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.
