Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis

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Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1 −/− mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, as well as variable expressivity of other ciliopathy features including cystic kidneys. Pcm1 −/− multiciliated ependymal cells and PCM1 −/− retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1 −/− RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromized early ciliogenesis. We show these molecular cascades are maintained in vivo , and we suggest that the cellular threshold to trigger ciliogenesis varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, inducing the departure of CP110 and CEP97 to initiate ciliogenesis.

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  1. eLife assessment

    This manuscript will be of interest to centrosome and cilia cell biologists. It evaluates the in vivo and in vitro role of PCM1, and by extension, centriole satellites in ciliogenesis. The major strength of this study is the detailed characterisation of Pcm1-/- mutant mice, which reveals a role for PCM1 in biogenesis of specific types of cilia, such as motile cilia on ependymal cells. The claims are generally well supported by the data, but the mechanistic basis for the cell-type specific requirement for PCM1 in ciliogenesis remains to be fully clarified.

  2. Reviewer #1 (Public Review):

    Overall, this is a well-written and well-executed study that addresses the in vivo and in vitro functions of PCM1, a key component and regulator of centriolar satellites previously implicated in centrosome and ciliary biogenesis and function. The authors first generated mice lacking PCM1 and through careful phenotypic characterization, they demonstrate a tissue- and cell-type specific role for PCM1 in ciliogenesis in vivo, including a role in ciliogenesis in multiciliated ependymal cells but not airway epithelial cells. Consistently, Pcm1-/- mice were demonstrated to display perinatal lethality and select ciliopathy phenotypes such as hydrocephalus. Using high resolution immunofluorescence imaging and electron microscopy, the authors provide evidence that PCM1 promotes early stages of ciliogenesis, specifically removal of the CP110 capping protein from the distal end of (mother) centrioles. They go on to investigate this in more detail using cultured mouse embryonic fibroblasts (MEFs) and RPE1 cells lacking PCM1. Intriguingly, they find that PCM1 is required for ciliogenesis in RPE1 cells but not in MEFs, even though CP110 levels at the mother centriole are elevated in both cell types when PCM1 is depleted. The authors propose that PCM1 promotes ciliogenesis in select cell types by "wicking away" CP110 from the mother centriole at the onset of ciliogenesis, and provide some additional evidence (e.g. co-immunoprecipitation and live cell imaging analysis) to support this model. The manuscript represents a significant amount of high-quality work, and most of the claims are justified by the data. However, the manuscript would be strengthened by addressing the following points:

    1. Based on their results, including the observation that CP110 and CEP97 centrosomal levels are increased in PCM1-/- cells, the authors propose that PCM1 promotes ciliogenesis by mediating removal/"wicking away" of CEP97 and CP110 from the mother centriole at the onset of ciliogenesis (Figure 9). Although this model could explain the authors' observations, alternative models should be considered. For example, an equally plausible mechanism is that PCM1 promotes centrosome/mother centriole recruitment of an E3 ligase that (negatively) regulates CP110. Indeed, the authors show in Fig. 4 that MEFs lacking PCM1 display reduced centrosome levels of the E3 ligase MIB1. This raises the question if MIB1 is also reduced at the centrosome in RPE1 cells lacking PMC1, and whether other E3 ligases known to promote CP110 removal/degradation are also decreased at the mother centriole of PCM1-/- cells. This includes EDD1/UBR5, which was previously implicated in CP110 removal from the mother centriole of RPE1 cells (Hossain et al. 2017; Goncalves et al., 2021), and which may be linked to centriolar satellites via CSPP-L (Shearer et al. 2018). Other relevant CP110 regulators to check include LUBAC and PRPF8, which may act in parallel with UBR5 to mediate CP110 removal from the mother centriole (Shen et al., 2021). The authors should at least discuss the possibility that PCM1 might affect the centrosome localization of these known CP110 regulators, if not address it experimentally. Finally, to confirm that reduced ciliogenesis in PCM1-/- cells is indeed due to increased levels of CP110 at the mother centriole, the authors could (partially) deplete CP110 from PCM1-/- RPE1 cells to investigate if this rescues the ciliogenesis phenotype of the mutant cells, e.g. as done recently by Goncalves et al. for CEP78-/- cells.

    2. Figure 5 supplement 1A, B; lines 232-242; 430-439: the authors report that Talpid3 localization at the centrosome in PCM1 mutant cells is equivalent to that of controls. However, when looking at Figure 5 supplement 1B it seems that Talpid3 levels at the centrosome may be slightly elevated at the centrosome in the mutant cells although the change is not statistically significant. I suggest the authors specifically state this in the text, given that previous work by Wang et al. (2016) indicated that PCM1 does have an effect on centrosomal Talpid3 levels. A change in Talpid3 centrosomal level could be very small, requiring larger sample size to reach statistical significance, and different experimental conditions (fixation, permeabilization, antibody dilution etc.) could also influence the results and explain the discrepancy between the authors' observations and those of Wang et al. (2016).

    3. Figure 5 supplement 1C, D: given that the authors´ results are in contrast to those of Wang et al. (2016), they should measure the actual fluorescence intensity of Centrobin at the mother centriole rather than just counting number of Centrobin foci, as they have done for e.g. CP110.

    4. The observed requirement for PCM1 in promoting ciliogenesis in RPE1 cells and not MEFs is puzzling, given that the authors still observed increased CP110 levels at the mother centriole in the Pcm1-/- MEFs. In the discussion (lines 464-473), the authors suggest that CP110 removal from the mother centriole may be more important for ciliogenesis in cells using the "extracellular" pathway of ciliogenesis compared to cells forming cilia via the "intracellular" pathway. However, mouse fibroblasts and RPE1 cells were shown to both form cilia via the "intracellular" pathway (e.g. see Ganga et al. 2021) thus this explanation seems insufficient to explain the observed differences between RPE1 cells and MEFs lacking PCM1. It would be helpful if the authors could comment on this.

  3. Reviewer #2 (Public Review):

    Centriole satellites are membraneless granules that surround the centrosome. Some proteins localize exclusively to centriole satellites, while others are present at both satellites and the centrosome. The function of centriole satellites is somewhat mysterious, but they have been implicated in ciliogenesis, autophagy, and mediating cellular stress responses. PCM1 is a core scaffolding protein essential for the assembly of centriole satellite and many studies have examined the role of centriole satellites in PCM1 depleted cell lines. However, the role of centrosome satellites at the organismal level has not been examined, and it remains unclear if the effects observed in cell lines are present across diverse cell types found in vivo.

    In this manuscript, Hall et al., examine the effect of PCM1 knockout in mice. Surprisingly, Pcm1-/- mice are viable but exhibit increased perinatal lethality. Mice lacking PCM1 also have many interesting phenotypes, including dwarfism, male infertility, hydrocephaly, and hydronephrosis. These phenotypes are consistent with defects occurring in both primary and motile cilia. The ciliogenesis deficits in Pcm1-/- mice must be relatively mild, as severe defects in cilia assembly result in embryonic lethality. Thus, centriole satellites are not required for cilia assembly in most cell types. Consistently, the authors show that Pcm1-/- MEFs have no apparent phenotypes in cilia assembly. Pcm1-/- multiciliated ependymal cells have a delay in ciliogenesis and defects in cilia beating. Surprisingly, given the array of interesting phenotypes to examine in the mice, the authors switch to characterizing PCM1-/- RPE1 cells. Unlike primary MEFs, PCM1-/- RPE1 cells show reduced ciliogenesis. The authors show that in RPE1 cells, PCM1 promotes the recruitment of preciliary vesicles to the mother centriole and helps remove the CP110/CEP97 centriole capping complex. The authors propose that CP110 and CEP97 are transported away from mother centrioles by centriole satellites. However, Pcm1-/- MEFs also fail to remove CP110 from the mother centriole, despite having no defects in ciliogenesis. Thus, CP110 removal is not universally required for ciliogenesis.

    This is an excellent manuscript that thoroughly examines the role of PCM1 both in vivo and in vitro. In my view, the major strength of this work lies in the examination of the impact of PCM1 loss in vivo. As a result, I was a little surprised the authors didn't focus more attention on the interesting phenotypes that arise in the Pcm1-/- mouse. The switch over to RPE1 cells is abrupt. Moreover, the phenotypes observed in this cell line are likely not occurring in most cell types in vivo, or else the expected organismal phenotypes would probably be even more severe. That notwithstanding, the RPE1 cell biology is rigorous, high quality, and the conclusions are well-justified. Overall, the work will be of broad interest to the centrosome/cilia community.

  4. Reviewer #3 (Public Review):

    The manuscript by Hall et al., first describes the global and multi-organs phenotype of PCM1-/- mice and then focus on the role of PCM1 in the process of basal body production/maturation in multiciliated cells and finally on the role of PCM1 in primary ciliogenesis on RPE1 and MEF cells. In multiciliated cells, they show that the absence of PCM1 delays basal body formation and that PCM1 is required for the formation of structurally normal cilia, and for their consecutive coordinated beating. As regards to primary ciliogenesis, they show that PCM1 is required to allow efficient ciliation in RPE1 but not in MEF cells. Notably, they reveal defects in the formation of the preciliary vesicle in RPE1 cells and propose that PCM1 restricts CP110 and Cep97 at the centrosomal centriole in both MEFs and RPE1.

    The study presented here represents a lot of nice work and highlights original data. However, in its present form, the study, which covers many aspects of the PCM1 mouse phenotype, is too fragmentary and does not allow to have, either a global view of the diversity of the phenotypes, or give mechanistic insight into one of the phenotypes. I would recommend the authors make two different papers on multiciliation and primary ciliogenesis, or try to test whether both type of ciliation are affected in a common way by the absence of PCM1. For instance, the title focuses only on the last part of the paper. Below are my comments.

    Global phenotype

    The authors convincingly show that the absence of PCM1 during development leads to perinatal lethality, hydrocephalus, cerebellar hypoplasia, oligospermia and cystic kidneys.

    Role of PCM1 in multiciliation

    The authors convincingly show that the absence of PCM1delays centriole amplification and therefore multiciliation which has never been shown before to my knowledge.

    They also propose that the basal bodies produced in absence of PCM1 show a problem of rotational polarity. This is not fully supported by the data. To confirm this observation, the authors should look at later time points as P3 is very early and the rotational polarity is progressively established after BB docking and the beginning of cilia beating. Also many more cells should be analyzed. Since this is a lot of work by EM, one should consider doing it by immunostainings as done in some other papers. Same comment for the absence of ciliary pocket in PCM1 KO. P3 is too early and since some cilia do not show a clear ciliary pocket, one should look in a sufficient number of EM sections.

    The defect in translational polarity is interesting and has never been described before. This phenotype is analyzed at P5 and should also be confirmed at later time point since the delay in multiciliation in the PCM1 KO may affect the number of cells with a terminal differentiated state and therefore bias the result. In fact, migration of BB is the last event occurring during multiciliation.

    The phenotype of cilia beating uncoordination is convincing and confirms what has been also described by Zhao et al., in 2021. The authors seem to propose a causality link between this phenotype and the proteomic study between WT and PCM1 KO in another MCC cell type: mTEC at ALID7. Since the difference resolve in these mTEC at ALID21, do the authors think the delay in cilia motility protein expression could explain a consecutive permanent problem of cilia beating coordination seen at later stages ? Also it is difficult to link these results with motility since motility is assessed in ependymal cilia and proteomic study in mTEC. One would like to know if motility is also affected in mTEC. And to use the proteomic study to propose an additional explanation of the one proposed by Zhao et al. showing that PCM1 depletion also deregulates the centriolar and ciliary targeting of satellites client proteins, a process that could affect cilia beating. The structural defects of cilia seen by the authors and by Zhao et al., are also one important piece of explanation.

    In vitro, MCC in PCM1 KO seem to display less cilia. Is this true in vivo in the brain? Since it is not obvious in vivo in the trachea, it would be nice to just address qualitatively whether this is the case in vivo in the brain. Also, are the number of BB affected ? Zhao et al., counted the number of BB in PCM1 siRNA treated cells and show no difference. If one would address how PCM1 affect the number of cilia, this is important to know whether less centrioles are produced or whether they fail to dock correctly at the plasma membrane. Since formation of the preciliary vesicle is affected in in RPE1 cells, it is tempting to speculate that a similar defect could arise in MCC and affect motile ciliogenesis. If the « number of cilia » phenotype is not true in vivo, one should also consider a culture artefact.

    Altogether, the phenotype on multiciliation needs to be strengthened to confirm the original results and to be put into the context of the previous study done in vitro (Zhao et al., 2021).

    Role of PCM1 in primary ciliogenesis

    Knockdown of different satellite components have been shown to affect primary ciliogenesis (Conkar et al., 2017; Kim et al., 2008; Klinger et al., 2014; Lee and Stearns, 2013; Mikule et al., 2007; Staples et al., 2014, Kurtulmus et al., 2016). More particularly cell type dependent variability of PCM1 suppression on ciliogenesis has previously been described (Odabasi et al., 2019; Wang et al., 2016). It appears necessary to clarify in one paragraph in the introduction this bibliographic context and to put forward the unresolved questions the present study proposes to address as well as the new insights it provides on the question.

    First, the two main phenotypes described here, e.g. defect in ciliary vesicle formation and defect in CP110 and Cep97 removal from the mother centrioles, are very similar to the phenotype described in WDR8 knock down (Kurtulmus et al., 2016). Is there any reason why the authors did not cite this study ? If not, and since WDR8 and PCM1 are interacting partners and are interdependent for their localization, I would suggest assessing whether PCM1 acts upstream or downstream of the WDR8-Cep135 axis. For example, I would suggest testing if WDR8 expression in PCM1 KO rescue the ciliary vesicle and CPP110/Cep97 phenotypes.

    The phenotype of preciliary vesicle formation defect in PCM1 KO is convincing in RPE1 cells. I would suggest to reproduce the MyoVa staining in MEFs to detect whether, in cells forming cilia in the absence of PCM1, the ciliary vesicles are forming properly. It may be a good control and also give insight into how PCM1 affects differentially ciliogenesis in different cell types. Also, the extent of TEM analysis is difficult to assess (I did not find the « n »). TEM is important to confirm the phenotype since MyoVa is an actin-based molecular motor that plays several roles in the final stages of secretory pathways.

    Then the authors propose that PCM1 promotes the transition zone formation and IFT recruitment. The data presented here support that PCM1 promotes TZ formation. However, since PCM1 absence compromises preciliary vesicle formation, one could conclude that TZ alterations are just a consequence of this defect. This needs to be discussed. Regarding recruitment of IFT and TZ components, the data presented here do not support that PCM1 promotes TZ components and IFT recruitment. In fact, TZ components are not absent in non ciliated RPE1 KO cells, just decreased, and they are present at normal levels in ciliated MEFs in absence of PCM1.

    The authors propose that centriolar satellites restrict CP110 and Cep97 levels at centrioles, which promotes ciliogenesis. Defect in the removal of CP110 and Cep97 from the mother centriole are very convincing in PCM1 KO both in RPE1 and MEFs. However, the causality link between this mother centriole maturation and ciliogenesis still needs to be tested since MEFs are able to ciliate in the absence of PCM1 and in the presence of CP110. Knock down of CP110 in PCM1 KO would be needed to accurately test this hypothesis. For example, in absence of WDR8, CP110 knock down does not rescue ciliogenesis defect probably because of the upstream defect of preciliary vesicle docking (Kurtulmus et al., 2016). This could be the case also here.

    Finally, the authors propose that PCM1 satellites transport CP110 and Cep97 away from the centriole. They nicely show that CP110 colocalize with satellites. By IP, they suggest that PCM1 and CP110 coIP which need to be further confirmed by another IP since the signal is really weak. They show that CP110 does not colocalize anymore to the satellites as soon as 1h after serum deprivation. If satellites were involved in removing CP110 from the mother centriole for ciliation, I would expect to see an increase in CP110 localization to the satellites, and not a decrease at this time point. The authors also measure an increase of CP110 and Cep97 at the centrioles in PCM1 KO, which would go in line with their hypothesis. However, this phenotype is the opposite of what was shown in Quarantotti 2019 in the same cell type where they show that upon PCM1 loss, CP110 was decreased at the centrosome. Together with the fact that the overaccumulation of CP110 and Cep97 illustrated by IF and measured is weak, more data are needed to support this phenotype. Altogether, the hypothesis that satellites are transporting CP110 and Cep97 away from the centrioles needs more data to be convincing.