponges silicification is mediated enzymatically by silicateins, or alternatively it is facilitated by collagen (Ehrlich et al. 2008; Ehrlich and Worch 2008;
Schröder et al. 2006; Sumper and Brunner 2008). However, the detection of LCPAs in sponges suggested, that there might be more similarities between the two systems than it was initially anticipated, perhaps arising from the chemistry of Si, potentially promoting convergent evolution. An interesting suggestion was made by (Likhoshway et al. 2006), namely that the ability to silicify evolved simultaneously in different taxa around 600 MYA due to the high silicate content of the sea water, which could have facilitated the incorporation of Si into the metabolic cycles of organisms such as poriferans and chrysophytes, also implying that the system present in diatoms did not evolve on its own and was acquired from chrysophytes instead. Indeed, similarities in the cellular and molecular mechanisms of biosilicification (diatom-like SITs, the presence of an SDV) have been identified (Likhoshway et al. 2006).
The discovery of a silicifier in the calcifier-dominated haptophyte clade, P. neolepis
(Yoshida et al. 2006) has also raised questions, about whether this system evolved de novo, was acquired via convergent evolution, or whether a horizontal transfer of genes from other silicifiers, e.g. diatoms has occurred. Recently, diatom-like SITs have also been detected in an opisthokont choanoflagellate (Marron et al. 2013) suggesting occurrence of horizontal gene transfer from SIT-possessing algae (e.g. diatoms) to choanoflagellates, which have previously been documented to acquire other algal genes
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Durak, G.M. Chapter I: Introduction in this manner (Marron et al. 2013; Nedelcu et al. 2008; Sun et al. 2010). It was further suggested by Marron et al. (2013) that biomolecules with a capacity to precipitate silica could exist in various taxa, however, capacity for biosilicification could only be attained, if Si-selective transport and concentration systems are in place, allowing Si substrate acquisition. Still, very little is known about the molecular mechanisms of silicification in different taxa, hence the theories proposed by both Likhoshway et al.
(2006) and Marron et al. (2013) remain to be proven with more research.
Given the above information and a unique opportunity to investigate the sole silicifying representative of the haptophyte algae the main aims of this project are to address the following questions:
1. Why and how is P. neolepis silicifying?
2. How does the P. neolepis silicification system compare to calcification in
coccolithophores and silica deposition systems in other taxa?
3. What is the evolutionary origin of the silicification system in P. neolepis?
These questions are further elaborated on and addressed in chapters II-V.
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Durak, G.M. Chapter II: General organism description
CHAPTER II: PRYMNESIUM NEOLEPIS: GENERAL ORGANISM DESCRIPTION, BIOLOGY
AND PHYSIOLOGY OF SILICIFICATION
II.1. INTRODUCTION
Haptophytes are a group of mainly marine algae characterised by the presence of a unique flagellum-like appendage, in some cases used for prey capture: the haptonema
(Kawachi et al. 1991). Haptophytes were estimated to have diverged from
Stramenopiles ca. 824 MYA, with extant groups appearing ca. 543 MYA (Liu et al.
2010). Haptophytes are divided into two classes: Pavlovophyceae and
Prymnesiophyceae. Pavlovophytes are represented by a single order, Pavlovales, characterised by asymmetrical cell shape sometimes covered with knob-shaped organic scales/modified hairs and unequal (anisokont) flagella (Cavalier-Smith 1994; Green
1980; Green and Hori 1994). Prymnesiophytes are divided into six orders, of which four
(Coccolithales, Isochrysidales, Syracosphaerales and Zygodiscales) have an ability to produce calcified scales (coccoliths) and two (Phaeocystales and Prymnesiales) do not calcify.
Diverged ca. 291 MYA the order Prymnesiales is represented by widely distributed, often toxic bloom forming flagellates such as Prymnesium and Chrysochromulina and an exclusive within the entire haptophyte clade, silica scale producing species (Fig. II.
1. A-C) (Lundholm and Moestrup 2006; Simonsen and Moestrup 1997; Weissbach and
Legrand 2012; Yoshida et al. 2006). This unique silicifier - Prymnesium neolepis - was previously known as Hyalolithus neolepis and was then reassigned into the Prymnesium genus based on additional molecular and taxonomical analyses (Edvardsen et al. 2011;
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Durak, G.M. Chapter II: General organism description
Yoshida et al. 2006). In contrast to the remainder of Prymnesium genus, comprising of highly motile flagellates with a flexible, non coiling haptonema, P. neolepis persists in a non-motile stage in culture (Fig. II. 1. A,C) (Green et al. 1982; Yoshida et al. 2006).
Non-motile P. neolepis cells possess two short, quiescent flagella, a long, stationary haptonema and a characteristic silica scale cover overlying organic scales (Fig. II. 1. C)
(Yoshida et al. 2006). The silica scales of P. neolepis are non-interlocking hat-shaped, with two apices, a recurved rim and regular perforations over the central part of the scale (Fig. II. 1. C). Both the silica scales and the significantly smaller, organic scales similar to those of closely related Prymnesium and Chrysochromulina sp. are produced intracellularly and then deposited outside the plasma membrane (Yoshida et al. 2006).
A motile phase in P. neolepis cultures was also observed. Motile cells are characterised by the presence of two long flagella and lack of silica scales. A cell cover consisting of organic scales of variable size, similar to those of closely related C. polylepis was observed instead (Yoshida et al. 2006). These flagellated cells appear in cultures maintained at 10 ºC under oligotrophic conditions and frequently revert back to the non- motile, silicified phase (Yoshida et al. 2006). It is currently unknown whether the non- motile stage is also dominant under environmental conditions, however, silica scales and whole silicified cells of the non-motile P. neolepis stage have been reported sporadically in samples from all over the world (Yoshida et al. 2006).
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Durak, G.M. Chapter II: General organism description
Fig. II. 1. A - P. parvum light microscopy image reproduced from the Encyclopaedia of
Life (© University of Liverpool), B - C. kappa, a close relative of Prymnesium: shadowcast electron microscopy image with a characteristic coiled haptonema, reproduced from Parke et al., (1955), C - SEM image of Prymnesium neolepis (after
Yoshida et al., 2006). Haptonemas were marked with arrows. Scale bars=5 µm, C. kappa cell size≈5 µm.
II.1.1. Putative capacity for silicification in Prymnesium sp.
Earlier, preliminary reports on Prymnesium sp. cyst formation in culture indicated, that cyst wall associated organic scales are covered with silica-containing fibrous material on their distal faces, hinting, that Prymnesium sp. might have a capacity to silicify (Fig.
II. 2. A,B) (Green et al. 1982; Pienaar 1980; Pienaar 1981). The presence of Si- containing fibrous deposits was suggested to be linked with small, oval bodies localized immediately below the plasma membrane and occasionally associating with organic scales (Pienaar 1980). However, the presence of Si detected in the cyst wall of
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Durak, G.M. Chapter II: General organism description
Prymnesium in this case could be an artefact of culturing conditions and will be discussed later in this chapter.
Fig. II. 2. Electron micrographs of Prymnesium sp.: A - organic scales with electron dense, fibrous, Si containing material (arrows), B - whole cyst (arrow: cyst wall composed of organic scales with fibrous deposits). Images modified from Pienaar
(1980).
This chapter presents studies of the physiology of silica scale production as well as the sequence of events involved in the formation of individual silica scales. The original morphological description of P. neolepis by Yoshida et al. (2006) is also supplemented with information on some biological and biochemical properties of this alga.
Comparisons in terms of cell morphology and scale formation with other members of
Prymnesium, closely related Chrysochromulina and biomineralizing coccolithophores and diatoms are made. This chapter also contributes the basis for further investigations undertaken in this work.
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Durak, G.M. Chapter II: General organism description
II.2. METHODS
All chemical reagents used in this investigation were obtained from Sigma-Aldrich, unless otherwise specified.
II.2.1. Culture conditions
Prymnesium neolepis strains TMR5, PZ241 and VF28 were obtained from Roscoff
Culture Collection and were originally isolated from the Mediterranean Sea (PZ241,
VF28) and from around Japan (TMR5). A diatomm Odontella sinensis strain PLY624 and a coccolithophore, Coccolithus pelagicus ssp braarudii strain PLY182g were obtained from the Plymouth Culture Collection of Marine Microalgae. Algae were maintained in aged filtered sea water (FSW) supplemented with f/2 media (Guillard and
Ryther 1962) under irradiance of 80-100 µmol/s/m2, at 18 ˚C for P. neolepis and 15 ˚C for the remaining strains. Additionally, P. neolepis and O. sinensis were supplemented with 100 µM silicate. Photoperiod used for maintenance of P. neolepis was set to 18:6 h light:dark and at 12:12 light:dark for other algal species. Dilute batch cultures were maintained in glass conical flasks, as cells would not grow well in plastic vessels and cultures reaching densities ≈105 cells/ml would rapidly collapse. Culture health and density were periodically monitored under a light microscope. Cell counts were done using a Sedgewick-Rafter cell counting chamber. Three samples per experimental repeat were aliquoted and counted.
Artificial Sea Water (ASW) used in high/low silica experiments was prepared according to a recipe by Dr. Alison Taylor (Table II. 1.). Chemicals were dissolved in distilled
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Durak, G.M. Chapter II: General organism description
water, autoclaved and 0.22 µm filtered NaHCO3 was added after the solution has cooled.
Table II. 1. Chemical composition of ASW.
Chemical Concentration [mM]
NaCl 450
MgCl2 30
MgSO4 16
KCl 8
CaCl2 10
HEPES 20
NaHCO3 2
II.2.2. DNA extraction, gene amplification, sequencing and molecular analysis
DNA extractions were made using DNAeasy blood and tissue Kit (Qiagen) following the manufacturer's instructions, or a standard phenol/chloroform protocol. Small subunit rRNA gene (18S, SSU) and the large subunit of the ribulose-bisphosphate carboxylase
(rbcL) marker genes were amplified using universal primers (Table II. 2.) and then sequenced for three P. neolepis strains: TMR5, VF28 and PZ241.
PCR conditions for gene amplification were as follows: 35 cycles, each at 95 °C for
5min, 95 °C for 30 s, and 1 min at 56 °C for rbcL and 58 °C for 18S, followed by 72 °C for 1 min. Prior to sequencing, PCR products were treated with ExoSapit (Affymetrix) according to manufacturer's instructions to remove excess primers, dNTPs, and single
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Durak, G.M. Chapter II: General organism description stranded DNA. Cleaned PCR products were then amplified again (PCR cycle: 96 °C for
1min, 96 ºC for 10 s, 55 °C for 5 s, individual annealing temperatures as specified in
PCR conditions above for 4 min) and sent off for sequencing, performed by Source
Bioscience (Cambridge).
Table II. 2. Primers used for amplification of SSU, LSU and rbcL genes.
Amplicon Loci Primers Primer Sequence 5'-3' Reference size (bp)
rbcL (Yoon et rbcL CCATATGC(CT)AAAATGGGATATTGG 673 090F al. 2002)
rbcL ATACATTTCTTCCATAGTTGC 770R
A18 (Medlin et SSU AACCTGGTTGATCCTGCCAGT 1688 DIR al. 1988)
A18 TCCTTCTGCAGGTTCACCTAC REV
A phylogenetic tree based on the rbcL gene and including haptophyte representatives was constructed to ascertain that the strains in culture were indeed P. neolepis.
Additionally, an SSU-based tree including P. neolepis, other haptophytes and representatives of heterokontophyte silicifiers was produced. In both cases trees have been constructed with a neighbour-joining method using Kimura 2-parameter model to compute evolutionary distances and with maximum likelihood method, using Kimura 2- parameter gamma distributed with invariant sites models in MEGA5 software (Kimura
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Durak, G.M. Chapter II: General organism description
1980; Tamura et al. 2011). A bootstrap of 1000 replicates was used in both cases. SSU and rbcL sequences of P. neolepis and relevant haptophytes and heterokontophytes were aligned with MAFFT (Multiple Alignment using Fast Fourier Transform) online utility. P. neolepis rbcL sequence alignment along with additional sequences obtained from GenBank and used for rbcL and SSU alignments were supplemented as electronic appendices 1-3.
II.2.3. Confocal microscopy
Silica scale formation was tracked with a fluorescent probe, LysoTracker yellow HCK-
123 (Invitrogen), visualised with argon laser at λ=488 nm excitation, and emission collected between λ=500-550 nm. Chlorophyll autofluorescence was collected simultaneously between λ=650-710 nm. Calcite was imaged with a He/Ne laser with
λ=633 nm excitation and emission filter set between 650-700 nm. FITC-Concanavalin-
A (Invitrogen) used for targeting polysaccharides and binding to β 1,3- and β 1,4- linkages between glucose and mannose backbone (Mandal et al. 1994) was imaged using an argon laser, with excitation and emission filters set to λ=488 nm and λ=500-
550 nm respectively.
LysoSensor Yellow/Blue DND-160 (PDMPO, Invitrogen) also used for tracking the new silica scale deposition and silica scale reflectance were imaged using multiphoton excitation with a Mai Tai pulsed infra-red laser (Spectraphysics) set to λ=740 nm.
Emission was collected between λ=435-485 nm and λ=500-550 nm for PDMPO and with a 560 nm long pass filter for silica scale reflectance. All imaging was carried out on a Zeiss LSM 510 meta confocal laser scanning microscope.
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Durak, G.M. Chapter II: General organism description
Time lapses: time frames were taken every 4 mins for time-lapse 1, and at 150 s intervals for time-lapse 2. Time frames for time-lapses 3,4 and 5 were taken every 45 s.
List of all time-lapses with their brief description is attached in appendix II.1. All time- lapses were supplied as an electronic appendix.
II.2.4. Differential Interference Contrast (DIC)/light microscopy imaging
DIC images were taken with a Nikon Eclipse Ti microscope equipped with a
Photometric Evolve EMCCD camera. Images were taken every 45 s for all time lapses.
Transmitted light imaging was done using a Nikon Diaphot 300 microscope with an attached Photometrics CoolSnapEZ CCD camera.
II.2.5. NaOH etching of biosilica material
P. neolepis biosilica purified with the KMnO4 method detailed in section II.2.6. and was subsequently etched with 0.5-10 M NaOH for a period of 4-6 h to gain insights into silica deposition pattern in silica scales. Samples were then washed 3x with deionised water and kept at -20 °C until SEM analysis.
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Durak, G.M. Chapter II: General organism description
II.2.6. Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Energy Dispersive Microanalysis (EDX)
Samples were filtered onto a 0.45 µm filter which was dried or placed directly onto the stub, sputter coated with gold or chromium for imaging and with carbon for EDX analysis. Imaging was carried out using a JEOL JSM-7001F Field Emission Microscope at 5-15 kV accelerating voltage. Images were acquired with JEOL JSM-6610LV and
JEOL JSM 5600 Scanning Electron Microscopes at 15 kV. EDX analysis was carried out using a JEOL 6100 Scanning Electron Microscope and with JSM-7001F Field
Emission Microscope at 7-15 kV, using Oxford instruments Inca and Aztec X-Ray micro-analysis suites respectively.
TEM imaging of non-mineral material on copper grids (Agar) stained with 2% uranyl acetate for 30 s was carried out using a JEOL 1400 Transmission Electron Microscope.
II.2.7. Fourier Transform Infrared Spectroscopy (FTIR)
Biosilica samples from P. neolepis and O. sinensis were purified using an SDS/EDTA treatment as detailed in section IV.2.1. and analyzed with a Bruker IFS-66 FT-IR spectrometer attached to a Bruker Hyperion 1000 microscope operating in transmission mode. Spectra were recorded between 600 cm-1 and 4000 cm-1 at 4 cm-1 resolution, with
32 scans/spectrum. The baseline was corrected using the "rubberband correction" method with a standard preset, included in Bruker OPUS software. Silica gel was obtained by adding 0.5 M sulphuric acid to a saturated solution of sodium metasilicate.
Gel was then washed 3x with deionised water and dried in a flow hood prior to FT-IR analysis.
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Durak, G.M. Chapter II: General organism description
II.2.8. Light: dark experiment: flow cytometry assessment of silica scale production
1 ml of concentrated exponentially growing culture (density between 1-1.5x105 cells/ml) was added to 4 ml of fresh f/2 media in FSW at pH 8.20, with 100 µM Si supplement. Cells were then labelled with HCK-123 (1 µM) and incubated in light at
100 µmol/s/m2 and in dark for 10 h. Cells were then harvested by centrifugation and washed 3x with 2% SDS with 0.1 M Tris/EDTA (pH 8.00), and incubated for 15 mins with intermittent vortexing to remove cellular debris. Samples were then pelleted with a bench top centrifuge at max rpm, and washed 3x with deionised water. Samples were analysed using an Accuri C6 flow cytometer set to record side scatter and green fluorescence (SSC vs. FL1A), gated for fluorescent and non-fluorescent particles.
Experiments were repeated five times in triplicate. Ratios of fluorescent (new) vs. non- fluorescent (scales produced prior to the treatment), serving as a proxy of silicification in treatments were compared using a student's t-test for independent samples (Statsoft:
Statistica).
II.2.9. High/Low Si experiment
Initial attempts to grow P. neolepis in low silica (< 3 µM, below quantification level with the silicomolybdate assay) ASW in batch cultures were unsuccessful. No growth was observed and within a few days of the start of the experiment cells could no longer be found in culture upon examination with light microscopy. Therefore, a short-term experiment aimed to investigate P. neolepis response to low Si conditions was performed. Cells grown in FSW f/2 media without silicate enrichment were settled in glass bottom petri dishes (5 control and 5 low Si dishes in total) and washed 5x with
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Durak, G.M. Chapter II: General organism description low Si (< 5 µM) f/2 ASW. Media were then replaced with 100 µM Si and f/2 ASW for control dishes and low Si f/2 ASW for the low Si experiment. Dishes were then incubated for 18h with 1 µM PDMPO at 18 ºC with a 16:8 h light:dark cycle at 150
µmol/s/m2. After the period of incubation, 10 images/dish were taken for estimation of cell density. 1ml sample was aliquoted from each dish for subsequent determination of
Si concentration in the media post incubation (refer to section II.2.10. for detailed method description). Cells were then lysed with 0.5 ml of 10% H2O2 and left for 20 min to allow the silica scales to settle on the bottom of the dish. 10 pictures/dish were taken for estimation of silica scale production. Imaging was carried out using a Zeiss
Confocal microscope as specified in section II.2.3.
Images were then processed with Image J software by thresholding grey values to remove background fluorescence and select for values above a certain threshold, corresponding to scale fluorescence. This allowed calculation of the number of particles and the total area of particles in each image, an average number of scales per cell and average scale area/cell.
II.2.10. Si concentration determination: silicomolybdate assay
Silica concentrations were determined following a colorimetric method modified from
Strickland and Parsons (1968). This assay relies on formation of a silicomolybdic acid complex via reaction of ammonium heptamolybdate with silicic acid. This complex is subsequently reduced to eliminate interference from phosphorous, resulting in formation of molybdenum blue complex, allowing absorption corresponding to silica concentrations to be measured (Coradin et al. 2004; Strickland and Parsons 1968).
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Durak, G.M. Chapter II: General organism description
Samples for the analysis were prepared as follows: 0.3 ml of the molybdate reagent was added to a 0.75 ml sample. Sample was then vortexed and incubated for 10 min at room temperature. Next, 0.45 ml of the reducing reagent was added and the sample was vortexed. The sample was then incubated for 3 h with occasional vortexing. Absorbance at 810 nm was then determined with a Varian Cary 100 Bio UV-Visible
Spectrophotometer. Calibration curves were based on a series of dilutions of standard silicate solution in synthetic seawater (1/1000, 1/500, 1/100, 1/50) and distilled and synthetic seawater blanks, all set up in 4 replicates. Reagents used in the analysis are listed in Table II. 3.
Table II. 3. List of reagents used in the silicomolybdate assay and their composition.
Solution/volume Chemicals
Molybdate reagent 4 g (NH4)6Mo7O24*4H2O, 12 ml 12 M HCl (500 ml)
Reducing reagent 10 ml of metol-sulphite solution (6 g Na2SO3, 10 g Metol,
(30 ml) filtered), 6 ml oxalic acid at 71.44 g/l, 6 ml 0.5 M H2SO4
Synthetic Seawater 25 g NaCl, 8 g MgSO4*7H2O (1 l)
Silicate standard 0.96 g Na2SiF6 (1 l)
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Durak, G.M. Chapter II: General organism description
II.2.11. Ge supplementation of P. neolepis cultures and biosilica material purification for EDX analysis
Cultures were set up in triplicate and maintained as specified in section II.2.1., in f/2 supplemented filtered sea water with 100 µM Si enrichment in the control and 50 µM Si with 3 µM Ge or 50 µM Ge without Si supplementation in test cultures. Growth was monitored during the experiment, and once cell counts were no longer possible due to cells aggregating together, cultures were harvested and biosilica material was purified for SEM analysis as specified below.
Cell culture pellets were treated with KMnO4/HCl, following a modified method for diatom frustule cleanup by Diana Sarno, obtained during a phytoplankton identification course hosted by the MBA and SAHFOS. P. neolepis pellets were washed 3x with deionised water, resuspended in 500 µl of deionised water and transferred to 3 ml glass bottles. 0.5 ml of saturated KMnO4 was then added, the bottle contents were vortexed and left to incubate at room temperature overnight. Solutions were then transferred to glass test tubes, placed in a heating block at 90 ºC and 2 ml of concentrated HCl was added. Samples were then boiled until the colour changed to clear yellow. Samples were transferred to 50 ml tubes containing 45 ml of deionised water to neutralise them and centrifuged for 20 min at 6000x g. Pellets were washed 3x with deionised water and stored at -20 ºC until analysis with SEM and EDX as specified in section II.2.6.
Additionally, P. neolepis responses to 10, 20 and 50 µM Ge supplementation were monitored using fluorescent dye HCK-123, and visualised with a confocal microscope as specified in section II.2.3. For this experiment, samples were set up in duplicate on glass bottom dishes, incubated for 18 h with Ge concentrations specified above and
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Durak, G.M. Chapter II: General organism description maintained in conditions as specified in section II.2.1. Control samples were supplemented with 100 µM silicate.
II.3. RESULTS
II.3.1. Molecular identification of P. neolepis strain
To ensure, that the newly isolated strains in culture were in fact P. neolepis, amplification and sequencing of 2 marker genes: rbcL (all three strains) and of SSU gene for strain TMR5, has been carried out. Phylogenetic analysis of sequences obtained from P. neolepis in this study, and those downloaded from GenBank confirmed, that all three strains examined (TMR5, VF28 and PZ241) are in fact P. neolepis (Fig. II. 3., Fig. II. 4.). Although all strains are strongly clustered together with a strong bootstrap support, small differences between strains on both rbcL and SSU genes have been recorded (Table II. 4.). These are likely due to different geographical locations of sites from which the sequenced strains were collected. Strains PZ241 and
VF28 were originally collected from the Mediterranean Sea, and are identical on the rbcL gene, whereas TMR5 and NIES1393, both collected around Japan are different from the Mediterranean strains and less dissimilar between each other (Table II. 4., Fig.
II. 3.).
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Durak, G.M. Chapter II: General organism description
Table II. 4. A summary of the degree of homology between rbcL and SSU gene sequences obtained from P. neolepis strains isolated from different locations.
Strain Gene % Identity Strain Colleciton Site
TMR5 vs. PZ241 rbcL 99.85 TMR5 Japan
TMR5 vs. VF28 rbcL 99.85 NIES1393 Japan
TMR5 vs. NIES1393 rbcL 99.26 PZ241 Mediterranean Sea
PZ241 vs. VF28 rbcL 100.00 VF28 Mediterranean Sea
PZ241 vs. NIES1393 rbcL 99.11
VF28 vs. NIES1393 rbcL 99.11
TMR5 vs. NIES1393 SSU 98.95
The phylogenetic position of P. neolepis strains examined in this study is consistent with that described by Edvardsen et al. (2011) and the previous study by Yoshida et al.
(2006), placing P. neolepis within the order Prymnesiales of the haptophyte clade (Fig.
II. 3.). The distant relationship between P. neolepis, clustering within the haptophyte lineage and silicifiers of the heterokontophyte lineage is depicted in Fig. II. 4.
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Durak, G.M. Chapter II: General organism description
Fig. II. 3. Unrooted maximum likelihood phylogenetic tree of selected haptophytes based on the rbcL gene sequences. Numbers at nodes indicate bootstrap support.
Branches marked with: > 90% and > 70% bootstrap support from both maximum likelihood and neighbour joining methods. Isoc.=Isochrysidales. Prymnesium neolepis grouping marked with a red bracket. Scale bar indicates a distance of 0.05 substitutions per site.
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Durak, G.M. Chapter II: General organism description
Fig. II. 4. Unrooted maximum likelihood phylogenetic tree constructed for haptophytes and heterokontophyte silicifiers based on SSU rRNA sequences. Numbers at nodes indicate bootstrap support. Branches marked with: > 90% and > 70% bootstrap support from both maximum likelihood and neighbour joining methods.
Bacill.=Bacillariophyceae. P. neolepis strains marked with a red bracket. Scale bar indicates a distance of 0.05 substitutions per site.
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Durak, G.M. Chapter II: General organism description
II.3.2. P. neolepis: general morphology
All morphological and experimental analyses were conducted on P. neolepis strain
TMR5 as it proved to be the easiest one to maintain in culture. In order to gain general information on P. neolepis morphology and cell properties, various microscopy techniques, enabling immediate and continuous observations have been employed.
P. neolepis strain TMR5 cells are spherical to ellipsoidal, on average 11 µm in diameter
(n=25, SD=1.39). Cells are strongly polarized and covered with a layered silica scale covering, which can exceed 30 µm in diameter (Fig. II. 5. A,B). The posterior, non- flagellar pole of the cell is vacuolar, devoid of pigment and characterized by a distinct, rough surface covered in "bumps" (Fig. II. 5. A,B, see Fig. II. 7.). The posterior pole also contains the developing scales (Fig. II. 8. A,B, Time-lapses 1,2). The anterior part of the cell is occupied by a large chloroplast with finger-like projections, and contains most of the main cell organelles (see Fig. II. 7., Yoshida et al., 2006). The haptonema and two short flagella are quiescent and protrude from the centre of the chloroplast (Fig.
II. 7.).
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Durak, G.M. Chapter II: General organism description
Fig. II. 5. P. neolepis: A - light microscopy image of a naked and silicified cells
(haptonema marked with an arrow), B - DIC image with marked posterior pole of the cell (small arrow) and haptonema (thick arrow), C - rhizopodial-amoeboid cell. A - scale bar corresponds to 5 µm, B,C scale bars=10 µm.
Time-lapse DIC observations of P. neolepis revealed, that spheroid cells exhibit frequent metaboly, especially at the posterior, non-flagellar pole of the cell (Time-lapse
3,4). They are also able to assume amoeboid shape and move by crawling (Fig. II. 5. C).
Cells can subsequently return to their usual, spheroid form (Time-lapse 3). The cues for this shape transition have not been identified, however, amoeboid cells were observed to occur more frequently in cultures in stationary to senescent phases.
Cells characterised by the presence of 2 haptonemas were also encountered (Fig. II. 6.
A,B). These were found to be capable of phagocytosis via pseudopodium-like protrusion emerging from the posterior pole of the cell, preying on their conspecifics
(Time-lapse 5). This cell type was found to occur more frequently in cultures reaching stationary phase. Additionally, cells can almost cease to silicify in culture due to unknown factors. Increased number of naked or poorly silicified cells was observed in cultures in stationary phase, which was probably related to a decrease in Si availability for uptake from the media. Late exponential and senescent phases in P. neolepis
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Durak, G.M. Chapter II: General organism description cultures are also characterized by cells aggregating together into palmelloid assemblages lodged in a mucous-like material.
Fig. II. 6. DIC images of P. neolepis: A - cell with two haptonemas. B - double haptonema cell feeding on remnants of another P. neolepis cell (Time-lapse 5). Scale bars=10 µm.
II.3.3. In-vivo visualization of silicification in P. neolepis: silica scale formation and exocytosis
Intracellular silica polymerization has previously been visualized in organisms such as diatoms and sponges using two fluorescent probes: [2-(4-pyridyl)-5-[[4-(2- dimethylaminoethylaminocarbamoyl)methoxy]phenyl]-oxazole] (PDMPO) and
LysoTracker Yellow HCK-123 (HCK-123) (Schröder et al. 2004a; Shimizu et al. 2001).
PDMPO is an acidotropic fluorescent pH indicator with dual excitation and emission
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Durak, G.M. Chapter II: General organism description dependent on the pH, emitting strong green fluorescence in the presence of polysilicic acid (Diwu et al. 1999; Shimizu et al. 2001). Like PDMPO, HCK-123 is a dye selectively accumulating in acidic cellular compartments, such as the SDV, and is incorporated into the newly forming silica structure (Desclés et al. 2008). In this study, these two fluorescent probes were successfully adapted for labelling the silica in a haptophyte P. neolepis, enabling detailed investigation of the localization and mechanism of silica scale formation (Fig. II. 7.).
Fig. II. 7. Confocal microscopy 3D projection of a Z-stack of P. neolepis cells with
HCK-123 labelled scales (green) accumulated at the posterior pole of the cell. Note haptonemas (arrows) projecting from the middle of the finger-like chloroplast (red) at the cells' anterior. Scale bar=5 µm.
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Durak, G.M. Chapter II: General organism description
Confocal microscopy investigations in conjunction with silica-labelling fluorophores revealed that cells maintain between 2-3 scales intracellularly at one time and secrete them sequentially at the posterior pole of the cell (Time-lapses 1,2,3, Fig II. 8. A,B).
Silica scales are always maintained with the concave side of the scale directed towards the cell membrane when in the cell (Fig. II. 8. A,B, Fig. II. 9., Time-lapse 1).
Fig. II. 8. 3D projections of Z-stacks of P. neolepis collected with a confocal microscope, showing cell polarity relative to the site of silica scale production:
A - PDMPO-labelled cell with 2 intracellular scales (green, arrows) localized in the vacuolar, posterior region of the cell (chlorophyll autofluorescence in red). B - tubulin immunofluorescence labelled cell with visible flagella (small arrows) and the haptonema (thick arrow) at the anterior pole of the cell and HCK-123 labelled intracellular scales (green) localized on the posterior pole of the cell (see sections
III.2.3. and III.2.7. for methodology pertaining to tubulin immunolabelling and imaging). Scale bars= 5 µm.
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Durak, G.M. Chapter II: General organism description
To further investigate the mechanism of scale secretion, time lapse DIC microscopy observations were made. These revealed, that scale liberation is preceded by the mature scale migrating close to the cell membrane at the posterior, vacuolar part of the cell.
Then, the cytoplasm filling the concave part of the scale is moved to the side, forming an invagination, to allow the scale to break through the membrane with one side of the rim. This phase is accompanied by localized protoplast contractions. The scale is then pushed out of the cell at a slight angle and inverted, so that the concave side of the scale is again directed towards the cell membrane. Scale secretion events were captured in
Time-lapse 4 and summarized in Fig. II. 9. Time between scale secretion events was highly variable (from 6-78 min) and averaged at 39 min (n=22, SD=17.99).
Additionally, some cells do not silicify for prolonged periods of time. The averaged time between secretion events obtained can be potentially skewed towards longer intervals due to scales exiting the cell at different points, sometimes out of focus and therefore impossible to capture.
Fig. II. 9. Time course of a silica scale secretion event: 0 - mature scale migrating towards the cell membrane (arrow), 10 - removal of cytoplasm and forming of an invagination, 12- early stage of scale exocytosis starting from the rim of the scale, 14 - scale further pushed out at an angle relative to the plasma membrane, 15-inversion of the liberated scale, 17 - scale positioning on the cell surface bar=5 µm. Time indicated in minutes at the top of each image.
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Durak, G.M. Chapter II: General organism description
The sequence of events during silica scale formation was further examined with SEM imaging of purified P. neolepis biosilica, visualising scales captured at different developmental stages. The first stage of the formation appears to be deposition of a ring structure delimiting the size of newly forming scale (Fig. II. 10. A). Subsequently, thread-like structures projecting from the rim are formed, and give rise to a number of loop-like structures (Fig. II. 10. A). Deposition of loophole structures continues until the internal part delimited by the initial ring structure is densely packed (Fig. II. 10. B).
This stage is also accompanied by thickening of the original ring structure, forming the rim of the scale. The last stage of the silica scale growth appears to be deposition of an additional layer of silica on top of the whole structure, decreasing pore size arising from previously deposited loops and sealing gaps between them (Fig. II. 10.).
Fig. II. 10. Silica scales at different developmental stages: A-basal rim with loops beginning to form, B-scale with secondarily thickened rim and most of the loops formed, C-complete scale. Scale bars=1 µm.
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Durak, G.M. Chapter II: General organism description
To ensure that these findings were not artefacts of abnormal scale formation, slow etching of silica scales with alkali was applied. SEM imaging of etched biosilica samples corroborated the existence of thread-like precursor structure anchored to the rim of the scale, and forming loophole pattern underlying pores present in the convex side of the scale (Fig. II. 11.).
Fig. II. 11. SEM images of a P. neolepis biosilica sample etched with 0.5 M NaOH for a period of 6 h, revealing loophole pattern projecting from the rim towards the centre of the scale (A) and the primary ring structure underlying the rim of the scale (B). Images were collected at different locations within the same sample.
II.3.4. Morphological observations of silica scales
Silica scales produced by P. neolepis varied in shape from elliptical to round, with two apices on the convex side and a re-curved rim (Fig. II. 12., inset). Additionally, scales were often bent around the longitudinal axis to a variable degree, much like thin potato crisps. Average scale dimensions measured 6.8 x 4.4 µm (n=100, SD=1.58 and
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Durak, G.M. Chapter II: General organism description
SD=0.88 for length and width respectively). Individual scales varied greatly in size, with scale length and width ranging from 2.8-14.6 µm and 2.1-6.7 µm respectively.
Fig. II. 12. SEM image of KMnO4/HCl cleaned P. neoelpis silica scales. Inset: TEM cross-section of an individual scale: scale rim indicated by black arrows, two apices on the convex side marked with red arrows. TEM sections of P. neolepis scales were kindly supplied by Mr. Glenn Harper (University of Plymouth).
P. neolepis does not appear to arrange scales in any specific pattern, and the loose, non- interlocking and non-adhering sphere of scales surrounding the cell arises most likely from random protoplast rotations in suspension (Fig. II. 13. B). If the cell is stationary, i.e. resting on a flat surface, scales accumulate at the posterior pole of the cell (Fig. II.
7.). Cells secrete scales continuously and accumulate multiple layers of scales, often thicker at the posterior, secretory pole of the cell. The loose silica scale-cell association
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Durak, G.M. Chapter II: General organism description is easily perturbed by pipetting of media or stirring, which often results in the scale covering being partially or completely removed (Fig. II. 13. A).
Fig. II. 13. P. neolepis: A - polarized light micrograph of a cell with a disturbed scale cover. B -c onfocal microscopy 3D reconstruction of a cell with uniformly distributed scale cover (white: reflectance of silica scales, red: chlorophyll autofluorescence). Scale bars=5 µm.
II.3.5. Non-biomineralised elements of P. neolepis cell covering
Previous electron microscopy investigations on P. patellifera and P. parvum detected a layer of material deposited on top of the plasma membrane, underlying the organic scale layer, which was suggested to be composed of glycocalyx (Green et al. 1982).
Similarily, coccolith bearing haptophytes possess a layer of columnar (fibrillar) material overlying the plasma membrane, suggested to play a role in securing the biomineralised elements to the cell surface (Drescher et al. 2012; Rowson et al. 1986; Taylor et al.
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Durak, G.M. Chapter II: General organism description
2007). Additionally, coccoliths and entire coccospheres have previously been shown to be coated with polysaccharide-containing organics, labelling with a fluorophore conjugated lectin - Concanavalin A (Con-A) (Fig. II. 14. C,D) (Godoi et al. 2009;
Hirokawa et al. 2005; Marsh 2003). Therefore, Con-A was used as a preliminary investigative tool to probe the loosely associated silica scale cover for the presence of potentially polysaccharide-containing matrix.
Con-A labelling and subsequent imaging of P. neolepis with a confocal microscope revealed that a thick, on average 1.8 µm layer (n=30, SD=0.8) of potentially polysaccharide, mannose-containing material is indeed covering the cell (Fig. II. 14.
A,B). As the silica scale covering does not adhere closely to the plasma membrane and is not uniform in thickness, this material is proposed to provide a matrix, in which layers of organic and silica scales are lodged, therefore maintaining the loose, non- interlocking scale covering around the cell (Fig. II. 13. B). Unlike calcified coccoliths of coccolithophores, silica scales making up the cell cover in P. neolepis are not covered with this material (Fig. II. 14. A-C). However, scales which have been shed often display strong fluorescence upon Con-A labelling, suggesting that they can be coated secondarily (Fig. II. 14. A, arrows).
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Durak, G.M. Chapter II: General organism description
Fig. II. 14. 3D projections of confocal microscopy Z-stacks of Concanavalin-A labelled cells of P. neolepis: A - 3D reconstruction with labelled detached scales (arrows), B - individual slice showing irregularities and partial detachment of the Con-A labelled cell coating. C - decalcified C. pelagicus cells with holes in polysaccharide covering marking spaces where coccoliths were attached, D - individual C. pelagicus coccoliths.
P. neolepis was additionally labelled with PDMPO (in blue) to visualize cell body. Con-
A is shown in green, chlorophyll autofluorescence in red. Scale bars=5 µm.
In addition to silica scales, P. neolepis also deposits much smaller organic scales, previously reported to form a layer under the silica scales (Yoshida et al. 2006). Organic scales produced by P. neolepis strain TMR5 were ellipsoidal, with serrated edges which
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Durak, G.M. Chapter II: General organism description were absent from organic scales of strain NIES 1393, and radially arranged ridges (Fig.
II. 15. A,B). Organic scales were on average 1.06 µm in length and 0.95 µm in width
(n=10, SD=0.13, 0.1 for length and width respectively) . The imaging resolution did not allow for any further investigation into additional patterning potentially marking scales, and was not pursued any further.
Fig. II. 15. A,B-TEM images of uranyl-acetate stained organic scales with a clearly visible radial ridge pattern. B-scale with prominent serrated edges. Scale bar=1 µm.
II.3.6. FT-IR characterization of chemical structure of P. neolepis biosilica
In order to obtain information on the structure of biosilica produced by P. neolepis and to enable comparisons with the dominant silicifiers, diatoms, Fourier transform infrared spectroscopy (FT-IR) has been applied. This tool was also used to obtain preliminary information on the presence of organic matter potentially occluded within the purified silica scales. FT-IR transmission spectra of biosilica produced by P. neolepis and a
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Durak, G.M. Chapter II: General organism description diatom, O. sinensis were collected and compared against each other and a poorly condensed silica gel. As expected, all three spectra exhibited peaks characteristic of amorphous, hydrated silica.
The FT-IR spectrum of biogenic amorphous silica can be divided into two main regions:
(1) > 2500 cm-1, with the main broad peak between 2800-3800 cm-1 corresponding to
-1 OH stretching vibrations of adsorbed/molecular H2O, (2) a region < 1300 cm , representing the major vibrational modes of silica, detailed in Fig. 16. Based on the shape of the peak around 1100 cm-1 (peak 3, Fig. II. 16.), silica gel represented the least and P. neolepis biosilica represented the most ordered amorphous silica structures (very sharp peak compared to that of O.sinensis and silica gel) (Chukin and Malevich 1977).
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Durak, G.M. Chapter II: General organism description
Fig. II. 16. FT-IR transmission spectra of purified P. neolepis (red), Odontella sinensis
(black) biosilica and a silica gel (green). Peaks are assigned as follows: 1:~800 cm-1 symmetric SiO bond stretching (Stolen and Walrafen 1976), 2:~960 cm-1 Si-OH vibrations, OH vibrates as a single mass, antisymmetric SiO stretching of O3SiOH
(Hartwig and Rahn 1977; Stolen and Walrafen 1976). 3,4: 1000-1400 cm-1-a peak with a shoulder corresponding to deformation vibrations of the SiOH hydroxyl, 5-1538 cm-1 amide II band (Wellner et al. 1996), 6-≈1643 cm-1 H-O-H bending-molecular water deformation (Davis and Tomozawa 1996; Little 1966). 7-absorption bands between
2800-3000 cm-1 assigned to organic matter (δ CH aliphatic) (Landais et al. 1993), 8:
~2800-3800 cm-1, OH stretching of the adsorbed/molecular water (Chukin and
Malevich 1977).
No peaks in the FT-IR spectrum of P. neolepis biosilica at wave numbers corresponding to vibrations of groups indicative of the presence of organic fraction, as visible around
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Durak, G.M. Chapter II: General organism description
2800-3000 cm-1 (CH stretching bands) and the amide II band at 1538 cm-1 in biosilica isolated from O. sinensis were observed (Fig. II. 16., peaks 5,7). However, subsequent dissolution of purified silica scales with acidified ammonium fluoride revealed presence of associated soluble and insoluble organics (see sections IV.3.4. and IV.3.8. for details).
II.3.7. Physiology of silicification in P. neolepis
The main questions regarding the physiology of silicification in P. neolepis were aimed at establishing whether it is essential for this organism to silicify and what are the consequences of limiting Si availability and inhibiting the process in terms of growth and silica structure formation. The possibility of light dependency of silica biomineralisation in this species was also investigated. However, experimental verification of the above questions proved to be very difficult and in many cases impossible due to technical issues arising from poor culture growth and cells often not withstanding experimental conditions due to cell fragility.
II.3.8. Dependency of silicification on light availability
Biomineralisation processes in photosynthetic phytoplankton are known to be to some extent dependent on light availability, often via secondary metabolic and cellular processes or energetic requirements. In calcifying coccolithophores, biomineralisation occurs mainly during the day and has been suggested to be potentially dependent on energy derived from photosynthesis (Paasche 1999; Paasche 2001; Sekino and Shiraiwa
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1996; Taylor et al. 2007). In diatoms, silicification is directly correlated with cell division and also tends to be more prevalent during the light phase (Chisholm et al.
1978; Martin-Jézéquel et al. 2000). To determine if similar light dependencies might be linked with silica deposition in P. neolepis, a simple experiment involving a 10h incubation in light and dark conditions was conducted. Silica scales deposited during the experiment were labelled with Lysotracker HCK-123, allowing their quantification with flow cytometry. Statistical analysis of the resulting ratios of labelled vs. unlabelled scales, providing a proxy for silicification rate revealed no statistical difference (t=-
0.42, df=28, p=0.68, n=15) in the silicification rates between light and dark conditions
(Fig. II. 17.). This result indicates strongly that light is not essential for silicification in
P. neolepis, and there is no short-term dependence on light for scale production in this silicifier.
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Durak, G.M. Chapter II: General organism description Box Plot of Var2 grouped by Var1 Light Dark II 2v*30c 0.50
0.45
0.40
0.35
0.30
Ratio
0.25
0.20
0.15
0.10 Light Dark Mean Mean±SE Var1 Mean±SD
Fig. II. 17. Box-whisker plots of the newly deposited to old scale ratios as measured with flow cytometry (n=15).
II.3.9. Dependency of silica scale production on silica availability
Although originally the impact of silica starvation on growth was to be determined, measurement of growth under Si-replete conditions proved to be impossible. Attempts to grow P. neolepis in ASW (artificial sea water) with Si concentrations < 3 µM were unsuccessful. Cells introduced into these cultures as an inoculum could no longer be observed with a light microscope after 2-3 days from inoculation and control cultures supplemented with 100 µM Si in ASW grew poorly and frequently collapsed. Therefore a short term response to low Si conditions was investigated instead. For this purpose, a method utilizing confocal microscopy in conjunction with silica labelling fluorescent dyes was developed. The method involved incubation of P. neolepis cells in high
(supplemented with 100 µM Si) and low (< 3 µM) Si ASW with f/2 media in the presence of a silica-labelling fluorescent probe for a period of 18 h. Cell counts were
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Durak, G.M. Chapter II: General organism description made at the end of the incubation period on random fields of view, directly on the glass bottom dishes (Fig. II. 18. A). Cells were then lysed in order to enable quantification of silica scales produced during the experiment, for which purpose images of random fields of view were taken (Fig. II. 18. B).
Verification of the method: cell counts per field of view were variable both within and between dishes (Fig. II. 19. A,B), which was compensated for by taking 10 counts per dish, and increasing the number of replicates to 5. This variability was most likely caused by washing cells with treatment media directly on the dish. Cell counts were averaged to obtain cell density per dish, which was used in further analysis.
Fig. II. 18. Confocal microscopy images of PDMPO stained P. neolepis cells (18 h incubation in 100 µM Si in ASW with f/2 nutrients). A-example image used for cell counts. Green channel was switched off for easier image processing. B - example image of silica scales post H2O2 lysis (all channels except green were switched off for clarity).
Scale bars=20 and 10 µm for A and B respectively.
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Results obtained from this experiment indicated, that P. neolepis possess an efficient mechanism for Si extraction from the media, as average Si concentration in control samples decreased from 100 µM (plus > 3 µM of unavoidable Si contamination introduced with ASW) to 29.05 µM (n=4, SD=1.26) at the end of the incubation period.
Additionally, cells were still able to produce considerable amounts of silica scales in low Si conditions (Fig. II. 20. A,B), with Si concentrations at the end of the experiment averaged at 5.87 µM (n=4, SD=1.82).
Fig. II. 19. Validation of the method used for cell counts in the low Si experiment:
A - variability of cell counts between all replicates in all dishes (D=dish). B - cell count variability within 1 dish ("replicate" on x-axis refers to a replicate of a cell count carried out within one dish).
Overall, limiting Si supply resulted in a lower average number of scales/cell (51.49% decrease), scale area/cell (46.97% decrease) compared to the control (Fig. 20. A,B). The effect of limiting Si supply on the scale size (area), was quite minor and a 11.11%
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Durak, G.M. Chapter II: General organism description decrease in area/scale in the treated vs. control samples was recorded, indicating, that limiting Si supply has little effect on the size of scales produced. Qualitative inspection of scales produced under the low Si treatment indicated no morphological departures from those produced in the control.
Fig. II. 20. Quantification of silica scales produced after 18 h incubation period under low Si and control conditions. Box-whisker plots of: A - scale area/cell, B - average number of scales/cell. Box plots are based on data averaged per dish.
II.3.10. Silicon metabolism in P. neolepis: disruption with germanium
Ge acts as a competitive inhibitor of Si metabolism, with growth and silicification inhibitory effects becoming apparent once a species-specific threshold ratio of Ge:Si in the media is approached (Azam and Chisholm 1976; Azam et al. 1974; Lewin 1966).
Ge was previously shown to interfere with growth of some algae, especially silicifiers, when introduced to cultures at concentrations between 2.1-14.3 µM (Lewin 1966;
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Markham and Hagmeier 1982). Diatoms were shown to be especially prone to germanium induced growth inhibition due to Ge interfering with cell wall formation and respond to ratios as low as 0.02 Ge:Si, a property that has been long used to eliminate diatom contamination of non-silicifying species in algal culture collections (Azam and
Chisholm 1976; Azam et al. 1973; Lewin 1966; Markham and Hagmeier 1982). Ge uptake and co-deposition with silica frustules in diatoms was also shown to result in malformations (Azam et al. 1973).
Here Ge was used to determine, if (1) silicification in P. neolepis can be inhibited and with what consequences, (2) if Ge can be taken up and co-incorporated into silica structures and (3) whether it affects the morphology of silica scales. In order to answer these questions, growth experiments, confocal laser scanning microscopy in conjunction with fluorescent silica markers, SEM imaging and EDX analysis were carried out.
Methodological limitations relating to Si assay development did not allow for Si concentration measurement in these early experiments, however, special care was taken to keep Si contamination in the media as low as possible.
II.3.11. Inhibition of silica scale production in P. neolepis with Ge: CLSM observations
Results of incubation of P. neolepis with a range of Ge concentrations revealed, that as in diatoms, Si metabolism can be blocked with Ge, resulting in inhibition of silica deposition. Multiple experiments involving cell treatment with 10, 20 and 50 µM Ge consistently indicated, that cells were unable to produce and deposit silica scales under these Ge concentrations. At 10 µM Ge partially formed internal scales were still present in some cells (Fig. II. 21. B), however at 50 µM Ge intracellular scale formation was no longer observed (Fig. II. 21. C). Incubation with 10 µM Ge also resulted in some cell
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Durak, G.M. Chapter II: General organism description mortality as determined by visual inspection, with the effects becoming much more severe at 50 µM Ge, where very few cells remained intact after 18 h incubation period
(Fig. II. 21. A-C).
Fig. II. 21. A - silicified cells in control, B - non-silicifying cells post 10 µM Ge treatment with partially formed intracellular scales marked with arrows, C - few surviving, non-silicifying cells post 50 µM Ge treatment. Scale bars=10 µm (A,C) and
20 µm (B). Images representative of 18 h incubation experiments conducted in duplicate. HCK-123 labelling of silica shown in green, chlorophyll autofluorescence in red.
II.3.12. Ge/Si co-feeding of P. neolepis: growth
Since the potency of Ge toxicity to Si metabolism depends on concentration of both Ge and Si, a much lower (3 µM) Ge concentration, supplemented with 50 µM Si was chosen for the next experiment. The aim of this experiment was to determine if Ge/Si
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Durak, G.M. Chapter II: General organism description co-feeding could result in Ge co-incorporation into the silica structure and if so, whether it caused any adverse effects on silica scale morphology. Results of this experiment revealed, that at these Ge and Si concentrations culture growth was not inhibited nor decreased (Fig. II. 22.). In a growth experiment where cells were dosed with 50 µM
Ge/Si however no growth was observed. After 2 days cells could no longer be found in these cultures when examined with a light microscope.
Fig. II. 22. A-Growth curve for 3 µM Ge supplemented and control P. neolepis cultures.
Error bars=SD based on 3 cell counts per each replicate (n=3 replicates). Growth rates calculated for the mid-exponential phase between day 8-10 were at µcontrol=4.1 and
µGe=3.9.
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Durak, G.M. Chapter II: General organism description
II.3.13. Ge/Si co-feeding of P. neolepis: Ge incorporation into silica scales
To determine if formation of silica scales was adversely affected by Ge, SEM analysis of biosilica obtained from the 3 µM Ge/50 µM Si treated culture was carried out. No morphological abnormalities were visible in the material examined (relevant images attached in the appendix II.2.), and silica scales produced in Ge treated samples were not significantly different from the controls in terms of length or width (t=1.64 for length and t=-0.243 for width, df=198, p>0.01). Ge co-incorporation into scales was confirmed with EDX analysis (Fig. II. 23.), indicating that the Si transporters and further metabolic pathways are not strictly Si specific and are not significantly impaired at low Ge concentrations.
Fig. II. 23. EDX spectrum of purified P. neolepis silica scales obtained from a culture dosed with 3 µM Ge and 50 µM Si. High C peak is caused by the nitrocellulose filter on which the sample was deposited.
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Durak, G.M. Chapter II: General organism description
II.4. DISCUSSION
II.4.1. P. neolepis strain TMR5: morphological and biological characteristics and comparisons with closely related algae
Analysis of rbcL and SSU gene sequences indicated, that some genetic variability between P. neolepis strains from different geographical locations exists. In terms of morphology, P. neolepis exhibits traits characteristic of other members of Prymnesium genus. P. neolepis cells are polar, with the posterior, non-flagellar pole being dominated by vacuoles and vesicles, which in other Prymnesium and closely related
Chrysochromulina species contain storage metabolites (Green et al. 1982; Parke et al.
1955). Additionally, some morphological variation between different strains was noted.
Compared to P. neolepis strain NIES 1393, organic scales produced by strain TMR5 were larger (1.06 x 0.95 vs. 0.6 x 0.4 µm for TMR5 and NIES 1393 respectively), and possessed serrated edges absent in NIES 1393 (Yoshida et al. 2006). This is not unusual, as members of Prymnesium genus are known to produce several types of organic scales (Green et al. 1982).
As previously observed for cultures of other Prymnesium and Chrysochromulina species, approaching stationary phase and senescence in P. neolepis is marked by cells becoming more irregular and amoeboid in shape, increasingly forming gelatious palmelloids towards senescence (Conrad 1941; Green et al. 1982; Parke et al. 1955). In natural marine microalgal populations, aggregation of phytoplankton was suggested to be a response to limited nutrient supply, increasing sinking rates to reach deeper, higher in nutrients layers within the water column (Bernard 1963; Smetacek 1985; Thornton
2002). Senescence in P. neolepis is also characterised by cells becoming increasingly
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Durak, G.M. Chapter II: General organism description phagotrophic, however, unlike in continuously mixotrophic flagellates of Prymnesium and Chrysochromulina, this behaviour appears to be related to nutrient limitation. In all the aforementioned algae, phagotrophy always occurs at the non-flagellar pole of the cell, often via pseudopodial cell protrusions (Jones et al. 1993; Legrand et al. 2001;
Parke et al. 1955; Tillmann 1998). This mixotrophic behaviour was previously suggested to be a mechanism allowing for N and P supplementation in normal conditions and conferring an advantageous compensation mechanism when nutrients become limited (Legrand et al. 2001; Nygaard and Tobiesen 1993), which appears consistent with the increased incidence of phagotrophy towards culture senescence in P. neolepis. No cells ingesting conspecifics were observed during the exponential growth phase, however, no queries into bacterial ingestion during this growth phase were made at this time either. Thus, a possibility that the fully silicified, non-nutrient limited P. neolepis could be capable of feeding on bacteria remains unresolved.
II.4.2. Organic and biomineralised cell covering
The thick, irregular Concanavalin-A-staining, putatively polysaccharide containing deposit covering the plasma membrane in P. neolepis is suggested to act as a matrix in which silica scales are lodged, thus maintaining them in a spherical formation surrounding the cell surface. A similar role was attributed to a layer of extracellular, columnar deposits in coccolithophores, which have been suggested to act as an adhesive, directly connecting organic scales and coccoliths to the plasma membrane
(Drescher et al. 2012; Manton and Leedale 1969; Rowson et al. 1986).
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Durak, G.M. Chapter II: General organism description
Biosilica scales of P. neolepis consist of an amorphous, although relatively well organised hydrated silica phase similar to that found in diatoms, in which FT-IR frustule signatures are species-specific (Kammer et al. 2010; Vardy and Uwins 2002). The lack of specific signatures produced by organics, of which presence in biosilica was later confirmed with EDX and SDS-PAGE analysis (see Chapter IV for details) could have been due to signal levels falling below detection limits of the machine used.
The loosely associated, delicate scale covering of P. neolepis does not appear to have a structural role, as has been suggested for biomineralised cell covering in diatoms or coccolithophores, however it could potentially act as grazing deterrent (Hamm et al.
2003; Young 1994). As silica scales in this species are embedded in a thick layer of the diffuse, extracellular deposit, forming a "shell" at a distance from the protoplast, it is possible that this structure plays a role in maintaining a privileged environment. This was previously suggested as one of potential functions of coccospheres forming analogical, but more compact and resilient structures in coccolithophores (Paasche
2001). The formation of a loose silica scale cover in P. neolepis might also serve to regulate buoyancy of cells in suspension in a manner similar to that previously suggested for coccoliths and diatom frustules, thickening in response to nutrient limitation to sink into deeper, nutrient-rich waters (De la Rocha et al. 2000; M Franck et al. 2000; Young 1987). The shape and patterning of P. neolepis silica scales can be speculated to have similar optical properties and functions as the nanopatterned diatom frustules. Both organisms produce silica structures characterised by a periodic nature of the pore pattern, which in diatoms was suggested to confer photonic crystal properties to the silica cell wall, potentially serving to enhance light acquisition and therefore photosynthesis (Fuhrmann et al. 2004; Yamanaka et al. 2008). However, given the loose and disorganised scale arrangement on the P. neolepis cell surface, such a role is unlikely.
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Durak, G.M. Chapter II: General organism description
II.4.3. Silica scale formation
Organic, unmineralized scales formed in Golgi vesicles and liberated near flagellar roots are typical of haptophytes (Green et al. 1982; Manton 1966; Manton and Leedale 1969;
Parke et al. 1955). In calcifying coccolithophores, organic scales usually co-occur with mineralized coccoliths in a layered arrangement, with organic scales underlying cocccoliths (Inouye and Pienaar 1984; Manton and Leedale 1969). A similar arrangement is found in P. neolepis. Organic scales serve as a baseplate for the mineralized coccoliths (with the exception of Emiliania huxleyi) (Inouye and Pienaar
1984; Klaveness 1972; Manton and Leedale 1969; Pienaar 1994). This is in contrast with biomineralised scales produced by P. neolepis. Organic scales produced by this species are much smaller and solid, not matching the perforated, concave silica scales morphologically. These observations are further supported by SEM imaging of silica scales at various stages of development, where no associated baseplate structure was observed under the forming loophole pattern of scales. Etching of the silica scale material further confirmed the absence of an underlying organic baseplate. Instead, P. neolepis biosilica is associated with a filamentous, insoluble, likely cellulose-containing organic phase, potentially serving as a matrix for sequential silica deposition processes
(see sections II.3.3. and IV.3.8. for details). This suggests, that silica scales do not arise from biomineralisation of an organic scale as it is the case in most coccolithophores, but are separate structures.
The lack of homology between organic and silica scales is further supported by the formation site of silica scales being different to that of organic scales and coccoliths.
Biosilica scales of P. neolepis are formed in the posterior, vacuolar region of the cell, in
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Durak, G.M. Chapter II: General organism description a compartment previously proposed to be a Silica Deposition Vesicle (SDV) similar to that of diatoms, as opposed to a Golgi-derived vesicle in which organic scales and coccoliths are formed (Billard 2004; Manton 1966; Yoshida et al. 2006). Furthermore, as with unmineralized haptophyte scales, coccoliths are secreted at the anterior pole of the cell, near the flagellar root (Manton 1967; Taylor et al. 2007). In contrast, silica scale secretion in P. neolepis occurs at the posterior, non-flagellar pole of the cell, co- localized with silica scale formation site instead. The silica scale formation process in P. neolepis appears to bear some similarities to frustule formation in diatoms, where an organic, chitin-containing matrix is mineralized within a specialized SDV and then secreted (see Chapter I for a review) (Durkin et al. 2009; Kröger and Poulsen 2008a;
Tesson and Hildebrand 2010). Unlike frustule formation in diatoms, which is closely related and synchronised with the cell cycle to ensure that each daughter cell receives one newly formed frustule valve and derives the other from the parent cell and then restricts silicification to girdle bands following cell division (Graham et al. 2009), silica scale formation in P. neolepis appears to be a continuous process occurring during the entire cell cycle.
II.4.4. Physiology of silicification in P. neolepis
Silicification in marine microalgae is an energetically costly process, involving specialised Si transport systems and metabolism (Hildebrand 2005; Martin-Jézéquel et al. 2000). The uniformity of silicification rates in P. neolepis under light and dark conditions indicates, that it is a continuous process not dependent on light, and therefore energy derived directly from photosynthesis. This also suggests that the energy required for silica scale formation is obtained from respiration instead, as was previously shown
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Durak, G.M. Chapter II: General organism description for diatoms (Martin-Jézéquel et al. 2000; Sullivan 1976). Silicification in P. neolepis also persists through cell division, as dividing cells were observed to usually contain 2-3 intracellular scales.
Attempts to grow P. neolepis at low Si concentrations (< 3 µM) failed, suggesting that
Si is important for normal growth of this organism. Subsequent efforts to assess the ability of P. neolepis to deposit silica scales under low Si concentrations suggests, that an efficient Si-selective, most likely active transport system, enabling substrate acquisition and concentration is present. The decrease in the scale area/cell and the number of scales/cell in low Si treatments without a considerable decrease in average scale area indicates that Si limitation decreases silica scale production without a concomitant decrease in size. Additionally, the slight increase in Si concentration after incubation in low Si media (from < 3 µM to ≈5 µM) indicates, that the elaborate, putatively polysaccharide-containing matrix maintaining the loose association between cells and silica scales might have contributed to Si carryover and retention from pre- experimental, Si-containing culture media. This also further supports the role of the putative polysaccharide and siliceous cell covering in stabilizing the microenvironment around the cell.
II.4.5. Ge inhibition of silicification
Similar to diatoms, Ge was found to act as a competitive inhibitor of Si in P. neolepis, resulting in a complete inhibition of silicification at Ge concentrations ≥ 10 µM. At higher concentrations (≥ 10 µM), Ge becomes increasingly toxic to P. neolepis, resulting in cell mortality, potentially due to disruption of Si metabolism. This, combined with the lack of growth and subsequent cell death observed for growth
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Durak, G.M. Chapter II: General organism description experiments in low Si media and those involving Ge/Si co-feeding at 50 µM Ge/Si suggests, that Si metabolism is essential for normal growth of P. neolepis cultures. Due to methodological inconsistencies between previous studies investigating the use of Ge for elimination of diatoms from algal cultures (i.e. insufficient information on the method of media preparation pertaining to Si concentration, dissolution of the poorly soluble GeO2 directly in sea water etc.), comparisons between different algae, especially diatoms and P. neolepis in terms of sensitivity to Ge were not possible.
Treatments at lower (3 µM) Ge concentrations resulted in Ge incorporation into silica scales without causing silica scale malformation, analogous to previous observations documented for diatom frustules produced in Ge/Si co-feeding experiments at low Ge concentrations (Jeffryes et al. 2008). This also suggests that the Si transport system is not strictly Si-specific, and facilitates transport of analogous but larger ions, such as germanic acid.
II.4.6. P. neolepis: the only silicifying haptophyte described to date?
Findings by Pienaar (1980, 1981), previously mentioned in the introduction and pertaining to the presence of silica associated with the fibrous, organic deposits in
Prymnesium sp. could potentially be an artefact of an interaction of the aforementioned material with an excess of silicic acid in the media. Controlled, biologically mediated silica precipitation has received a great deal of attention over the past two decades due to its potential application in materials science, it is now known, that it can be facilitated by a wide range of different organic compounds. These include polycationic polysaccharides, polypeptides, highly acidic proteins or polyamine/quaternary ammonium group containing organic compounds amongst many others (see sections
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Durak, G.M. Chapter II: General organism description
I.3.5-1.3.7, I.6. for details), which are ubiquitously distributed amongst taxa (Gautier et al. 2008a; Hedrich et al. 2013; Kröger et al. 1999; Shchipunov et al. 2005; Wenzl et al.
2004; Wenzl et al. 2008). It is therefore not implausible, that the organic material coating of the cyst cover-forming scales, which themselves are often associated with polysaccharides, could facilitate precipitation of excess Si out of the growth media. This is also a likely scenario, as culture growth media are commonly prepared and autoclaved in glass bottles, significantly increasing the concentration of free silicic acid, often precipitating in a form of needle-like glass structures (Lohmiller and Lipman
1998). Additionally, silica aggregates precipitated during the autoclaving process could simply get lodged in the organic, fibrous coating of the outermost scales of a cyst, contributing to detection of silica in this material.
In the light of the above information, silica association with the organic deposits covering Prymnesium cysts is unlikely to be a product of an active silicification process occurring within cells. These preliminary observations remain highly speculative until further, more thorough inquiries are made. Thus, P. neolepis remains the only haptophyte described to date, capable of actively producing fully biomineralised scales composed of silica. Therefore, a unique opportunity to investigate the process of biosilicification, potentially unique to haptophytes, and comparisons of mineralized and unmineralized cell covers between haptophytes and other silicifying algae is presented.
II.5. SUMMARY
P. neolepis is the only obligate silicifier within the haptophyte clade described to date.
Silicification in P. neolepis occurs in a compartment of origin different to that of other biomineralizing haptophytes yet putatively similar to that of diatoms. The organic
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Durak, G.M. Chapter II: General organism description matrix underlying silica scales is non-homologous with organic scales in contrast to the case in coccolithophores. The site of silica scale exocytosis is also different to that of coccoliths, further indicating that silica scales are structures formed in a process fundamentally different to coccolithogenesis in calcifying coccolithophores.
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
CHAPTER III: THE ROLE OF CYTOSKELETON IN ALGAL BIOMINERALISATION
III.1. INTRODUCTION
Extracellular structures in haptophytes, such as organic scales and mineralized coccoliths are produced in specialised vesicles originating from the Golgi body
(Graham et al. 2009; Schmid 1987). In calcifying coccolithophores, biomineralised scales are deposited in specialized coccolith vesicles (CVs) in a sequential process, involving finely controlled nucleation and growth of calcite crystals on an organic baseplate, serving as a biotemplate (see Chapter I for more details) (Young and
Henriksen 2003). The CV itself has been suggested to be externally moulded during coccolithogenesis by an interaction with a fibrillar structure of putative cytoskeletal composition adjacent to the CV, although little is known about the exact processes involved (Didymus et al. 1994; Drescher et al. 2012; Langer et al. 2010; Marsh 1999;
Marsh et al. 2002; Westbroek et al. 1984). Similarly, the presence of specialised vesicles and their close interaction with the cytoskeleton during silica cell wall morphogenesis has been observed and well documented in diatoms of heterokontophyte origin (Schmid 1987; Tesson and Hildebrand 2010). In both cases mineral deposition proceeds in specialised, membranous compartments and entails a complex interplay of the entire cellular machinery, where the role of cytoskeleton can be assigned to three main types of tasks: (1) intracellular transport of vesicles, membranes and other building blocks required, (2) active shaping of biomineralised structures during deposition, (3) secretion of complete structures and their localization on the cell surface.
The capacity of cytoskeleton microfilaments to facilitate these processes arises from
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes intrinsic structural properties of actin and tubulin microfilaments, enabling dynamic interaction with each other as well as with other molecules in the cell (Aylett et al.
2011).
The cytoskeleton in Eukaryotes consists of actin microfilaments, microtubules and intermediate filaments, serving a vital role in maintenance of cell shape, movement, molecule delivery and localization, cell division, genetic material segregation or cytoplasmic streaming (Desai and Mitchison 1997; Kueh and Mitchison 2009; Verchot-
Lubicz and Goldstein 2010). Actin microfilaments (F-actin) and tubulin protofilaments
(building blocks of microtubules, MTs) are polar polymers of G-actin and αβ-tubulin monomers respectively (Wickstead and Gull 2011). Due to the filaments' polar structure, addition of monomer units is in general favoured at the barbed end in actin and plus end in tubulin, whereas depolymerisation processes are favoured at the pointed and minus ends for actin and tubulin filaments respectively (Fig. III. 1. A,B) (Allen and
Borisy 1974; Desai and Mitchison 1997; Le Clainche and Carlier 2008; Small et al.
1978).
Both actin and tubulin can use NTP (nucleoside triphosphate) hydrolysis to generate dynamic instability to shrink/grow, initiate localized polymerization/depolymerisation, treadmilling, or bending in order to e.g. shape membranes, move molecules, organelles or vesicles (Löwe and Amos 2009). They can also create directional intracellular tracks on which a range of motor proteins, such as microtubule-associated kinesins and dyneins or F-actin-associated myosins can traffic molecules to target sites (Fig. 1A,B)
(Gross et al. 2007; Löwe and Amos 2009; Vale 2003).
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Fig. III. 1. Mechanism of motor protein interaction with MTs and F-actin: A-myosin V moving towards the barbed and myosin VI-towards the pointed end of F-actin. B- kinesin and dynein motors walking towards plus and minus ends of a MT respectively
(Gross et al. 2007).
Historically, an array of cytoskeleton disruptive chemicals has been used to probe functions of cytoskeleton by specifically disrupting selected cytoskeletal components within a cell (Peterson and Mitchison 2002). Use of such substances will not only cause disruption of the microfilaments targeted, but will also secondarily impair intracellular transport facilitated by motor proteins associated with them. Depending on the type and dose of the drug used, cytoskeleton disruptors can cause microfilament capping and stabilization, filament shortening, dissociation of supramolecular structures or almost complete depolymerisation (Jordan et al. 1992; Margolis et al. 1980; Peterson and
Mitchison 2002). This in turn depends on the inhibitor binding site in the filament or
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes association with its monomeric subunits, making the choice of the right inhibitor and dosage critical to interpretation of the response produced.
To date, only one investigation has endeavoured to probe the specific role of actin and tubulin microfilaments in coccolithogenesis, using cytoskeleton inhibitors in Emiliania huxleyi to confirm their involvement in coccolith morphogenesis (Langer et al. 2010).
No investigations at all have been made concerning silicification within the haptophyte clade, represented by a single, silica scale producing species - Prymnesium neolepis
(Yoshida et al. 2006). Therefore this study aims to interrogate and compare these two fundamentally different biomineralisation systems of the haptophyte algae. To achieve this, two inhibitors, latrunculin B and nocodazole have been employed. The former binds to actin monomers and facilitates F-actin depolymerisation, whereas the latter binds to microtubules capping them, concomitantly binding to tubulin monomers preventing their incorporation into filaments, and at micromolar concentrations used for the purpose of this study causes MT depolymerisation (Jordan et al. 1992; Margolis et al. 1980; Morton et al. 2000; Spector et al. 1999). Effects of individual system component disruption on biomineralisation in terms of morphology of the biomineralised structure, quantitative mineral deposition and secretory abilities were compared. Additionally, the haptophyte silicification system present in haptophytes has been compared with that of the most prominent and thoroughly investigated heterokontophyte silicifiers - diatoms.
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III.2. METHODS
III.2.1. Culture conditions and cytoskeleton inhibitor dosing
Coccolithus pelagicus ssp braarudi (strain PLY 182g) and Prymnesium neolepis (strain
TMR5) were cultured as described in Chapter II. Cytoskeleton inhibitor doses used are specified in Table III. 1. The concentrations correspond to the lowest effective concentrations as determined in a preliminary cytoskeleton inhibitor trial on C. pelagicus, where latrunculin B concentrations between 0.1-100 µM and nocodazole concentrations between 0.1-10 µg/ml were tested. As DMSO was used as a solvent for cytoskeleton inhibitors, an additional control with DMSO concentration corresponding to the highest amount of DMSO introduced to treatments with cytoskeleton inhibitors was added.
Table III. 1. Cytoskeleton inhibitor and control DMSO doses.
DMSO Chemical: Control latrunculin B nocodazole Control
Dose: - 0.25% 1 µM 5 µg/ml
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
III.2.2. Monitoring of culture health under experimental conditions
Fv/Fm ratios, indicating photosystem II efficiency were recorded using a Z985 Cuvette
AquaPen (Qubit Systems) before and after the experimental period for both P. neolepis and C. pelagicus. Dark adaptation times prior to Fv/Fm measurement were 20 min and
30 min for P. neolepis and C. pelagicus respectively.
Additionally, 1 µM SYTOX Green (Invitrogen) was used to determine if the P. neolepis cell membrane integrity and health were compromised during the treatment. Cells were subjected to treatment with cytoskeleton inhibitors, transferred onto glass bottom dishes and incubated for 15 min with 1 µM SYTOX Green. Cells were imaged with a confocal laser scanning microscope as specified in section III.2.3.. Images were then analyzed, and 25 cells per replicate from each treatment were grouped into three categories: unlabelled, weakly labelled and strongly labelled. The first two categories were considered viable cells. Each treatment was replicated three times and a total of 75 cells/treatment was analysed. Chi-square tests were used to determine if there were any differences between treatments.
III.2.3. Confocal microscopy observations of P. neolepis under cytoskeleton inhibitor treatment
5 ml of P. neolepis culture was aliquoted into 7 ml bijou bottles, supplemented with f/2 media and dosed with cytoskeleton inhibitors for a period of 24 h. Cells were incubated in cytoskeleton inhibitor for 2.5 h prior to addition of the fluorescent marker to ensure disruption had occurred prior to measuring silica scale formation. Once the initial inhibitor incubation period was over, 1 µM Lysotracker Yellow HCK 123 (HCK 123)
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes or LysoSensor DND-160 Yellow/Blue (PDMPO) were added to all treatments. Cells were then carefully transferred onto poly-L-lysine coated glass bottom dishes and imaged with a confocal microscope. Additionally, latrunculin B treated cells were imaged as Z-stacks of whole cells and then selective cell lysis with a Mai Tai pulsed multiphoton infra-red laser (Spectraphysics) set to 740 nm was administered in order to inspect the silica scale content of individual cells. Additionally, scale length, measured along the longest axis of each individual scale under DMSO (24 h incubation), nocodazole and control treatments (5 h incubation) was recorded. Variation in scale length between treatments was assessed using a two sample Kolmogorov-Smirnov two sample test and a t-test (Statsoft: Statistica).
Imaging: Texas Red-labelled tubulin antibodies were imaged using excitation with a
λ=543 nm He/Ne laser, with emission filters set to λ=565-615 nm. HCK 123 and
SYTOX Green were excited with λ=488nm (Argon laser) and emission filters were set to λ=500-550 nm and λ=650-710 nm for HCK 123 and chlorophyll autofluorescence respectively. Calcite was imaged using excitation with a He/Ne laser at λ=633 nm, and emission was collected between λ=650-700 nm. PDMPO excitation and emission filters were set as specified in section II.2.3. All confocal images were taken with a Zeiss LSM
510 Meta microscope.
III.2.4. Flow cytometry monitoring of silica scale production in P. neolepis
1ml of concentrated exponentially growing culture was added to 4 ml of fresh FSW (pH
8.20), supplemented with f/2 nutrients and 100 µM Si and yielding cell densities between 1-1.5x105 cells/ml. Samples were then dosed with 5 µg/ml nocodazole or 1 µM
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes latrunculin B and 0.25% DMSO, corresponding to the amount of DMSO present in the nocodazole solution. Overall, DMSO concentration in all treatments did not exceed
0.35%. Additional 0.1% DMSO treatment, only containing DMSO introduced with the fluorescent dye, was set up to confirm, that the higher DMSO concentrations used in the experiment had no adverse effects on scale production.
Samples were incubated with cytoskeleton inhibitors/DMSO for 2.5 h to allow cytoskeleton disruption. Cells were then labelled with 1µM LysoTracker Yellow HCK
123, and incubated for 24 h. Cell count and Fv/Fm ratio immediately before and 26.5 h post dosing with cytoskeleton inhibitors were recorded to monitor culture health. Cells were left to silicify over a 24 h period (18:6 light:dark cycle). Cells were then harvested by centrifugation, washed 3x with 2% SDS solution with 0.1 M Tris/EDTA (pH 8.00) to remove cellular debris and then washed again 3x with deionised water. Samples were then analysed using Accuri C6 flow cytometer set to record side scatter and green fluorescence (SSC vs. FL1A), gated for fluorescent and non-fluorescent particles.
Experiments were repeated five times in triplicate in order to ensure, that consistent response was exhibited both within and between experiments. Student's t-test for independent samples was used to determine differences between treatments (Statsoft:
Statistica).
III.2.5. C. pelagicus: Differential Interference Contrast (DIC) imaging of cytoskeleton inhibitor treated cells
C. pelagicus cells were decalcified by decreasing the pH with HCl down to pH 4.00 for
3 min. pH was then brought back up to 8.20 with NaOH, and the cells were inspected under a microscope to ensure complete decalcification. 1ml of cells was then aliquoted
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes onto poly-L-lysine coated glass bottom dishes, supplemented with 2 ml of fresh f/2 media and dosed with inhibitors as listed in table 1. Images were taken 24h after inhibitor application. Cytoskeleton inhibitors were then washed off 3x with fresh f/2 media and cells were allowed to recover to ensure, that the malformations were due to inhibition of the respective cytoskeletal components and not imminent cell death, resulting from cytoskeleton inhibitor poisoning. Images were taken again 24 and 48 h post cytoskeleton inhibitor removal. DIC images were taken with a Nikon Eclipse Ti microscope equipped with a Photometrics Evolve EMCCD camera.
III.2.6. C. pelagicus: Electron Microscopy imaging of cytoskeleton inhibitor treated coccoliths
C. pelagicus samples: 15 ml samples were supplemented with 5 ml of filtered sea water
(FSW) and f/2 media, and dosed with cytoskeleton inhibitors as specified in "culture conditions and cytoskeleton inhibitor treatment" section. Cells were then incubated in inhibitros for 1 h and subsequently decalcified at pH 4.0 for 3 minutes. Samples were inspected under a light microscope to ensure complete decalcification. Cells were incubated with cytoskeleton inhibitors for 24 h following decalcification. Samples were then pelleted and 1ml of NaOCl was added to each sample. Samples were vortexed and placed on a rocking table for an hour to dissociate organic matter and prevent the sample from settling. Samples were then vortexed again, pelleted and NaOCl was removed. The remaining pellet was washed 3x with deionised water adjusted to pH
10.00 with NH4OH. Samples were then filtered onto a 0.22 µM filter membranes, put on a stub and sputter coated with gold-palladium. Imaging was done with a JEOL JSM-
7001F Field Emission Microscope and JEOL JSM-6610LV Scanning Electron
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Microscope at 15 kV accelerating voltage. The images were then analysed and 100 coccoliths per replicate, giving a total n=300 per three replicates, were examined and assigned into one of three categories: complete, partially formed/slightly malformed and malformed coccoliths. Chi-square tests were then performed on data obtained to determine, whether there were differences in frequencies of occurrence of coccoliths from different categories between treatments.
III.2.7. Tubulin Immunofluorescence labelling
Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich) was used as primary anti-α-tubulin antibody, and Texas Red conjugated anti-mouse IgG raised in goat (Thermo scientific, #31660) was used as a secondary antibody.
Pre-labelling treatment:
P. neolepis cultures were placed in 7 ml bijou bottles and dosed with 1µM HCK 123 and 5 µg/ml nocodazole for 5 h to disrupt the tubulin network and to stain internal silica scales. Normal, control samples without the tubulin inhibitor were also examined.
C. pelagicus cells were decalcified with 25 mM EGTA solution in Ca2+ free artificial sea water, according to a protocol modified from Taylor et al. (2007), dosed with nocodazole and incubated for 5 h. Decalcification with low pH proved to be a much quicker and more efficient method and was therefore used in subsequent experiments instead of EGTA based protocol.
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Fixing and labelling: cells were settled onto a poly-L-lysine coated glass bottom dish, washed 3x with ASW (see section II.2.1. for details) and then fixed in 2% glutaraldehyde with 1.7% BSA (bovine serum albumin) in artificial sea water (ASW, recipe specified in section II.2.1.) for 10 min. Cells were then washed 3x with
ASW/1.7% BSA with 0.5% glutaraldehyde and incubated for 5 min. Solution was then removed and replaced with 0.05% Triton X-100 in ASW for 10 min. Cells were then washed 3x for 5 min with ASW/1.7% BSA and incubated for 20 min. 1/50 primary anti-
α-tubulin antibody was then added and cells were incubated on bench overnight. Cells were then rinsed 3x with ASW/1% BSA, and secondary, Texas Red-conjugated antibodies in 1/150 dilution. Samples were then left to incubate for 2.5 h, washed with
ASW/1.7% BSA 3x and viewed under a confocal microscope.
The experiment was repeated three times with three replicates per experiment, in order to ensure the consistency of the labelling pattern. Cells were also treated with secondary antibodies alone to control for non-specific antibody staining.
III.3. RESULTS
III.3.1. Assays to determine effects of cytoskeleton inhibition on culture health
In order to determine the roles of actin and tubulin in calcification and silicification in haptophytes, cytoskeleton inhibitors latrunculin B (actin) and nocodazole (tubulin) were used. Effects of both inhibitors are rapid, and reversible, once chemicals are removed from the system (De Brabander et al. 1976; Morton et al. 2000). To determine the
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes involvement of different cytoskeleton components, it was crucial to ensure that cells were healthy and viable whilst under cytoskeleton inhibitor treatment. Failing that would introduce effects non-specific to cytoskeleton inhibition and resulting from poor health and death of cultures.
Photopigment degradation, especially chlorophyll a, is one of the first signs of cell death (Berges and Falkowski 1998; Franklin et al. 2012). Chlorophyll fluorescence is a parameter often used as a proxy for chlorophyll concentration, and arises mainly from photosystem II (PSII) fluorescence (Krause and Weis 1991; Kromkamp and Forster
2003). Photosynthetic capacity of PSII, measured as the Fv/Fm ratio, has therefore been established as general health and stress indicator (Berges et al. 1996; Boyd and
Abraham 2001; Kromkamp and Forster 2003; Schagerl and Möstl 2011). Fv/Fm represents the maximum potential photochemical efficiency of PSII (photosynthetic quantum yield), where Fv is the difference between maximum and minimum fluorescence and Fm is the maximum fluorescence in a dark adapted sample (Kromkamp and Forster 2003).
This parameter has been measured for both P. neolepis and C. pelagicus before and after the cytoskeleton inhibitor treatment.
III.3.2. Effects of cytoskeleton inhibitors on P. neolepis health
Results of post treatment screening of P. neolepis cultures revealed, that the Fv/Fm ratio was only slightly diminished in latrunculin B treated cells (Table III. 2.), thus indicating that overall cultures were in good health post treatment.
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In addition to PSII monitoring, cell mortality assessed with cell counts before and after sample incubation with cytoskeleton inhibitors was also screened, and did not exceed
15% in all treatments This was considered acceptable, as in addition to the innate fragility of P. neolepis, cytoskeleton inhibitors, incapacitating cell division and preventing compensation for natural cell mortality were used. Additionally, 4.5% and
6.5% cell mortality was recorded for Control + 5 µl DMSO (accounting for the amount of DMSO introduced with the dye Lysotracker HCK123 in the flow cytometry experiments) and the DMSO control (Table II. 2.), indicating a slightly detrimental effect of the solvent on cultures.
Table III. 2. Photosynthetic efficiency and cell viability counts recorded for P. neolepis
26.5 h post cytoskeleton inhibitor treatment. Measurements were taken from triplicate samples of each treatment and averaged.
Control DMSO latrunculin B nocodazole Control with 5 µl Control [1 µM] [5 µg/ml] DMSO
average Fv/Fm 0.73 0.71 0.71 0.65 0.71
SD 0.03 0.01 0.01 0.03 0.02
Mortality 26.5 h
post treatment 0 4.5 6.5 14.6 13.9
[%]
Rescue of cytoskeleton inhibitor effects following removal of chemicals was not possible in P. neolepis due to cell fragility, rendering cells unable to withstand repeated
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes washing. Therefore another method of culture health evaluation was employed. This involved assessment of plasma membrane integrity, as it is one of the main criteria of cell viability and can be verified with a fluorescent DNA-binding probe, SYTOX Green
(Darzynkiewicz et al. 1994). This fluorescent marker is only able to penetrate compromised plasma membranes, and has been previously applied in health assessment of a wide range of unicellular organisms, including phytoplankton (Reavie et al. 2010;
Ribalet et al. 2007; Sato et al. 2004).
Following a prior study on phytoplankton mortality involving SYTOX Green application, three categories of cells have been distinguished based on the intensity of labelling: 1-viable cells (no staining), 2-weakly loaded cells considered to be of reduced viability, 3-strongly stained, dead cells (Fig. III. 2.) (Veldhuis et al. 2001).
Fig. III. 2. SYTOX Green labelled P. neolepis cells representative of the three cell viability categories: A - live cell with no labelling, B - live cell with weak, partial labelling (reduced viability), C,D - strongly labelled cells that are dead or leaky/approaching apoptosis (4 - D a partially lysed cell). Scale bar≈5 µm.
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45% of cells in P. neolepis controls showed weak staining with SYTOX Green, with further 12% exhibiting strong labelling diagnostic of leaky and dead cells (Table III. 3.), indicating fragility of this species. The control, DMSO and latrunculin B treatment population structure upon labelling was not statistically different, and comprised of approx. 43% unlabelled cells, 35% presenting with weak labelling and strongly labelled cells contributing approx. 12% of the total cells examined (Table III. 3.). The proportion of unlabelled cells in nocodazole treatment decreased significantly (20%), whereas the number of weakly stained cells amounted to 38%, which was not statistically different from the control sample (Table III. 3.). The percentage of strongly labelled cells increased significantly (41%), indicating, that nocodazole had some detrimental effects on cells. It is worth noting however, that all weakly labelled and a high proportion of strongly labelled cells in the nocodazole treatment had intact, healthy looking chloroplasts. This indicates, that the increased labelling might be a result of cells becoming more "leaky" due to microtubule depolymerization rather than dying/approaching apoptosis, which would be in agreement with the lack of effect of nocodazole on PSII efficiency. Therefore, the effects of cytoskeleton inhibitors on culture health following treatment were considered minor and culture health was deemed satisfactory.
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Table III. 3. Results of χ2 test between DMSO and nocodazole treatment labelled with
SYTOX Green. Examples of cell labelling on which category assignment was based are provided in Fig. III. 2. A-C. Results based on an experiment conducted in triplicate, n=75.
χ2 DMSO Control nocodazole
Labelling vs. Df p sample sample
nocodazole [%] [%]
None 9.82 1 <0.05 42.67 20.00
Weak 0.28 1 >0.05 45.33 38.67
Strong 53.78 1 <0.05 12.00 41.33
Whole sample 63.88 2 <0.05
III.3.3. Effects of cytoskeleton disruptors on silica scale morphogenesis in P. neolepis
Silica scales produced under experimental conditions were tracked with a fluorescent marker LysoTracker Yellow HCK 123 (HCK 123) and LysoSensor DND-160
Yellow/Blue (PDMPO), introduced into samples pre-incubated with cytoskeleton inhibitors for 2.5 h. Confocal microscopy imaging of cells post 24 h incubation with latrunculin B and fluorescent marker revealed that inhibition of actin polymerisation interfered with scale secretion. This resulted in retention of newly formed scales within cells (Fig. III. 3. A). Virtually no scales were secreted under this treatment, and cells accumulating an abnormally high number of silica scales were observed (Fig. III. 3. B).
Normally, P. neolepis maintains 2-3 scales intracellularly at one time (see Chapter II for
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes details). However, up to 8 scales were found within cells post latrunculin B treatment, using cell lysis with a multiphoton laser at 740 nm to confirm the number of internal scales (Fig. III. 3. A,B). The percentage of cells over-accumulating scales and their exact numbers as well as scale length were not possible to determine due to scales overlapping in the cell as well as their displacement, floating and loss of fluorescence following cell lysis with a laser. Despite the inability of cells to secrete completed silica scales, no obvious visible morphological aberrations in the overall scale shape were observed. This suggests that actin plays a role in secretion of scales but not their formation.
Fig. III. 3. Confocal microscopy images representative of P. neolepis cells following a
24 h treatment with 1 µM latrunculin B. A - cells pre-lysis showing signs of silica scale over-accumulation and the lack of extracellular scales (individual optical slice), B - the same cells lysed to demonstrate over-accumulated scales, C-DMSO control (B,C - 3D projections of Z-stacks). Scales produced during the treatment were labelled with
PDMPO (green), chlorophyll autofluorescence is shown in red. Intracellular scales marked with arrows (A). Scale bars correspond to 5 µm (A,B) and 2 µm (C). A total of
87 individual cells was examined (16-27 cells per each of the four replicates).
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Microtubule disruption did not have as severe an effect on scale secretion as interference with actin polymerization. Scales produced under nocodazole treatment were significantly smaller and appeared more round compared to controls (Fig. III. 4.
A-D, Fig. III. 5. B, Table III. 4.).
Fig. III. 4. 3D projections of confocal microscopy generated Z-stacks of P. neolepis after 24 h treatment with cytoskeleton inhibitors and a fluorescent marker HCK 123:
A,B - nocodazole (scale bar=5 µm), C - DMSO (scale bar=2 µm), D - control (scale bar=5 µm). Silica scales deposited during treatment labelled with HCK 123 (green), chlorophyll autofluorescence in red.
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Only scale length, measured along the longest axis of the scale could be determined due to the scales' flattened shape and consequent frequent positioning at an angle, rendering simultaneous measurement of width and length not possible.
Scales produced by cells treated with tubulin disruptor were on average 1.3 µm shorter than those produced in the DMSO treatment, measuring 3.21 µm and 4.52 µm respectively (Table III. 4.). DMSO and control scale lengths were not statistically different from each other (t=1.07, p=0.29, df=118) (Table 4).
Table III. 4. T-test comparisons of P. neolepis scales between different cytoskeleton inhibitor treatments (5 h for nocodazole and control, 24 h for DMSO). Experiments were conducted in triplicate, ntotal=60.
Mean scale Difference Treatment SD t df P length [µm] of means
DMSO vs. 1.31 -5.83 118 <0.05 nocodazole
Control 4.69 0.89
DMSO 4.45 0.85
nocodazole 3.21 1.52
The difference in the mean scale length in nocodazole treated samples was not caused by a uniform decrease in scale length, but resulted from formation of frequently occurring, abnormally small scales between 0.5-2 µm in length (Fig. III. 5. A,B). Also, the frequency of occurrence of scales above 4µm was considerably lower than in the control and DMSO, contributing to lowering of the mean scale length produced by cells
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes treated with nocodazole (Fig.III. 5. A). The overall distribution of scale length upon tubulin disruption was significantly different to that of DMSO (two sample
Kolmogorov-Smirnov test, p < 0.001, n=60). Distributions of scale length in control and
DMSO treatment were not statistically different (two sample Kolmogorov-Smirnov test, p > 0.1, n=60)
A 60
50
40
30
20 Frequency [%] Frequency 10
0 0-1 1.01-2 2.01-3 3.01-4 4.01-5 5.01-6 6.01-7
Size class [µm]
Fig. III. 5. A - frequency distribution of P. neolepis scales according to different size classes (red-nocodazole, black-control, gray-DMSO). B - confocal microscopy 3D projection of a Z-stack of a nocodazole treated P. neolepis cells immunolabelled with anti-α-tubulin antibodies. Silica scales produced under treatment were labelled with
Lysotracker HCK123, shown in green. Abnormally small, round scales typical of the nocodazole treatment are marked with white arrows. An intracellular scale of normal morphology is indicated with a red arrow. Such abnormally small scales are absent from controls (see Fig. III. 4. C,D and Fig. III. 7. for images representative of control cells).
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III.3.4. Quantification of cytoskeleton inhibitor effects on silica scale production in P. neolepis
To quantify the cytoskeleton inhibitor effects on silicification in P. neolepis, flow cytometry measurements in conjunction with fluorescent labelling of silica scales produced during treatment were used. As cells were lysed prior to flow cytometry analysis, both intracellular and extracellular scales deposited during incubation with cytoskeleton inhibitors were quantified. The ratio of fluorescent (new) vs. non- fluorescent (scales produced prior to labelling) scales was used as a proxy for silicification rate.
Flow cytometry analysis of HCK 123 labelled scales after treatment with cytoskeleton inhibitor for 24 h revealed that both nocodazole and latrunculin B caused a severe decrease in silicification rate (Fig. III. 6. A,B, Table III. 5.). DMSO and control samples were not significantly different, indicating, that DMSO did not have an inhibitory effect on silica scale production (Fig. III. 6. A, Table III. 5.). As the DMSO treatment corresponded to the highest amount of DMSO used as a solvent in cytoskeleton inhibitor dosed samples, it was used as a reference for analysis of data obtained from actin and tubulin inhibitor treatments (Table III. 5.).
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Fig. III. 6. Box-whisker plots of fluorescent vs. non-fluorescent scales ratios post 24h treatments with: A - latrunculin B, nocodazole, DMSO and control. B - latrunculin B and nocodazole (expanded y-axis).
A 78.9% decrease in average silicification rate in latrunculin B, and a 71.5% decrease in nocodazole treated cells relative to DMSO treatment was observed. Additionally, a significant decrease in silicification rate in the latrunculin B treatment relative to nocodazole was found (Table III. 5.), consistent with confocal microscopy observations
(Fig. III. 3, Fig. III. 4.).
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Table III. 5. Results of the t-test comparisons of the fluorescent vs. non-fluorescent scales ratio between 24 h Control, DMSO, latrunculin B and nocodazole treatments.
Experiment conducted 5 times in triplicate, n=15.
Difference Treatment t df P of means
DMSO vs. nocodazole 0.142 10.754 28 <0.05
DSMO vs. latrunculin B 0.157 12.387 28 <0.05
nocodazole vs. latrunculin B 0.015 2.097 28 <0.05
III.3.5. P. neolepis: immmunofluorescence imaging of the MT cytoskeleton
Eukaryote microtubules (MTs) are assembled from protofilaments, arising from linear α and β tubulin heterodimer polymerization (Wickstead and Gull 2011). To investigate the MT arrangement in P. neolepis, secondary immunofluorescence was employed. The method relies on binding of primary anti-α-tubulin antibodies, and then their visualisation with secondary antibody-fluorophore conjugates, targeted to primary antibodies. Immunofluorescence labelled samples were examined with confocal laser scanning microscopy. Due to the small size of individual microtubules and background fluorescence due to cell fixation with glutaraldehyde, only higher order structures such as cables and networks were detectable (Tagliaferro et al. 1997). Imaging of labelled control sample cells did not show any MT- intracellular silica scale associations (Fig.
III. 7.).
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Fig. III. 7. Confocal microscopy 3D projection of a Z-stack of P. neolepis tubulin- immunolabelled control cells.1: karyokinetic spindle between dividing cells 2-4: intracellular scales (tubulin: yellow, silica scales in green). Scale bar=5 µm.
Imaging of the nocodazole treated samples confirmed, that the tubulin polymerization inhibitor was effective and disrupted the microtubular network, as evident in Fig. III. 8., where the mitotic spindle between two dividing cells has been dissociated and only rudimentary spindle microtubules remained discernible. Virtually no MT structures apart from the haptonema and flagella persisted post nocodazole treatment. The remaining vestigial microtubular associations appeared severely disrupted (Fig. III. 8.).
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Fig. III. 8. P. neolepis: 3D confocal microscopy projection of a Z-stack of anti-α-tubulin immunolabelled nocodazole treated cells. 1: a malformed, intracellular scale, 2- haptonema and a flagellum, 3-intracelllular scale, 4-remnants of a dissociated karyokinetic spindle (tubulin: yellow, silica scales in green). Scale bar=5 µm.
A further control using secondary antibodies only (Texas-red conjugated antibodies specific to anti-mouse IgG), yielded non-specific labelling patterns in control samples
(Fig. III. 9.), indicating, that patterning obtained from un-dosed and nocodazole dosed cells was a result of antibody-binding with α-tubulin and not a result of non-specific binding.
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Fig. III. 9. Confocal microscopy image of a P. neolepis immunofluorescence labelling control: cells incubated with secondary antibodies only, exhibiting a non-specific labelling pattern (yellow). HCK123 scales labelled in green.
Imaging of the actin component of the cytoskeleton with Texas Red conjugated-
Phalloidin-a fungus derived toxin binding specifically to actin was also attempted. The attempt was, however, unsuccessful, perhaps due to low F-actin content or its degradation, and was not pursued further.
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III.3.6. Effects of cytoskeleton inhibition on culture health of C. pelagicus
Culture health in C. pelagicus was assessed in two ways: (1) measurement of photosynthetic efficiency following a 25 h cytoskeleton inhibitor treatment and (2) the ability to recalcify following removal of cytoskeleton inhibitors after the 25 h incubation period.
Photosystem II efficiency in C. pelagicus was not affected by cytoskeleton inhibitors, and only a small decrease in average Fv/Fm ratio was recorded for the latrunculin B treatment. However, given that cells were able to re-calcify normally (see below), this small decrease in Fv/Fm was deemed negligible (Table III. 6.).
Table III. 6. Photosynthetic efficiency recorded post 25 h cytoskeleton inhibitor treatment of C. pelagicus.
DMSO Control latrunculin B nocodazole Control
average 0.62 0.61 0.58 0.61 Fv/Fm
SD 0.01 0.01 0.02 0.01
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
III.4. Effects of cytoskeleton disruptors on biomineralisation in C. pelagicus: light microscopy imaging
In order to investigate the in-vivo effects of actin and tubulin disruption on C. pelagicus coccolithogenesis and its subsequent recovery from cytoskeleton inhibitors, differential imaging contrast light microscopy imaging (DIC) was employed. Cells were decalcified
1 h prior to the experiment (Fig. III. 10.), to enable tracking of coccoliths produced during the subsequent 24 h incubation with cytoskeleton inhibitors. Chemicals were then removed and replaced with fresh f/2 media in filtered sea water, and cells were imaged again at 24 and 48 h intervals to monitor recovery.
Fig. III. 10. DIC of decalcified C. pelagicus cells at the beginning of the experiment.
Scale bar=10 µm.
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Disruption of actin polymerization in C. pelagicus resulted in a complete cessation of coccolith secretion, and hence new coccolith production (Fig. III. 11. C). Coccoliths visible in some cells post treatment most likely represent those present in cells prior to dosing with latrunculin B (Fig. III. 10.). A number of cells without any intracellular coccoliths was also observed, and could be a result of actin inhibition or an intrinsic mechanism halting coccolithogenesis.
Cells treated with nocodazole were still able to produce and secrete coccoliths, however, coccoliths were malformed and often detached from cells (Fig. III. 11. D, Fig. III. 12.).
These malformations were further investigated with SEM.
Both control and DMSO treated cells had re-deposited nearly full coccospheres 24 h post treatment, indicating no adverse effects of DMSO on coccolithogenesis (Fig. III.
11. A,B).
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Fig. III. 11. DIC images of cytoskeleton inhibitor treated C. pelagicus: A - D:24 h post treatment: A - control, B-DMSO, C-latrunculin B,
D - nocodazole treatment. E-H - 48 h post cytoskeleton inhibitor removal: E - control, F - DMSO, G - latrunculin B, H - nocodazole. Each treatment was conducted in triplicate, images are representative of a response in all replicates. Intracellular coccoliths visible in latrunculin B treated cells are a pre-treatment artefact. Scale bars=10 µm.
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Cells were successfully recovered post 24 h cytoskelton inhibitor treatment and resumed calcification once the inhibitors have been removed (Fig. III. 11. E-H). Control, DMSO and most of the nocodazole treated cells reassembled a full coccosphere 48 h post experiment. The effect of latrunculin B on coccolithogenesis was more severe than that of nocodazole, however, cells resumed calcification as both coccoliths adhering to individual cells and a large amount of shed coccoliths was observed post 48 h latrunculin B removal (Fig. III. 11. G, Fig. III. 12.). The accumulation of shed coccoliths was a common feature between all treatments and consistently comprised of a high proportion of malformed coccoliths, which may have been the cause of their detachment/failure to attach to the cell surface in the first instance.
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Fig. III. 12. DIC image of C. pelagicus post 48 h recovery from latrunculin B treatment.
Image was collected from the centre of the dish, where all shed coccoliths would accumulate during sample transport. Note that the vast majority of shed coccoliths are malformed (marked with thin arrows, normal coccolith indicated with a thick arrow).
Similar images were obtained from all treatments.
III.4.1. SEM analysis of cytoskeleton inhibitor-induced coccolith malformations in C. pelagicus
Intracellular and extracellular coccoliths produced during treatment with cytoskeleton inhibitors were isolated and examined with SEM, to further investigate malformations visible on previously collected DIC images.
Coccoliths produced post cytoskeleton inhibitor treatments presented various states of completion and malformation and were therefore divided into three categories based on
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes their morphology, summarised in Fig. III. 13. A-D. Coccoliths classed as partially malformed/incomplete were characterised by a largely complete structure with some slight aberrations to the shape of crystal deposited (Fig. III. 13. B). The malformed coccoliths were characterised by completely distorted crystal structure and often resembled disorganised blocks of calcite aggregated into a larger "lump" structure (Fig.
III. 13. C,D).
Fig. III. 13. FESEM images of C. pelagicus coccoliths used for morphological aberration ranking of coccoliths produced in controls, DMSO and nocodazole treatments: A - normal coccolith, B - partially malformed/incomplete coccolith, C, D - malformed coccoliths. Scale bars correspond to 1 µm.
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Statistical analysis of frequency of occurrence of coccoliths belonging to three distinct classes between control and DMSO treatment confirmed, that these two samples were not significantly different from each other (χ2(df=2, n=300)=0.16, p > 0.2). The DMSO treatment data were then used as a more suitable control against the dataset obtained from the nocodazole treatment due to solvents' presence in the inhibitor-treated sample.
The Chi-square comparison between the DMSO and nocodazole treatment revealed, that significantly more malformed and partially formed coccoliths were produced in nocodazole treated sample (Table III. 7.). Malformed and partially formed coccoliths accounted for 44.3% and 28% of the total number of coccoliths analysed in the nocodazole sample, whereas in the DMSO treatment, corresponding numbers were much lower at 15.6% and 18.6% respectively. Normal coccoliths in the nocodazole treatment comprised only 27.7% of the total sample, whereas the majority of coccoliths produced in the DMSO sample (65.7%) presented as normal.
Table III. 7. χ2 statistic results of DMSO and nocodazole treatments, n=300.
Coccolith DMSO sample nocodazole sample
2 category χ Df p [%] [%]
Normal 66.0 1 <0.05 65.70 27.70
Partially formed 16.7 1 <0.05 15.60 28.00
Malformed 144.0 1 <0.05 18.60 44.30
Whole sample 226.6 2 <0.05
Coccoliths obtained from latrunculin B treatment were not included into the statistical analysis, as virtually no coccoliths were observed to have been secreted during the DIC
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes imaging. Therefore, the images obtained from this treatment are either of coccoliths that were almost complete/at various stages of completion prior to dosing with latrunculin B and/or of those, that were being deposited during the treatment. It is therefore not possible to determine, if coccoliths were formed during the treatment. However, it appears that coccoliths extracted post incubation with latrunculin B, that were present in cells at various stages of formation prior to inhibitor treatment (or those that were being produced during the treatment) within cells have also been affected and severely malformed (Fig. III. 14. A-C). This indicates, some degree of calcification still occurred after latrunculin treatment had begun.
Fig. III. 14. A-C - malformed/incomplete coccoliths representative of latrunculin B treatment. As determined with DIC imaging, these coccoliths were mostly intracellular at the time of extraction. Scale bars correspond to 1 µm.
III.4.2. C. pelagicus: immunofluorescence imaging of the MT cytoskeleton
Confocal microscopy imaging of the α-tubulin-immunolabelled C. pelagicus cells revealed the presence of MT networks close to the CV, creating an ovoid structure
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes beneath the maturing coccolith (Fig. III. 15.:1,2). No intimate MT-CV association was observed, likely due to the resolution limitation arising from imaging of very fine structures with high background fluorescence levels.
Fig. III. 15. 3D projections of confocal microscopy Z-stacks of anti-α-tubulin immunolabelled C. pelagicus control cells. A: 1-3-tubulin network around the forming coccolith (tubulin: yellow, calcite reflectance in white). B-Same cells with the white channel switched off for clarity.
The extensive MT network in C. pelagicus cells was severely disrupted and reduced post 24 h nocodazole treatment (Fig. III. 16. A,B). Virtually no discernible organised
MT features were visible post treatment, however, some remains of the MT structures were still present in some cells (Fig. III. 16. A,B).
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Fig. III. 16. 3D projections of confocal microscopy Z-stacks of anti-α-tubulin immunolabelled C. pelagicus after 5 h treatment with nocodazole. A-1,3- coccolith vesicles with a forming coccolith. B-same cells with white channel turned off, with remnants of tubulin cables near the coccolith of cell 1 (α-tubulin: yellow, calcite reflectance in white). Scale bar=5 µm.
Incubation of C. pelagicus with secondary antibody only did not yield any specific labelling patterns (Fig. III. 17. A,B).
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Fig. III. 17. 3D confocal microscopy projections of Z-stacks of C. pelagicus nonspecific labelling control (secondary antibodies only, shown in yellow). A - nonspecific staining only, forming coccoliths in white. B - same image with the white channel switched off for clarity.
III.5. DISCUSSION
III.5.1. Cytoskeleton inhibitors and culture health
Monitoring of the P. neolepis and C. pelagicus culture viability during incubation with cytoskeleton inhibitors was essential for establishing that the effects observed resulted from disruption of the target cytoskeleton components and were not an artefact of poor culture health. Screening of PSII did not indicate any significant departures in the Fv/Fm ratios from the control in C. pelagicus and only a small decrease in PSII efficiency was found in latrunculin B treated P. neolepis cultures, potentially caused by higher sensitivity of P. neolepis to disruption of actin-dependent photosynthesis regulation or
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes via secondary effects arising from interruption of other vital, actin-based cellular processes (Takagi 2003).
SYTOX Green screening of P. neolepis control cultures indicated that high percentages of cells with reduced viability were normally present in exponentially growing cultures and were therefore considered to arise from intrinsic fragility of P. neolepis. High percentage of cells with reduced viability is also normally found in field phytoplankton samples (30-60% of the population) as well as in cultures of other fragile algal species
(Veldhuis et al. 2001; Zetsche and Meysman 2012). The significant decrease in the share of unlabelled cells, with a concomitant significant increase in strongly labelled cells in nocodazole treatment could be an artefact resulting from an increased permeability of the plasma membrane due to the presence of DMSO, which is known to permeabilize and thin membranes, combined with effects of a potent microtubule inhibitor (Gurtovenko and Anwar 2007). This seems likely, as the efficiency of PSII was not affected by this drug.
The mortality recorded for cytoskeleton inhibitor and DMSO treatments in P. neolepis was considered minor and a likely result of cell loss to autolysis, which could not have been compensated by cell division, as both latrunculin B and nocodazole disrupt cytoskeleton integrity, prompting mitosis suppression (Gachet et al. 2001; Jordan et al.
1992). In the case of DMSO, it is more likely that it had a minor, negative effect on cell growth rather than mortality, potentially by secondarily altering water and ion transport in cells (Gurtovenko and Anwar 2007).
Calcification recovery assay in C. pelagicus showed that coccolithogenesis was re- established in all treatments after inhibitor removal. The slower recovery of cells following latrunculin B treatment might be either a result of the presence of residual inhibitor due to entrapment in cell coating or a slower recovery of actin cytoskeleton.
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Overall, the combined results indicate that both P. neolepis and C. pelagicus cells remained viable and in good healthy during cytoskeleton inhibitor treatments and responses observed were specific to disruption of target elements of cytoskeletal network.
III.5.2. The role of actin in biomineralisation
Upon incubation with latrunculin B, P. neolepis cells were unable to secrete scales, indicating the presence of an actin-dependent mechanism responsible for silica scale exocytosis. However, P. neolepis cells were still capable of forming scales internally, where they accumulated, although there was a significant decrease in silicification rate, as determined with flow cytometry. This, along with no obvious scale malformations suggests, that the silica biodeposition process in this haptophyte is not strictly actin dependent and the main factor limiting scale formation when actin is disrupted is the cells' physical capacity to maintain multiple intracellular scales. As cells were still able to deposit scales it appears, that impairing actin cytoskeleton did not majorly interfere with intracellular transport of components required for silica scale biomineralisation to be carried out.
In contrast to the above response of P. neolepis, incubation of C. pelagicus with latrunculin B had more severe effects. In this case, the entire process of coccolithogenesis and coccolith secretion was brought to a complete halt following F- actin disruption. This indicates, that actin-dependent intracellular transport of vesicles and substrates, facilitated by motor proteins is essential for coccolithogenesis to proceed in this species. The severe intracellular coccolith malformations observed post actin
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes inhibitor treatment suggest, that actin may be involved in actively shaping the growing coccolith. However, it cannot be ruled out that the short term exposure to low pH during decalcification could have contributed to malformations observed. The pH shock would have caused a temporary cell acidification, coupled with a decrease in calcification rate for a short period of time, until the cells have re-adjusted pH and re-established normal calcification rate (Taylor et al. 2011). The consequence of this short-term interference with the calcification system is an increased malformation of coccoliths formed during and directly after the decalcification event, contributing to the 19% of malformed coccoliths recorded in control and DMSO samples. Additionally, a small percentage of malformed coccoliths is naturally produced in coccolithophore cultures (GMD, personal observation; Langer et al., 2010).
Compared to the previous study by Langer et al. (2010), involving actin disruption in another coccolithophore, Emiliania huxleyi, effects of actin disruption in C. pelagicus were much more severe, causing a complete inhibition of coccolithogenesis. In the study by Langer et al. (2010), treatment with cytochalasin B did not completely stop coccolith formation, but induced a 50% increase in malformed coccolith production instead. This divergence in response might arise from the fact, that unlike latrunculins, cytochalasin B does not depolymerize F-actin, but caps the actively growing barbed ends, preventing both polymerization and depolymerisation, inducing filament shortening (MacLean-Fletcher and Pollard 1980; Morton et al. 2000; Theodoropoulos et al. 1994). It also promotes isotropic F-actin network disruption, creating focal F-actin accumulations, however it does not affect filament annealing or monomer addition at the pointed end of actin microfilaments (Forscher and Smith 1988; MacLean-Fletcher and Pollard 1980; Schliwa 1982). Additionally, at the concentration used by Langer et al. (2010), cells were still able to divide, suggesting, that although severely impaired, F-
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes actin was still present and probably still able to facilitate motor-protein or microfilament polymerization/depolymerization driven intracellular transport to some extent.
Considering the above, it is conceivable, that the severe coccolith malformations in latrunculin B treated C. pelagicus are at least partially due to cells actively forming coccoliths upon drug addition, resulting in a progressive F-actin inhibition and depolymerisation as the drug takes effect, causing coccolith malformation and ultimately bringing coccolithogenesis to a stop.
The observed inhibition of biomineralised scale secretion in both P. neolepis and C. pelagicus post latrunculin B treatment indicates, that this process, along with subsequent scale positioning on the cell surface is actin-based in both organisms. This is consistent with previous reports on transport of organelles, secretory vesicles and vacuoles in plant, animal and fungal cells, which have been show to be driven by myosin-facilitated trafficking along actin cables, and rapid membrane movement in plants (Hammer and Sellers 2011; Schott et al. 2002; Tabb et al. 1998; Vale 2003).
III.5.3. The role of tubulin in biomineralisation
Treatment of P. neolepis and C. pelagicus with nocodazole caused severe disintegration and almost complete depolymerisation of MTs in both species, as confirmed with immunofluorescence imaging. Disruption of the microtubular cytoskeleton caused a significant decrease in scale production in P. neolepis, indicating its heavy involvement in silica biomineralisation in this species. The observed decrease in silicification rates could result from disruption of three main mechanisms essential for biomineralisation to proceed and mediated by MTs: (1) intracellular transport, (2) active, mechanical
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes shaping of the growing biomineral structure and (3) secretion of mature biomineralised elements.
The observed decrease in silicification rate in P. neolepis could potentially arise from substrate limitation, as like F-actin, MTs form extensive and dynamic intracellular networks, serving as tracks for intracellular transport-mediating motor proteins, or direct attachment sites for the cargo (Vale 2003). MT-based motor proteins, such as kinesins and dyneins are responsible for transporting both non-membranous molecules, such as mRNA as well as membranous vacuoles, vesicles, lysosomes, organelles, or endoplasmic reticulum (ER) (Brendza et al. 2000). Therefore, disruption of microtubular network impairs both MT and motor protein mediated transport of specific substrates into the biomineralising compartments such as CV or SDV. It also affects distribution and dynamics of the ER and the Golgi body, structures that are both heavily involved in biomineralisation in haptophytes, resulting in the adverse effects of tubulin disruption on both silica scale and coccolith formation (Billard 2004; Lippincott-
Schwartz et al. 1995; Wubbolts et al. 1999). Due to the profound role of MTs in intracellular transport, MT disruption could also affect the general cell health, potentially introducing secondary effects leading to reduction of cell capacity for biomineralisation and further contributing to the observed decrease in silica scale production in P. neolepis. The frequent manifestation of abnormally small, scales and overall shift towards production of smaller scales post nocodazole treatment in P. neolepis also indicates, that MTs-although not forming structures visible with immunofluorescence or TEM-are involved in maintaining the size and shape of the newly forming silica scale in this species. This might be achieved either via direct interaction with the SDV or by acting in concert with actin cytoskeleton, with scale size potentially limited by substrate availability due to disruption of the intracellular, tubulin-based transport system (Rodriguez et al. 2003; Yoshida et al. 2006).
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Depolymerisation of the microtubular network in C. pelagicus caused a significant increase in mild to severe coccolith malformation, consistent with findings by Langer et al. (2010). In fact, only 28% of coccoliths obtained post tubulin disruption in this species were fully developed. The arrangement of calcite under nocodazole treatment became severely distorted, indicating that MTs are likely to be involved in delimiting and guiding crystal growth via mechanical control over the CV, as evident from coccoliths resembling disarrayed blocks of crystals obtained from this treatment (Fig.
13B-D). Indeed, extensive MT networks have been confirmed to be localized in close proximity to the maturing CV, and could be utilised for substrate transportation into the
CV (Fig. 15A,B). Existence of fine microtubular structures directly associated with the growing coccoliths and not resolved with immunofluorescence is also plausible. Based on the severe malformation cases, it is also possible, that formation of the organic template involved in guiding nucleation of crystals during coccolithogenesis could be affected, resulting in displacement of crystal nucleation centres, accounting for the complete lack of organisation of the developing structure. In addition to the increased production of aberrant coccoliths, the frequency of occurrence of slightly malformed and incomplete coccoliths also increased post nocodazole treatment, indicating increased premature secretion to be related to MT network disruption in C. pelagicus.
This is in contrast with the findings of Langer et al. (2010), who concluded, that this process was cytoskeleton independent and might be due to the specifics of the cytoskeleton inhibitor used for the purpose of this study.
It will be important to determine whether the calcification rate in C. pelagicus has also been affected by MT disruption, as over 70% of coccoliths from the nocodazole treatment were to some extent malformed, and detached from cells. Consequently, very few coccoliths were seen attached to the cell surface under this treatment, with the
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes majority of coccoliths accumulating at the bottom of the dish, making visual estimations impossible. As similar accumulations of shed, malformed coccoliths were seen in all treatments and after recovery experiments, it has been concluded, that aberrations in coccolith shape interfere with its adherence to the cell surface.
III.5.4. Differential role of cytoskeleton in silicification in haptophyte and
Bacillariophyte (diatom) algae
Silicification in diatoms requires heavy involvement of both the actin and the tubulin cytoskeleton, actively controlling frustule morphogenesis by mechanical moulding of the SDV (Tesson and Hildebrand 2010; Van De Meene and Pickett-Heaps 2002). Actin has been identified as the major component involved in control over silica deposition during diatom valve formation at the micro and meso scale as well as the overall valve shape, and positioning of structures such as e.g. the raphe (Cohn et al. 1989; Tesson and
Hildebrand 2010; Van De Meene and Pickett-Heaps 2002). In contrast, in the haptophyte, P. neolepis, actin appears to be involved in exocytosis rather than formation of the silica scales, as treatment with an actin inhibitor latrunculin B did not produce any visible aberrations in the overall shape of scales deposited. Microtubules in diatoms are responsible for localising the SDV formation site, and frustule formation cannot be initiated if MTs are absent (Van De Meene and Pickett-Heaps 2002). Disruption of MTs during valve morphogenesis results in severe distortions, as the microtubular cytoskeleton in diatoms is responsible for strengthening and positioning of specific structures of the frustule (Van De Meene and Pickett-Heaps 2002). In P. neolepis, although no SDV-MT associations could be resolved with immunofluorescence, MTs are involved in controlling the overall shape of silica scales. This is potentially achieved
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes by delimiting the shape of the SDV. Disruption of MTs in this species results in production of dwarf scales and alteration of the shape, producing more round morphologies. In this case scale production was severely decreased, but nucleation of new scales was not completely inhibited like initiation of frustule formation in diatoms.
III.6. CONCLUSIONS
Differential roles in biomineralisation for actin and tubulin in the silicification and calcification in haptophytes and bacillariophytes have been established. Actin inhibition in haptophytes prevents secretion of the biomineralised structure, however, the entire biomineralisation process is halted in the calcifier, whilst the silicifier is still able to proceed with siliceous scale deposition until its cell capacity is reached. Effects of cytoskeleton inhibition on formation of mineralized scales were mainly caused by direct, mechanical interaction of the cytoskeleton with the vesicle, in which the structure was being deposited and by the intracellular transport, limiting substrate delivery and capacity for structure exocytosis.
The cytoskeleton appears to be more heavily involved in morphogenesis of biomineralised scales in C. pelagicus than in P. neolepis, as cytoskeleton disruption resulted in severe coccolith malformations. In P. neolepis silica scale morphology was not as severely affected, although production of abnormally small silica scales was observed post microtubule disruption (see Table III.8. for a summary of the effects of cytoskeleton inhibitors on P. neolepis and C. pelagicus). The differences in the use of cytoskeletal elements for biomineralisation in these two haptophytes are likely to be related to the differences in the formation site of biomineralised structures (Golgi-
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes derived CV in C. pelagicus and an SDV in P. neolepis), the absence of an organic scale serving as a baseplate in P. neolepis and a different secretion site of the completed structure (see Chapter II for details on silica scale formation process).
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Durak, G.M. Chapter III: The role of cytoskeleton in biomineralisation in haptophytes
Table III. 8. Summary of the effects of cytoskeleton inhibitors on P. neolepis and C. pelagicus.
Cytoskeleton Effects
inhibitor P. neolepis C. pelagicus
Exocytosis of the biomineralized scales inhibited
Silica scales formed and accumulated intracellularly until Latrunculin B internal storage capacity exceeded. Coccolith formation ceased (actin inhibitor) Significant decrease in silica scale production.
No visible malformations to the silica scales Coccoliths malformed
The size distribution of the silica scales shifted significantly Significant increase in production of malformed coccoliths.
Nocodazole towards smaller sizes. Scale morphology more round, Frequent detachment of coccoliths from the cell surface, likely
(MT- inhibitor) frequent occurrence of abnormally small "dwarf" scales due to the malformations
Significant decrease in silica scale production
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
CHAPTER IV: BIOCHEMICAL ANALYSIS OF PRYMNESIUM NEOLEPIS SILICA SCALES
IV.1. INTRODUCTION
Silica biomineralisation mechanisms have been of great interest due to their potential for extrapolation and subsequent use in materials science, as well as their relevance from the evolutionary point of view (i.e. evolution of different biomineralisation systems in eukaryotes). Therefore, they have been investigated in a variety of organisms
(see Chapter I for an extensive review). Most research efforts within this area have been focused on the silicification systems of two evolutionarily distinct groups of organisms: the diatoms (unicellular algae) and sponges (simple metazoans). The former utilise silica to deposit a rigid external cell wall, termed the frustule, whereas the latter produce silica spicules, maintaining structural support of the organism amongst other functions
(Uriz et al. 2003). In both cases silica structures produced contain an organic phase directly involved in silica precipitation and organics acting as a scaffolding for the developing structures (Pickett-Heaps 1990; Schröder et al. 2007). Silicification systems employed by these two groups of organisms are, however, quite different in terms of the major organic fractions involved in silica deposition and their mode of activity, i.e. enzymes in sponges vs. polycationic polypeptides in diatoms (see sections I.3. and I.6. for detailed information). Nonetheless, they also share common traits. These include the presence of an organic matrix underlying the biosilica structures, acting as a template in conjunction with the remaining organic fraction capable of active precipitation of silica
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales out of solution, such as long chain polyamines (LCPAs) (e.g. Matsunaga, Sakai et al.
2007; Sumper, Kröger et al. 2004).
In order to investigate the biosilica associated organic fraction in different taxa a variety of approaches for extraction from the silica matrix have been developed. The oldest and still popular methods for biosilica dissolution are based on the use of HF acid (Ehrlich et al. 2010). These methods however have significant drawbacks, in that HF has been found to cleave disulfide, O-glycosidic and phosphate ester bonds leading to deposphorylation of peptides and proteins recovered after treatment (Fuchs and Gilvarg
1978; Mort and Lamport 1977). Another popular method of silica dissolution, commonly used for extraction of the organic matrix from sponge spicules is the use of alkali such as NaOH (Ehrlich et al. 2010). Alkali can be used to etch biosilica at various concentrations and with addition of other chemicals, such as sodium dodecyl sulfate and other anionic biosurfactants (Ehrlich et al. 2010; Ehrlich et al. 2006). However, despite the attempts to reduce the detrimental effects that this treatment has on the organic phase, NaOH based methods cause protein denaturation (Ehrlich et al. 2010).
Finally, a relatively mild treatment involving acidified ammonium fluoride (NH4F) solution, which dissolves biosilica via conversion into ammonium hexafluorosilicate can also be applied (Ehrlich et al. 2010; Kröger et al. 2002). This method is often used as an alternative to the harsh HF or alkali treatments, and was successfully applied to extract relatively intact proteins and LCPAs from diatom biosilica (Kröger et al. 2002;
Swift and Wheeler 1992).
Overall, as each silica dissolution method has its own constraints and only allows to retrieve biomolecules which are not specifically affected by it, the NH4F based method was deemed to be the best choice for extraction of biosilica-associated organics from the silicifier investigated in this study - P. neolepis.
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
Prymnesium neolepis is a marine haptophyte alga, producing small (≈1-1.5 µm), unmineralized organic scales as well as larger (≈2-17 µm) silica scales (see section I.3. for detailed description). To date, it is the only haptophyte alga known to deposit silica structures rather than calcite-mineral phase utilized by the dominant biomineralizing group within this clade-coccolithophores. As such, it presents a unique and interesting opportunity to investigate the biochemical composition and mechanisms of silicification within another evolutionarily distinct, calcifier-dominated group of organisms. This would enable further comparisons between the systems as well as provide an insight into the evolution of the ability to silicify within the algal-and to some extent-within the metazoan clades.
Therefore, the objectives of this chapter are to:
1. Verify the presence of silica-scale associated organics.
2. To characterize the organics, if present.
3. To draw comparisons between the silicification systems present in P. neolepis,
diatoms and sponges.
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
IV.2. METHODS
IV.2.1. Silica scale purification
The silica scale purification protocol was modified from a procedure for the isolation of diatom frustules (Kröger et al. 1999). Cells were harvested by low pressure suction pump filtration, removed from filters with a pipette and pelleted by centrifugation. An excess of 2% SDS in 100 mM EDTA, 0.1 M Tris at pH 8.00 was added, the solution was vortexed and left for 30-60 min, with occasional vortexing in order to dissociate the protoplast debris from scales. Samples were then centrifuged at 6000x g for 10-15 min, supernatant was discarded and the procedure was repeated 5-7 times. Samples were then sonicated for 10-15 min, vortexed, centrifuged at 6000x g and the supernatant was discarded. Samples were then boiled in the SDS solution at 95 ºC for 10 min to further remove the organic phase from silica, vortexed and centrifuged. Samples were washed with the SDS solution or sonicated again if required based on a visual assessment with a microscope. Once sample purity was deemed satisfactory (i.e. no pigments or any other visible protoplast contamination was observed), the samples were washed 5x with deionised water and sub-sampled for further sample purity verification using a FESEM and TEM. This involved further subsampling of the material onto stubs/copper grids and screening for the presence of cellular debris and organic scales. Once samples were confirmed to consist of silica scales only and the absence of protoplast debris and organic scales were confirmed, samples were frozen until further processing.
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IV.2.2. Glycerol cushion separation of the purified silica scales and further assessment of sample purity
A portion of the biosilica material purified as described above was further separated on a glycerol cushion in order to verify if samples were contaminated with organic scales produced by P. neolepis (Yoshida et al. 2006). 176.5 mg of silica scales were resuspended in 2.5 ml of deionised water and 500 µl of the suspension was overlaid on top of 4 ml of 50% glycerol solution and spun down at 3200x g for 2 minutes. The supernatant was collected into a separate tube, and the pellet was resuspended in 500 µl of deionised water and spun down through a glycerol cushion again. The procedure was repeated 4 times. The pellet was then washed 5x with deionised water and stored in the freezer. The supernatant collected was also pelleted for 15 min at 6000x g, washed 5x with deionised water and frozen. Both the purified silica scale pellet (termed "scale fraction") and the supernatant (termed "supernatant fraction"), which should contain the organic scales (if present), were then sub-sampled for FESEM examination. The procedure yielded 103 mg of the silica scale fraction and 34 mg of the supernatant fraction, in which all organic scales (if present) should be retained along with lighter silica scale material.
The material was then treated with ammonium fluoride to extract the NH4F soluble and insoluble organic fractions. The NH4F soluble fractions were then run on a 16%
Schägger gel (Schägger and von Jagow 1987) and compared. The insoluble material was washed 5x with deionised water and examined with a FESEM and TEM to reassess sample purity and the absence of organic scale contaminants.
In order to further ensure the purity of biosilica material, 30 mg of biosilica scales were incubated on a rocking table for a period of 20 h with 100 mM EDTA (pH 8.00) at 4 ºC, to extract any proteins that were accessible prior to silica dissolution (method modified
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales from Kröger et al., 1997). After the incubation period was over, the sample was centrifuged for 1min at max rpm, the supernatant was transferred to a 3 kDa cutoff filtration column (Amicon) and concentrated to 500 µl. The sample was then washed once with 5ml of 50 mM EDTA, lyophilized and resuspended in 100 µl of deionised water. Sample was then run on a 16% Schägger gel and stained with EZBlue gel staining reagent (Sigma-Aldrich) to confirm the absence of protein contamination.
IV.2.3. Ammonium fluoride extraction of the organic phase
In order to dissolve the silica component of scales, 2 ml of 10 M NH4F was added to 30-
100 mg biosilica sample and vortexed until the pellet was dissolved. 0.5 ml of 6 M HCl was then added to the mixture, vortexed, and the pH was adjusted to between 4-5 with 6
M HCl. The sample was incubated on bench for 30 min. Mixture was then centrifuged for 15 min at max rpm and the supernatant was transferred to a 3 kDa cutoff filtration column (Amicon) to concentrate and desalt protein. The NH4F insoluble pellet was washed twice with 50 mM ammonium acetate and stored in the freezer. The supernatant from the insoluble phase washes was added to the amicon column with the rest of the supernatant and concentrated to 1ml. The concentrate was washed once with 500 mM and 200 mM NH4Ac (ammonium acetate) and 3x with 50 mM NH4Ac. 5 ml of each solution was used per wash. The sample was then further concentrated to 150-400 µl.
All flow through was collected and stored in the freezer for further analysis. Protocol was modified from (Kröger et al. 1999).
Fig. IV. 1.. shows the above silica scale purification steps and the fractions obtained post NH4F treatment of the biosilica.
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
Fig. IV. 1. A schematic summarising processing of the P. neolepis biosilica in terms of fractionation post SDS/EDTA purification of scales and the subsequent phase separation post NH4F extraction.
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
IV.2.4. Separation of the putative polyamine fraction from the NH4F flow-through on an ion exchange column
The flow through from the NH4F extraction described above was diluted (4.5:100) with deionised water and passed through an 8 ml liquid chromatography column (Sigma) containing 2 ml of high S strong cation exchange resin (Bio-Rad). The column was previously washed 10x with deionised water, the resin was added and then the system was flushed with 10 ml of deionised water, 10 ml of 2 M ammonium acetate and then again washed 3x with deionised water. The diluted flow-through sample was then fed into the column, the whole process being gravity-driven. Once the sample has passed through the column, the resin was washed 3x with 1ml of 200 mM NH4Ac and eluted
4x with 1ml of 2 M ammonia, the eluant was neutralised with acetic acid and lyophilized (sample referred to as "ammonium eluate"). Resin was then eluted again 4x with 1ml of 2 M NaCl, 100 mM NH4OH, 50 mM NH4Ac, neutralised with acetic acid and dialysed 1x against 5 l of 50 mM ammonium acetate in a 1 kDa dialysis tube, collected into a tube and lyophilized (sample referred to as "dialysate"). Both samples were then resuspended in 100 µl of deionised water and examined on a 16% Schägger gel.
IV.2.5. Polyacrylamide gel analysis
All NH4F soluble organic phase extracts were run on a 16% Schägger gel (Schägger and von Jagow 1987) in order to retain both high and low molecular weight components.
The gels were then stained with Coomassie Brilliant Blue (colloidal or R-250, Sigma-
Aldrich), Stains-all (Sigma-Aldrich) as per manufacturer's instructions and silver
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales stained following a protocol modified from (Chevallet et al. 2006). Sequential staining with Coomassie Brilliant Blue, Stains-all and silver stain was also used to enhance the sensitivity of labelling (Goldberg and Warner 1997). Unstained Broad Range Page
Ruler (Fermentas) and Mark 12 protein standard (Invitrogen) were used as a size reference for Coomassie and sliver staining. Pre-stained Ladder (Thermo-Scientific) was used with Stains-all. Additionally, a standard 12% SDS-PAGE was used for diatom protein digest analysis.
IV.2.6. Trypsin digestion of the NH4F soluble organic extract
In order to determine if the organic fraction obtained as the NH4F soluble phase consisted of protein, a trypsin digest was conducted, where 10 µl of the NH4F soluble extract was treated with 2 µg of TPCK (tosyl phenylalanyl chloromethyl ketone ) treated trypsin in a 15 µl reaction, buffered with 100 mM Tris-Cl at pH 8.80. A control without trypsin was also included. Samples were incubated at 37 °C overnight and analysed on a 16% Schägger-PAGE.
Controls for the digest consisted of 15µl reactions containing 2 µg of TPCK treated trypsin buffered with 100 mM Tris-Cl at pH 8.80 and 2 µg of diatom proteins-silaffin rSilC and cingulin rmCinY3 (kindly provided by Prof. Kröger) and one reaction only containing trypsin. Samples were incubated at 37 °C overnight and analysed on 12%
SDS-PAGE gel.
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
IV.2.7. Amino acid analysis
Aminoacid analysis was carried out using a protocol modified from Bidlingmeyer et al.
(1984). 10 mg of purified biosilica material and an equivalent amount of deionised water for a blank control were transferred into 2 ml glass tubes, covered with Parafilm and lyophilized. 100 µl of 6 M HCl (ultrapure, Pierce) was added into each sample. 100
µl of phenol (100%, ultrapure) was then added to prevent oxygen degradation of the amino acids. Samples were then transferred into hydrolysis vials, frozen with liquid nitrogen and evacuated with a vacuum pump for 3 min and nitrogen was pumped into the vials The procedure was repeated 3x to eliminate oxygen. The samples were then thawed and incubated at 110 °C for 24 h. The hydrolyzed samples were transferred into
Eppendorf tubes, vacuum dried and stored at -20 °C until analysis.
The samples were then PITC (phenylisothiocyanate) derivitized and subjected to a
HPLC (high-performance liquid chromatography) analysis using Dionex UltiMate®
3000 HPLC (Thermo Scientific) against appropriate amino acid standards prepared fresh before the analysis.
IV.2.8. Mass spectrometry analysis
Mass spectrometry analysis of the putative polyamine phase separated on the cation exchanger was kindly provided by Prof. Kröger. A replicate sample of the polyamine extract and the excised protein bands from the NH4F-soluble extract visualized on a
16% Schägger gel with EZBlue (Sigma-Aldrich) (one band at≈63 kDa and two bands around 55 kDa) were sent for mass spectrometry analysis to Alta Bioscience (UK). To aid the mass spectrometry identification of the proteins, the P. neolepis transcriptome
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
(kindly supplied by Dr. I. Probert) was translated into protein sequences in all 6 possible open reading frames and forwarded to Alta Bioscience.
Protein samples were analysed using an Orbitrap Mass Spectrometer (Thermo
Scientific), following a standard LC-MS/MS (liquid chromatography–mass spectrometry/mass spectrometry) analysis of trypsin digested samples. Peptide sequences obtained were then compared against the transcriptome data translated into protein sequences. DNA sequences corresponding to the peptide data generated by the
MS were recovered from the transcriptome, translated into protein sequences with
BioEdit software and blasted against the NCBI protein database in order to look for homologous sequences in other taxa. Amino acid composition of the two protein sequences retrieved were generated with BioEdit software.
IV.2.9. FESEM, TEM and EDX analysis of the NH4F insoluble extract
The NH4F insoluble extract was washed 5x with deionised water and 5x with absolute ethanol (molecular grade) before being deposited on an aluminium stub in order to prevent appearance of drying artefacts on the organic structures. FESEM/TEM imaging and EDX analysis were conducted as described in section II.2.6.
IV.2.10. Confocal microscopy imaging of the NH4F insoluble extract
Alexa-Fluor conjugated Wheat Germ Agglutinin (WGA, Invitrogen), Calcofluor White
(CW, Sigma) and Concanavalin-A (Con-A, Invitrogen), targeting β 1,4 N-acetyl glucosamine linkages (WGA), β 1,3- and β 1,4- linkages (CW) and internal and non-
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales reducing, terminal α-D-mannosyl/glucosyl groups in polysaccharides (Con-A)
(Harrington and Hageage 2003; Mandal et al. 1994; Nagata and Burger 1974) were selected to provide preliminary information on the presence of polysaccharides in the
NH4F insoluble extract. Concentrations and incubation times used to label the extract are specified in Table IV. 1.
Table IV. 1. Concentrations and incubation times of fluorophores used.
Dye Concentration Incubation time [min]
WGA 5µg/ml 20
CW 10µg/ml 20
Con-A 20µg/ml 60
Additionally, controls with whole cells and whole scale extracts were incubated with the dyes. Imaging was done on a Zeiss LSM 510 meta confocal laser scanning microscope.
Alex-fluor WGA and Con-A were imaged using an argon laser, with excitation and emission filters set to λ=488 nm and λ=500-550 nm respectively. CW was imaged using a multiphoton excitation via a Mai Tai pulsed infra-red laser (Spectraphysics), set to
λ=740 nm excitation and λ=435-485 nm emission.
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IV.3. RESULTS
IV.3.1. Amino acid analysis of purified P. neolepis biosilica
In order to provide preliminary information on the presence and nature of the organic component associated with silicified scales, the amino acid content of purified biosilica was analysed using HCl/phenol digestion. This approach allows to confirm the likely presence of proteins in the silica material and indicates the gross amino acid composition of the silica scale associated organic fraction.
The amino acid analysis of the purified whole scales resulted in recovery of 13 different amino acids, indicating that an organic phase was indeed present and putatively contained protein. Biosilica was confirmed to be proline rich, relative to other amino acid residues detected (Fig. IV. 2.).
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Fig. IV. 2. HPLC chromatogram of amino acids of the whole scales. Abbreviations of the amino acid residues are as follows: D - aspartic acid, E - glutamic acid, O - glutamine, S - serine, G - glycine, T - threonine, A- alanine, P - proline, V - valine, I - isoleucine, L - leucine, F - phenylalanine, K- lysine.
IV.3.2. NH4F soluble phase extract: putative protein and polyamine phase
In order to recover whole proteins from P. neolepis biosilica without hydrolysing them, dissolution of silica with acidified NH4F was applied. The resulting NH4F soluble fraction, containing silica-associated organic phase extract was run on a 16% Schägger gel, yielding 2 bands between 50-70 kDa (one singlet and one doublet) and a broad band around 2.5 kDa (Fig. IV. 3. A-C). The broad band around 2.5 kDa has only labelled with Coomassie Blue, suggesting binding by charge in absence of protein and was predicted to likely contain polyamines as it was the case with diatoms (Fig. IV. 3.
A-C) (Prof. Kröger, personal communication). The two bands between 50-70 kDa
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales labelled pink with Stains-all, indicating presence of potentially slightly acidic proteins
(Fig. IV. 3. C).
Fig. IV. 3. NH4F soluble extract run on a 16% Schägger gel, subsequently stained with:
A - Coomassie Blue, B - silver stain, C - Stains-all. Molecular weight markers are indicated on the left hand side lane on gels.
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IV.3.3. Sample purity verification: Schägger-PAGE analysis of the NH4F soluble phase generated from glycerol cushion-separated silica scales and EDTA extraction of protein from whole scales
In addition to the silica scales, P. neolepis also produces non-silicified, organic scales which could potentially contaminate the biosilica fraction and contribute to the organics recovered. Separation of the biosilica material on the glycerol cushion aimed to fractionate the heavier silica scales separate from the lighter, organic scales/remnants of organic debris and shards of silica scales by density, with the lighter (organic) fraction being unable to penetrate into the cushion. Thus, the pure "silica scale" phase at the bottom of the cushion and the overlying "supernatant" phase, containing all putative organic contamination and lighter siliceous material were obtained. No organic scale/debris contamination was detected upon TEM and FESEM screening of subsamples of the above fractions (appendix IV.1.). TEM and FESEM screening of the
NH4F insoluble extract obtained from the "supernatant" fraction, in which contamination was expected to be more evident due to the "enrichment" in the putative organic contamination and subsequent demineralization did not show any evidence of contamination either.
Analysis of the NH4F soluble extract of the "silica scale" and "supernatant" fractions yielded identical Schägger-PAGE labelling pattern (Fig. IV. 4.). This further suggested, that the "silica scale" and "supernatant" fractions were unlikely to be contaminated with organic scales produced by P. neolepis, as the banding pattern would differ between the two extracts if organic scales/contaminants were responsible. Thus the banding visible on the Schägger-PAGE represent an NH4F soluble organic phase intimately associated with the silica scales.
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Fig. IV. 4. Coomassie Blue staining of the NH4F soluble extract of the silica scale fraction run on a 16% Schägger gel (lane 1) and the supernatant fraction (lane 2).
Schägger-PAGE analysis of the EDTA extract from the whole scales did not detect the presence of any protein, further confirming that the protoplast and organic scale contamination was absent from the biosilica material.
IV.3.4. Trypsin digest of the NH4F soluble extract: verification of presence of protein
In order to confirm, that bands between 50-70 kDa consisted of protein, a trypsin digestion of the NH4F soluble extract was carried out. Upon trypsin digestion, the two bands between 50-70 kDa disappeared (Fig. IV. 5. A,B). The broad band below 2.5 kDa however persisted post digest, suggesting that it might not consist of protein (Fig. IV. 5.
A,B). Both diatom rmCinY3 and rSilC proteins were digested by trypsin (Fig. IV. 5. C).
Trypsin itself has undergone self digestion and no band was observed for controls containing trypsin only (Fig. IV. 5. A-C).
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Fig. IV. 5. A - Coomassie stained 16% Schägger gel: lane1-trypsin digested NH4F soluble extract, lane 2 - non-digested NH4F extract control, lane 3 - non-digested rmCinY3 control, lane 4 - trypsin, B - gel A stained with silver stain on top of
Coomassie Blue and Stains-all, C - Coomassie Blue stained 12% SDS-PAGE gel: lane
1: trypsin digested rSilC, lane 2 - trypsin digested rmCinY3, lane 3-non-digested rSilC control, lane 4 - non-digested rmCinY3 control, lane 5 - trypsin.
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IV.3.5. Cation-exchange liquid chromatography column samples: purification of putative LCPAs from the NH4Fextraction flow through
LCPAs purification via cation exchange was possible on account of their positive charge, after the preliminary separation from larger proteins was performed on a size seclusion column (Kröger et al. 2000). The putative LCPA material retained in the
NH4F flow through fraction, subsequently bound to the HighS cation exchange resin and eluted with ammonia yielded one broad band around 2.5 kDa upon Coomassie Blue staining, suggesting putative presence of LCPAs (Fig. IV. 6.). The purpose of the second wash with NaCl/NH4OH/NH4Ac solution was to elute any biomolecules that did not elute with ammonia. After dialysing the salts out of the second eluant ("dialysate" sample) however, no bands were observed.
Fig. IV. 6. Coomassie stained 16% Schägger gel with samples isolated on a cation exchanger: lane 1 - ammonium elute, lane 2 - dialysate.
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IV.3.6. Mass spectrometry analysis of the protein extracts
The results of mass spectrometry analysis of trypsin digested protein samples yielded sixteen unique peptide matches to a sequence from the P. neolepis peptide translation of the transcriptome, named comp1037 (Table IV. 2.). The corresponding nucleotide sequence was then retrieved from the transcriptome, translated into a peptide sequence and blasted against the NCBI protein database, revealing some degree of homology with lipocalin like proteins (Table IV. 2.). The amino acid sequence of comp1037, along with an alignment with NCBI-derived, partially homologous sequences are attached as appendix IV.2. and IV.3. respectively.
Table IV. 2. Summary of putative properties of the comp1037 peptide recovered from the transcriptome following MS analysis of trypsin digested protein samples extracted from P. neolepis biosilica.
Length MW E Protein (amino Identity Coverage (kDa) value acids)
38%-Lipocalin like protein
from Karlodinium veneficus 1e-8 28%
comp1037 294 31.6 26%-Apolipoprotein
D/lipocalin from 1e-5 68%
Osteococcus tauri
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Putative amino acid composition of comp1037 is summarised in Fig. IV. 7. Comp1037
appears to be rich in proline and lysine, which characteristics are common in proteins
with silica precipitating properties (Kauss et al. 2003; Kröger et al. 1999).
Amino Acid Composition P1F3_comp1037_c0_seq1: 0 to 881: Frame 3 294 aa
18
16
14
12
10 Mol % Mol 8
6
4
2
0 Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr Amino Acid
Fig. IV. 7. Amino acid composition of comp1037 peptide retrieved from P. neolepis
transcriptome. Abbreviations are as follows: Ala - alanine, Cys - cysteine, Asp - aspartic
acid, Glu - glutamic acid, Phe - phenylalanine, Gly - glycine, His - histidine, Ile -
isoleucine, Lys - lysine, Leu - leucine, Met - methionine, Asn - aspargine, Pro - proline,
Gln - glutamine, Arg - arginine, Ser - serine, Thr - threonine, Val - valine, Trp -
tryptophan, Tyr - tyrosine.
IV.3.7. Mass spectrometry analysis of the putative polyamine phase isolated on the
cation exchanger
The ammonia eluate sample isolated on a cation exchanger contained molecules in the
mass range of 500-800 Da (Fig. IV. 8. A,B). Each peak represents a molecular ion with
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales a single positive charge. Two main series ("triplets") of peaks with mass to charge ratio
(m/z) values of 559.6 (a)/573.6 (b) /587.6 (c) in triplet 1 and 630.7 (a')/ 644.7 (b')/ 658.7
(c') in triplet 2 were recorded. The mass differences of 14 within each triplet suggested, that the three peaks are methylation isoforms of each other. The mass differences of 71 between the pairs a-a', b-b', and c-c' suggested presence of N-methyl propyleneimine units, a pattern previously found in diatom LCPAs (Kröger et al. 1999).
A
Gregg Hill P5814 es297514 24 (0.841) Cm (20:37) TOF MS ES+ 346.3 100 7.91e3
332.3 261.3 316.3 B 214.2 245.3 417.4
% 403.4 275.3 401.4 630.7 644.7 228.2 347.4 299.3 431.5 488.5 557.6 573.6 185.7 474.5 645.7 200.2 587.6 616.6 701.7 715.7 150.2 529.6 674.7 731.8 0 m/z 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
Fig. IV. 8. A,B - mass spectra of the ammonia eluate isolated from the NH4F flow through on a cation exchanger. A - spectrum obtained by Prof. Kröger, B - spectrum of a replicate sample obtained by Alta Bioscience.
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Collision induced fragmentation (CID) of individual ions revealed the presence of multiple N-methyl propyleneimine units, as two series of peaks with mass differences of
71 were present between neighbouring peaks in each series (228.2/299.3/370/441.4 and
313.3/384.4/455.4). This confirms, that the parent molecule of m/z=573.6 contained multiple linearly linked N-methyl propyleneimine units (Fig. IV. 9.). Fragmentation analysis with other peaks yielded analogous results, confirming the presence of N- methylated oligopropyleneimines (data not shown).
Fig. IV. 9. Collision induced fragmentation spectrum of the m/z=574.6 peak.
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IV.3.8. FESEM and EDX and confocal analysis of the NH4F insoluble extract, detection of polysaccharides with fluorescent markers
The NH4F insoluble extract was imaged in order to verify if any remnants of organic structures potentially underlying silica scales were present post demineralization, as was previously found in e.g. sponges and diatoms (Scheffel et al. 2011; Uriz et al. 2003).
The FESEM images of the ethanol washed NH4F insoluble extract revealed that the material is represented by two distinct morphological components: a fibrous one (Fig.
IV. 10. A) and a globular one (Fig. IV. 10. B). The majority of the extract consisted of the fibrous component.
Successful labelling of the NH4F insoluble extract with both WGA (Wheat germ agglutinin) and CW (Calcofluor white) (Fig. IV. 10. C,D), indicated putative presence of chitin or a similar polysaccharide. The whole cells and purified scales displayed very little and no fluorescence respectively when the above fluorescent dyes were used, indicating that non-specific labelling was unlikely (data not shown). The NH4F insoluble extract did not label with Con-A.
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Fig. IV. 10. FESEM images of the NH4F insoluble biosilica-associated organic phase:
A - fibrous component , B - globular component. Scale bars correspond to 100 nm.
C - D: confocal images of the same extract: C - labelled with WGA, D -labelled with calcofulor white. Scale bars correspond to 5 and 10 µm respectively.
The EDX analysis of the NH4F insoluble extract indicated presence of carbon, oxygen, sulphur, phosphorus and calcium in the extract, confirming presence of organic material
(Fig. IV. 11.). Some trace remains of the mineral phase were also detected. Both fibrous and globular components of the extract returned similar spectra.
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
Fig. IV. 11. EDX analysis of the NH4F insoluble extract. The high aluminium signal is due to the stub, the copper signal is also most likely a contamination.
IV.4. DISCUSSION
The biochemical analysis approaches adopted in this investigation have been previously used to successfully identify and characterise a variety of organic, silica precipitating components in organisms such as silicifying sponges and diatoms (Ehrlich et al. 2010;
Kröger et al. 1999; Kröger et al. 1997; Müller et al. 2003). Indeed, they have also yielded good results when applied to investigation of the biosilica produced by P. neolepis. Preliminary amino acid analysis of the P. neolepis silica scales indicated that the material indeed contains a proline rich organic fraction, suggesting the presence of proteins. Subsequent NH4F extraction and SDS-PAGE analysis further confirmed the
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales presence of silica scale associated proteins and LCPAs. An insoluble organic fraction potentially acting as an organic scaffolding in an intact scale has also been recovered.
IV.4.1. NH4F soluble organic extract: silica-associated proteins
The disappearance of the singlet and doublet bands between 50-70 kDa upon trypsin digestion confirmed, that these bands generated from the NH4F soluble extract consisted of protein (Fig. IV. 5. A,B). The mass spectrometry analysis of the aforementioned bands in conjunction with transcriptome and NCBI blast analysis generated a putative biosilica associated protein candidate, exhibiting a weak homology with lipocalin-like proteins, which are ubiquitously expressed in both prokaryotes and eukaryotes and are responsible for transport of small hydrophobic molecules (Flower et al. 1993). This finding suggests a potential existence of a novel biosilica-associated type of proteins specific to silicifying haptophytes. Although based on the results obtained in this study further description of comp1037 peptide was not possible, these preliminary data constitute a valuable basis for further investigation into the nature and identity of silica scale-associated protein fraction in P. neolepis.
IV.4.2. LCPAs
The persistence of the broad band around 2.5 kDa after treatment with trypsin indicated, that this fraction either did not contain any protein or that the cleavage sites for trypsin
(peptide bonds between the carboxyl group of arginine and lysine and the amino group of the adjacent amino acid), were not accessible to the enzyme (Simpson 2006).
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
Additionally, the broad band around 2.5 kDa corresponded to a size region similar to the previously described for silica precipitating LCPAs, isolated from diatoms and sponges (Matsunaga et al. 2007; Sumper and Kröger 2004). Indeed, subsequent separation of the NH4F flow through on a high S cation exchanger and an MS analysis confirmed, that the material appearing on the Schägger gel around 2.5 kDa consisted of
LCPAs. The MS analysis indicated the presence of linearly linked N-methylated oligopropyleneimine repeats (n=4-9 repeats) with the [m+H]+ ion unit value identical with the diatom oligo-propyleneimine modified lysine residues that form side chains in silaffins (Kröger et al. 1999). To date, the LCPAs found in diatoms have been found to contain putrescine, propylenediamine and spermidine as a base molecule (Kröger and
Poulsen 2008b), whereas the sponge LCPAs contain butaneamine and N-linearly linked propyleneimine sulfate chains (Kröger et al. 1999; Matsunaga et al. 2007; Sumper and
Kröger 2004) (Fig. IV. 12. A-C). Hence the LCPAs found in P.neolepis are the first putatively lysine-based LCPAs reported to date. It is possible however, that degradation of the protein occurred prior to extraction, and the LCPAs isolated are a degradation product of a protein similar to a diatom silaffin, which is modified with LCPA side- chains (Kröger et al. 1999). This could have happened due to the biosilica material remaining in culture solution for extended periods of time as a result of the slow growth of the species, and therefore its accessibility to proteolytic enzymes potentially present in the media. However, the former case, indicating free LCPAs seems more likely, as the amount of the polyamine material extracted per sample vastly exceeded that of protein material, and some remnants of degraded protein material would be expected to still be evident on a gel upon silver staining. Instead, the characteristic pattern of one singlet and one doublet band around 50-70 kDa along with a broad band around 2.5 kDa was consistently found post NH4F extraction of three different biosilica samples. Also, the LCPAs in e.g. diatoms have been shown to vary both within and between species in
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
terms of the chain length, attachment moieties and the degree of methylation,
characteristics that are thought to be responsible for morphogenesis of the species-
specific frustule ornamentation (Sumper and Kröger 2004; Sumper and Lehmann 2006),
hence the presence of a lysine based LCPA in P. neolepis would not be surprising. At
this stage, further analysis is required to show categorically whether these are indeed
non-protein bound LCPAs or degradation products of a larger protein, with the first step
in this direction involving molecular identification and biochemical characterization of
the biosilica associated proteins extracted from P. neolepis. In both cases it is a
significant find, as the polyamine fraction found in P. neolepis biosilica contributes a
novel type of a polyamine, putatively involved in silicification or a new type of a non-
protein bound polyamine. It is the first report of a polyamine component putatively
involved in biosilicification within the haptophyte clade, indicating that convergent
evolution of silica biomineralisation might have occurred in poriferan, heterokontophyte
and haptophyte clades.
Fig. IV. 12. A - sponge LCPA structure after Matsunaga et al. (2007), B - schematic
structure of a putrescine based diatom LCPA (putrescine moiety in blue), after Kröger,
Poulsen (2008), C-polyamine-modified lysine from a diatom sillaffin (lysine moiety in
gray), after (Kröger and Poulsen 2008b). Propyleneimine unit repeats are indicated by C "n" number.
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
IV.4.3. NH4F insoluble organic extract
The EDX analysis of the NH4F insoluble material, indicating the presence of C, O, Ca,
P, S and Si (Fig. IV. 11.) provided additional confirmation of the presence of an organic fraction, consisting potentially of carbohydrates with some residual mineral fraction.
The presence of proteins in the extract is also likely, despite the absence of N from the spectra obtained. N is very difficult to detect by EDX analysis due to the detectors' construction, even when the N content of the sample is expected to be very high (Laskin et al. 2003). Also, an amino acid analysis of the NH4F insoluble extract yielded similar results to the whole scale extract, indicating presence of at least peptide components
(Prof. Kröger, personal communication).
The fibrous material revealed by FESEM of the NH4F insoluble extract (Fig. IV. 10. A) is hereby hypothesised to act as an organic matrix, as it is the case with diatoms and sponges. The fibrous material is thus proposed to form a scaffolding, on which the loop structures are deposited during silica scale morphogenesis (see section II.3.3. for details). The globular fraction of the same material (Fig. IV. 10. B) consists of organics of unknown function, occluded during scale formation and then aggregated once the mineral phase was removed. The EDX spectra obtained from the extract, along with the information obtained in previous studies involving diatoms, where chitin was found to constitute a major organic matrix component within girdle bands (Durkin et al. 2009), prompted a preliminary screening for polysaccharides (chitin in particular) in the extract. The material stained with WGA, which has previously been used for detection of chitin in diatom frustules by Durkin and Mock et al. (2009) and binds specifically to
β 1,4 N-acetylglucosamine polymers (Nagata and Burger 1974) as well as with CW, which binds to β-1,3- and β-1,4-linked polysaccharides, such as chitin and cellulose
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales
(Fig. 10), (Harrington and Hageage 2003). Thus preliminary information on the putative presence of chitin in the P. neolepis biosilica has been gained. The presence of chitin should be further verified i.e. using a highly chitin-specific Chitin Binding Probe (CBP).
The lack of labelling with Con-A, which binds to internal and non-reducing terminal α-
D-mannosyl and α-D-glucosyl groups (Mandal et al. 1994) indicates absence of mannose-backbone containing polysaccharides or their inaccessibility for binding.
Further characterization of the NH4F insoluble organic phase associated with P. neolepis silica scales should focus on providing more information on the nature of the organic matrix underlying the silica scales in terms of its structure, composition and capacity to actively precipitate silica.
IV.5. SUMMARY
The biochemical investigation of the P. neolepis biosilica resulted in detection of closely associated organics, fractioning into a soluble and insoluble phase upon NH4F demineralization of the material. The NH4F soluble fraction was found to contain potentially novel, silica-associated proteins as well as a new type of lysine-based polyamine, contributing the first report of putatively silica-precipitating components present within a haptophyte alga. The fibrous fraction of the NH4F insoluble organic extract was proposed to serve as a matrix underlying biosilica structure-a feature shared also by the diatom and sponge systems. It is also suggested to contain chitin. The presence of LCPAs, silica-associated proteins and an insoluble organic matrix as common components involved in silica biomineralisation in diatoms, haptophytes and sponges, provides an indication, that the silica biomineralisation systems have been
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Durak, G.M. Chapter IV: Biochemical analysis of P. neolepis silica scales acquired via means of convergent evolution, which topic will be further addressed in
Chapter V.
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Durak, G.M. Chapter V: General Discussion
CHAPTER V: GENERAL DISCUSSION AND CONCLUDING REMARKS
V.1. INTRODUCTION
In this study physiological and biochemical aspects of silicification in P. neolepis were successfully identified, allowing for comparisons between other biomineralizing algal taxa such as diatoms and coccolithophores. I have shown that P. neolepis is so far the only obligate silicifier within the haptophyte clade, in which formation of biomineralised elements of cell covering fundamentally differs from that of calcifying members of this taxon, bearing some similarities to the silicification system of heterokontophytes instead. Differences in the formation and exocytosis site, cytoskeleton involvement and structure morphogenesis between silica scales and calcitic coccoliths were revealed (see summary in Table V.1.), indicating that the biomineral structures produced in these two systems as well as the systems themselves are most likely non-homologous Biochemical analyses of P. neolepis biosilica revealed that the organic matter .associated with silica scales contained soluble and insoluble proteins, LCPAs and putatively organic template-forming chitin, hinting that silica biomineralisation in haptophytes, heterokontophytes and silicifying sponges share some common features and mechanisms involved in controlled silica precipitation, of which evolutionary significance is discussed further in this chapter.
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Durak, G.M. Chapter V: General Discussion
Table V. 1. Table summarizing differences between the formation of the silica scales and coccoliths.
Silica scale Coccolith
Material Silica (SiO2*nH2O) calcite (CaCO3)
Organic, likely chitin-containing, filamentous matrix underlying the silica Basis of the Organic baseplate homologous with an scale; presence of novel, Si-associated lipocalin-like proteins and LCPAs structure organic scale putatively involved in silica precipitation
Formation site SDV-like compartment localized in the posterior, vacuolar area of the cell Golgi-derived coccolith vesicle (CV)
Secretion site Posterior of the cell Anterior of the cell, near the flagellar root
The role of actin in Non-essential for silica scale formation, essential for exocytosis of a Essential for both coccolith formation and
structure formation complete scale exocytosis
The role of Likely involved in transport of substrates required for silica scale Play a very important role in correct microtubules in formation. Also involved in defining the overall shape of the silica scales morphogenesis of the coccoliths structure formation
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Durak, G.M. Chapter V: General Discussion
V.1.1. Origins of photosynthetic Eucarya
Photosynthetic eucaryotes are hypothesized to have originated ca. 1.5 BYA (billion years ago), acquiring a plastid via primary endosymbiosis of a cyanobacterium, however some estimates suggest that this event could have occurred even 2 BYA
(Douzery et al. 2004; Yoon et al. 2004). Chromalveolates are thought to have subsequently differentiated via secondary endosymbiotic event, in which a non- photosynthetic eukaryote engulfed a photosynthetic eukaryote, estimated to have taken place ca. 1.4 BYA (Fig. V. 1.) (Bhattacharya et al. 2007; Keeling 2010; Yoon et al.
2002). It has been generally accepted that green and red algae and higher plants are a product of a primary endosymbiotic event involving a non-photosynthetic eukaryote and a cyanobacterium (Fig. V. 1.) (Graham et al. 2009). The reminder of biomineral forming algae were derived from a non-photosynthetic eukaryote engulfing a red alga
(Fig. 1) (Graham et al. 2009).
However, recent studies by Moustafa et al 2009, involving analysis of whole diatom genomes indicated that diatoms were derived from two secondary endosymbiotic events, of which the first involved a green, prasinophyte-like alga. The red alga, previously accepted to be the plastid donor in the remaining algal lineages (apart from the green algae) did not undergo endosymbiosis until much later (Graham et al. 2009;
Moustafa et al. 2009). This chain of endosymbiotic events was then indicated as a common sequence of events leading to differentiation of all Chromalveolates (Fig. V.
1.) (Hohmann-Marriott and Blankenship 2011; Prihoda et al. 2012). In addition to assimilation of genetic material in the course of endosymbiotic events, a number of genes in algae, e.g. in diatoms was also acquired via HGT (horizontal gene transfer) from bacteria and Archaea (Prihoda et al. 2012).
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Durak, G.M. Chapter V: General Discussion
Fig. V. 1. The hypothetical chain of evolutionary events leading to plastid acquisition and subsequent lineage differentiation in eukaryotes (after Hohmann-Marriot and
Blankenship 2011).
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Durak, G.M. Chapter V: General Discussion
V.1.2. Evolution of silicification in Eucarya
The evolution of silicification in eukaryotes dates back to Neoproterozoic/Cambrian periods (710-680 MYA), which are characterized by supersaturation of the ocean with respect to monosilicic acid, where silica biomineralisation was first used in skeleton formation by benthic demosponges (Kooistra et al. 2007; Love et al. 2009; Maliva et al.
1989; Müller et al. 2007a). Silica biomineralisation systems were subsequently developed in radiolarians in Ordovician and then in diatoms, of which radiation period falls in Cretaceous (Kooistra et al. 2007; Maliva et al. 1989). The extensive proliferation of diatoms during Cretaceous and Tertiary resulted in a progressive depletion of Si from the euphotic zone, which in turn caused other silicifiers, such as sponges to decline due to the ensuing Si limitation (Harper and Knoll 1975; Maldonado et al. 1999). Changes in Si availability leading to its limitation due to biological uptake led to extinction of some silicifiers and generated selective pressure forcing development of active Si transport and concentration mechanisms in others (Raven and Waite 2004; Siever
1992). Therefore, silicifiers themselves constituted the driving force behind the evolution of efficient Si-specific transport systems via removal of Si from the surface ocean in a process of coevolution of organisms with their environment (Raven and
Giordano 2009). To date, almost all contemporary natural waters except some silica- rich hot springs are undersaturated with silicic acid (Channing and Edwards 2004).
Therefore capacity to actively transport and accumulate Si is essential for silica biomineralizing organisms.
Another theory pertaining to the evolution of Si-based systems and Si incorporation into cellular processes of algae has been put forward by Darley and Volcani (1969). In their view Si could have acted as an “inorganic enzyme”, providing bioactive surfaces within
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Durak, G.M. Chapter V: General Discussion the cell that then could be utilized to proceed with other vital reactions involved in prevention of cell aging. Due to the resulting Si accumulation in the cell, the excess was hypothesized to be sequestered in the endoplasmic reticulum and finally to be removed into acidic vacuole, where it would then autopolymerize. As solid SiO2 could no longer serve as a reaction site, it was extruded from the cell that would then have to replenish
Si from external source, thus developing a continuous requirement for Si supply that is evident in i.e. diatoms (Darley and Volcani 1969).
V.1.3. Evolution of other biomineralisation systems in Eucarya
The most common and widespread types of biomineralisation products of the extant eukaryotes are represented by calcium carbonates, calcium phosphates and silica (Knoll
2003). The ability to deposit these biominerals is widespread across the phyla, which has been hypothesised to be a consequence of two mechanisms: (1) HGT and (2) paraphyletic, independent evolution of biomineralisation systems partially based on common biochemical pathways which developed early within the eukaryotes (Knoll
2003). A large number of theories concerning the evolution of biomineralisation in
Eucarya has been proposed over the years. Some of them indicated the necessity of rigorous control of the intracellular concentrations of vital elements such as Ca and Fe that were present at very high extracellular concentrations at that time, as a driving force for further development of these systems and their subsequent adaptation for biomineral deposition (Weiner and Dove 2003). This theory was further explored by Westbroek and Marin (1998) in an endeavour to explain a phenomenon of molluscan nacre and coral induced bone regeneration and their biocompatibility with human tissues (Atlan et al. 1997; Dupoirieux et al. 1994; Silve et al. 1992). In the scenario proposed by
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Durak, G.M. Chapter V: General Discussion
Westbroek and Marin (1998) multiple taxa inherited biochemical components from a common, non-mineralized ancestor and subsequently independently adapted them to form biomineralised skeletal elements. Therefore, the polyphyletic character of calcareous biomineralisation systems is a result of independent development of the basic, common physiological and biochemical properties of cells, such as the ability to bind Ca2+ ions and regulate internal inorganic carbon chemistry with carbonic anhydrase and other enzymes, enabling supply of ions required for mineral deposition, which properties are widespread in all Eucarya (Aizawa and Miyachi 1986; Sanders et al.
1999; Westbroek and Marin 1998). The authors then suggest that the common physiological and biochemical basis originated in a common, unmineralized eukaryote ancestor during the Neoproterozoic. As Neoproterozoic oceans were oversaturated with
CaCO3, organisms inhabiting this environment had to develop efficient systems preventing spontaneous CaCO3 precipitation on the body surface, which provided the basis for subsequent evolution of mechanisms allowing for localization and precise control over biomineralisation of evolutionarily beneficial calcareous elements such as skeletons (Grotzinger and Knoll 1995; Knoll and Swett 1989; Westbroek and Marin
1998).
Additionally, in a "grand unified theory of biomineralisation" proposed by Kirschvink and Hagadorn (2000) magnetite biomineralisation is argued to represent a
"protobiomineralisation" system, of which understanding could provide a template for elucidating vacuolar-based biomineralisation processes in metazoans. Magnetite biomineralisation is estimated to have evolved 2 BYA in early eukaryotic ancestors, which putatively acquired it via endosymbiosis of magnetotactic bacteria (Kirschvink and Hagadorn 2000). Kirschvink and Hagadorn's theory introduces magnetite
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Durak, G.M. Chapter V: General Discussion biomineralisation as a missing link between biomineralisation systems and a precursor on which the extant biomineralisation systems are based.
V.2. SILICIFICATION IN P. NEOLEPIS: EVOLUTIONARY CONTEXT
The peculiar capacity of P. neolepis to biomineralise silica could be a result of a number of different processes that occurred during the evolution of the Prymnesiales. Based on the available information on the evolution of biomineralisation systems in Eucarya, four likely scenarios leading to acquisition of the ability to silicify in P. neolepis can be proposed: the system could have been (1) inherited from diatoms or a common silicifying ancestor of haptophytes and heterokontophytes or (2) result from a de-novo convergent evolution. In another scenario (3), the system could have been developed following acquisition of crucial components via HGT, in a process similar to that which was suggested to have occurred in choanoflagellates after acquisition of SITs (Marron et al. 2013). Another, potential theory (4) can be proposed based on the information concerning algal evolution, which involved a series of endosymbiotic events and consequently resulted in (at least partial) assimilation of the genetic material of the endosymbiont into the host genome. This process could have contributed vital components such as Si transporters, later adapted for biomineralisation purposes (e.g. a non-silicifying green, prasinophyte alga Platymonas was found to have a capacity for active Si uptake, the mechanism, acquisition route and incidence of this trait in prasinophytes are however unknown) (Nelson et al. 1984). This process could be further facilitated by HGT and as previously suggested by Westbroek and Marin (1998), could involve a further adaptation and development of a pre-existing biochemical machinery, inherited or acquired via HGT, ultimately allowing for biomineralisation. In this
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Durak, G.M. Chapter V: General Discussion scenario (4) I propose that basic biochemical components which could potentially be developed into calcification or silicification system were present in ancestral haptophytes before the ability to biomineralise was acquired. During the course of evolution, potentially facilitated by events such as HGT, environmental pressure and other specific variables affecting different haptophyte lineages lead to development of one system and not the other (or none at all), giving rise to calcifying clades such as coccolithophores, the silicifier, P. neolepis (and potentially more, undescribed silicifying haptophytes) and non-biomineralizing members. However, at this stage features supporting this theory, such as e.g. the presence of Si precipitating LCPAs in P. neolepis as well as in a variety of organisms (e.g. sponges, diatoms) could also be applied in favour of another theory proposed. As LCPAs are found in broad distribution of prokaryotic and eukaryotic species, the existing polyamine metabolism could have been adapted for Si precipitation via convergent evolution (Cohen 1998). The fact that
LCPAs retrieved from P. neolepis were different from those found in diatoms and sponges could therefore support either of these two theories. The presence of a novel, silica-associated protein in P. neolepis, which displays no homology with Si-associated proteins isolated from diatoms or sponges could in turn point towards either de-novo system evolution, convergent evolution or adaptation of a pre-existing component (the
Si-associated protein isolated from P. neolepis shares some homology with lipocalins).
No evidence for HGT in P. neolepis were found during this study. Therefore, in spite of the novel and evolutionarily very important information obtained in this study, it is still not possible to determine which exact mechanisms were involved in development of silicification system in haptophytes.
The polyphyletic nature of silicification systems in different taxa combined with recurring common elements such as the aforementioned LCPAs, SITs, presence of an organic matrix (containing either chitin or collagen) etc. or the complete de-novo
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Durak, G.M. Chapter V: General Discussion evolution of the ability to deposit Si via cooptation of the pre-existing mechanisms (e.g. as in the case of plants, where acquaporins were adapted for Si transport) further indicate, that processes involved in evolution of Si biomineralisation in Eucarya were highly complex and are therefore very difficult to resolve (Knoll 2003; Ma et al. 2006;
Marron et al. 2013; Raven and Waite 2004). Ultimately all silicification systems described to date are unified by the chemistry of silicic acid and consequently bear similarities in the form of e.g. active groups precipitating silica (eg. amines in collagen and LCPAs) and the presence of an organic matrix on which Si is deposited in a controlled manner (e.g. chitin, collagen, proteins such as frustulins) (Durkin et al. 2009;
Ehrlich and Worch 2008; Kröger and Poulsen 2008a; Matsunaga et al. 2007; Poll et al.
1999). Paradoxically, despite the vast amount of information available on different biomineralisation systems, our knowledge on their in-vivo mechanisms and evolution is overall still very limited. However, an array of genetic and biochemical tools are now available for a slow but methodical investigation of biomineralisation systems used by extant organisms, in an effort to finally unravel their evolutionary origins and cellular basis.
V.3. SUMMARY
Overall this study provided valuable insights into the biomineralisation systems utilized by marine haptophyte algae. A potential evolutionary scenario for development of silicification in haptophytes has been proposed. This study provided important information pointing towards common physiological and biochemical aspects of biomineralisation in algae. Novel information on the physiological and biochemical mechanisms of silicification in haptophytes also provides a highly valuable basis for
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Durak, G.M. Chapter V: General Discussion further research into the evolution of biomineralisation in algae and the remaining
Eucarya.
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Durak, G.M. List of appendices
LIST OF APPENDICES
Appendix II.1.
List of time-lapses:
Time-lapse 1: confocal microscopy time lapse of HCK-123 labelled P. neolepis cells with scales developing in the posterior pole of the cells (in green). Cell secreting a scale is marked with an arrow. Chloroplast autofluorescence in red. Recording time: 76 min.
Time-lapse 2: confocal microscopy time lapse of PDMPO labelled P. neolepis cells with multiple intracellularly developing scales visible after cells are lysed (scales in green and indicated with arrows, chloroplast autofluorescence in red). Recording time:
23 min.
Time-lapse 3: DIC time lapse of a P. neolepis cell exhibiting metaboly and crawling behaviour, subsequently returning to its original spheroid shape. Recording time: 140 min.
Time-lapse 4: DIC time lapse of P. neolepis cells secreting scales. Recording time: 150 min.
Time-lapse 5: DIC time lapse of a P. neolepis cell with two haptonemas feeding on remnants of a conspecific. Recording time: 142min.
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Appendix II.2.
Fig. 1. SEM image of silica scales purified from cultures supplemented with 3 µM Ge and 50 µM Si, showing no morphological aberrations.
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Durak, G.M. List of appendices
Appendix IV.1.
Fig. 1. FESEM image of a non-purified P. neolepis sample with visible organic scales
(arrows).
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Durak, G.M. List of appendices
Fig. 2. FESEM image of a purified P. neolepis biosilica sample with no visible organic contamination (i.e. remnants of the protoplast, organic scales).
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Fig. 3. TEM image of a uranyl acetate stained NH4F insoluble organic phase isolated from purified P. neolepis biosilica, showing no signs of contamination with organic scales.
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Durak, G.M. List of appendices
Appendix IV.2.
Comp1037 peptide sequence.
>comp1037
CKYFMPSPPLPPFPPSPPSPPPKPKPKPKPKPPLEESSQSSSPFHVISKTTPTAPVAK
PPLECPPVKTVPVLEFDEFAEKGWWFVQKQMPTAIETTKLSYCVYHHYLLVGG
TTESGKLTLNMFADKTALKKANGEGASFTDYITTDDKYRSEGGLCLFEGKKTA
KVQFGMCDGEKRGAFFVIDFDSKEGWAVVSGGQPTIKTKTPGPKGGCKTGDGF
LDAGLWILTKDPLPEFWIVETAVAAASKAGFDIEVLDDVMHEGCDYFMPSPPIP
PGSPPAPPTSPLPPSSPSPPSPP
Appendix IV.3.
Alignment of comp1037 and weak homology-exhibiting NCBI -derived sequences.
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Durak, G.M. List of appendices
10 20 30 40 50 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | |ABV22241.1| lipocalin-like M ------T ------V L S G A R M |CAL58014.1| Apolipoprotein M ------T R R L R G H H A Q R A V A R L G A V A L A L A L T Comp 1037 P. neolepis C K Y F M P S P P L P P F P P S P P S P P P K P K P K P K P K P P L E E S S Q S S S P F H V I S K T
60 70 80 90 100 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | |ABV22241.1| lipocalin-like L R I S A V F A V S S A I Q C P P P D F K T Q G E L E G G F D L KW Y T S S K - W Y I Q Q Q M I I S |CAL58014.1| Apolipoprotein R S H A F V L G V E A S E E C ------A R V E P V D P F D L D A Y V E A E - W Y V A A Q K P T S Comp 1037 P. neolepis T P T A P V - - A K P P L E C P P ------V K T V P V L E F D E F A E K GWW F V Q K Q M P T A
110 120 130 140 150 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | |ABV22241.1| lipocalin-like Y L P A D Y L N C V T A E Y S L L D S P T L L G ------Y N I K V M N H A E N K D G K P L G P L |CAL58014.1| Apolipoprotein Y Q P T R D L F C V R A N Y T V V D E R T I S I ------W N T A - - - N R D G V D G S P R N A D Comp 1037 P. neolepis I E T T K L S Y C V Y H H Y L L V G G T T E S G K L T L N M F A D K T A L K K A N G E G A S F T D Y
160 170 180 190 200 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | |ABV22241.1| lipocalin-like T T I C A K V V N E ------T A G K L E V S P C F L P T F L A G P YW I V A F D K E A G - |CAL58014.1| Apolipoprotein G R F K L R G L I E ------D P N M P S K I A V G M R F L P R F L Y G P YW V V A T D V S P G D Comp 1037 P. neolepis I T T D D K Y R S E G G L C L F E G K K T A K V Q F G M C D G E K - - R G A F F V I D F D S K E G -
210 220 230 240 250 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | |ABV22241.1| lipocalin-like ------W A L V S G G A P K N Q G T N - - - - - G C Q T G T G T N D S G L W I F T R K P D |CAL58014.1| Apolipoprotein A E F D E R G Y S W A I I S G G Q P T I S R G N - - - - - G L C E P S G - - - - G L W L F V R D P E Comp 1037 P. neolepis ------W A V V S G G Q P T I K T K T P G P K G G C K T G D G F L D A G L W I L T K D P L
260 270 280 290 300 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | |ABV22241.1| lipocalin-like R D D A L L Q K V R G I A A A K G F D V S V F R D T D E S T C R S ------|CAL58014.1| Apolipoprotein V S E E V V S K M K E K C E S L G I D P D V L I P V T Q E G C S F ------Comp 1037 P. neolepis P E F W I V E T A V A A A S K A G F D I E V L D D V M H E G C D Y F M P S P P I P P G S P P A P P T
310 . . . . | . . . . | . . . . |ABV22241.1| lipocalin-like ------L Q S P L V V |CAL58014.1| Apolipoprotein ------P T L P - - - Comp 1037 P. neolepis S P L P P S S P S P P S P P
Fig. 1. Alignment of Comp1037 protein isolated from P. neolepis biosilica with NCBI-derived sequences of a lipocalin-like protein
from Karlodinium veneficum and an apolipoprotein/lipocalin protein sequence from Ostreococcus tauri.
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Durak, G.M. List of references
REFERENCES
Addadi, L., Joester, D., Nudelman, F., and Weiner, S. (2006). "Mollusk Shell Formation: A Source of New Concepts for Understanding Biomineralization Processes." Chemistry – A European Journal, 12(4), 980-987. Adl, S. M., Simpson, A. G. B., Lane, C. E., Lukeš, J., Bass, D., Bowser, S. S., Brown, M. W., Burki, F., Dunthorn, M., Hampl, V., Heiss, A., Hoppenrath, M., Lara, E., le Gall, L., Lynn, D. H., McManus, H., Mitchell, E. A. D., Mozley-Stanridge, S. E., Parfrey, L. W., Pawlowski, J., Rueckert, S., Shadwick, L., Schoch, C. L., Smirnov, A., and Spiegel, F. W. (2012). "The Revised Classification of Eukaryotes." Journal of Eukaryotic Microbiology, 59(5), 429-514. Aizawa, K., and Miyachi, S. (1986). "Carbonic anhydrase and CO< sub> 2 concentrating mechanisms in microalgae and cyanobacteria." FEMS Microbiology letters, 39(3), 215-233. Aizenberg, J., Sundar, V. C., Yablon, A. D., Weaver, J. C., and Chen, G. (2004). "Biological glass fibers: correlation between optical and structural properties." Proceedings of the National Academy of Sciences of the United States of America, 101(10), 3358-3363. Allen, C., and Borisy, G. G. (1974). "Structural polarity and directional growth of microtubules of Chlamydomonas flagella." Journal of Molecular Biology, 90(2), 381-402. Atlan, G., Balmain, N., Berland, S., Vidal, B., and Lopez, É. (1997). "Reconstruction of human maxillary defects with nacre powder: histological evidence for bone regeneration." Comptes Rendus de l'Académie des Sciences-Series III-Sciences de la Vie, 320(3), 253-258. Aylett, C., Löwe, J., and Amos, L. A. (2011). "New insights into the mechanisms of cytomotive actin and tubulin filaments." Int Rev Cell Mol Biol, 292, 1-71. Azam, F., and Chisholm, S. (1976). "Silicic acid uptake and incorporation by natural marine phytoplankton populations." Limnol. Oceanogr., 21(3). Azam, F., Hemmingsen, B., and Volcani, B. (1973). "Germanium incorporation into the silica of diatom cell walls." Archiv für Mikrobiologie, 92(1), 11-20. Azam, F., Hemmingsen, B. B., and Volcani, B. E. (1974). "Role of silicon in diatom metabolism." Archives of Microbiology, 97(1), 103-114. Bansal, V., Rautaray, D., Ahmad, A., and Sastry, M. (2004). "Biosynthesis of zirconia nanoparticles using the fungus Fusarium oxysporum." Journal of Materials Chemistry, 14(22), 3303-3305. Berges, J. A., Charlebois, D. O., Mauzerall, D. C., and Falkowski, P. G. (1996). "Differential effects of nitrogen limitation on photosynthetic efficiency of photosystems I and II in microalgae." Plant Physiology, 110(2), 689-696. Berges, J. A., and Falkowski, P. G. (1998). "Physiological stress and cell death in marine phytoplankton: Induction of proteases in response to nitrogen or light limitation." Limnology and Oceanography, 129-135. Bergquist, P. R. (1978). Sponges: Univ of California Press. Bernard, F. (1963). "Vitesses de chute en mer des amas palmelloides de Cyclococcolithus. Ses consequences pour le cycle vital des mers cbaudes." Pelagos, 1, 5-34.
- 209 -
Durak, G.M. List of references
Bhattacharya, D., Archibald, J. M., Weber, A. P., and Reyes-Prieto, A. (2007). "How do endosymbionts become organelles? Understanding early events in plastid evolution." Bioessays, 29(12), 1239-46. Bidle, K. D., and Azam, F. (1999). "Accelerated dissolution of diatom silica by marine bacterial assemblages." Nature, 397(6719), 508-512. Billard, C., Inouye, I. (2004). "Coccolithophores: From Molecular Processes to Global Impact", in H. R. Thierstein and J. R. Young, (eds.). Springer. Boyd, P. W., and Abraham, E. R. (2001). "Iron-mediated changes in phytoplankton photosynthetic competence during SOIREE." Deep Sea Research Part II: Topical Studies in Oceanography, 48(11–12), 2529-2550. Brendza, R. P., Serbus, L. R., Duffy, J. B., and Saxton, W. M. (2000). "A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein." Science, 289(5487), 2120-2122. Brinker, C. J., and Scherer, G. W. (1990). Sol-gel science: the physics and chemistry of sol-gel processing: Academic Pr. Buitenhuis, E. T., De Baar, H. J. W., and Veldhuis, M. J. W. (1999). "Photosynthesis and calcification by Emiliania huxleyi (Prymneisophyceae) as a function of inorganic carbon species." Journal of Phycology, 35(5), 949-959. Callot, G., Chauvel, A., Arvieu, J.C., Chamayou, H. (1992). "Mise en évidence de kaolinite et de silice dans les structures cellulaires de l'épiderme et du cortex de racines de palmier en forêt d'Amazonie." Bull. Soc. Bot. Fr, 139, 7-14. Carlisle, E. (1981). "Silicon: A requirement in bone formation independent of vitamin D1." Calcified Tissue International, 33(1), 27-34. Carlisle, E. M. (1988). "Silicon as a trace nutrient." Science of The Total Environment, 73(1–2), 95-106. Cattaneo-Vietti R., B. G., Cerrano C., Sa M., Benatti U., Giovine M., Gaino E. . (1996). "Optical fibres in an Antarctic sponge." Nature, 383, 397–398. Cavalier-Smith, T. (1994). "Origin and relationships of Haptophyta." SYSTEMATICS ASSOCIATION SPECIAL VOLUME, 51, 413-413. Cha, J. N., Shimizu, K., Zhou, Y., Christiansen, S. C., Chmelka, B. F., Stucky, G. D., and Morse, D. E. (1999). "Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro." Proceedings of the National Academy of Sciences, 96(2), 361-365. Channing, A., and Edwards, D. (2004). "Experimental taphonomy: silicification of plants in Yellowstone hot-spring environments." Transactions of the Royal Society of Edinburgh: Earth Sciences, 94(4), 503-521. Charnot, Y., Peres, G. (1971). "Contribution a l'etude de la regulation endocrinienne du metabolisme silicique." Anal. Endocrinol., 32, 397–402. Chen, C.-h., and Lewin, J. (1969). "Silicon as a nutrient element for Equisetum arvense." Canadian Journal of Botany, 47(1), 125-131. Chevallet, M., Luche, S., and Rabilloud, T. (2006). "Silver staining of proteins in polyacrylamide gels." Nature protocols, 1(4), 1852-1858. Chiba, Y., Mitani, N., Yamaji, N., and Ma, J. F. (2009). "HvLsi1 is a silicon influx transporter in barley." The Plant Journal, 57(5), 810-818. Chiovitti, A., Harper, R. E., Willis, A., Bacic, A., Mulvaney, P., and Wetherbee, R. (2005). "Variations in the substituted 3-linked mannans closely associated with the silicified walls of diatoms." Journal of Phycology, 41(6), 1154-1161. Chisholm, S., Azam, F., and Eppley, R. (1978). "Silicic acid incorporation in marine diatoms on light: dark cycles: Use as an assay for phased cell division." Limnol. Oceanogr, 23(51), 8-529. Chukin, G., and Malevich, V. (1977). "Infrared spectra of silica." Journal of Applied Spectroscopy, 26(2), 223-229.
- 210 -
Durak, G.M. List of references
Cohen, S. S. (1998). A Guide to the Polyamines: Oxford University Press New York. Cohn, S., Nash, J., and Pickett-Heaps, J. (1989). "The effect of drugs on diatom valve morphogenesis." Protoplasma, 149(2-3), 130-143. Conrad, W. (1941). "Notes protistologiques: XXI. Sur les Chrysomonadines à trois fouets. Aperçu synoptique." Bulletin-Institut Royal des Sciences Naturelles de Belgique= Mededelingen-Koninklijk Belgisch Instituut voor Natuurwetenschappen, 17(45). Coradin, T., Eglin, D., and Livage, J. (2004). "The silicomolybdic acid spectrophotometric method and its application to silicate/biopolymer interaction studies." Journal of Spectroscopy, 18(4), 567-576. Curnow, P., Senior, L., Knight, M. J., Thamatrakoln, K., Hildebrand, M., and Booth, P. J. (2012). "Expression, Purification, and Reconstitution of a Diatom Silicon Transporter." Biochemistry, 51(18), 3776-3785. Darley, W. M., and Volcani, B. E. (1969). "Role of silicon in diatom metabolism: A silicon requirement for deoxyribonucleic acid synthesis in the diatom Cylindrotheca fusiformis Reimann and Lewin." Experimental Cell Research, 58(2–3), 334-342. Darzynkiewicz, Z., Li, X., and Gong, J. (1994). "Assays of cell viability: discrimination of cells dying by apoptosis." Methods Cell Biol, 41, 15-38. Davis, K., and Tomozawa, M. (1996). "An infrared spectroscopic study of water-related species in silica glasses." Journal of non-crystalline solids, 201(3), 177-198. De Brabander, M. J., Van de Veire, R. M., Aerts, F. E., Borgers, M., and Janssen, P. A. (1976). "The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro." Cancer Res, 36(3), 905-16. De la Rocha, C. L., Hutchins, D. A., Brzezinski, M. A., and Zhang, Y. (2000). "Effects of iron and zinc deficiency on elemental composition and silica production by diatoms." Marine Ecology Progress Series, 195, 71-79. Del Amo, Y. D., and Brzezinski, M. A. (1999). "THE CHEMICAL FORM OF DISSOLVED SI TAKEN UP BY MARINE DIATOMS." Journal of Phycology, 35(6), 1162-1170. Desai, A., and Mitchison, T. J. (1997). "Microtubule polymerization dynamics." Annual review of cell and developmental biology, 13(1), 83-117. Desclés, J., Vartanian, M., El Harrak, A., Quinet, M., Bremond, N., Sapriel, G., Bibette, J., and Lopez, P. J. (2008). "New tools for labeling silica in living diatoms." New Phytologist, 177(3), 822-829. Didymus, J. M., Young, J. R., and Mann, S. (1994). "Construction and Morphogenesis of the Chiral Ultrastructure of Coccoliths from the Marine Alga Emiliania huxleyi." Proceedings of the Royal Society of London. Series B: Biological Sciences, 258(1353), 237-245. Diwu, Z., Chen, C.-S., Zhang, C., Klaubert, D. H., and Haugland, R. P. (1999). "A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells." Chemistry & biology, 6(7), 411-418. Douzery, E. J., Snell, E. A., Bapteste, E., Delsuc, F., and Philippe, H. (2004). "The timing of eukaryotic evolution: does a relaxed molecular clock reconcile proteins and fossils?" Proceedings of the National Academy of Sciences of the United States of America, 101(43), 15386-15391. Drescher, B., Dillaman, R. M., and Taylor, A. R. (2012). "Coccolithogenesis In Scyphosphaera apsteinii (Prymnesiophyceae)." Journal of Phycology, 48(6), 1343-1361.
- 211 -
Durak, G.M. List of references
Dupoirieux, L., Costes, V., Jammet, P., and Souyris, F. (1994). "Experimental study on demineralized bone matrix (DBM) and coral as bone graft substitutes in maxillofacial surgery." International journal of oral and maxillofacial surgery, 23(6), 395-398. Durkin, C. A., Mock, T., and Armbrust, E. V. (2009). "Chitin in Diatoms and Its Association with the Cell Wall." Eukaryotic Cell, 8(7), 1038-1050. Earnshaw, A., and Greenwood, N. (1997). Chemistry of the Elements: Butterworth- Heinemann. Edvardsen, B., Eikrem, W., Throndsen, J., Sáez, A. G., Probert, I., and Medlin, L. K. (2011). "Ribosomal DNA phylogenies and a morphological revision provide the basis for a revised taxonomy of the Prymnesiales (Haptophyta)." European Journal of Phycology, 46(3), 202-228. Ehrlich, H., Demadis, K. D., Pokrovsky, O. S., and Koutsoukos, P. G. (2010). "Modern views on desilicification: biosilica and abiotic silica dissolution in natural and artificial environments." Chemical reviews, 110(8), 4656-4689. Ehrlich, H., Ereskovskii, A., Drozdov, A., Krylova, D., Hanke, T., Meissner, H., Heinemann, S., and Worch, H. (2006). "A modern approach to demineralization of spicules in glass sponges (Porifera: Hexactinellida) for the purpose of extraction and examination of the protein matrix." Russian Journal of Marine Biology, 32(3), 186-193. Ehrlich, H., Heinemann, S., Heinemann, C., Simon, P., Bazhenov, V. V., Shapkin, N. P., Born, R., Tabachnick, K. R., Hanke, T., and Worch, H. (2008). "Nanostructural organization of naturally occurring composites-part I: silica- collagen-based biocomposites." Journal of Nanomaterials, 2008, 53. Ehrlich, H., and Worch, H. (2008). "Collagen: A Huge Matrix in Glass Sponge Flexible Spicules of the Meter-Long Hyalonema sieboldi", Handbook of Biomineralization. Wiley-VCH Verlag GmbH, pp. 22-41. Epstein, E. (1994). "The anomaly of silicon in plant biology." Proceedings of the National Academy of Sciences, 91(1), 11-17. Fichtinger-Schepman, A. M. J., Kamerling, J. P., Versluis, C., and Vliegenthart, J. F. (1981). "Structural studies of the methylated, acidic polysaccharide associated with coccoliths of< i> Emiliania huxleyi(lohmann) kamptner." Carbohydrate Research, 93(1), 105-123. Flower, D. R., North, A. C., and Attwood, T. K. (1993). "Structure and sequence relationships in the lipocalins and related proteins." Protein Science, 2(5), 753- 761. Forscher, P., and Smith, S. J. (1988). "Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone." J Cell Biol, 107(4), 1505-16. Franklin, D. J., Airs, R. L., Fernandes, M., Bell, T. G., Bongaerts, R. J., Berges, J. A., and MAlin, G. (2012). "Identification of senescence and death in Emiliania huxleyi and Thalassiosira pseudonana: Cell staining, chlorophyll alterations, and dimethylsulfoniopropionate (DMSP) metabolism." Limnology and Oceanography, 57(1), 305. Fuchs, E., and Gilvarg, C. (1978). "The use of hydrofluoric acid in the identification of lipid intermediates in peptidoglycan synthesis." Analytical biochemistry, 90(2), 465-473. Fuhrmann, T., Landwehr, S., El Rharbi-Kucki, M., and Sumper, M. (2004). "Diatoms as living photonic crystals." Applied Physics B, 78(3-4), 257-260. Gachet, Y., Tournier, S., Millar, J. B., and Hyams, J. S. (2001). "A MAP kinase- dependent actin checkpoint ensures proper spindle orientation in fission yeast." Nature, 412(6844), 352-355.
- 212 -
Durak, G.M. List of references
Gautier, C., Abdoul-Aribi, N., Roux, C., Lopez, P. J., Livage, J., and Coradin, T. (2008a). "Biomimetic dual templating of silica by polysaccharide/protein assemblies." Colloids and Surfaces B: Biointerfaces, 65(1), 140-145. Gautier, C., Abdoul-Aribi, N., Roux, C., Lopez, P. J., Livage, J., and Coradin, T. (2008b). "Biomimetic dual templating of silica by polysaccharide/protein assemblies." Colloids Surf B Biointerfaces, 65(1), 140-5. Godoi, R. H. M., Aerts, K., Harlay, J., Kaegi, R., Ro, C.-U., Chou, L., and Van Grieken, R. (2009). "Organic surface coating on Coccolithophores-Emiliania huxleyi: Its determination and implication in the marine carbon cycle." Microchemical journal, 91(2), 266-271. Goldberg, H. A., and Warner, K. J. (1997). "The Staining of Acidic Proteins on Polyacrylamide Gels: Enhanced Sensitivity and Stability of “Stains-All” Staining in Combination with Silver Nitrate." Analytical Biochemistry, 251(2), 227-233. Graham, L. E., Graham, J. M., and Wilcox, L. W. (2009). Algae: Pearson College Division. Green, J. (1980). "The fine structure of Pavlova pinguis Green and a preliminary survey of the order Pavlovales (Prymnesiophyceae)." British Phycological Journal, 15(2), 151-191. Green, J., Hibberd, D., and Pienaar, R. (1982). "The taxonomy of Prymnesium (Prymnesiophyceae) including a description of a new cosmopolitan species, P. patellifera sp. nov., and further observations on P. parvum N. Carter." British Phycological Journal, 17(4), 363-382. Green, J., and Hori, T. (1994). "Flagella and flagellar roots." SYSTEMATICS ASSOCIATION SPECIAL VOLUME, 51, 47-47. Grégoire, C., Rémus-Borel, W., Vivancos, J., Labbé, C., Belzile, F., and Bélanger, R. R. (2012). "Discovery of a multigene family of aquaporin silicon transporters in the primitive plant Equisetum arvense." The Plant Journal, 72(2), 320-330. Gross, S. P., Vershinin, M., and Shubeita, George T. (2007). "Cargo Transport: Two Motors Are Sometimes Better Than One." Current Biology, 17(12), R478-R486. Grotzinger, J. P., and Knoll, A. H. (1995). "Anomalous carbonate precipitates: is the Precambrian the key to the Permian?" Palaios, 578-596. Guillard, R. R. L., and Ryther, J. H. (1962). "Studies od marine planktonic diatoms: I. Cyclotella nana Husedt, and Detonula Confervacea (Cleve) Gran." Canadian journal of microbiology, 8(2), 229-239. Gurtovenko, A. A., and Anwar, J. (2007). "Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide." J Phys Chem B, 111(35), 10453-60. Hamm, C. E., Merkel, R., Springer, O., Jurkojc, P., Maier, C., Prechtel, K., and Smetacek, V. (2003). "Architecture and material properties of diatom shells provide effective mechanical protection." Nature, 421(6925), 841-843. Hammer, J. A., and Sellers, J. R. (2011). "Walking to work: roles for class V myosins as cargo transporters." Nature Reviews Molecular Cell Biology, 13(1), 13-26. Harper, H. E., and Knoll, A. H. (1975). "Silica, diatoms, and Cenozoic radiolarian evolution." Geology, 3(4), 175-177. Harrington, B. J., and Hageage, G. J. (2003). "Calcofluor White: A Review of its Uses and Applications in Clinical Mycology and Parasitology." Lab Medicine, 34(5), 361-367. Hartwig, C. M., and Rahn, L. A. (1977). "Bound hydroxyl in vitreous silica." The Journal of Chemical Physics, 67, 4260.
- 213 -
Durak, G.M. List of references
Hattori, T., Inanaga, S., Tanimoto, E., Lux, A., Luxová, M., and Sugimoto, Y. (2003). "Silicon-Induced Changes in Viscoelastic Properties of Sorghum Root Cell Walls." Plant and Cell Physiology, 44(7), 743-749. Hedrich, R., Machill, S., and Brunner, E. (2013). "Biomineralization in diatoms— phosphorylated saccharides are part of Stephanopyxis turris biosilica." Carbohydrate Research, 365(0), 52-60. Heinemann, S., Heinemann, C., Ehrlich, H., Meyer, M., Baltzer, H., Worch, H., and Hanke, T. (2007). "A Novel Biomimetic Hybrid Material Made of Silicified Collagen: Perspectives for Bone Replacement." Advanced Engineering Materials, 9(12), 1061-1068. Henriksen, K., Stipp, S. L. S., Young, J. R., and Marsh, M. E. (2004). "Biological control on calcite crystallization: AFM investigation of coccolith polysaccharide function." American Mineralogist, 89(11-12), 1709-1716. Hildebrand, M. (2000). "Silicic acid transport and its control during cell wall silicification in diatoms", in E. Bäuerlein, (ed.), Biomineralization: From Biology to Biotechnology and Medical Applications. Weinheim, Germany: Wiley-VCH. Hildebrand, M. (2005). "Silicic Acid Transport and Its Control During Cell Wall Silicification in Diatoms", Biomineralization. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 159-176. Hildebrand, M., Frigeri, L. G., and Davis, A. K. (2007). "SYNCHRONIZED GROWTH OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) PROVIDES NOVEL INSIGHTS INTO CELL‐WALL SYNTHESIS PROCESSES IN RELATION TO THE CELL CYCLE1." Journal of Phycology, 43(4), 730-740. Hildebrand, M., Higgins, D. R., Busser, K., and Volcani, B. E. (1993). "Silicon- responsive cDNA clones isolated from the marine diatom Cylindrotheca fusiformis." Gene, 132(2), 213-218. Hinsinger, P., Jaillard, B., and Dufey, J. E. (1992). "Rapid Weathering of a Trioctahedral Mica by the Roots of Ryegrass." Soil Sci. Soc. Am. J., 56(3), 977- 982. Hirokawa, Y., Fujiwara, S., and Tsuzuki, M. (2005). "Three Types of Acidic Polysaccharides Associated with Coccolith of Pleurochrysis haptonemofera: Comparison with Pleurochrysis carterae and Analysis Using Fluorescein- Isothiocyanate-Labeled Lectins." Marine Biotechnology, 7(6), 634-644. Hohmann-Marriott, M. F., and Blankenship, R. E. (2011). "Evolution of photosynthesis." Annual review of plant biology, 62, 515-548. Hurd, D. C., Pankratz, H. S., Asper, V., Fugate, J., and Morrow, H. (1981). "Changes in the physical and chemical properties of biogenic silica from the central equatorial Pacific; Part III, Specific pore volume, mean pore size, and skeletal ultrastructure of acid-cleaned samples." American Journal of Science, 281(7), 833-895. Iler, R. K. (1979). The chemistry of silica: solubility, polymerization, colloid and surface properties, and biochemistry: Wiley New York. Inouye, I., and Pienaar, R. N. (1984). "New observations on the Coccolithophorid Umbilicosphaera sibogae var. foliosa (Prymnesiophyceae) with reference to cell covering, cell structure and flagellar apparatus." British Phycological Journal, 19(4), 357-369. Jeffryes, C., Gutu, T., Jiao, J., and Rorrer, G. L. (2008). "Two-stage photobioreactor process for the metabolic insertion of nanostructured germanium into the silica microstructure of the diatom Pinnularia sp." Materials Science and Engineering: C, 28(1), 107-118.
- 214 -
Durak, G.M. List of references
Jones, H. L. J., Leadbeater, B. S. C., and Green, J. C. (1993). "Mixotrophy in marine species of Chrysochromulina (Prymnesiophyceae): ingestion and digestion of a small green flagellate." Journal of the Marine Biological Association of the United Kingdom, 73(02), 283-296. Jordan, M. A., Thrower, D., and Wilson, L. (1992). "Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis." Journal of cell science, 102(3), 401-416. Kammer, M., Hedrich, R., Ehrlich, H., Popp, J., Brunner, E., and Krafft, C. (2010). "Spatially resolved determination of the structure and composition of diatom cell walls by Raman and FTIR imaging." Analytical and Bioanalytical Chemistry, 398(1), 509-517. Kauss, H., Seehaus, K., Franke, R., Gilbert, S., Dietrich, R. A., and Kröger, N. (2003). "Silica deposition by a strongly cationic proline‐rich protein from systemically resistant cucumber plants." The Plant Journal, 33(1), 87-95. Kawachi, M., Inouye, I., Maeda, O., and Chihara, M. (1991). "The haptonema as a food-capturing device: observations on Chrysochromulina hirta (Prymnesiophyceae)." Phycologia, 30(6), 563-573. Keeling, P. J. (2010). "The endosymbiotic origin, diversification and fate of plastids." Philos Trans R Soc Lond B Biol Sci, 365(1541), 729-48. Kimura, M. (1980). "A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences." J Mol Evol, 16(2), 111-20. Kinrade, S. D., Gillson, A.-M. E., and Knight, C. T. G. (2002). "Silicon-29 NMR evidence of a transient hexavalent silicon complex in the diatom Navicula pelliculosa." Journal of the Chemical Society, Dalton Transactions, 0(3), 307- 309. Kirschvink, J. L., and Hagadorn, J. W. (2000). "10 A grand unified theory of biomineralization." The Biomineralisation of Nano-and Micro-Structures. Wiley-VCH Verlag GmbH, Weinheim, Germany, 139-150. Klaveness, D. (1972). "Coccolithus huxleyi (Lohmann) Kamptner. 1. Morphological investigations on the vegetative cell and the process of coccolith formation." Protistologica (Paris. 1965), 8(3). Knoll, A., and Swett, K. (1989). "Carbonate deposition during the late Proterozoic Era: an example from Spitsbergen." American Journal of Science, 290, 104-132. Knoll, A. H. (2003). "Biomineralization and evolutionary history." Reviews in Mineralogy and Geochemistry, 54(1), 329-356. Kooistra, W., Gersonde, R., Medlin, L. K., and Mann, D. G. (2007). "The origin and evolution of the diatoms: their adaptation to a planktonic existence." Evolution of primary producers in the sea, 207-249. Krasko, A., Lorenz, B., Batel, R., Schröder, H. C., Müller, I. M., and Müller, W. E. (2000). "Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin." European Journal of Biochemistry, 267(15), 4878-4887. Krause, G. H., and Weis, E. (1991). "Chlorophyll Fluorescence and Photosynthesis: The Basics." Annual Review of Plant Physiology and Plant Molecular Biology, 42(1), 313-349. Kröger, N., Bergsdorf, C., and Sumper, M. (1994). "A new calcium binding glycoprotein family constitutes a major diatom cell wall component." EMBO J, 13(19), 4676-83. Kröger, N., Deutzmann, R., Bergsdorf, C., and Sumper, M. (2000). "Species-specific polyamines from diatoms control silica morphology." Proceedings of the National Academy of Sciences, 97(26), 14133-14138.
- 215 -
Durak, G.M. List of references
Kröger, N., Deutzmann, R., and Sumper, M. (1999). "Polycationic Peptides from Diatom Biosilica That Direct Silica Nanosphere Formation." Science, 286(5442), 1129-1132. Kröger, N., Lehmann, G., Rachel, R., and Sumper, M. (1997). "Characterization of a 200-kDa diatom protein that is specifically associated with a silica-based substructure of the cell wall." Eur J Biochem, 250(1), 99-105. Kröger, N., Lorenz, S., Brunner, E., and Sumper, M. (2002). "Self-Assembly of Highly Phosphorylated Silaffins and Their Function in Biosilica Morphogenesis." Science, 298(5593), 584-586. Kröger, N., and Poulsen, N. (2008a). "Diatoms-from cell wall biogenesis to nanotechnology." Annu Rev Genet, 42, 83-107. Kröger, N., and Poulsen, N. (2008b). "Diatoms-from cell wall biogenesis to nanotechnology." Annual review of genetics, 42, 83-107. Kröger, N., and Wetherbee, R. (2000). "Pleuralins are involved in theca differentiation in the diatom Cylindrotheca fusiformis." Protist, 151(3), 263-73. Kromkamp, J. C., and Forster, R. M. (2003). "The use of variable fluorescence measurements in aquatic ecosystems: differences between multiple and single turnover measuring protocols and suggested terminology." European Journal of Phycology, 38(2), 103-112. Kueh, H. Y., and Mitchison, T. J. (2009). "Structural Plasticity in Actin and Tubulin Polymer Dynamics." Science, 325(5943), 960-963. Landais, P., Rochdi, A., Largeau, C., and Derenne, S. (1993). "Chemical characterization of torbanites by transmission micro-FTIR spectroscopy: origin and extent of compositional heterogeneities." Geochimica et cosmochimica acta, 57(11), 2529-2539. Langer, G., de Nooijer, L. J., and Oetjen, K. (2010). "On the role of the cytoskeleton in coccolith morphogenesis: the effectof cytoskeleton inhibitors." Journal of Phycology, 46(6), 1252-1256. Laskin, A., Iedema, M. J., and Cowin, J. P. (2003). "Time-Resolved Aerosol Collector for CCSEM/EDX Single-Particle Analysis." Aerosol Science and Technology, 37(3), 246-260. Le Clainche, C., and Carlier, M. F. (2008). "Regulation of actin assembly associated with protrusion and adhesion in cell migration." Physiol Rev, 88(2), 489-513. Lee, M., and Li, C.-W. (1992). "The origin of the silica deposition vesicle of diatoms." Bot Bull Acad Sin, 33, 317-325. Legrand, C., Johansson, N., Johnsen, G., Borsheim, K., and Graneli, E. (2001). "Phagotrophy and toxicity variation in the mixotrophic Prymnesium patelliferum (Haptophyceae)." Limnology and Oceanography, 46(5), 1208-1214. Lewin, J. (1966). "Silicon Metabolism in Diatoms. V. Germanium Dioxide, a Specific Inhibitor of Diatom Growth 1." Phycologia, 6(1), 1-12. Li, C.-W., Chu, S., and Lee, M. (1989). "Characterizing the silica deposition vesicle of diatoms." Protoplasma, 151(2), 158-163. Likhoshway, Y. V., Masyukova, Y. A., Sherbakova, T. A., Petrova, D. P., and Grachev, M. A. (2006). "Detection of the gene responsible for silicic acid transport in chrysophycean algae." Dokl Biol Sci, 408, 256-60. Lippincott-Schwartz, J., Cole, N. B., Marotta, A., Conrad, P. A., and Bloom, G. S. (1995). "Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic." J Cell Biol, 128(3), 293-306. Little, L. H. (1966). Infrared spectra of adsorbed species: Academic Press. Liu, H., Aris-Brosou, S., Probert, I., and de Vargas, C. (2010). "A Time line of the Environmental Genetics of the Haptophytes." Molecular Biology and Evolution, 27(1), 161-176.
- 216 -
Durak, G.M. List of references
Lohmiller, J., and Lipman, N. (1998). "Silicon Crystals in Water of Autoclaved Glass Bottles." Contemp Top Lab Anim Sci, 37(1), 62-65. Love, G. D., Grosjean, E., Stalvies, C., Fike, D. A., Grotzinger, J. P., Bradley, A. S., Kelly, A. E., Bhatia, M., Meredith, W., and Snape, C. E. (2009). "Fossil steroids record the appearance of Demospongiae during the Cryogenian period." Nature, 457(7230), 718-721. Löwe, J., and Amos, L. A. (2009). "Evolution of cytomotive filaments: The cytoskeleton from prokaryotes to eukaryotes." The International Journal of Biochemistry & Cell Biology, 41(2), 323-329. Lowenstam, H. A., and Weiner, S. (1989). On biomineralization: Oxford University Press, USA. Lundholm, N., and Moestrup, Ø. (2006). "The biogeography of harmful algae", Ecology of harmful algae. Springer, pp. 23-35. M Franck, V., Brzezinski, M. A., Coale, K. H., and Nelson, D. M. (2000). "Iron and silicic acid concentrations regulate Si uptake north and south of the Polar Frontal Zone in the Pacific Sector of the Southern Ocean." Deep Sea Research Part II: Topical Studies in Oceanography, 47(15), 3315-3338. Ma, J., and Takahashi, E. (2002). Soil, fertilizer and plant silicon research in Japan: Elsevier Science BV. Ma, J. F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Katsuhara, M., Ishiguro, M., Murata, Y., and Yano, M. (2006). "A silicon transporter in rice." Nature, 440(7084), 688-691. Ma, J. F., and Yamaji, N. (2006). "Silicon uptake and accumulation in higher plants." Trends in Plant Science, 11(8), 392-397. Ma, J. F., Yamaji, N., Mitani, N., Tamai, K., Konishi, S., Fujiwara, T., Katsuhara, M., and Yano, M. (2007). "An efflux transporter of silicon in rice." Nature, 448(7150), 209-212. MacLean-Fletcher, S., and Pollard, T. D. (1980). "Mechanism of action of cytochalasin B on actin." Cell, 20(2), 329-341. Maldonado, M., Carmona, M. C., Uriz, M. J., and Cruzado, A. (1999). "Decline in Mesozoic reef-building sponges explained by silicon limitation." Nature, 401(6755), 785-788. Maliva, R. G., Knoll, A. H., and Siever, R. (1989). "Secular change in chert distribution: a reflection of evolving biological participation in the silica cycle." Palaios, 519- 532. Mandal, D. K., Bhattacharyya, L., Koenig, S. H., Brown, R. D., Oscarson, S., and Brewer, C. F. (1994). "Studies of the Binding Specificity of Concanavalin A. Nature of the Extended Binding Site for Asparagine-Linked Carbohydrates." Biochemistry, 33(5), 1157-1162. Manton, I. (1966). "Observations on scale production in Prymnesium parvum." Journal of cell science, 1(3), 375-380. Manton, I. (1967). "Further observations on the fine structure of Chrysochromulina chiton with special reference to the haptonema,‘peculiar’Golgi structure and scale production." Journal of cell science, 2(2), 265-272. Manton, I., and Leedale, G. (1969). "Observations on the microanatomy of Coccolithus pelagicus and Cricosphaera carterae, with special reference to the origin and nature of coccoliths and scales." Journal of the Marine Biological Association of the United Kingdom, 49(01), 1-16. Manton, I., and Leedale, G. F. (1963). "Observations on the micro-anatomy of crystallolithus hyalinus gaarder and markali." Archiv für Mikrobiologie, 47(2), 115-136.
- 217 -
Durak, G.M. List of references
Margolis, R. L., Rauch, C. T., and Wilson, L. (1980). "Mechanism of colchicine-dimer addition to microtubule ends: implications for the microtubule polymerization mechanism." Biochemistry, 19(24), 5550-5557. Märkel, K., Röser, U., Mackenstedt, U., and Klostermann, M. (1986). "Ultrastructural investigation of matrix-mediated biomineralization in echinoids (Echinodermata, Echinoida)." Zoomorphology, 106(4), 232-243. Markham, J. W., and Hagmeier, E. (1982). "Observations on the effects of germanium dioxide on the growth of macro-algae and diatoms." Phycologia, 21(2), 125-130. Marron, A. O., Alston, M. J., Heavens, D., Akam, M., Caccamo, M., Holland, P. W. H., and Walker, G. (2013). "A family of diatom-like silicon transporters in the siliceous loricate choanoflagellates." Proceedings of the Royal Society B: Biological Sciences, 280(1756). Marsh, M. E. (1999). "Coccolith crystals ofPleurochrysis carterae: Crystallographic faces, organization, and development." Protoplasma, 207(1-2), 54-66. Marsh, M. E. (2003). "Regulation of CaCO3 formation in coccolithophores." Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 136(4), 743-754. Marsh, M. E., Ridall, A. L., Azadi, P., and Duke, P. J. (2002). "Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite." Journal of Structural Biology, 139(1), 39-45. Martin-Jézéquel, V., Hildebrand, M., and Brzezinski, M. A. (2000). "Silicon metabolism in diatoms: implications for growth." Journal of Phycology, 36(5), 821-840. Matsunaga, S., Sakai, R., Jimbo, M., and Kamiya, H. (2007). "Long-Chain Polyamines (LCPAs) from Marine Sponge: Possible Implication in Spicule Formation." ChemBioChem, 8(14), 1729-1735. Medlin, L., Elwood, H. J., Stickel, S., and Sogin, M. L. (1988). "The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions." Gene, 71(2), 491-499. Medlin, L. K. (2011). "A review of the evolution of the diatoms from the origin of the lineage to their populations." The Diatom World, 93-118. Milligan, A. J., and Morel, F. M. (2002). "A proton buffering role for silica in diatoms." Science, 297(5588), 1848-1850. Mitani-Ueno, N., Yamaji, N., Zhao, F.-J., and Ma, J. F. (2011). "The aromatic/arginine selectivity filter of NIP aquaporins plays a critical role in substrate selectivity for silicon, boron, and arsenic." Journal of Experimental Botany. Mitani, N., and Ma, J. F. (2005). "Uptake system of silicon in different plant species." Journal of Experimental Botany, 56(414), 1255-1261. Mitani, N., Yamaji, N., Ago, Y., Iwasaki, K., and Ma, J. F. (2011). "Isolation and functional characterization of an influx silicon transporter in two pumpkin cultivars contrasting in silicon accumulation." The Plant Journal, 66(2), 231- 240. Mizutani, T., Nagase, H., Fujiwara, N., and Ogoshi, H. (1998). "Silicic Acid Polymerization Catalyzed by Amines and Polyamines." Bulletin of the Chemical Society of Japan, 71(8), 2017-2022. Montpetit, J., Vivancos, J., Mitani-Ueno, N., Yamaji, N., Rémus-Borel, W., Belzile, F., Ma, J., and Bélanger, R. (2012). "Cloning, functional characterization and heterologous expression of TaLsi1, a wheat silicon transporter gene." Plant Molecular Biology, 79(1-2), 35-46. Mort, A. J., and Lamport, D. T. (1977). "Anhydrous hydrogen fluoride deglycosylates glycoproteins." Analytical biochemistry, 82(2), 289-309.
- 218 -
Durak, G.M. List of references
Morton, W. M., Ayscough, K. R., and McLaughlin, P. J. (2000). "Latrunculin alters the actin-monomer subunit interface to prevent polymerization." Nature cell biology, 2(6), 376-378. Moustafa, A., Beszteri, B., Maier, U. G., Bowler, C., Valentin, K., and Bhattacharya, D. (2009). "Genomic footprints of a cryptic plastid endosymbiosis in diatoms." Science, 324(5935), 1724-1726. Müller, W., Li, J., Schröder, H., Qiao, L., and Wang, X. (2007a). "The unique skeleton of siliceous sponges (Porifera; Hexactinellida and Demospongiae) that evolved first from the Urmetazoa during the Proterozoic: a review." Biogeosciences Discussions, 4(1), 385-416. Müller, W. E., Boreiko, A., Wang, X., Krasko, A., Geurtsen, W., Custodio, M. R., Winkler, T., Lukic-Bilela, L., Link, T., and Schroder, H. C. (2007b). "Morphogenetic activity of silica and bio-silica on the expression of genes controlling biomineralization using SaOS-2 cells." Calcified Tissue International, 81(5), 382-93. Müller, W. E., Krasko, A., Le Pennec, G., and Schroder, H. C. (2003). "Biochemistry and cell biology of silica formation in sponges." Microsc Res Tech, 62(4), 368- 77. Müller, W. E., Wang, X., Schröder, H. C., Korzhev, M., Grebenjuk, V. A., Markl, J. S., Jochum, K. P., Pisignano, D., and Wiens, M. (2010). "A cryptochrome based photosensory system in the siliceous sponge Suberites domuncula (Demospongiae)." FEBS Journal, 277(5), 1182-1201. Müller, W. E. G., Belikov, S. I., Tremel, W., Perry, C. C., Gieskes, W. W. C., Boreiko, A., and Schröder, H. C. (2006). "Siliceous spicules in marine demosponges (example Suberites domuncula)." Micron, 37(2), 107-120. Müller, W. G., Rothenberger, M., Boreiko, A., Tremel, W., Reiber, A., and Schröder, H. (2005). "Formation of siliceous spicules in the marine demosponge Suberites domuncula." Cell and Tissue Research, 321(2), 285-297. Nagata, Y., and Burger, M. M. (1974). "Wheat Germ Agglutinin: molecular characteristics and specificity for sugar binding." Journal of Biological Chemistry, 249(10), 3116-3122. Nedelcu, A. M., Miles, I. H., Fagir, A. M., and Karol, K. (2008). "Adaptive eukaryote- to-eukaryote lateral gene transfer: stress-related genes of algal origin in the closest unicellular relatives of animals." Journal of Evolutionary Biology, 21(6), 1852-1860. Nelson, D. M., Riedel, G. F., Millan-Nunez, R., and Lara-Lara, J. R. (1984). "SILICON UPTAKE BY ALGAE WITH NO KNOWN Si REQUIREMENT. I. TRUE CELLULAR UPTAKE AND pH-INDUCED PRECIPITATION BY PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) AND PLATYMONAS SP. (PRASINOPHYCEAE)1." Journal of Phycology, 20(1), 141-147. Nygaard, K., and Tobiesen, A. (1993). "Bacterivory in algae: a survival strategy during nutrient limitation." Limnology and Oceanography, 273-279. Otzen, D. (2012). "The role of proteins in biosilicification." Scientifica, 2012. Outka, D. E., and Williams, D. C. (1971). "Sequential Coccolith Morphogenesis in Hymenomonas carterae." Journal of Eukaryotic Microbiology, 18(2), 285-297. Paasche, E. (1999). "Reduced coccolith calcite production under light-limited growth: a comparative study of three clones of Emiliania huxleyi (Prymnesiophyceae)." Phycologia, 38(6), 508-516. Paasche, E. (2001). "A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation, and calcification-photosynthesis interactions." Phycologia, 40(6), 503-529.
- 219 -
Durak, G.M. List of references
Pak, J., and Yoo, H. (2013). "Facile synthesis of spherical nanoparticles with a silica shell and multiple Au nanodots as the core." Journal of Materials Chemistry A, 1(17), 5408-5413. Parke, M., and Adams, I. (1960). "The motile (Crystallolithus hyalinus Gaarder & Markali) and non-motile phases in the life history of Coccolithus pelagicus (Wallich) Schiller." Journal of the Marine Biological Association of the United Kingdom, 39(02), 263-274. Parke, M., Manton, I., and Clarke, B. (1955). "Studies on marine flagellates II. Three new species of Chrysochromulina." Journal of the Marine Biological Association of the United Kingdom, 34(03), 579-609. Perry, C. C. (2003). "Silicification: The Processes by Which Organisms Capture and Mineralize Silica." Reviews in Mineralogy and Geochemistry, 54(1), 291-327. Peterson, J. R., and Mitchison, T. J. (2002). "Small molecules, big impact: a history of chemical inhibitors and the cytoskeleton." Chemistry & biology, 9(12), 1275- 1285. Pickett-Heaps, J., Schmid, A-M. M,. Edgar, L. A. (1990). "The cell biology of diatom valve formation." Prog. Phycol. Res., 7, 1–168. Pienaar, R. N. (1980). "Observations on the structure and composition of the cyst of Prymnesium (Prymnesiophyceae)." Proceedings of the Southern African Electron Microscopy Society, 10, 73-74. Pienaar, R. N. (1981). "Ultrastructural studies on the cysts of Prymnesium (Prymnesiophyceae)." Phycologia, 20, 112. Pienaar, R. N. (1994). "Ultrastructure and calcification of coccolithophores." Coccolithophores. Cambridge University Press, Cambridge, 13-37. Poll, W. H. v. d., Vrieling, E. G., and Gieskes, W. W. C. (1999). "Location and expression of frustulins in the pennate diatoms Cylindrotheca fusiformis, Navicula pelliculosa, and Navicula salinarium (Bacillariophyceae)." Journal of Phycology, 35(5), 1044-1053. Preisig, H. (1994). "Siliceous structures and silicification in flagellated protists." Protoplasma, 181(1-4), 29-42. Preisig, H. R., Vors, N., and Hfillfors, G. (1991). "Diversity ofheterotrophic heterokontflagellates", in L. J. Patterson D J, (ed.), The biology of free living heterotrophic flagellates. Oxford: Clarendon Press, pp. 361-399 Prihoda, J., Tanaka, A., de Paula, W. B. M., Allen, J. F., Tirichine, L., and Bowler, C. (2012). "Chloroplast-mitochondria cross-talk in diatoms." Journal of Experimental Botany, 63(4), 1543-1557. Raven, J., and Giordano, M. (2009). "Biomineralization by photosynthetic organisms: evidence of coevolution of the organisms and their environment?" Geobiology, 7(2), 140-154. Raven, J. A. (1983). "The transport and function of silicon in plants." Biological Reviews, 58(2), 179-207. Raven, J. A., and Waite, A. M. (2004). "The evolution of silicification in diatoms: inescapable sinking and sinking as escape?" New Phytologist, 162(1), 45-61. Reavie, E. D., Cangelosi, A. A., and Allinger, L. E. (2010). "Assessing ballast water treatments: Evaluation of viability methods for ambient freshwater microplankton assemblages." Journal of Great Lakes Research, 36(3), 540-547. Ribalet, F., Wichard, T., Pohnert, G., Ianora, A., Miralto, A., and Casotti, R. (2007). "Age and nutrient limitation enhance polyunsaturated aldehyde production in marine diatoms." Phytochemistry, 68(15), 2059-2067. Richthammer, P., Bormel, M., Brunner, E., and van Pee, K. H. (2011). "Biomineralization in diatoms: the role of silacidins." ChemBioChem, 12(9), 1362-6.
- 220 -
Durak, G.M. List of references
Rodriguez, O. C., Schaefer, A. W., Mandato, C. A., Forscher, P., Bement, W. M., and Waterman-Storer, C. M. (2003). "Conserved microtubule–actin interactions in cell movement and morphogenesis." Nature cell biology, 5(7), 599-609. Rowson, J. D., Leadbeater, B. S. C., and Green, J. C. (1986). "Calcium carbonate deposition in the motile (Crystallolithus) phase of Coccolithus pelagicus (Prymnesiophyceae)." British Phycological Journal, 21(4), 359-370. Sanders, D., Brownlee, C., and Harper, J. F. (1999). "Communicating with calcium." The Plant Cell Online, 11(4), 691-706. Sandgren, C. D. (1983). "Survival strategies of chrysophycean flagellates: reproduction and the formation of resistant resting cysts", in G. A. Fryxell, P. S. o. America, B. S. o. A. S. Section, and B. S. o. A. B. Section, (eds.), Survival strategies of the algae: [symposium heldin Vancouver, British Columbia, July 15, 1980, was sponsored by the Phycological Society of America and the Systematic and Phycological Sections of the Botanical Society of America]. Cambridge: Cambridge University Press. Sangster, A., Hodson, M., and Tubb, H. (2001). "Silicon deposition in higher plants." Studies in Plant Science, 8, 85-113. Sato, M., Murata, Y., Mizusawa, M., Iwahashi, H., and Oka, S.-i. (2004). "A simple and rapid dual-fluorescence viability assay for microalgae." Microbiol Cult Coll, 20(2), 53-59. Schagerl, M., and Möstl, M. (2011). "Drought stress, rain and recovery of the intertidal seaweed Fucus spiralis." Marine Biology, 158(11), 2471-2479. Schägger, H., and von Jagow, G. (1987). "Tricine-sodium dodecyl sulfate- polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa." Analytical biochemistry, 166(2), 368-379. Scheffel, A., Poulsen, N., Shian, S., and Kröger, N. (2011). "Nanopatterned protein microrings from a diatom that direct silica morphogenesis." Proceedings of the National Academy of Sciences, 108(8), 3175-3180. Schliwa, M. (1982). "Action of cytochalasin D on cytoskeletal networks." J Cell Biol, 92(1), 79-91. Schloßmacher, U., Wiens, M., Schröder, H. C., Wang, X., Jochum, K. P., and Müller, W. E. G. (2011). "Silintaphin-1 – interaction with silicatein during structure- guiding bio-silica formation." FEBS Journal, 278(7), 1145-1155. Schmid, A.-M. (1987). "Wall Morphogenesis in Centric Diatoms", in W. Wiessner, D. Robinson, and R. Starr, (eds.), Algal Development. Springer Berlin Heidelberg, pp. 34-41. Schmid, A.-M. (1994). "Aspects of morphogenesis and function of diatom cell walls with implications for taxonomy." Protoplasma, 181(1-4), 43-60. Schmid, A.-M., and Schulz, D. (1979). "Wall morphogenesis in diatoms: Deposition of silica by cytoplasmic vesicles." Protoplasma, 100(3-4), 267-288. Schott, D. H., Collins, R. N., and Bretscher, A. (2002). "Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length." J Cell Biol, 156(1), 35-9. Schröder, H., Brandt, D., Schloßmacher, U., Wang, X., Tahir, M., Tremel, W., Belikov, S., and Müller, W. G. (2007). "Enzymatic production of biosilica glass using enzymes from sponges: basic aspects and application in nanobiotechnology (material sciences and medicine)." Naturwissenschaften, 94(5), 339-359. Schröder, H., Perović-Ottstadt, S., Wiens, M., Batel, R., Müller, I., and Müller, W. G. (2004b). "Differentiation capacity of epithelial cells in the sponge Suberites domuncula." Cell and Tissue Research, 316(2), 271-280. Schröder, H. C., Boreiko, A., Korzhev, M., Tahir, M. N., Tremel, W., Eckert, C., Ushijima, H., Müller, I. M., and Müller, W. E. G. (2006). "Co-expression and
- 221 -
Durak, G.M. List of references
Functional Interaction of Silicatein with Galectin: MATRIX-GUIDED FORMATION OF SILICEOUS SPICULES IN THE MARINE DEMOSPONGE SUBERITES DOMUNCULA." Journal of Biological Chemistry, 281(17), 12001-12009. Schröder, H. C., Krasko, A., Le Pennec, G., Adell, T., Wiens, M., Hassanein, H., Muller, I. M., and Muller, W. E. (2003). "Silicase, an enzyme which degrades biogenous amorphous silica: contribution to the metabolism of silica deposition in the demosponge Suberites domuncula." Prog Mol Subcell Biol, 33, 249-68. Schröder, H. C., Perovic-Ottstadt, S., Rothenberger, M., Wiens, M., Schwertner, H., Batel, R., Korzhev, M., Muller, I. M., and Muller, W. E. (2004a). "Silica transport in the demosponge Suberites domuncula: fluorescence emission analysis using the PDMPO probe and cloning of a potential transporter." Biochem J, 381(Pt 3), 665-73. Schröder, H. C., Wang, X., Tremel, W., Ushijima, H., and Muller, W. E. G. (2008). "Biofabrication of biosilica-glass by living organisms." Natural Product Reports, 25(3), 455-474. Sekino, K., and Shiraiwa, Y. (1996). "Evidence for the involvement of mitochondrial respiration in calcification in a marine coccolithophorid, Emiliania huxleyi." Plant and Cell Physiology, 37(7), 1030-1033. Shchipunov, Y. A., Kojima, A., and Imae, T. (2005). "Polysaccharides as a template for silicate generated by sol–gel processes." Journal of colloid and interface science, 285(2), 574-580. Sheppard, V., Poulsen, N., and Kröger, N. (2010). "Characterization of an endoplasmic reticulum-associated silaffin kinase from the diatom Thalassiosira pseudonana." Journal of Biological Chemistry, 285(2), 1166-1176. Shimizu, K., Cha, J., Stucky, G. D., and Morse, D. E. (1998). "Silicatein α: cathepsin L- like protein in sponge biosilica." Proceedings of the National Academy of Sciences, 95(11), 6234-6238. Shimizu, K., Del Amo, Y., Brzezinski, M. A., Stucky, G. D., and Morse, D. E. (2001). "A novel fluorescent silica tracer for biological silicification studies." Chemistry & biology, 8(11), 1051-1060. Siever, R. (1992). "The silica cycle in the Precambrian." Geochimica et cosmochimica acta, 56(8), 3265-3272. Silve, C., Lopez, E., Vidal, B., Smith, D. C., Camprasse, S., Camprasse, G., and Couly, G. (1992). "Nacre initiates biomineralization by human osteoblasts maintained in vitro." Calcified Tissue International, 51(5), 363-369. Simonsen, S., and Moestrup, Ø. (1997). "Toxicity tests in eight species of Chrysochromulina (Haptophyta)." Canadian Journal of Botany, 75(1), 129-136. Simpson, R. J. (2006). "Fragmentation of Protein Using Trypsin." Cold Spring Harbor Protocols, 2006(5), pdb.prot4550. Simpson, T. L. (1984). The cell biology of sponges: Springer-Verlag. Small, J. V., Isenberg, G., and Celis, J. E. (1978). "Polarity of actin at the leading edge of cultured cells." Nature, 272(5654), 638-639. Smetacek, V. (1985). "Role of sinking in diatom life-history cycles: ecological, evolutionary and geological significance." Marine Biology, 84(3), 239-251. Spector, I., Braet, F., Shochet, N. R., and Bubb, M. R. (1999). "New anti-actin drugs in the study of the organization and function of the actin cytoskeleton." Microscopy Research and Technique, 47(1), 18-37. Stolen, R., and Walrafen, G. (1976). "Water and its relation to broken bond defects in fused silica." The Journal of Chemical Physics, 64, 2623. Strickland, J., and Parsons, T. (1968). " A practical handbook of seawater analysis." Bull. Fish. Res. Bd. Can, 167, 311.
- 222 -
Durak, G.M. List of references
Sullivan, C. (1977). "Diatom mineralization of silicic acid. II. Regulation of Si(OH) 4 transport rates during cell cycle of Navicula pelliculosa." Journal of Phycology, 13(1), 86-91. Sullivan, C., and Volcani, B. (1973). "Role of silicon in diatom metabolism III. The effects of silicic acid on DNA polymerase, TMP kinase and DNA synthesis in< i> Cylindrotheca fusiformis." Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis, 308(2), 212-229. Sullivan, C. W. (1976). "Ditaom mineralization of silicic acid. I Si(OH)4 transport characteristics in Navicula pelliculosa." Journal of Phycology, 12(4), 390-396. Sumerel, J. L., Yang, W., Kisailus, D., Weaver, J. C., Choi, J. H., and Morse, D. E. (2003). "Biocatalytically Templated Synthesis of Titanium Dioxide." Chemistry of Materials, 15(25), 4804-4809. Sumper, M. (2004). "Biomimetic Patterning of Silica by Long-Chain Polyamines." Angewandte Chemie International Edition, 43(17), 2251-2254. Sumper, M., and Brunner, E. (2008). "Silica Biomineralisation in Diatoms: The Model Organism Thalassiosira pseudonana." ChemBioChem, 9(8), 1187-1194. Sumper, M., Brunner, E., and Lehmann, G. (2005). "Biomineralization in diatoms: characterization of novel polyamines associated with silica." FEBS letters, 579(17), 3765-3769. Sumper, M., and Kröger, N. (2004). "Silica formation in diatoms: the function of long- chain polyamines and silaffins." Journal of Materials Chemistry, 14(14), 2059- 2065. Sumper, M., and Lehmann, G. (2006). "Silica Pattern Formation in Diatoms: Species- Specific Polyamine Biosynthesis." ChemBioChem, 7(9), 1419-1427. Sun, G., Yang, Z., Ishwar, A., and Huang, J. (2010). "Algal Genes in the Closest Relatives of Animals." Molecular Biology and Evolution, 27(12), 2879-2889. Swift, D. M., and Wheeler, A. (1992). "EVIDENCE OF AN ORGANIC MATRIX FROM DIATOM BIOSILICA1." Journal of Phycology, 28(2), 202-209. Tabb, J. S., Molyneaux, B. J., Cohen, D. L., Kuznetsov, S. A., and Langford, G. M. (1998). "Transport of ER vesicles on actin filaments in neurons by myosin V." Journal of cell science, 111(21), 3221-3234. Tagliaferro, P., Tandler, C. J., Ramos, A. J., Pecci Saavedra, J., and Brusco, A. (1997). "Immunofluorescence and glutaraldehyde fixation. A new procedure based on the Schiff-quenching method." Journal of Neuroscience Methods, 77(2), 191- 197. Takagi, S. (2003). "Actin-based photo-orientation movement of chloroplasts in plant cells." Journal of Experimental Biology, 206(12), 1963-1969. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011). "MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods." Molecular Biology and Evolution, 28(10), 2731-2739. Taylor, A. R., Chrachri, A., Wheeler, G., Goddard, H., and Brownlee, C. (2011). "A voltage-gated H+ channel underlying pH homeostasis in calcifying coccolithophores." PLoS biology, 9(6), e1001085. Taylor, A. R., Russell, M. A., Harper, G. M., Collins, T. f. T., and Brownlee, C. (2007). "Dynamics of formation and secretion of heterococcoliths by Coccolithus pelagicus ssp. braarudii." European Journal of Phycology, 42(2), 125-136. Tesson, B., and Hildebrand, M. (2010). "Extensive and Intimate Association of the Cytoskeleton with Forming Silica in Diatoms: Control over Patterning on the Meso- and Micro-Scale." PLoS ONE, 5(12), e14300.
- 223 -
Durak, G.M. List of references
Thamatrakoln, K., and Hildebrand, M. (2007). "Analysis of Thalassiosira pseudonana Silicon Transporters Indicates Distinct Regulatory Levels and Transport Activity through the Cell Cycle." Eukaryotic Cell, 6(2), 271-279. Thamatrakoln, K., and Hildebrand, M. (2008). "Silicon uptake in diatoms revisited: a model for saturable and nonsaturable uptake kinetics and the role of silicon transporters." Plant Physiol, 146(3), 1397-407. Theodoropoulos, P. A., Gravanis, A., Tsapara, A., Margioris, A. N., Papadogiorgaki, E., Galanopoulos, V., and Stournaras, C. (1994). "Cytochalasin B may shorten actin filaments by a mechanism independent of barbed end capping." Biochem Pharmacol, 47(10), 1875-81. Thornton, D. (2002). "Diatom aggregation in the sea: mechanisms and ecological implications." European Journal of Phycology, 37(2), 149-161. Tillmann, U. (1998). "Phagotrophy by a plastidic haptophyte, Prymnesium patelliferum." Aquatic Microbial Ecology, 14, 155-160. Uriz, M.-J. (2006). "Mineral skeletogenesis in sponges." Canadian journal of zoology, 84(2), 322-356. Uriz, M.-J., Turon, X., Becerro, M. A., and Agell, G. (2003). "Siliceous spicules and skeleton frameworks in sponges: Origin, diversity, ultrastructural patterns, and biological functions." Microscopy Research and Technique, 62(4), 279-299. Vale, R. D. (2003). "The molecular motor toolbox for intracellular transport." Cell, 112(4), 467-80. Van De Meene, A. M. L., and Pickett-Heaps, J. D. (2002). "Valve morphogenesis in the centric diatom Proboscia alata Sundstrom." Journal of Phycology, 38(2), 351- 363. Van den Hoek, C., Mann, D., and Jahns, H. M. (1995). Algae: Cambridge University Press. van der Wal, P., de Jong, E. W., Westbroek, P., de Bruijn, W. C., and Mulder-Stapel, A. A. (1983). "Polysaccharide localization, coccolith formation, and golgi dynamics in the coccolithophorid Hymenomonas carterae." Journal of Ultrastructure Research, 85(2), 139-158. Vardy, S., and Uwins, P. (2002). "Fourier Transform Infrared Microspectroscopy as a Tool to Differentiate< i> Nitzschia closterium and< i> Nitzschia longissima." Applied spectroscopy, 56(12), 1545-1548. Vaulot, D. (1985). Cell Cycle Controls in Marine Phytoplankton, Ph.D., Massachusetts Institute of Technology/Woods Hole Oceanographic Institution. Vaulot, D., Olson, R., Merkel, S., and Chisholm, S. (1987). "Cell-cycle response to nutrient starvation in two phytoplankton species, Thalassiosira weissflogii and Hymenomonas carterae." Marine Biology, 95(4), 625-630. Veis, A. (2003). "Mineralization in Organic Matrix Frameworks." Reviews in Mineralogy and Geochemistry, 54(1), 249-289. Veldhuis, M., Kraay, G., and Timmermans, K. (2001). "Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth." European Journal of Phycology, 36(2), 167-177. Verchot-Lubicz, J., and Goldstein, R. (2010). "Cytoplasmic streaming enables the distribution of molecules and vesicles in large plant cells." Protoplasma, 240(1- 4), 99-107. Wang, X., Schloßmacher, U., Wiens, M., Batel, R., Schröder, H. C., and Müller, W. E. G. (2012). "Silicateins, silicatein interactors and cellular interplay in sponge skeletogenesis: formation of glass fiber-like spicules." FEBS Journal, 279(10), 1721-1736. Wang, X., Wiens, M., Schröder, H. C., Schloßmacher, U., Pisignano, D., Jochum, K. P., and Müller, W. E. G. (2011). "Evagination of Cells Controls Bio-Silica
- 224 -
Durak, G.M. List of references
Formation and Maturation during Spicule Formation in Sponges." PLoS ONE, 6(6), e20523. Weiner, S., and Dove, P. M. (2003). "An Overview of Biomineralization Processes and the Problem of the Vital Effect." Reviews in Mineralogy and Geochemistry, 54(1), 1-29. Weissbach, A., and Legrand, C. (2012). "Effect of different salinities on growth and intra- and extracellular toxicity of four strains of the haptophyte Prymnesium parvum." Aquatic Microbial Ecology, 67(2), 139-149. Wellner, N., Belton, P. S., and Tatham, A. S. (1996). "Fourier transform IR spectroscopic study of hydration-induced structure changes in the solid state of omega-gliadins." Biochemical Journal, 319(Pt 3), 741. Wenzl, S., Deutzmann, R., Hett, R., Hochmuth, E., and Sumper, M. (2004). "Quaternary Ammonium Groups in Silica‐Associated Proteins." Angewandte Chemie International Edition, 43(44), 5933-5936. Wenzl, S., Hett, R., Richthammer, P., and Sumper, M. (2008). "Silacidins: Highly Acidic Phosphopeptides from Diatom Shells Assist in Silica Precipitation In Vitro." Angewandte Chemie, 120(9), 1753-1756. Werner, D. (1966). "Die Kieselsäure im stoffwechsel von Cyclotella cryptica Reimann, Lewin und Guillard." Arch. Mikrobiol., 55, 278–308. Werner, D. (1967). "Hemmung der Chloropyllsynthese und der NADP+-abängingen glycerinaldehyd-3-phosphat-dehydrogenase durch germaniumsäure bei Cyclotella cryptica." Arch. Mikrobiol., 57, 51-60. Westbroek, P., de Jong, E. W., van der Wal, P., Borman, A. H., de Vrind, J. P. M., Kok, D., de Bruijn, W. C., and Parker, S. B. (1984). "Mechanism of Calcification in the Marine Alga Emiliania huxleyi [and Discussion]." Philosophical Transactions of the Royal Society of London. B, Biological Sciences, 304(1121), 435-444. Westbroek, P., and Marin, F. (1998). "A marriage of bone and nacre." Nature, 392(6679), 861-862. Wickstead, B., and Gull, K. (2011). "The evolution of the cytoskeleton." The Journal of cell biology, 194(4), 513-525. Wiens, M., Bausen, M., Natalio, F., Link, T., Schlossmacher, U., and Müller, W. E. G. (2009). "The role of the silicatein-α interactor silintaphin-1 in biomimetic biomineralization." Biomaterials, 30(8), 1648-1656. Wiens, M., Schröder, H.-C., Wang, ., Link, T., Steindorf, D., and M ller, W. E. G. (2011). "Isolation of the Silicatein-α Interactor Silintaphin-2 by a Novel Solid- Phase Pull-Down Assay." Biochemistry, 50(12), 1981-1990. Wiens, M., Wang, X., Unger, A., Schröder, H. C., Grebenjuk, V. A., Pisignano, D., Jochum, K. P., and Müller, W. E. (2010). "Flashing light signaling circuit in sponges: Endogenous light generation after tissue ablation in Suberites domuncula." Journal of Cellular Biochemistry, 111(6), 1377-1389. Wu, B., and Beitz, E. (2007). "Aquaporins with selectivity for unconventional permeants." Cell Mol Life Sci, 64(18), 2413-21. Wubbolts, R., Fernandez-Borja, M., Jordens, I., Reits, E., Dusseljee, S., Echeverri, C., Vallee, R. B., and Neefjes, J. (1999). "Opposing motor activities of dynein and kinesin determine retention and transport of MHC class II-containing compartments." Journal of cell science, 112(6), 785-795. Xiao, S., Hu, J., Yuan, X., Parsley, R. L., and Cao, R. (2005). "Articulated sponges from the Lower Cambrian Hetang Formation in southern Anhui, South China: their age and implications for the early evolution of sponges." Palaeogeography, Palaeoclimatology, Palaeoecology, 220(1–2), 89-117.
- 225 -
Durak, G.M. List of references
Yamaji, N., and Ma, J. F. (2007). "Spatial Distribution and Temporal Variation of the Rice Silicon Transporter Lsi1." Plant Physiology, 143(3), 1306-1313. Yamaji, N., Mitatni, N., and Ma, J. F. (2008). "A Transporter Regulating Silicon Distribution in Rice Shoots." The Plant Cell Online, 20(5), 1381-1389. Yamanaka, S., Yano, R., Usami, H., Hayashida, N., Ohguchi, M., Takeda, H., and Yoshino, K. (2008). "Optical properties of diatom silica frustule with special reference to blue light." Journal of Applied Physics, 103(7), 074701-074701-5. Yoon, H. S., Hackett, J. D., and Bhattacharya, D. (2002). "A single origin of the peridinin-and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis." Proceedings of the National Academy of Sciences, 99(18), 11724-11729. Yoon, H. S., Hackett, J. D., Ciniglia, C., Pinto, G., and Bhattacharya, D. (2004). "A molecular timeline for the origin of photosynthetic eukaryotes." Mol Biol Evol, 21(5), 809-18. Yoshida, M., Noël, M.-H., Nakayama, T., Naganuma, T., and Inouye, I. (2006). "A Haptophyte Bearing Siliceous Scales: Ultrastructure and Phylogenetic Position of Hyalolithus neolepis gen. et sp. nov. (Prymnesiophyceae, Haptophyta)." Protist, 157(2), 213-234. Young, J. R. (1987). "Possible functional interpretations of coccolith morphology." Abh. Geol. B.-A, 39, 305-313. Young, J. R. (1994). "functions of coccoliths", in A. Winter, Siesser, W.G. , (ed.), Coccolithophores. Cambridge: Cambridge University Press, pp. 63-82. Young, J. R., Bergen, J. A., Bown, P. R., Burnett, J. A., Fiorentino, A., Jordan, R. W., Kleijne, A., Van Niel, B., Romein, A. T., and Von Salts, K. (1997). "Guidelines for coccolith and calcareous nannofossil terminology." Palaeontology, 40(4), 875-912. Young, J. R., Davis, S. A., Bown, P. R., and Mann, S. (1999). "Coccolith Ultrastructure and Biomineralisation." Journal of Structural Biology, 126(3), 195-215. Young, J. R., and Henriksen, K. (2003). "Biomineralization Within Vesicles: The Calcite of Coccoliths." Reviews in Mineralogy and Geochemistry, 54(1), 189- 215. Zetsche, E.-M., and Meysman, F. J. R. (2012). "Dead or alive? Viability assessment of micro- and mesoplankton." Journal of Plankton Research, 34(6), 493-509. Zhou, Y., Shimizu, K., Cha, J. N., Stucky, G. D., and Morse, D. E. (1999). "Efficient Catalysis of Polysiloxane Synthesis by Silicatein α Requires Specific Hydroxy and Imidazole Functionalities." Angewandte Chemie International Edition, 38(6), 779-782.
- 226 -
Durak, G.M. List of references
- 227 -