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  1. Evaluation Summary:

    This manuscript is of interest to developmental and cell biologists exploring cilia dynamics and ciliopathies. The authors address the molecular mechanisms by which INPP5E, a phosphoinositide phosphatase essential for regulating cilia function, is targeted to the primary cilium of cultured mammalian cells. Using immunoprecipitation, ciliary localization, phosphatase activity assays in combination with structural modelling, the authors identify four motifs important for ciliary localization of INPP5E, and uncover several novel and important interactions with other ciliary proteins providing a likely mechanism for ciliary targeting. The claims are generally well supported by the data, but some additional data acquisition and analysis are required to fully support the authors' conclusions and provide a conceptual advance in the field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

  2. Reviewer #1 (Public Review):

    Overall this is a well-written manuscript that dissects the complex molecular mechanisms by which INPP5E is targeted to the primary cilium. INPP5E is a well-studied ciliary phosphoinositide phosphatase that is mutated in several ciliopathies such as Joubert Syndrome. Despite intense investigation, the precise mechanisms by which INPP5E is targeted to the primary cilium were so far unclear. By combining cell-based analysis of various INPP5E mutant constructs with biochemical assays and structure prediction, the authors convincingly show that ciliary targeting of INPP5E requires its folded catalytic domain and is controlled by four ciliary localization signals, two of which were not described previously. The experiments are generally well carried out and the claims are justified by the data. The manuscript would be strengthened by a quantitative analysis of some of the key data. Specifically:

    1. For all IFM figures comparing cilia localization of various EGFP-INPP5E mutants, the authors should provide information about how many times experiments were repeated, and how many cells/cilia were analyzed in each case. In Figures 2d and 3c, the authors do provide a quantitative analysis of cilia localization of selected mutants, but the number of cells examined is not stated in the legend, nor in the Methods section. Furthermore, it is unclear whether relative expression levels and/or stability of the different INPP5E mutant constructs in RPE1 cells are similar. This is important to clarify as it could influence their cilia localization.

    2. Figure 5g and line 368: based on the IP in Figure 5g the authors conclude that co-IP of ARL13B with INPP5E is "somewhat reduced" with deltaCLS2 and deltaCLS3. However, judging from Figure 5g it also seems that the IP of the latter two is less efficient than that of the other INPP5E mutants, which could explain the apparently reduced co-IP of ARL13B with deltaCLS2 and deltaCLS3. It would be desirable if authors could somehow quantify the IP results, e.g. by measuring band intensities and quantifying ARL13B levels in each IP pellet relative to the amount of EGFP-INPP5E levels in the same IP pellets. The same applies to Figures 6a, 6d and 7a where the amount of EGFP-INPP5E IPed seems to vary quite a bit between mutants. In particular, the band intensities for the deltaCLS2 and deltaCLS3 mutants seem quite low in the EGFP blots of these figures compared to the other mutants.

    3. Figure 6e: the specific bands corresponding to WT(1-1460) and NT(WW/mut) CEP164-EGFP are not clearly apparent in this figure. The former seems largely to be absent in the EGFP blot (?) and the latter shows several bands. It would be important to clarify. this point

  3. Reviewer #2 (Public Review):

    Cilleros-Rodriguez & Martin-Morales et al. address questions surrounding the recruitment of the phosphoinositide 5-phosphatase INPP5E to primary cilia. Utilizing structural analyses, site-directed mutagenesis, co-localization and binding studies, the authors show that ciliary targeting of INPP5E requires the folded INPP5E catalytic domain in addition to four ciliary localization signals (CLSs), two of which were newly identified here. They further examine the roles these INPP5E CLSs play in cilia localization and INPP5E/cilia protein interactions, revealing a level of co-operativity and redundancy among the four signals. The ciliary phosphoinositide phosphatase INPP5E hydrolyzes ciliary transition zone phosphoinositides and is mutated in ciliopathies such as Joubert syndrome (JBTS). The authors additionally show that some INPP5E gene mutations associated with JBTS prevent INPP5E ciliary recruitment. Collectively, these data reveal an interesting degree of complexity in targeting INPP5E to cilia that requires a high degree of regulation.


    The authors use an extensive and systematic approach to identify and test a substantial number of INPP5E mutants to define novel CLSs and reveal important residues required for INPP5E ciliary localization.

    The authors use a methodical approach using INPP5E CLS deletion mutants to examine INPP5E interaction with ciliary protein binding effectors to delineate possible molecular mechanisms governing INPP5E ciliary localization.

    Together, these data reveal valuable insight into the complex regulation of INPP5E recruitment to cilia, which will be important for developmental and cell biologists.


    The data presented are mostly sound with appropriate controls; however, further data acquisition and analysis are required to support the conclusions made by the authors.

    INPP5E plays an essential role in cilia dynamics, regulating processes that include phosphoinositide and protein composition, cilia-dependent signaling pathways, ciliogenesis and cilia stability. Functional examination of the INPP5E CLSs would provide stronger evidence of their biological significance and provide a conceptual advance in the cilia field essential to development and disease. This is particularly important considering that INPP5E mutations associated with JBTS have variable effects on INPP5E cilia localization.

    Examination of INPP5E recruitment to cilia by immunofluorescence requires expansion considering the publication of a recent article in the field (doi:10.3389/fcell.2021.634649) that explores possible preparation artifacts affecting INPP5E cilia localization.

    Extended mechanistic examination is also required to support the authors' conclusions that they have identified the molecular mechanisms governing INPP5E ciliary targeting by each CLS. Immunoprecipitation studies alone are insufficient to support these claims. Alterations of immunoprecipitation are not necessarily indicative of alterations of binding without validation of protein folding and direct binding studies. Additional functional studies would increase confidence that these ciliary protein interactions are important in this context.

    Key questions that would provide a conceptual advance in the cilia field have not been addressed in this manuscript and would provide further evidence to support claims regarding the biological significance of the INPP5E CLSs. The manuscript would be improved with additional functional studies, for example, examination of the role the CLSs play in cilia protein or phosphoinositide composition, cilia-dependent signaling pathways, cilia assembly or disassembly.

    Indirect immunofluorescence analysis in fixed cells has been used extensively here to demonstrate the identification and characterization of cilia localization signals in INPP5E. A recent publication in this field has reported possible artifactual INPP5E localization issues dependent upon fixation conditions (doi:10.3389/fcell.2021.634649). Could the authors assess their data in the context of this new report? Could the authors repeat a part of their principal localization experiments with alternative fixation conditions to address any possible artifactual issues with INPP5E ciliary localization? This would increase confidence in the co-localization studies. At a minimum, this needs to be addressed in the manuscript discussion.

    The claim that the authors have elucidated the mechanisms of INPP5E ciliary targeting requires further evidence that has not been addressed in the immunoprecipitation studies described within the manuscript. Are these protein interactions essential for INPP5E cilia localization? What are the functional consequences of these interactions? Are these direct or indirect interactions? The authors use the terminology "decreased binding" when actually referring to "decreased immunoprecipitation". This language needs to be clarified in the text as a reduction in co-immunoprecipitation could also be due to protein misfolding in the mutants. The authors need to validate that the mutant proteins are correctly folding and confirm immunoprecipitation data with direct binding studies before a claim of "decreased binding" can be substantiated.

    In conclusion, the questions asked in this manuscript could provide an impactful advance in the cilia field, but the data collected to date do not fully support the authors' conclusions.

  4. Reviewer #3 (Public Review):

    This is an excellent manuscript that provides a lot of new and important information regarding INPP5E and the mechanisms of ciliary targeting. The authors show that ciliary targeting of INPP5E is not a simple 1-motif mechanism and identify 4 motifs (CLS1-4) located on the same side of the protein structure that are all required for different aspects of ciliary targeting through interactions with numerous different ciliary players. The combination of structural modeling with interaction studies, ciliary localization and phosphatase assays to assess the proper folding of the catalytic domain allows for a very thorough investigation of each of the four CLS motifs. This is combined with a large number of carefully designed mutations to probe the function of different CLS motifs to provide a very complete study.

    The work is well carried out and the manuscript is scholarly written with a high degree of clarity. I am sure it will have a high impact on the ciliary community.