The inner scaffold protects from centriole fracture

Institut, Villigen, Switzerland 3 Swiss Light Source, Paul Scherrer Institut, 5232 Villigen, Switzerland 4 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France. 5 Biozentrum, University of Basel, 4056 Basel, Switzerland 15 * Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK † These authors contributed equally to this work. § Correspondence to: virginie.hamel@unige.ch and paul.guichard@unige.ch

cartwheel on the proximal end, which is crucial for centriole assembly (10, 11) or the distal appendages at the very distal region, essential for membrane docking during ciliogenesis (12).
The central core region of the centriole is defined by the presence of a circular inner scaffold that maintains the cohesion of microtubule triplets under compressive forces (Le Guennec et al, in press). Using cryo-tomography, we recently showed that the inner centriole scaffold forms an 10 extended helix covering ~70% of the centriole length and that is rooted at the inner junction between the A and B microtubules (Fig. 1A, B). This connection consists of a stem attaching the neighboring A and B microtubules and three arms extending from the same stem toward the centriolar lumen (Le Guennec et al, in press) ( Fig. 1A, B). The stem of the inner scaffold has been detected in Paramecium tetraurelia, Chlamydomonas reinhardtii and human centrioles, 15 suggesting that it represents an evolutionary conserved structural feature.
The molecular identity of some components of the inner scaffold has been uncovered using Ultrastructure Expansion Microscopy (U-ExM), which allows nanometric localization of proteins within structural elements (13). Notably, the centriolar proteins POC1B, FAM161A, POC5 and Centrin-2 have been shown to localize to the inner scaffold along the microtubule 20 blades in human cells (Le Guennec et al, in press). Moreover, these proteins form a complex that can bind to microtubules through the microtubule-binding protein FAM161A (14) (Le Guennec et al., in press). Importantly, a subset of these proteins has been shown to be important for centriole elongation (15) as well as for centriole and basal body integrity (16,17). This observation highlights the prominent role of the inner scaffold structure in providing stability to the entire centriolar microtubule wall organization. However, this hypothesis has not been challenged up to now and the exact contribution of the inner scaffold to microtubule triplets cohesion and how the inner scaffold is connected to the microtubule blade is unknown. 5 We recently identified the conserved proteins POC16/WDR90 as proteins localizing to the central core region in both Chlamydomonas reinhardtii and human centrioles (9), which is also covered by the inner scaffold structure. Impairing POC16 or WDR90 functions has been found to affect ciliogenesis, suggesting that POC16/WDR90 stabilizes the microtubule wall, thereby ensuring proper flagellum or cilium assembly (Hamel et al, 2017). Interestingly, POC16 10 has been proposed to be at the inner junction between the A and B microtubules (18) through its homology with FAP20, an axonemal microtubule doublet inner junction protein of Chlamydomonas reinhardtii flagella (19)(20)(21). As the stem connects the A-and B-microtubules interface, these observations suggest that POC16/WDR90 may connect the inner scaffold to the microtubule triplet through this stem structure, thus ensuring centriole cohesion (Fig. 1C). 15 In this study, using a combination of cell biology, biochemistry and Ultrastructure Expansion Microscopy (U-ExM) approaches, we establish that the conserved POC16/WDR90 proteins localize on the centriolar microtubule wall in the central core region of both Chlamydomonas and human cells centrioles. We further demonstrate that WDR90 is a microtubule-binding protein that recruits inner scaffold components and that loss of this protein 20 leads to a slight centriole elongation, impairment of the canonical circular shape of centrioles as well as centriolar fracture in both species. Our results highlight that the essential role of POC16/WDR90 is in recruiting the inner scaffold and maintaining the architecture of centrioles.

POC16/WDR90 is a conserved microtubule wall component of the central core region
To test the hypothesis that POC16/WDR90 is a microtubule triplet component, we analyzed its distribution using U-ExM. We observed that the endogenous POC16 longitudinal 5 fluorescence signal is restricted to the central core region as compared to the tubulin signal depicting total centriolar length (Fig. 1D, F, G). From top viewed centrioles, we measured both POC16 and tubulin maximal intensity signals from the exterior to the interior of the centriole and quantified the shift between x-values ( Fig. 1E, H, shift between POC16 and tubulin Δ = 0 nm).
We concluded that POC16 localizes precisely on the microtubule wall in the central core region 10 of Chlamydomonas centrioles. As a control, we could recapitulate the internal localization along the microtubule wall of POB15, another central core protein (Fig. 1I-M) as previously reported using immunogold-labeling (9). Furthermore, the POC16 human homolog WDR90 localizes, similarly to POC16, on the centriolar microtubule wall, demonstrating the evolutionary conserved localization of POC16/WDR90 on microtubule triplets in the central core region of 15 centrioles ( Fig. 1N-R).
Next, we compared the relative position of WDR90 to previously described inner scaffold components (Fig. 1R-T). We found that while WDR90 precisely localizes to the centriolar microtubule wall (Fig. 1R, x-value for maximal fluorescent signal shift between WDR90 and tubulin: Δ= 2nm), POC1B, FAM161A, POC5 and Centrin-2 signals were shifted 20 towards the centriole lumen in comparison to the tubulin signal, as previously reported (Fig. 1S, Δ= 14 to 30nm) (Le Guennec et al, in press).

POC16/WDR90 is an evolutionary conserved microtubule associated protein that forms a complex with FAM161A and POC5
Proteins of the POC16/WDR90 family consist of an N-terminal DUF667-containing domain (domain of unknown function), homologous to the ciliary inner junction protein FAP20 ( Fig. S1A) (Yanagisawa et al., 2014), which is followed by multiple WD40 repeats that form β- 5 propeller structures ( Fig. 2A and Fig. S1B) (22).
First, we wanted to probe the evolutionary conservation of POC16/WDR90 family members as centriolar proteins. To this end, we raised an antibody against Paramecium tetraurelia POC16 and confirmed its localization at centrioles similarly to what we found in Chlamydomonas reinhardtii and human cells (Fig. S1C) (Hamel et al., 2017). 10 Further driven by its predicted homology to FAP20 and the underlying hypothesis that POC16/WDR90 proteins might be joining A and B microtubules as well as by their precise localization on the microtubule wall, we first set out to understand the structural homology between the predicted structures of POC16-DUF667 domain to the recently published near atomic structure of FAP20 from flagella microtubule doublets (23) (Fig. S2A-C). Strikingly, we 15 observed high similarities between the two structures, suggesting similar biological functions at the inner junction. Moreover, we fitted POC16 model prediction into FAP20 cryo-EM density map and found a good concordance, further hinting for a conserved localization at the level of the microtubule triplet (Fig. S2D).
Prompted by this result, we then tested whether POC16/WDR90 proteins can bind 20 microtubules both in vivo and in vitro. To do so, we overexpressed the N-terminal part of WDR90 and crPOC16 comprising the DUF667 domain (WDR90-N(1-225) and CrPOC16(1-295), respectively, fused to GFP in U2OS cells and found that this region is sufficient to decorate cytoplasmic microtubules in vivo ( Fig. 2B and S3A). We next tested whether overexpressing such a WDR90-N-terminal fragment could stabilize microtubules. To this end, we analyzed the microtubule network in cells overexpressing mCherry-WDR90-N after depolymerizing microtubules through a cold shock treatment ( Fig. S3B-D). We found that while low expressor cells did not maintain a microtubule network, high expressor cells did. This suggests that 5 WDR90-N can stabilize microtubules. In contrast, we observed that full-length WDR90 fused to GFP only anecdotally binds microtubules in vivo, possibly due to an autoinhibited conformation of the full-length protein and/or to interacting partners preventing microtubule binding in cells ( Fig. S3E, Fig. 2H).
Next, we determined whether different POC16/WDR90 N-terminal domains directly bind 10 to microtubules in vitro and whether this function has been conserved in evolution. Bacterially expressed, recombinant POC16/WDR90 DUF667 domains from seven different species were purified and their microtubule interaction ability was assessed using a standard microtubulepelleting assay ( Fig. S1A and Fig. 2C). We found that the POC16/WDR90 DUF667 domain directly binds to microtubules in vitro. This interaction was further confirmed using negative 15 staining electron microscopy, where we could observe recombinant WDR90-N localizing on in vitro polymerized microtubules (Fig. 2E).
We next investigated whether POC16/WDR90 could also interact with free tubulin dimers, considering that closure of the inner junction between the A and B microtubules necessitates two microtubule/tubulin-binding sites as recently reported for FAP20 (23). We 20 observed that all POC16/WDR90 orthologs directly interact with tubulin dimers, generating oligomers that pellet under centrifugation (Fig. 2D). We then tested whether the DUF667 domain could still interact with tubulin once bound to microtubules. We subsequently incubated either 8 WDR90-N or crPOC16(1-295) pre-complexed with microtubules with an excess of free tubulin and analyzed their structural organization by electron microscopy (Fig. 2E, F and Fig. S3F, G).
We observed an additional level of decoration due to the simultaneous complexion of the DUF667 domains with tubulin and microtubules (Fig. 2E, F and Fig. S3F, G). Furthermore, we revealed a 8.5nm periodical organization of tubulin-WDR90-N oligomers on microtubules (Fig.   5 2G), similar to the FAP20 decoration observed on the microtubule doublet structure in cryo-EM (23).
Based on these results, we concluded that POC16/WDR90 is an evolutionary conserved microtubule/tubulin-interacting protein with the capacity to connect microtubules, a functional prerequisite for an inner junction protein that simultaneously interacts with the A and B 10 microtubules.
We next wondered whether POC16/WDR90 could mediate the interaction between microtubule triplets and the underlying inner scaffold. To this end, we took advantage of the strong cellular microtubule decoration of the inner scaffold component FAM161A in human cells when overexpressed ((24), Le Guennec et al. in press), in contrast to GFP-WDR90 (Fig.   15 2H-K). Concomitant overexpression of both proteins leads to a redistribution of GFP-WDR90 on microtubules, suggesting that FAM161A and WDR90 are part of the same molecular complex ( Fig. 2L, M). We also observed that GFP-WDR90 co-localizes with mCherry-POC5 when coexpressed and that both redistribute to small and large condensates within the cell, possibly reflecting an interaction (Fig. 2N-Q). As expected, we observed centriolar co-localization of 20 GFP-WDR90 with the two overexpressed inner scaffold proteins.
Taken together, these data demonstrate that WDR90 is a microtubule/tubulin-binding protein that localizes to the central core region of the centriolar microtubule wall. Moreover, we found that WDR90 interacts with inner scaffold components, suggesting that WDR90 might bridge the centriolar microtubule wall with the underlying inner scaffold structure.

WDR90 is recruited in G2 during centriole core elongation
We next assessed whether WDR90 recruitment at centrioles is correlated with the 5 appearance of inner scaffold proteins during centriole biogenesis. Centrioles duplicate only once per cell cycle during S phase, with the appearance of one procentriole orthogonally to each of the two mother centrioles. Procentrioles then elongate during the following G2 phase of the cell cycle, acquiring the inner scaffold protein POC5 that is critical for the formation of the central and distal parts of the nascent procentriole (Azimzadeh et al., 2009). We followed endogenous 10 WDR90 localization across the cell cycle by analyzing synchronized human RPE1 cells fixed at given time points and stained for either Centrin-2 or HsSAS-6, both early protein marker of duplicating centrioles (10, 15) ( Fig. 3 and Fig. S4A, B). We found that while Centrin-2 and HsSAS-6 are recruited as expected early on during procentriole formation in S phase, WDR90 starts appearing only in early G2 when procentriole elongation starts ( Fig. 3A-F). Signal 15 intensity analysis over the cell cycle further demonstrates that WDR90 appears on procentrioles in early G2 and reaches full incorporation by the end of G2, similarly to the inner scaffold protein POC5 (25) (Fig. 3G, H).
Moreover, we noticed that beside its centriolar distribution, WDR90 localizes also to centriolar satellites, which are macromolecular assemblies of centrosomal proteins scaffolded by 20 the protein PCM1 and involved in centrosomal homeostasis (26) (Fig. S4C-H). We tested whether WDR90 satellite localization depends on the satellite protein PCM1 by depleting PCM1 using siRNA and assessing WDR90 distribution. We found that in absence of PCM1, WDR90 is solely found at centrioles (Fig. S4E-H), demonstrating that WDR90 satellite localization is PCM1-dependent.
Altogether, these data establish that WDR90 is a centriolar and satellite protein that is recruited in G2 of the cell cycle, during procentriole elongation and central core/distal formation, similar to the recruitment of the inner scaffold protein POC5.

WDR90 is important to recruit Centrin-2 and POC5
To better understand the function of WDR90, we analyzed cycling human cells depleted for WDR90 using siRNA and co-labeled WDR90 with either the early centriolar marker Centrin-2 or the protein POC5. As previously shown (9), WDR90 siRNA-treated cells showed 10 significantly reduced WDR90 levels at centrosomes in comparison to control cells (Fig. S5A, C).
Moreover, we observed an asymmetry in signal reduction at centrioles in WDR90-depleted cells, with only one of two Centrin-2 positive centrioles still associated with WDR90 in G1 and early S-phase (69% compared to 10% in controls) and one of four Centrin-2 positive centrioles in S/G2/M cells (77% compared to 0% in controls, Figure S5B). As the four Centrin-2 positive dots 15 indicate duplicated centrioles, this result suggests that the loss of WDR90 does not result from a duplication failure (Fig. S5B). We postulate that the remaining WDR90 signal possibly corresponds to the mother centriole and that the procentriole is depleted from WDR90 ( Fig.   S5E), similarly to what has been observed for the protein POC5 (Azimzadeh et al., 2009). We further conclude that WDR90 is stably incorporated into centrioles, in agreement with its 20 possible structural role.
We also noted that the intensity of the Centrin-2 and POC5 signals were markedly reduced upon WDR90 siRNA treatment ( Fig. S5D-K). Indeed, we found that only 39% of WDR90-depleted cells displayed 2 POC5 dots in G1 (negative for HsSAS-6 signal) in contrast to the 86% of control cells with 2 POC5 dots (Fig. S5H). Moreover, 68% of control cells had 2 to 4 POC5 dots in S/G2/M (associated with 2 HsSAS-6 dots) in contrast to 29% in WDR90-depleted condition (Fig. S5H). The HsSAS-6 signal was not affected in WDR90-depleted cells, confirming that initiation of the centriole duplication process is not impaired under this condition  To ascertain this phenotype, we generated a stable cell line expressing a siRNA-resistant version of WDR90 fused to GFP in its N-terminus. We found that expression of GFP-WDR90RR significantly rescued the loss of Centrin-2 and POC5 at centrioles (Fig. 3I-L).
Taken together, these results indicate that the depletion of WDR90 leads to a decrease in Centrin-2 and POC5 localization at centrioles but does not affect the initiation of centriole 15 duplication nor the recruitment of the distal cap protein CP110.

Chlamydomonas POC16 is crucial to maintain centriole core integrity
To investigate the structural role of POC16/WDR90 proteins on centrioles, we initially turned to Chlamydomonas reinhardtii poc16m504 mutant, which we previously reported to 20 display flagella defects with 80% of mutant cells bearing 0, 1 or impaired flagella ( Fig. S6A-C) (9). We confirmed, by performing immunofluorescence analysis of wild-type and poc16m504 Chlamydomonas cells co-stained for POC16 and tubulin, that the overall POC16 levels at centrioles were reduced (Fig. 4A, B). Moreover, we noticed that 52% of poc16m504 centrioles had only one POC16 dot and 25% had none as compared to the 2 POC16 dots in the wild-type ( Fig. 4C). In contrast, by staining for the cartwheel component Bld12 (11), we found that the fluorescent signal was similar to wild-type in this background, suggesting that the proximal region of the centriole is not affected (Fig. S6D-F). 5 To assess whether the ultrastructure and in particular the central core region of centrioles in poc16m504 cells was defective, we analyzed this mutant using electron microscopy of resinembedded specimens ( Fig. 4D-H). We first noticed that the poc16m504 mutant displayed shorter centrioles with an average length of 370 nm (+/-7 nm) compared to 460 nm (+/-9 nm) in the wild type (Fig. 4D, E). Moreover, we found that the stellate fibers present in the transition zone 10 of wild-type centrioles (Fig. 4D, white star) (27), are ectopically localized to the central core region of poc16m504 mutants in 46% of the cases (Fig. 4D, F-H, red star). This additional localization of stellate fibers has previously been described for the δ-tubulin mutant uni-3, which also displays defective microtubule triplets (28). However, in contrast to uni-3, we noted that microtubule triplets were apparently not affected in the poc16m504 mutant (Fig. 4G). 15 In addition, we observed a loss of the inner scaffold structure in comparison to wild-type centrioles, which normally appears as a circular line in electron micrographs (29) and which is missing in the poc16m504 mutant (Fig. 4G, arrows of insets). This suggests either that the loss of the inner scaffold allows for the ectopic localization of the stellate fibers within the central core region of centrioles, or that the presence of the stellate fibers impairs the structure of the inner 20 scaffold. Furthermore, in one instance we observe a centriole with a broken microtubule wall at the level of the central core region, suggestive of centriole fracture (Fig. 4H, arrow).
To better characterize this phenotype, we turned to U-ExM that allows visualization of centrioles ultrastructure in a more quantitative manner in the context of the whole organism (13).
While the procentrioles looked intact, confirming that proximal assembly initiation is not affected in this mutant, 55% of the poc16m504 mutants displayed defective mature centrioles Altogether, these results demonstrate that the inner scaffold structure of the central core region of poc16m504 mutants is destabilized, highlighting the role of POC16 in maintaining the structural 10 integrity of the microtubule wall in this region of the centriole.

WDR90 depletion leads to a loss of inner scaffold components and to centriole fracture
Based on the above findings, we wondered whether WDR90 depletion might lead to a loss of all inner scaffold components as well as to a centriole architecture destabilization. We 15 tested this hypothesis by analyzing centrioles from WDR90-depleted cells using U-ExM (Fig. 5).
As expected, we observed a strong reduction of WDR90 at centrioles, with a reminiscent asymmetrical signal in one of the two mature centrioles (Fig. 5A, B). Unexpectedly, we found that WDR90-depleted centrioles exhibited a slight tubulin signal increase (502 nm +/-65 Prompted by the revealed connection between WDR90 and some inner scaffold proteins, we next analyzed whether the localization of the four described inner scaffold components POC1B, FAM161A, POC5 and Centrin-2 would be affected in WDR90-depleted cells. We found that the localization of these four proteins in the central core region of centrioles was markedly reduced in WDR90-depleted centrioles (Fig. 5D, E). Instead of covering ~60% of the entire centriolar lumen, we only observed a ~20% remaining belt, positive for inner scaffold components at the proximal extremity of the core region (Fig. 5E, F and Fig. S7A, B), suggesting 5 that their initial recruitment may not be entirely affected. Another possibility would be that the incomplete depletion of WDR90 would allow partial localization of inner scaffold components.
It should also be noted that Centrin-2, which displays a central core and an additional distal tip decoration (Le Guennec et al, in press), was affected specifically in its inner core distribution ( Fig. 6D, arrow, Fig. S7A, B). 10 The discovery of the inner scaffold within the centriole led to the hypothesis that this structure is important for microtubule triplet cohesion and thus overall centriole integrity (Le Guennec et al, in press). Remarkably, we found that upon WDR90 depletion, 10% of cells had their centriolar microtubule wall broken, indicative of fracture and loss of centriole integrity (15 out of 150 centrioles, Fig. 6G, H, Movies S1 and S2). The break occurred mainly above the 15 remaining belt of inner scaffold components, possibly reflecting a weakened microtubule wall in the central and distal region of the centriole (Fig. 6G). We also noticed that the perfect cylindrical shape (defined as roundness) of the centriolar microtubule wall was affected with clear ovoid-shaped or opened centrioles, illustrating that loss of the inner scaffold leads to disturbance of the characteristic centriolar architecture (Fig. 6H, Fig. S7C and Movies S1, S2). 20 Collectively, these data demonstrate that WDR90 is crucial to ensure inner core protein localization within the centriole, to maintain microtubule wall integrity and overall centriole roundness and stability (Fig. 6I).

What maintains centriole barrel cohesion and roundness is a fundamental open question.
Centrioles experience various forces, while performing their functions in cell division or cilia beating, that they must resist (8, 30, 31). Centrioles are microtubule barrel structures held 5 together by the A-C linker at their proximal region and a recently discovered inner scaffold in the central/distal region (Le Guennec et al, in press). The presence of such an extended scaffold covering 70% of the centriolar length has led to the hypothesis that this structure is important for maintaining centriole cohesion (Le Guennec et al, in press). Our work demonstrates that POC16/WDR90 family proteins, which are important for cilia and flagella formation, constitute 10 an evolutionary conserved central core microtubule triplet component that is essential for maintaining the inner centriolar scaffold. Their depletions lead to centriole destabilization by impairment of microtubule triplet cohesion, microtubule triplet breakage and loss of their canonical circular shape, thus revealing the crucial function of the inner scaffold.
We first demonstrate that POC16/WDR90 is a component of the microtubule triplet 15 restricted to the central core region. In addition and based on the sequence and structural similarity to the DUF667 domain of FAP20 that composes the inner junction in flagella, we propose that POC16/WDR90 localizes at the inner junction of the A and B microtubule of the centriolar microtubule triplet. In POC16/WDR90, this DUF667 domain is followed by a WD40 domain sharing a homology with the flagellar inner B microtubule protein FAP52/WDR16 (21) 20 leading us to postulate that the WD40 domains might also be located inside the B microtubule.
However, whether this is the case remains to be addressed in future studies.
Our work further demonstrates that WDR90 is recruited to centrioles in G2 phase of the cell cycle concomitant with centriole elongation and inner central core assembly. We found that WDR90 depletion does not impair centriole duplication nor microtubule wall assembly, as noted by the presence of the proximal marker HsSAS-6 and the distal cap CP110. In stark contrast, WDR90 depletion leads to a strong reduction of inner scaffold components at centrioles, leading 5 to centriole fracture.
Although several examples of centriole cohesion loss have been demonstrated in the past, the molecular mechanisms of centriole disruption are not understood. For instance, Delta-and Epsilon-tubulin null human mutant cells were shown to lack microtubule triplets and have thus unstable centrioles that do not persist to the next cell cycle (32). Remarkably, these centrioles 10 can elongate with a proper recruitment of the cartwheel component HsSAS-6 and the distal marker CP110 but fails to recruit POC5, a result that is similar to our findings with WDR90 depleted cells. As Delta-and Epsilon-tubulin null human mutant cells can solely assemble microtubule singlets (32), we speculate that WDR90 might not be recruited in these centrioles, as the A microtubule and B microtubule inner junction would be missing. As a consequence, the 15 inner scaffold proteins, such as POC5, will not be recruited, leading to the observed futile cycle of centriole formation and disintegration (32). It would therefore be interesting to study the presence of WDR90 in these null mutants as well as the other components of the inner scaffold in the future.
Our work also established that POC16 and WDR90 depletion affects centriole length 20 both in Chlamydomonas reinhardtii and human cells. While we observed shorter centrioles in poc16m504 mutants and the opposite, longer centrioles, in WDR90-depleted cells, these results emphasize the role of POC16/WDR90 in overall centriole length regulation and suggest an unexpected role of the inner scaffold structure in centriole length control. The observed discrepancy between the two phenotypes could arise from species difference or from the fact that provided expertise and help for the work performed in Paramecium tetraurelia ( Figure S1C).

Competing interests:
Authors declare no competing interests. Data and materials availability: 5 All data is available in the main text or the supplementary materials.

Materials and Methods
Figures S1-S7 Tables S1-S7 Movies S1-S2              For further experiments, U2OS:GFP-WDR90 cell line was grown in the medium specified above supplemented with 1µg/mL puromycin.

Ultrastructure Expansion Microscopie (U-ExM)
In cellulo and isolated Chlamydomonas centrioles preparation was previously described Guennec et al. in press). Gels were washed 3x10min in PBS-T prior to secondary antibodies incubation (1:400) for 3hrs at 37°C and 3x10min washes. A second round of expansion was done 3x150mL ddH20 before imaging.
Human U2OS cells were grown on 12mm coverslips and processed as previously described (see Le Guennec et al. in press). Coverslips were incubated for 5 hours in 2X AA/FA at 37°C.
Denaturation was performed for 1h30 at 95° and gels were processed as above. Specifically, staining against WDR90 (1:100) was performed overnight at 37°C.
Imaging was performed on a Leica SP8 microscope using a 63X 1.4NA oil objective with Lightening mode at max resolution to generate deconvolved images. 3D stacks were acquired with 0.12µm z-interval and an x, y pixel size of 35nm. Cloning of the GFP-WDR90 construct used in Fig. 2 was done as follows: Human WDR90 was cloned by nested RT-PCR using total RNAs extracted from human RPE1 cells. Three different fragments corresponding to aa. 1-578, 579-1138, 1139-1748 of WDR90 (based on Genebank sequence NP_660337) were amplified and cloned separately using the pCR Blunt II Topo system (Thermo Fisher Scientific). The full coding sequence was then reconstituted in pCR Blunt II by two successive cloning steps using internal Nru I and Sal I, introduced in the PCR primers and designed in order not to modify WDR90 aa sequence. WDR90 coding sequence was then cloned into a modified pEGFP-C1 vector (Clontech) containing Asc I and Pac I restriction sites.

Immunofluorescence in Human cells
Cells grown on a 15 mm glass coverslips (Menzel Glaser) were pre-extracted for 15sec in PBS

Microtubule binding assay
Taxol-stabilized microtubules (MTs) were assembled in BRB80 buffer (80 mM PIPES-KOH pH6.8, 1 mM MgCl 2 , 1 mM EGTA) from pure bovine brain tubulin at 1 mg/mL (Centro de Investigaciones Biológicas, Madrid, Spain). 50 µL of stabilized MTs were incubated with 20µL of protein at 1 mg/mL for 2 hours at room temperature. After centrifugation on a taxol-glycerol cushion (8'000 rpm, 30°C, 20min) the supernatant and the pellet were analyzed by Coomasie stained SDS-PAGE gels. As a control, MTs alone and each protein alone were processed the same way.

Tubulin binding assay
Tubulin at 10 µM was incubated with a slight molar ratio excess of each protein construct (around 15 µM) in MES buffer for 15 min on ice. After centrifugation at 13'000 x g at 4°C for 20 min, the supernatant and the pellet were analyzed by Coomasie stained SDS-PAGE.

In vitro microtubules decoration and imaging
For simple decoration, Taxol-stabilized microtubules were nucleated as described (3) and subsequently exposed to recombinant WDR90-N(1-225) in a 1:1 molar ratio for 30min at room temperature. Five µL of protein complexes solution were blotted on Lacey carbon grids and stained with Uranyl Acetate (2%) for 3 then 30 seconds.
For double decoration, in vitro microtubules were incubated with WDR90-N(1-225) in a 1:1 molar ratio for 5min at room temperature prior to addition of 2X free tubulin for 30min at room temperature. Negatively stained grids were prepared as above. Similarly, double decorated microtubules were prepared for cryo-fixation.
Electron micrographs were acquired on a Technai 20 electron microscope (FEI Company) and analyzed using ImageJ.

Displacement assay
U2OS cells were transfected in a 6-well plate using jetPRIME reagent (Polyplus-transfection, cat-114-07) following the manufacturer's instructions, with 2.5µg total DNA of the following combinations: GFP-WDR90 alone, mCherry-FAM161A alone, GFP-WDR90 and mCherry-FAM161A, mCherry-POC5 alone and GFP-WDR90 and mCherry-POC5. The medium was changed 4-6 hours after transfection and expression of the fluorescent fusion proteins was allowed for 24 hours. Cells grown on coverslips were pre-extracted for 15sec in PBS supplemented with 0.5% triton prior to iced-cold methanol fixation for 3min. Coverslips were washed once in PBS, mounted and directly imaged in GFP and mCherry channels.
For quantification, cells were classified as "No MT" when no cytoplasmic microtubule pattern was observed, "Few MT" when only a subset of microtubules were decorated or when microtubules were partially decorated along their length, and "All MT" when we observed total coverage of the cytoplasmic microtubule network.

Mitotic shake off
RPE1 p53-cells were seeded in T300 flasks the day before shake off. Flasks were shaken vigorously to detach mitotic cells collected in medium. Cells were pelleted by centrifugation for 5min at 1000 rpm and suspended in 10nM EdU containing medium prior to seeding in 6 well plates onto 15mm coverslips. Cells were fixed at different time points and processed in parallel for immunofluorescence or FACS analysis. Imaging was performed on a Zeiss LSM700 confocal microscope with a PlanApo 63x oil immersion objective (NA 1.4) and optical sections were acquired every 0.33 nm, then projected together using ImageJ.

Electron microscopy on Chlamydomonas reinhardti
For sample preparation, cells were pelleted for 5min at 500g, fixed in 2.5% glutaraldehyde/TAP Roundness was calculated on perfect top views of centrioles by connecting tubulin peaks on ImageJ.

Statistical analysis
The comparison of two groups was performed using a two-sided Student's t-test or its non parametric correspondent, the Mann-Whitney test, if normality was not granted either because not checked (n < 10) or because rejected (D'Agostino and Pearson test). The comparisons of more than two groups were made using one or two ways ANOVAs followed by post-hoc tests (Holm Sidak's) to identify all the significant group differences. N indicates independent biological replicates from distinct sample. Every experiment, except for resin electron microscopy, was performed at least 3 times independently. Data are all represented as scatter or aligned dot plot with centerline as mean, except for percentages quantifications, which are represented as histogram bars. The graphs with error bars indicate 1 SD (+/-) and the significance level is denoted as usual (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All the statistical analyses were performed using Excel or Prism7 (Graphpad version 7.0a, April 2, 2016).

Protein alignement
The protein sequences were aligned using Clustal Omega and the secondary structure elements were predicted using Phyre 2, PONDR and XtalPred-RF.

3D modelisation
The Chlamydomonas POC16 model was prepared using Phyre2 (Kelley 2015 Nature Protocols) and refined against the FAP20 cryo-EM map EMD_20858 using phenix.real_space_refine (Afonine 2018 ActaD). Superposition of the POC16 model excluding flexible loops against FAP20 was done using COOT (Emsley 2010 ActaD) and yielded a rmsd value of 1.6 Angs. The figures were prepared using ChimeraX (Goddard 2018 Protein Science).

PtPOC16 antibody purification
To generate anti-PtPOC16 antibody, a fragment encoding amino acids 2-210 was used for rabbit immunization (Eurogentec). Antibodies were subsequently affinity-purified over a column of              Table S4. Inner scaffold proteins length coverage.