Submitted to Open Biology: FOR REVIEW ONLY
A centriolar FOP-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes
Journal: Open Biology
Manuscript ID RSOB-17-0218.R2
Article Type: Research
Date Submitted by the Author: 31-May-2018
Complete List of Authors: Harmer, Jane; Lancaster University, Divison of Biomedical and Life Sciences; University of Huddersfield School of Applied Sciences, Department of Biological Sciences Towers, Katie; OXford Brookes University, Department of Biological and Medical Sciences Addison, Max; Lancaster University, Divison of Biomedical and Life Sciences Vaughan, Sue; OXford Brookes University, Department of Biological and Medical Sciences Ginger, Michael; University of Huddersfield School of Applied Sciences, Department of Biological Sciences McKean, Paul; Lancaster University, Divison of Biomedical and Life Sciences
Subject: cellular biology, microbiology
basal body, ciliogenesis, FOP, Trypanosoma brucei, cell morphogenesis, Keywords: paraflagellar rod
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1 A centriolar FOP-like protein required for paraflagellar rod assembly, but
2 not axoneme assembly in African trypanosomes
3
4 Jane Harmer1,4, Katie Towers2, Max Addison1, Sue Vaughan2, Michael L. Ginger3*, Paul G.
5 McKean1*
6
7 1Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University,
8 Lancaster, LA1 4YQ, UK
9 2Department of Biological and Medical Sciences, Faculty of Health and Life Science, Oxford Brookes
10 University, Gipsy Lane, Oxford, OX3 0BP, UK
11 3Department of Biological and Geographical Sciences, School of Applied Sciences, University of
12 Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK
13 4Present address: Department of Biological and Geographical Sciences, School of Applied Sciences,
14 University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK
15
16 Authors for correspondence:
17 Michael L. Ginger email: [email protected]
18 Paul G. McKean email: [email protected]
19 *MLG and PGM are co senior authors
20 Running head: Trypanosome FOPL function
21 Subject Area: cellular biology/microbiology
22 Keywords: basal body; ciliogenesis; FOP; Trypanosoma brucei, cell morphogenesis, paraflagellar
23 rod
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24
25 Abstract
26 Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organising centres
27 (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the
28 axoneme extends. We report conservation of FOP family proteins, TbFOPL and TbOFD1, in the
29 evolutionarily divergent sleeping sickness parasite Trypanosoma brucei, showing in contrast to
30 mammalian cells where FOP is essential for flagellum assembly, depletion of a trypanosome FOP
31 homologue, TbFOPL, affects neither axoneme nor flagellum elongation. Instead, TbFOPL depletion
32 causes catastrophic failure in assembly of a lineage specific, extra axonemal structure, the
33 paraflagellar rod (PFR). That depletion of centriolar TbFOPL causes failure in PFR assembly is
34 surprising since PFR nucleation commences ~2 m distal from the basal body. When over expressed
35 with a C terminal myc epitope, TbFOPL was also observed at mitotic spindle poles. Little is known
36 about bi polar spindle assembly during closed trypanosome mitosis, but indication of a possible
37 additional MTOC function for TbFOPL parallels MTOC localisation of FOP like protein
38 TONNEAU1 in acentriolar plants. More generally, our functional analysis of TbFOPL emphasizes
39 significant differences in evolutionary cell biology trajectories of FOP family proteins. We discuss
40 how at the molecular level FOP homologues may contribute to flagellum assembly and function in
41 diverse flagellates.
42
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43 1. Introduction
44 Coupled, N terminally located TOF LisH motifs define a small family of eukaryotic proteins, the FOP
45 family; members of which are required for ciliogenesis in flagellate eukaryotes, and cortical
46 cytoskeleton organisation in plant cells. Family members conserved amongst flagellate eukaryotes are
47 FOP (standing for FGFR1 oncogene partner), OFD1 (mutated in orofaciodigital syndrome 1), and
48 FOR20 (or FOP related protein of 20kDa) (1 8). With regard to localisation, in animal cells, OFD1,
49 FOR20, and FOP are all found at the base of cilia associated with basal bodies (or centrioles), either at
50 the level of the triplet microtubule barrel or the transition zone. The microtubule axoneme (the
51 defining structure of all eukaryotic flagella or cilia) extends from the basal body; the transition zone
52 defines the most proximal region of the flagellum and exhibits its own particular architecture, where
53 Y shaped projections link axoneme outer doublet microtubules to the flagellar membrane (9). FOP,
54 OFD1 and FOR20 are all required for the formation of a primary cilium; assembled by many types of
55 animal cell in response to appropriate environmental cues (10). Roles for FOR20 and OFD1 in cilia
56 assembly have been described in the ciliates Paramecium tetraurelia and Tetrahymena thermophila
57 (5, 11, 12), but we are not aware of any reports regarding functional studies of candidate OFD1 and
58 FOP orthologues in other flagellate protists or fungi.
59 One member of the FOP protein family is also conserved in at least one group of aflagellate
60 eukaryotes, land plants. The protein TONNEAU1 (or TON1), which is most similar to FOP, interacts
61 with at least one classic protein found at microtubule organising centres, centrin, and is required for
62 organisation of cortical microtubules during cell elongation and division (13). Absence of
63 TONNEAU1 from Arabidopsis, an aflagellate angiosperm, or the evolutionarily more basal bryophyte
64 moss Physcomitrella patens, which deploys flagellate motile sperm for reproduction, results in
65 organelle mis positioning and defective development (13, 14). Another group of organisms in which a
66 cortical based microtubule cytoskeleton exerts an overarching and relatively well understood effect on
67 cell morphogenesis and division are the flagellate trypanosomatids (15 17). Long known as the
68 aetiological agents of a variety of serious tropical diseases (e.g. African sleeping sickness, Chagas
69 disease, leishmaniasis), the parasitic trypanosomatid family belong to the excavate group of protists;
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70 which is widely recognised as evolutionarily divergent in comparison with other eukaryotic groups
71 (18, 19).
72 Flagellum assembly and function has been widely studied in trypanosomatids; many facets of this
73 biology are conserved with other flagellate eukaryotes, but there are also notable differences. Visually
74 the most notable difference is the presence of a complex paraflagellar rod (PFR). Within the flagellar
75 compartment, trypanosomatids and their nearest relatives build an elaborate extra axonemal PFR
76 structure, consisting of a trilaminar lattice composed of proximal, intermediate and distal domains
77 (20). The PFR is attached to outer doublet microtubules 4 7 of a canonical ‘9+2’ microtubule
78 axoneme and runs alongside the axoneme for much of the length of the flagellum. The PFR is
79 essential for flagellar beating and thus cell motility (21), and in Trypanosoma species PFR assembly
80 is required for attachment of the flagellum to the cell body, which in turn is required for normal cell
81 morphogenesis (22 23). In T. brucei, the PFR is present and attached to the axoneme from the point
82 where the flagellum exits the cell body (~2µm distant from the basal body), however, structural and/or
83 molecular cues that define assembly and asymmetric PFR attachment are not understood.
84 At a molecular level, in addition to obvious proteomic differences relating to PFR assembly, there are
85 examples of conserved proteins which have an essential flagellum assembly role that are somewhat
86 different in trypanosomatids; for instance, the tubulin cofactor C (TBCC) domain containing protein
87 RP2 (24, 25). Here, the catalytic TBCC domain of the trypanosome RP2 protein uniquely lies
88 downstream of twinned TOF LisH motifs essential for centriolar targeting of FOP family proteins
89 (24); whereas human RP2 is targeted to the basal body by a post translation N acyl modification (26).
90 As part of our ongoing studies looking at the role of TbRP2 in T. brucei flagellum assembly, we
91 turned our attention to the function of other trypanosomatid proteins that encode N terminal TOF
92 LisH motifs. We report here the conservation of OFD1 and FOP like proteins in trypanosomatid
93 protists, and our unexpected observation that a T. brucei FOP like protein is essential for assembly of
94 the extra axonemal PFR, but not the axoneme itself. Our data illustrates unexpected functional and
95 evolutionary diversity in the role of conserved centriole targeted proteins in eukaryotic flagellum
96 assembly and function.
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97
98 2. Materials and Methods
99 2.1 Cell culture and transfection
100 Procyclic T. brucei (927Smox (27) and S427) were cultured in SDM 79 medium supplemented with
101 10% v/v foetal bovine serum and haemin (28). Constitutive expression of YFP or GFP tagged
102 proteins and RNAi experiments were performed in 927Smox cells whereas myc epitope tagged
103 protein was constitutively expressed in a 427 genetic background. Logarithmic phase cells were
104 transfected and stable transformants selected using 10 µg ml 1 blasticidin (following transfection with
105 pENT6B derived endogenous tagging plasmids), 50 µg ml 1 hygromycin (following transfection with
106 pPOT endogenous tagging DNA or pDEX377 derived expression plasmids) or 3 µg ml 1 phleomycin
1 107 (following transfection with p2T7177 derived RNAi plasmids) (29, 30). 2 µg ml of puromycin was
108 used for the routine culture of 927Smox. Transgenic cultures were kept free of selectable markers for
109 at least 48hrs prior to the start of experiments. RNAi was induced by the addition of doxycycline to a
110 final concentration of 1µg ml 1.
111
112 2.2 Plasmid constructs
113 Fusion proteins were expressed using pEnT or pDEX based vector systems (30) or the PCR only
114 tagging approach (pPOT; (29). For constitutive expression of N terminal YFP tagged TbFOPL,
115 TbOFD1 and TbFLAM3 (31) from endogenous chromosomal loci, DNA sequences corresponding to
116 open reading frames (orf) and 3’ intergentic regions (igr) were amplified by PCR. Resultant
117 amplicons were digested by XbaI/XhoI (orf) and XhoI/BamHI (igr) prior to 3 way ligation into
118 Xba1/BamHI digested pEnT6B Y. Plasmids were linearised with XhoI prior to transfection. For
119 constitutive expression of C terminal GFP tagged TbFOPL from endogenous chromosomal loci,
120 pPOTv2 plasmid DNA was PCR amplified (29) and the resultant amplicon used directly for
121 transfection.
122 For expression of a myc epitope tagged TbFOPL the coding sequence minus stop codon was
123 amplified using a two step PCR reaction (in order to remove an internal XhoI site in the TbFOPL
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124 coding sequence), where the product of the first reaction was used as template for the second. The
125 resultant PCR amplicon was digested with HindIII and XhoI prior to being cloned into a HindIII/XhoI
126 digested pDEX377 myc vector (24). For TbFOP and TbOFD1 RNAi orf sequences were amplified
127 and the resultant amplicon cloned between opposing head to head T7 RNA polymerase promoters in
128 p2T7 177 vector, pre digested with BamHI and XhoI. Both pDEX and p2T7 derived plasmids were
129 linearised with NotI prior to transfection. Molecular masses for YFP and myc tagged proteins were
130 confirmed by immunoblotting (see results); correct genomic integration of DNA conferring
131 expression of TbFOPL::GFP was confirmed by Southern blotting (not shown).
132
133 2.3 Microscopy and immunoblotting
134 Cells were settled onto coverslips and either fixed directly with 3.7% paraformaldehyde or detergent
135 extracted for 30 seconds with 1% Nonidet P40 in 0.1M PIPES, 2mM EGTA, 1mM MgSO4, 0.1mM
136 EDTA, pH 6.9 prior to fixation. Fixed cells were placed in methanol for 10mins prior to rehydration
137 in PBS. Indirect immunofluorescence using polyclonal antiserum raised against recombinant TbRP2
138 (24) and monoclonal antibodies L8C4 and L3B2 (recognising PFR and FAZ, respectively (32)),
139 YL1/2 (33))and anti myc was performed as described previously or as stated in the manufacturer’s
140 instructions (myc; AbCam). Images were captured using an Applied Precision DeltaVision
141 microscope with a Roper Scientific Photometrics Cool SNAP HQ camera at 60X magnification and
142 processed using associated SoftWorx software and Adobe Photoshop. Nuclei and kinetoplast counts
143 of L8C4 and DAPI labelled cells were determined using a Leica DM RXA2 microscope and
144 associated FW4000 software.
145 Protein samples were separated by SDS PAGE and immunoblotted onto Hybond P membrane
146 (Amersham Biosciences) using standard protocols. Membranes were probed with monoclonal
147 antibodies BB2 (34) to detect YFP::TbFOPL or KMX1 (35) for the detection of β tubulin as
148 previously described. HRP conjugated secondary antibodies were detected using Immobilon Western
149 Chemiluminescent HRP substrate (Millipore) and BioRad XRS imaging System.
150
151 2.4 Electron microscopy
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152 Fixation was by addition of glutaraldehyde (2.5% final concentration, 5 minutes) to cultures. Cell
153 pellets were re suspended in 0.1M PBS (pH 7.4) for 10 minutes, followed by 2.5% glutaraldehyde,
154 2% paraformaldehyde and 0.1% tannic acid in 0.1M phosphate buffer (pH 7.0) for 2 hours at room
155 temperature. Pellets were washed with 0.1M phosphate buffer (pH 7.0) and post fixed in 1% osmium
156 tetroxide in 0.1M phosphate buffer (pH 7.0) for 1 hour at room temperature. Samples were rinsed and
157 stained en bloc for 40 minutes in 2% uranyl acetate, dehydrated in an ascending acetone series and
158 embedded in Agar 100 resin (Agar Scientific). Thin sections were examined by electron microscopy
159 using a Hitachi H 7650, operated at 120 kV.
160
161 2.5 Bioinformatics
162 Protein sequences were aligned by Clustal Omega (36) and the STRING database (37) was used to
163 identify known, and predicted, interactions between human FOP and other proteins.
164
165 3. Results
166 3.1 Divergent FOP family proteins in trypanosomatids
167 Additional to TbRP2, three further genes in T. brucei encode proteins with coupled N terminal TOF
168 LisH motifs: Tb927.11.3090, Tb927.5.4090 and Tb927.10.3000. Syntenic orthologues of all three
169 genes are present in all trypanosomatid species for which genome sequences are available at
170 EuPathDB (38). Tb927.11.3090 encodes a FOR20 orthologue and localises to both pro and basal
171 bodies (4). In contrast, the predicted proteins encoded by Tb927.5.4090 and Tb927.10.3000 are not
172 immediately recognisable as orthologous to any particular FOP family protein. In that context, we
173 also note failure to correctly predict a trypanosome FOP orthologue in both a published
174 bioinformatics survey of centriole/basal body evolution, and within the phylogenomic co occurrence
175 survey that is a part of the ‘STRING’ programme (37, 39). Given the importance of a microtubule
176 corset in defining trypanosome cell morphology, and involvement of a FOP related protein to
177 organising the cortical cytoskeleton in acentriolar plant cells, we made no assumption regarding the
178 localisation of proteins encoded by Tb927.5.4090 and Tb927.10.3000. Thus, we expressed both as N
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179 terminal fusions with YFP from their endogenous chromosomal loci (and thus under the regulatory
180 control of the endogenous 3’ intergenic sequence; in trypanosomatids 3’ intergenic sequences are
181 accepted as exerting the dominant influence on gene expression). In these experiments, YFP
182 fluorescence was compared to the indirect immunofluorescence signal obtained using polyclonal
183 affinity purified anti TbRP2 antibody (24). As shown in Figure 1 and Supp. Figure 1, proteins
184 encoded by Tb927.5.4090 (Figure 1) and Tb927.10.3000 (Supp. Figure 1), and tagged at the N
185 terminus with YFP, each co localised with the mature basal body marker TbRP2 but were not
186 detectable at other microtubule organising centres (MTOCs) or other cellular locales at any point
187 during the cell cycle.
188 Returning to the interrogation of T. brucei FOP family candidature, BLAST analyses revealed human
189 OFD1 identified Tb927.10.3000 as the top hit, albeit with an e value below an e 10 threshold and
190 requiring the insertion of numerous gaps to produce an alignment with moderate identity and
191 similarity. These trypanosome and human proteins also differ in length by over 200 amino acids
192 (Supp. Figure 2A B). Nevertheless, gene specific RNAi provided further evidence for the TbOFD1
193 candidature of Tb927.10.3000 (Supp. Figure 2C M). In contrast, HsFOP and divergent FOP like
194 proteins from Tetrahymena (TTHERM_00537420, or TtFop1; (40); TTHERM_00305510;
195 TTHERM_00689980) fail to identify candidate orthologues from trypanosomatids with expectancy
196 values above even an e 05 threshold; here both differences in size and an overall shorter protein length
197 influence analysis outcomes. However, with the acceptance of three insertions, HsFOP and the
198 Tb927.5.4090 gene product align with reasonable identity and similarity along their length (Figure
199 2A). Based on our analyses we believe Tb927.5.4090 encodes a FOP related protein, but following
200 published reports of HsFOP and TtFOP1 (1, 40) we conservatively refer to the trypanosome gene as
201 FOP like (or TbFOPL). We also prefer the designation FOP like because of the surprising RNAi
202 phenotype described below.
203
204 3.2 TbFOPL protein is required for PFR assembly but not axoneme formation
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205 Following TbFOPL RNAi induction, levels of YFP::TbFOPL declined (Figure 2B) and abnormal
206 cells appeared within 24 h. By 48 h, cell growth had slowed and very few cells presented with a
207 normal morphology (Figure 2B C; Figures 3 4; Supp. Figure 3); cells at this stage varied in size and
208 distinct intra flagellar swelling was frequently observed (Figure 3). Swelling was typically observed
209 either at the very distal end of the flagellum (Fig 3D; arrow), or within the flagellum (Figure 3E G;
210 arrows). Cells decorated for immunofluorescence microscopy with the monoclonal antibody L8C4
211 (which detects PFR2; one of the two major proteins that form the paraflagellar rod) indicated the
212 variable width of the flagellum noted in the SEM images was the result of defective PFR assembly
213 (Figure 4). Instead of the uniform PFR2 signal seen along the length of the flagellum, from the point
214 of cell body exit in normal cells (Figure 4A), we typically observed cells where PFR2 signal was
215 absent, except for the accumulation of PFR2 protein at a point coincident with the end of the cell body
216 and/or the distal tip of the flagellum (Figure 4B H). We also observed cells where sometimes a faint
217 PFR2 signal was present in the proximal region of the flagellum but the signal was then lost,
218 indicating an initiation of PFR assembly but subsequent failure of PFR assembly within the same
219 flagellum (Figure 4B, 4F, 4H). In cells where the PFR of a pre existing flagellum was fully formed,
220 we saw no evidence for subsequent PFR loss, although assembly of PFR in new elongating flagella
221 was perturbed (Figure 4C). In such cells, an accumulation of PFR2 at the end of the cell body and/or
222 flagellar tip was often evident; in some of these cells, detachment of the flagellum from the cell body
223 was also evident in the regions lacking PFR2 (Figure 4D G). The representative images shown in
224 Supp. Figure 3 illustrate how PFR formation was compromised in virtually all (>95%) cells 48 hours
225 post RNAi induction. We reported previously that for calmodulin (CaM) RNAi mutants, where PFR
226 formation also fails completely, the default status is for flagellum cell body attachment and that
227 flagellum detachment occurs some time later (22). Examples of cells with attached flagella that lacked
228 PFR were observed in RNAi induced TbFOPL mutants (Figures 4C and 4H). However, by 48 hours
229 post induction of TbFOPL RNAi considerable heterogeneity in cell morphology was evident (Figure
230 4; Supplementary Figure 3). Included in this heterogeneity were ‘cells’ or, perhaps more accurately,
231 cell ‘slivers’ apparently lacking flagella and of varying size. These were the likely consequence of
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232 asymmetric cell division; the higher resolution afforded by SEM emphasised the irregularities in cell
233 morphogenesis that could occur following TbFOPL depletion (Figures 3D G).
234 Depletion of centriolar FOP in cultured mammalian cells results in loss of ciliogenesis (6, 7). In
235 contrast, flagellum assembly was maintained post TbFOPL RNAi induction, albeit with flagella that
236 were often detached from the cell body. Indication that an intact axoneme was assembled within these
237 flagella came first from comparison with T. brucei intraflagellar transport (IFT) RNAi mutants (41,
238 42), and TbOFD1 RNAi mutants (Supp. Figure 2). In these mutants elongation of a ‘flagellar sleeve’
239 of particularly narrow diameter (~70nm, cf axoneme diameter ~180 200nm) is seen. It reflects
240 flagellar membrane elongation in the absence of axonemal microtubule extension, and it is
241 conceivably a consequence of the IFT independent movement of the trypanosome ‘flagellar
242 connector’ (41), which guides flagellum elongation, cytotactic inheritance of organelles, and overall
243 cell morphogenesis in T. brucei (43). The diameter of flagella seen from SEM analysis of TbFOPL
244 RNAi mutants was indicative of IFT dependent axoneme elongation, rather than sleeve formation.
245 Nonetheless, mindful of the importance of FOP for axoneme formation in ciliated mammalian cells
246 (6, 7), we looked at axoneme ultrastructure in our TbFOPL RNAi mutants. In fifty transverse sections
247 (out of a total of 51 analysed), axoneme structure looked normal, irrespective of whether PFR was
248 absent, or the axoneme partially surrounded by an accumulation of PFR protein(s) (Figure 5B C).
249 This contrasts with loss of outer doublet and/or central pair microtubule integrity associated with T.
250 brucei flagellum RNAi mutants depleted for radial spoke, central pair, or nexin dynein regulatory
251 complex components, and selected, cultured, fixed, and prepared for electron microscopy using the
252 same protocols as the current study (44 46). This further emphasises TbFOPL is not required for
253 axoneme formation per se. TEM analysis revealed that PFR protein accumulated and assembled as an
254 amorphous structure rather than the elaborate ordered lattice observed in normal cells (compare Fig
255 5A with B, C and D). In the one section where a defect in axoneme structure was evident,
256 displacement of an outer doublet microtubule (Figure 5D) was conceivably the consequence of
257 excessive accumulation of PFR proteins.
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258 To interrogate further the observation of flagellum detachment from the cell body we queried the
259 localisation of flagellum attachment zone (FAZ) components following TbFOPL RNAi induction. On
260 the cell body side of the FAZ, we observed a normal localisation of FAZ1, a component of the fibres
261 radiating from membrane junctional complexes (47), even in the absence of flagellum attachment
262 (Figure 6A). This was consistent with initial flagellum cell body attachment in cells where no PFR is
263 built (22). On the intraflagellar side of the FAZ, we queried the localisation of the high molecular
264 weight protein TbFLAM3 (48 49). Here, the normal localisation was lost following RNAi induction,
265 with YFP::FLAM3 co localising with the aberrant intraflagellar accumulation of PFR2 at the distal
266 end of the cell body (Figure 6B). In these experiments detergent extracted cytoskeletons, rather than
267 intact cells, were examined; the retention of L8C4 signal and YFP fluorescence indicated a stable
268 association of bulky, amorphous ‘PFR’ components with the cytoskeleton rather than the more labile
269 accumulation of PFR components seen in some flagellar RNAi mutants (50).
270
271 3.3 Localisation and flagellum exclusion of TbFOPL::myc
272 With the TONNEAU1 connection to acentriolar plant MTOCs and the microtubule dominant
273 organisation of cell form in trypanosomes in mind, we questioned further the localisation of TbFOPL
274 by expression of protein tagged at the C terminus with GFP (TbFOPL::GFP) and myc tagged
275 (TbFOPL::myc) protein. TbFOPL::GFP expressed from an endogenous locus, albeit under the
276 regulation of a PFR2 3’ intergenic region, gave the same localisation pattern as YFP::TbFOPL. For
277 TbFOPL::myc, expression was driven from a strong RNA Polymerase I promoter. Over expression of
278 TbFOPL::myc was evident form the accumulation of epitope tagged protein in the cell body of whole
279 cells, in addition to mature basal bodies (Figure 7A). We included an over expression of TbFOPL to
280 question why a basal body localised protein was so critical for specifying correct assembly of an
281 extra axonemal structure ~2µm distal to the basal body. Specifically, we considered whether there
282 was a pool of intraflagellar TbFOPL not seen when analysing the localisation of fluorescent tagged
283 FOPL protein. There is indication that the transition zone limits protein access into the flagellum
284 compartment on the basis of size (where only small proteins, less than 4.5nm Stokes radii or 40kDa,
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285 enter the flagellum by diffusion; 51 ). TbFOPL::myc (predicted molecular mass ~30kDa) fell
286 comfortably beneath this threshold limit (cf YFP tagged TbFOPL, molecular mass >50kDa). Notably
287 TbFOPL::myc was excluded from the flagellum and the nucleus in whole cells (Figure 7A). This
288 leaves open the question of how a basal body localised protein, TbFOPL, critically influences the
289 asymmetric assembly of the extra axonemal PFR, which is built only after the flagellum exits its
290 flagellar pocket, a distance of ~2 microns from the point where the axoneme initially extends from its
291 basal body. Curiously, careful examination of detergent extracted cytoskeletons revealed
292 TbFOPL::myc localisation at the poles of the mitotic spindle in early mitotic cells (Figure 7C (iii iv)).
293 This was in addition to basal body localisation throughout the cell cycle. However, the nuclear signal
294 was absent from cells fixed later in mitosis (Figure 7C (v)). Re examination of YFP::TbFOPL
295 localisation did not reveal any indication of nuclear localisation. The severe morphological
296 abnormalities of TbFOPL RNAi induced cells meant it was not realistic to sensibly ascertain whether
297 spindle formation was also compromised by an absence of TbFOPL protein.
298
299 4. Discussion
300 The combination of N terminal localised TOF LisH motifs is a seldom used but highly effective
301 means of localising proteins to MTOCs: FOP family proteins including FOR20 and OFD1 are
302 centriolar proteins conserved in diverse eukaryotes, and in humans chromosomal translocation results
303 in TOF LisH dependent retargeting of the tyrosine kinase domain of the FGFR1 receptor to the
304 centrosome, and an atypical myeloproliferative disorder (1, 2).
305 In trypanosomatids, and their free living relative Bodo saltans, the presence of N terminal TOF LisH
306 motifs in the GTPase activating protein RP2 provides a lineage specific elaboration within the FOP
307 family, and ensures basal body localisation of the protein (in animals, basal body localisation of RP2
308 is dependent on N terminal acylation (26)). Curiously, although the evolutionary context is for N
309 terminal TOF LisH motifs in eukaryotes – the single exception that we found in our bioinformatics
310 analysis was of a gene model encoding a 688 amino acid protein with a candidate C terminal TOF
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311 LisH motif combination in the centric diatom Thalassiosira pseudonana (Accession No.
312 XP_002286300.1). In all four trypanosome proteins that utilise a TOF LisH motif combination, N
313 terminal fusion to YFP does not compromise localisation to mature basal bodies, or in the case of
314 TbFOR20 localisation to mature and associated pro basal bodies (4). Here, the more significant and
315 unanticipated characteristic of the trypanosome FOP protein family is that TbFOPL is not required for
316 axoneme elongation but is essential for the assembly of extra axonemal PFR. This contrasts strikingly
317 with the essentiality of the mammalian FOP homologue at the earliest stages of ciliogenesis (6, 7).
318 Similar to the T. brucei calmodulin (CaM) RNAi mutant, where PFR assembly is also totally
319 compromised (22), a failure of PFR assembly in the TbFOPL depleted cells leads to flagellum cell
320 body detachment and abnormalities in cell morphogenesis. However, TbCaM is present in both the
321 PFR lattice and struts linking the PFR and outer doublet microtubules of the axoneme. Thus, the
322 TbCaM PFR assembly defect can be readily explained. In contrast, TbFOPL appears not to be present
323 within the flagellum compartment, even when expressed from a strong transcription promoter, thus
324 raising the question as to how a basal body located protein is essential for PFR assembly.
325 We found no indication of problems in axonemal assembly within TbFOPL depleted trypanosomes –
326 flagellum length was normal and only seldom was there a discernible defect in axoneme
327 ultrastructure. This indicated that intraflagellar transport (IFT) was not lost, but rather that TbFOPL
328 deficiency caused a specific defect in PFR assembly. PFR assembly apparently initiates in some
329 TbFOPL RNAi induced cells but then subsequently fails: PFR material accumulates as large
330 unstructured deposits within or at the end of the flagellum in a majority of cells (~96% of flagella 48 h
331 post RNAi induction in which PFR2 could be detected by immunofluorescence using monoclonal
332 antibody L8C4; at 24 h post RNAi induction ~60% of flagella showed abnormal accumulation of
333 PFR2). Basal body localised TbFOPL could play a critical, direct basal body localised role in the
334 import of PFR specific cargo into the flagellum i.e. although major components such as PFR1, PFR2,
335 and FLAM3 are imported into the flagellum, it is possible that not all PFR components are imported
336 thereby compromising intraflagellar assembly of the PFR lattice. In this context, there is precedent for
337 selective transport of axonemal sub complexes and/or roles for cytoplasmic chaperones or other
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338 accessory assembly proteins in pre assembly of a variety of proteins associated with distinct axonemal
339 sub structures (e.g. dynein arms) prior to sub complex import into the flagellum (52 55). Indeed, in
340 immunofluorescence experiments using monoclonal antibody ROD1, which recognises an antigen
341 from the outer or most distal region of the PFR lattice, signal intensity was severely reduced on some
342 cells or non existent in others (data not shown); this was reminiscent of similar immunofluorescence
343 experiments in PFR deficient snl mutants (50).
344 Alternatively, the domain architecture of TbFOPL is not typically reflective of either an enzyme or a
345 chaperone. Rather its architecture and available experimental evidence are more consistent with roles
346 in scaffolding or mediation of protein protein interaction. For instance, in mammalian cells, FOP
347 recruits the centrosomal protein CEP19, which in turn interacts with the GTPase RABL2, and it is
348 RABL2 that regulates IFT B function and thereby cilium assembly (56). Similarly, centrosome
349 localised FOP is required for anchoring microtubules to subcellular structures and localisation of the
350 centrosomal protein EB1; a plus end microtubule binding protein that has critical functions in
351 regulating +end microtubule dynamics (57). Notwithstanding the possibility that the filament like
352 PFR lattice could be self assembling rather than chaperone or accessory protein dependent, TbFOPL
353 could feasibly play a role in recruiting another protein or proteins that act in PFR assembly and/or
354 intraflagellar PFR attachment. At this point our data is consistent with a direct or an indirect role for
355 TbFOPL in PFR assembly.
356 Presently, a final possibility to consider regarding how TbFOPL influences PFR assembly is a
357 possible dependency and/or interaction between TbFOPL and TbKIF9B, a trypanosomatid specific,
358 basal body and axoneme localised kinesin required for normal PFR construction (58). Although there
359 is similarity between the RNAi phenotypes of TbFOPL and TbKIF9B, the KIF9B phenotype is unique
360 in that in a majority of cells a PFR forms in patches along the length of the flagellum; in other cells a
361 PFR is either absent or accumulates in a single patch. Whether it is basal body and/or axoneme
362 localised KIF9B that is required for normal PFR construction is not known (58). Probing for potential
363 interaction between these proteins represents one avenue with which to move forwards to understand
364 the mechanism by which TbFOPL defines PFR formation.
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365 Additional to understanding how a trilaminar PFR lattice assembles, there is also the specification of
366 attachment of the PFR proximal region to outer doublets 4 7 of the axoneme. Cues that define the
367 asymmetric attachment of PFR to axoneme at a position several microns distant from the basal body
368 are unknown. Although structural asymmetries exist within basal/probasal bodies (e.g. 40, 59, 60), it
369 is difficult to understand how a centriolar located protein such as TbFOPL could influence the
370 asymmetric attachment of the PFR to the axoneme. We note, however, that in the ciliate Tetrahymena,
371 a FOP1 like protein (and polyglutamylated tubulin) is asymmetrically distributed around the basal
372 body; it is proposed asymmetric distribution of the FOP1 like protein and polyglutamylated tubulin
373 may stabilise basal bodies against mechanical forces generated during ciliary beating (40). From our
374 current DeltaVision imaging we have been unable to detect asymmetric localisation of TbFOPL or
375 polyglutamylated tubulin (using the anti polyglutamylation monoclonal antibody GT335) at the T.
376 brucei basal body, but nevertheless, the Tetrahymena example raises the possibility that asymmetric
377 distribution of basal body localised proteins such as FOP, could, either directly or via post
378 translational modification of axonemal microtubules, affect PFR attachment.
379 We find the possible nuclear localisation of TbFOPL::myc in early mitotic cells intriguing. Promoter
380 driven expression of TbFOPL::myc occurs throughout the cell cycle but nuclear acquisition of
381 TbFOPL::myc is cell cycle dependent. We are therefore inclined to believe recruitment to the nucleus
382 (and potentially to the MTOCs nucleating the mitotic spindle) is genuine. Although no discrete
383 structures, such as centrosomes or spindle pole bodies (seen in yeast cells), have been observed in T.
384 brucei, distinct ring like structures, that appear to nucleate spindle microtubules, can be visualised by
385 electron microscopy (Reviewed in (61)). Our observation of nuclear recruitment of TbFOPL indicates
386 that in addition to a critical basal body related function, the protein may also be required to establish
387 the acentriolar MTOCs responsible for spindle microtubule nucleation. This observation potentially
388 provides a parallel with the acentriolar MTOC localisation of another FOP family protein,
389 TONNEAU1, in plants (13). We have not observed nuclear recruitment of FOR20 and TbRP2 (i.e.
390 other trypanosome TOF LisH proteins) even after over expression (data not shown), and so
391 recruitment of TbFOPL to a nuclear MTOC does not appear to be a general feature of TOF LisH
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392 targeting in trypanosomatids. In common with many unicellular eukaryotes, T. brucei undergoes a
393 closed mitosis (62) and proteins involved in mitosis must be transported across the nuclear envelope.
394 However, any possibility TbFOPL is involved in MTOC function other than at basal bodies should be
395 balanced with the observation that, like other known trypanosomatid basal body proteins, a TbFOPL
396 orthologue is absent from Perkinsela, an acentriolar, aflagellate basal kinetoplastid that is an
397 endosymbiont within Paramoeba isolates (63). This could imply that TbFOPL is either not essential
398 for spindle assembly and/or function in trypanosomes or that mitosis in Perkinsela occurs
399 independently of FOPL.
400 In this, and previous work (4, 24), we have investigated all four TOF LisH motif containing proteins
401 expressed in T. brucei. All four proteins localise to the mature basal body. TbFOR20 additionally
402 locates to the probasal body and TbFOPL potentially to mitotic spindle poles. TbFOPL, TbOFD1 and
403 TbRP2 have distinct flagellum assembly related functions, but no apparent phenotype is detected in
404 FOR20 depleted cells. We have previously shown that RNAi mediated knock down of TbRP2 affects
405 flagellum assembly and it is proposed that TbRP2 acts as a GTPase activating protein with a role in
406 protein trafficking; human RP2 acts as a GAP for the small GTPase ARL3 (64). In T. brucei cells
407 depleted for OFD1, the short flagellum phenotype generated suggests that IFT mediated transport
408 may be compromised in these cells, consistent with a proposed IFT related role for mammalian
409 OFD1. In contrast, the protein we have identified as being most similar to mammalian FOP appears to
410 have a distinctive phenotype relating to PFR assembly, but without affecting axoneme assembly.
411 Studying FOP like function in trypanosomes affords a unique opportunity to study assembly of extra
412 axonemal structures. Finally, although the PFR is unique to trypanosomes and evolutionary close
413 relatives, extra axonemal structures are observed in flagella in a diversity of flagellated eukaryotes,
414 including Giardia intestinalis, Gymnodinium aureolum and other dinoflagellates. There are also
415 unusual MTOCs, some of which are thought to have a flagellar origin (e.g. the apical polar ring of
416 apicomplexans and their near relatives (65 68)). Determination of whether FOP related or other FOP
417 family proteins play roles in the assembly or function of these cytoskeletal structures offers intriguing
418 possibilities for future research.
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419
420 Authors’ contributions 421 P.G.M. and M.L.G. designed research. J.H. generated mutants and performed light microscopy with 422 M.A. providing additional contribution. K.T. and S.V. analysed mutant cells by SEM and TEM. J.H., 423 M.L.G. and P.G.M. wrote the manuscript. 424 Competing interests 425 No competing interests to declare. 426 Funding 427 This work was supported by grants from the Biotechnology and Biological Sciences Research Council 428 (BBSRC) (grant numbers BBG0210581, BBF0109311 to P.G.M. and M.L.G. and BB/100402/1 to 429 S.V). 430 Data accessibility 431 Datasets supporting this article are provided in the electronic supplementary material 432 Acknowledgements 433 We thank Keith Gull (University of Oxford) for the kind gift of antibodies BBA4, KMX1, ROD1, 434 L3B2 and L8C4. 435 436 Notes added in revision 437 Reference to TrypTag.org, the genome wide project to localise every protein coding gene product in 438 T. brucei (69), gives an indication of basal body localisation for N terminally mNeonGreen tagged 439 TbFOPL, but there is currently no localisation data is available for TbOFD1. mNeonGreen::TbKIF9B 440 localisation does not mirror precisely the published localisation: an additional, cell cycle stage 441 specific flagellar tip signal is reported alongside localisation to pro and mature basal bodies. This 442 potentially adds further complexity to understanding the mechanistic basis for KIF9B dependent PFR 443 assembly (58) or any hypothetical interaction or dependency between KIF9B and FOPL. 444
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645 69. Dean S, Sunter JD, Wheeler RJ 2017 TrypTag.org: A Trypanosome Genome wide Protein 646 Localisation Resource. Trends Parasitol. 33, 80 82. (doi: 10.1016/j.pt.2016.10.009) 647 648 649 Figure Legends
650 Figure 1. Localisation of Tb927.5.4090 gene product (TbFOPL) in procyclic T. brucei. (A-B) 651 Localisation of YFP::TbFOPL at the mature basal body in 1K1N (A) and 2K mitotic (B) cells. Images 652 show detection of YFP::TbFOPL relative to TbRP2 in whole cells (A-C); insets show the same 653 localisations at higher magnification. 6 Diamidino 2 phenylindole (DAPI) was used to detect nuclear 654 DNA (N) and the mitochondrial genome (or kinetoplast, K). (C) TbFOPL::GFP localisation at the 655 mature basal body in a 1K1N procyclic T. brucei cell; inset shows TbFOPL::GFP and TbRP2 656 localisation at higher magnification. (D-E) Retention of YFP::TbFOPL (D) and TbFOPL::GFP (E) in 657 detergent extracted cytoskeletons. Scale bars in all main panels indicate 5 µm and in the inset panels 1 658 µm. 659 660 Figure 2. RNAi knockdown of TbFOPL results in severe morphological defects. (A) Cartoon 661 representation of human FOP and T. brucei FOPL, showing insertions necessary to achieve maximal 662 alignment of amino acid sequences, and amino acid alignment of Homo sapiens FOP (Accession 663 number CAA77020.1) and T. brucei FOPL. (B) Effect of TbFOPL RNAi induction on trypanosome 664 growth (diamonds) compared to RNAi non induced controls (triangles); immunoblotting with 665 monoclonal antibody BB2 (detecting a N terminal Ty epitope) confirmed depletion of YFP::TbFOPL 666 post RNAi induction. Anti β tubulin antibody KMX1 was used as a loading control. (C) Effect of 667 TbFOPL RNAi induction on cell morphology with regard to normal kinetoplast nuclei number and 668 positioning and/or normal PFR assembly. 669 670 Figure 3. Induction of TbFOPL RNAi results in flagellum assembly and cell morphology 671 defects. (A-C) Scanning electron micrographs of TbFOPL RNAi non induced cells showing procyclic 672 cells at different stages of the cell division cycle. (D-G) Scanning electron micrographs of TbFOPL 673 RNAi induced cells showing distended regions within flagella (arrows), flagellar detachment and (F) 674 two flagella aberrantly emerging from the same flagellar pocket, thereby illustrating one extreme of 675 morphogenetic abnormality present. Scale bars in all main panels indicate 10 µm and in the inset 676 panel 1 µm. 677 678 Figure 4. Effect of TbFOPL RNAi on paraflagellar rod (PFR) assembly. (A) Immunolabelling 679 of PFR in a TbFOPL RNAi non induced cell. (B-H) Immunolabelling of PFR in TbFOPL RNAi 680 induced cells highlighting different failures in PFR assembly. The PFR specific antibody L8C4 was 681 used in all images to detect PFR2 protein. DAPI was used to detect nuclear DNA (N) and the
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682 mitochondrial genome (or kinetoplast, K). Grey arrows in B, C, and H indicate the position of the 683 anterior cell end; grey arrowheads indicate the distal tip of flagella, including ‘new’ elongating 684 flagella of cells in B and D. Purple arrows and arrowheads also denote positions of old and new 685 flagella, respectively. Red asterisks indicate flagella where PFR assembly has apparently commenced 686 prior to a subsequent failure. Cells in panels B F and H are from 24 h post RNAi induction; Cells in G 687 are from 48 h post RNAi induction. Scale bars indicate 10 µm. 688 689 690 Figure 5. Transmission electron microscopy of flagella following induction of TbFOPL RNAi. 691 (A) Transverse thin section through the flagellum of a TbFOPL RNAi non induced cell shows normal 692 arrangement of axoneme and PFR. (B) Absence of PFR assembly following TbFOPL RNAi; dynein 693 ATPases, radial spokes and central pair (CP) projections are all present. (C) Massive accumulation of 694 unstructured PFR material in association with an axoneme where dynein ATPases, radial spokes and 695 CP projections are present. (D) A rare example of loss of axoneme integrity following TbFOPL 696 RNAi. Scale bars indicate 100 nm. 697 698 Figure 6. Flagellum attachment zone integrity following TbFOPL RNAi. (A) Upper panel, 699 normal FAZ1 localisation on the intracellular face of the flagellum attachment zone (FAZ) in 700 detergent extracted cytoskeletons; lower panel, retention of FAZ1 localisation following TbFOPL 701 RNAi and flagellum detachment. (B) Upper panel, normal YFP::TbFLAM3 localisation on the 702 intraflagellar face of the FAZ in detergent extracted cytoskeletons; lower panel, mis localisation of 703 YFP::TbFLAM3 following TbFOPL RNAi. FAZ1 detected by monoclonal antibody L3B2, PFR was 704 detected using monoclonal antibody L8C4. Scale bars in all main panels indicate 10 µm and in the 705 inset panel 1 µm. 706 707 Figure 7. Localisation of TbFOPL::myc in procyclic T. brucei. (A) Basal body localisation and 708 cell body accumulation of TbFOPL::myc in whole cells. The immunoblot confirms the expected 709 molecular mass of TbFOPL::myc; 5 x 106 cell equivalents were loaded for SDS PAGE. (B) Mature 710 basal body localisation of TbFOPL::myc is retained in detergent extracted cytoskeletons. (C) 711 Additional localisation of TbFOPL::myc at spindle poles during early mitosis (arrows in cells (iii) and 712 (iv)); images shown are detergent extracted cytoskeletons. Scale bars in all main panels indicate 5 µm 713 and in the inset panel 1 µm. 714 715 Supplementary Data 716 Supplementary Figure 1. Localisation of Tb927.10.3000 gene product in procyclic T. brucei. (A-B) 717 YFP::Tb927.10.3000 is present at mature basal bodies throughout procyclic cell cycle. Monoclonal 718 antibody YL1/2 detected tyrosinated α tubulin prominent on subpellicular microtubules at the
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719 posterior pole of whole cells and TbRP2 (Reference 4 in the main text) at the mature basal body. Scale 720 bars in all main panels indicate 5 µm and in the inset panels 1 µm. 721 722 Supplementary Figure 2. Orthology of Tb927.10.3000 and HsOFD1. (A) Cartoon representation of 723 human and T. brucei OFD1 proteins, showing insertions necessary to achieve maximal alignment of 724 amino acid sequences. (B) Amino acid alignment of Homo sapiens (Accession number AC003037.1) 725 and T. brucei (Tb927.10.3000) OFD1. (C) Effect of Tb927.10.3000 RNAi induction on trypanosome 726 growth (triangles; solid line) compared to RNAi non induced controls (diamonds; dashed line); 727 immunoblotting with monoclonal antibody BB2 (detecting an N terminal Ty epitope) indicated 728 depletion of YFP::Tb927.10.3000 post RNAi induction. Polyclonal rabbit sera detecting trypanosome 729 adenylate kinase isoform F (Ginger ML et al. 2005 J. Biol. Chem. 280, 11781 9 doi: 730 10.1074/jbc.M413821200) was used as a loading control. (D) Preliminary analysis of cell morphology 731 following Tb927.10.3000 RNAi induction: cells were scored for normal morphology versus assembly 732 of an abnormal, short flagellum (at 24 h post RNAi induction n = 51 ‘non induced’ cells / n = 67 733 ‘induced’ cells; 48 h post induction n = 41 ‘non induced’ / n = 75 ‘induced’; 72 h post induction n = 734 39 ‘non induced’ / n = 95 ‘induced’; 96 h post induction n = 25 ‘non induced’ / n = 123 ‘induced’). 735 (E-M) Electron and fluorescence microscopy analysis of cell morphology: E G, TEM analysis 736 illustrating in short flagella the accumulation of electron dense material, potentially including 737 unassembled PFR components, around normal 9+2 axoneme architecture; H J, SEM analysis 738 illustrating the short flagellum phenotype of Tb927.10.3000 RNAi mutants; K L, fluorescence 739 microscopy illustrating mixed ‘short flagellum’ and ‘short cell’ phenotypes in Tb927.10.3000 RNAi 740 mutants (96 h post RNAi induction); M, normal cell morphologies in Tb927.10.3000 cells not 741 induced for RNAi against Tb927.10.3000. In K M, the PFR is immunolabelled with monoclonal 742 antibody L8C4 (red); DAPI (blue) was used to stain nuclear and kinetoplast DNA. 743 Assessment of OFD1 candidature. Mature basal body localisation of YFP::Tb927.10.3000 is 744 analogous to mature centriole localisation of human OFD1 (Singla V et al. (2010) Dev. Cell 18, 410 745 24 doi: 10.1016/j.devcel.2009.12.022). The short flagellum phenotype of the RNAi mutant resembled 746 published T. brucei intraflagellar transport (IFT) RNAi phenotypes: the short flagellum phenotype of 747 Tb927.10.3000 RNAi mutants is more similar in presentation to the phenotype arising from defective 748 retrograde IFT than it is to defective anterograde IFT mutants, which fail to elongate an axoneme 749 beyond the transition zone, and thus fail to build flagella (Absalon S et al. (2008) Mol. Biol. Cell 19, 750 929 44 doi: 10.1091/mbc.E07 08 0749; Davidge J et al. (2006) J. Cell Sci. 119, 3935 43 doi: 751 10.1242/jcs.03203). Flagellar membrane elongation or ‘flagellar sleeve’, seen in some T. brucei IFT 752 mutants (Davidge et al. 2006), was also evident in SEM micrographs of Tb927.10.3000 RNAi 753 induced mutants. Loss of YFP::Tb927.10.3000 beneath the threshold of detection by immunoblot was 754 evident 24 h post RNAi induction, but presentation of the short flagellum phenotype was never 755 evident across all cells in RNAi induced cultures even by 96 h post induction. We interpret such
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756 partial presentation of morphological phenotype, in contrast to the depletion of YFP::Tb927.10.3000 757 beneath a threshold level of detection, coupled to literature reports of OFD1 function in mammals, 758 including a requirement in murine embryonic stem cells for OFD1 in formation of centriole distal end 759 appendages and IFT88 recruitment, to suggest that protein encoded by Tb927.10.3000 provides 760 regulatory or indirect function(s) in IFT, rather than being a core part of the IFT machinery. In 761 summary, candidature of Tb927.10.3000 as an OFD1 ortholog based only on amino acid sequence 762 alignment with HsOFD1 is equivocal, but coupled to comprehensive preliminary RNAi phenotype 763 analysis evidence for OFD1 candidature is persuasive. 764 765 Supplementary Figure 3. Penetrance of TbFOPL RNAi. Representative fields of view for whole 766 cells show rapid loss of normal cell morphology and aberration of normal PFR biogenesis in 767 populations at 24 (A-C) and 48 (D-F) h post RNAi induction. Scale bars represent 10 m.
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