IMPRS-gBGC workshop: Analytical Techniques in Microscopy

Supervisors: Katrin Krause & Elke-Martina Jung

September 13th, 2016

09.00-10.00 L Introduction fungi and fluorescence microscopy

10.00-12.00 P (Immuno)fluorescence staining

13.00-16.00 D, P Fluorescence and laserscanning microscopy Mycena chlorophos, wikipedia.org Panellus stipticus, wikipedia.org

Introduction fungi

Katrin Krause, Elke-Martina Jung IMPRS-gBGC workshop: Analytical Techniques in Microbiology Characteristics

• ca 1.5 million species, only 75,000 species have been described • eukaryotes: one or more nuclei • no chlorophyll • C heterotroph (chemo-organotroph) • absorb soluble nutrients through cell wall and plasma membrane • aerob (soil living fungi) • mainly immobile • storage compounds: glycogen, mannitol, trehalose and lipids (!) • structural cell wall polymers: mostly chitin and β-1,3-glucan • spores Form of cells

Hyphae , hyphal network= Yeast (single cells)

apical & invasive growth

Candida albicans Structure of fungal

Aut=autolysis AVC=apical vesicle cluster Chlam=chlamydospore ER=endoplasmatic reticulum G=Golgi (equivalent) Gl=glycogen L=lipid M=mitochondria MT=microtubules MW=melanized wall N=nucleus P=plasmalemma R=ribosomes S=septum SP=septal plug V=vacuole W=wall Wo=Woronin body - aging of hyphae - 2-30 mm diameter, extension zone 40 mm/min - Spitzenkörper: actin microfilament cluster in AVC center

Neurospora crassa, Deacon. 2006. Fungal biology Structure of a budding yeast BS=bud scar ER=endoplasmatc reticulum G=Golgi L=lipid body M=mitochondrion N=nucleus SPB=spindle pool body V=vesicle Vac=vacuole W=wall

Saccharomyces cerevisiae, Deacon. 2006. Fungal biology Components of the wall & its architechture

Distingt layers in subapical region of N. crassa (a) b-1,3-glucan & b-1,6-glucan (b) Glucoprotein reticulum in protein (c) Discrete protein layer (d) Chitin microfibrills in protein (e) Plasma membrane Deacon. 2006. Fungal biology Müller & Löffler, 1982 Diversity in living strategies

symbionts plant parasites

Xanthoria parietina Ustilago maydis

saprobionts human pathogens

Cyathus striatus Cryptococcus neoformans Fungal kingdom

Dikarya Higher fungi True fungi Slime molds

Tree of life - based on rRNA (18S, 5.8S, 28S) - true fungi and -like organisms - Phylum Glomeromycota - Mitosporic fungi (deuteromycotina, „) not known sexual stage

http://www.bact.wisc.edu/themicrobialworld/Whittaker.jpg Slime molds – social amoebas

Dictyostelium discoideum

• single amoeba assembling in hunger signals

• formation of a mobile „snail stage“, which is forming a sorokarp (fruiting body)

• single cells, no plasmodia

• in focus of research in development of multi-cellular organisms Zygomycota

• non-septated, multi-nuclear hyphae • haploid genome Entomophthora aphidis (Entomophthorales) with aphids • cell wall: chitin/chitosan, b-1,3-glucan • no mobile cells (zoospores)

Rhizopus stolonifer (Mucorales) on tomato

Zygospore (sexual )

Rhizopus nigricans Glomeromycota

• relationship with other fungi unclear • AM fungi (arbuscular mycorrhiza) • associated with 70 % of plants • haustoria-like structures vesicle/arbuscules Mitrophora semilibera Ascomycota

• yeast - filamentous fungi

• 75 % of all described fungi

• septated hyphae, septae with pores

• cell wall: β-1,3-glucan and chitin

Hook formation Ascogonium Ascogenous hyphae Trichogyne Antheridium

Ascus

Development from a hook to an ascus with ascospores

• ca. 30,000 species (37 %)

• mainly terrestrial lifestyle

• cell wall: chitin, glucan

Agaricus campestris Classification of Basidiomycota

3 major subgroups

Uredinomycetes with rust fungi e.g. Puccinia graminis

Ustilaginomycetes with smut fungi e.g. Ustilago maydis

Hymenomycetes with mushrooms, puff balls, jelly fungi Sexual development of basidiomycetes Schizophyllum commune

(Kothe et al. 2003) In the main stage of life the mycelium of basidiomycota is a . Model system Schizophyllum commune (Gemeiner Spaltblättling)

A B C D E

0.5 cm Initials Primordia Fruiting bodies

Fruiting bodies Lamellae with lengthwise split gills

Schubert et al. 2004 Life cycle of ectomycorrhizal fungi

http://ecomicro.univ-lyon1.fr Types of mycorrhiza

Basidiowww.lh-lawe.de- & Ascomycota Glomeromycota

www.bio.vu.nlBasidiomycota

Basidiomycotaww.hinti.ch

www. www.museums.orgAscomycota BasidiomycotaLegambientearcipelagosca Schwantes, 1996 ne.it Ectomycorrhiza

DAPI stained nuclei

Kothe et al. 2013 Ectomycorrhiza formation

Wagner et al. 2016 N-terminus Hydrophobins c cc c c cc c C-terminus

Wessels, J. G. H. (1996) Fungal Hydrophobins: proteins that function at an interface, Wessels, J. G. H. (1999) Fungi in Their Own Right, Fungal Genetics and Biology 27, 134-145 Trends in Plant Science 1, 9-15

soluble monomers & stable aggregates in 2 % SDS bei 100 °C Function of hydrophobins

• protection (aerial mycelium, conidiospores)

• pseudoparenchyme formation (fruiting bodies, air channels)

• surface contact (pathogenesis, appressoria)

Host specific mycorrhiza “right” “false” Tricholoma & pine: rhodamine-labeled hydrophobin tree Tr. & spruce: rhodamine-labeled hydrophobin Function of hydrophobins

Wettability Sc3 wild type “knock-out”

BASF Production of hydrophobic surfaces Decontamination of crude oil 2015

Metals, (secondary) plant metabolites, xenobiotics

vacuole The ectomycorrhizal MATE transporter mte1 in yeast

bright field DAPI Anti His merge

mte1

wt

bar = 1 µm wood

Brown rot White rot Loss of stability and weight Structure is preserved cube breaks Bleached, lighter, fibrous and moldy Dark-brown color Demarcation lines

www.chmltech.com Brown rot - Cellulose degradation

Endo-b-1,4-glucanase

Exo-b-1,4-glucanase

http://www.pilzarbeitskreis.de

Schwefelporling (Laetiporus sulphureus) Lärchenporling (Laricifomes officinalis) Echter Hausschwamm (Serpula lacrymans)

b-1,4-glucosidase

Fritsche, 1990 White rot – Lignin degradation

D Trametes versicolor (Schmetterlingsporling) Phellinus (Feuerschwamm) Phanerochaete chrysosporium Fomes (Zunderschwamm) 3dimensional macro molecule from phenylpropanoid Schizophyllum commune units Pleurotus ostreatus (Austernseitling) A) Ether bonds, B) Biphenyl bonds, C) - Mushroom production Phenylcoumarin structure D) Lignin degrading enzyme system Polyporus species after Fritsche, 1990; http://www.gymbsb.de - Pencil production White rot by Schizophyllum commune

Propidium jodide stained hyphae on apple tree wood Contamination of environment = Use of bioremediation

-

(Gianfreda & Rao, 2004) Persistence time of bioactive xenobiotics

Fritsche, 1990 Bioremediation by basidiomycetes

• ubiquitously distributed • cheap substrates e.g. agricultural waste • filamentous growth allows good penetration into the soil • constitutive enzymes (expression in nutrient limitation)

(Gianfreda & Rao, 2004) Accumulation of polycyclic aromatic hydrocarbons (PAHs)

Wang et al. 2009 10 m M benzene (Ben), naphthalene (NP), anthracene (Ant), pyrene (Py), benzo[ a ]pyrene (BaP), and indeno[1,2,3-cd]pyrene (IND) for 3 h.

Fungal toxins

- N containing secundary metabolites ( N-heterocycles, oxolanes, peptides, N-N compounds), sesqui- and triterpenes, anthrachinones - poisonings & magical religous ceremonies - 95 % of lethal fungal poisonings in ME - death cap (Amanita phalloides)

Sudini et al. 2015 Aflatoxin of Aspergillus flavus on coconut medium Aflatoxin B1 Tetrapolar mating system A und B together: Clamp fusion, Fruiting body development, Spore formation

A genes: Clamp formation conjugated nuclear division

B genes: Nuclear migration Homogenous incompatibility: identical i. allele is inducing termination of karyogamy. Nuclear migration: control via B and pheromone response

A≠B≠ A=B= A=B≠ A≠B= nuclear migration with no morphological „flat“ swallen hyphae, pseudoclamps, Barrage clamp formation, changes no aerial mycelium, reaction dikaryon, spores 20>nuclei compatible incompatible semicompatible semicompatible

DAPI

DAPI Anti-tub Anti-tub Intact microtubules are nessessary & speed of 11 mm/sec In the main stage of life the mycelium of Basidiomycetes is a dikaryon with clamps.

1

2 cone hook 3

4

5 clamp Schubert et al. 2004 6

chitin Anti-tub Septated mycelium with complicatedly structured pores

Dolipore

http://images.encarta.msn.com/xrefmedia/dencmed/targets/illus/ilt/T012705A.gif, http://cgdc3.igmors.u-psud.fr/microbiologie/basidiomycetes_fichiers/image003.jpg Septa in fungal hyphae

Simple septa Dolipore-septa

parenthosome Vesicular

Woronin bodies Non-perforated

Perforated

Paxillus involutus, chitin staining

Weber. 1993. Allgemeine Mykologie Wide of pores 15-900 nm – nucleus can pass Fluorescent staining

cell structure reagents emission colour Filter Axioplan DNA (nucleus) DAPI 450 nm blue 02 Chitin (cell wall) Chalcofluor White 435 nm blue 02

Mitochondria MitoTracker 525 nm green 10 green 24

Actin RLP (Rhodamine 570 nm bright red 15 labelled phalloidin) dark red 24

Fluorescent staining

Calcofluor

DAPI + MitoTracker

DAPI Immunofluorescent staining

Direct: more speed (only one incubation) and simplicity several antibodies labelled with different fluorochromes reagents are more expensive and less …

Indirect: primary non-conjugated antibody second antibody conjugated to a fluorochrome (FITC-goat anti-mouse, ...) more fluorochrome molecules per antigen & higher specificity more difficult with more than one antibody (cross-reaction) Antibodies for Indirect immunfluorescence staining (IIF)

cell antibody 1 antibody 2 labelled colour structure with tubulin T9026 F4018 FITC green mouse goat actin A2066 C2306 Cy3 red rabbit sheep

12-43 LSM 780-Elyra Tubulin

• tubulin forms long bundles along longitudinal axis during the cell • microtubules are components of tip growth • pathway for intracellular movement of organelles and proteins

Alberts et al., 2002 Localisation of actin

• as plaques in filamentous cells • aggregation on cell tips and septa • part of the cytoskeleton, which allows motility

[Geitmann and Emons 2000] Immunofluorescent staining: anti-tub, anti-act & DAPI Immunofluorescent staining: anti-tub, anti-act & DAPI

[G.Steinberg 2005]

Model of the cytosceleton in filamentous fungi Autofluorescence

Spontaneous emission of light after excitation: • porphyrins (red) • lipids (lemon – dark yellow) • chalk (white) • dermoxanthon (Cortinarius: yellow green)

• aesculin (blue) http://www.chemie.uni-jena.de/institute/oc/weiss/naturstoffe.htm# • protoberberin alcaloids (yellow) • keratin (blue white) • collagen (weak blue) • elastin (white) • cartilage (light blue)

http://www.chemie.uni-jena.de/institute/oc/weiss/naturstoffe.htm# Autofluorescence

• often emission in different wavelengths with lower intensity • use negative controls (without 1st or 2nd antibody) • use other filter sets (fluorescence microscopy) or specific wavelengths (laser scanning microscopy)

bright field FITC stained hydrophobin

DAPI staining of DNA/RNA autofluorescence Bioluminescent fungi

• 75 species of (Basidiomycota) with one exceptional ascomycete • luciferase: production of light by the oxidation of fungal luciferin sesquiterpene panal • greenish light at 520–530 nm

Mycena chlorophos, wikipedia.org Panellus stipticus, wikipedia.org Thank you for your attention! Questions?

Practical