A hierarchical pathway for assembly of the distal appendages that organize primary cilia
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eLife assessment
This important and detailed study presents the most comprehensive view of the functional organization and requirements for a mother centriole's distal appendage in primary cilia assembly published to date. Crispr-knockouts and super-resolution microscopy analysis of the distal appendage proteins provides convincing evidence to support the claims of the authors. This work will be of high value to cell biologists and biophysicists working on the structure and function of the centrosome as well as human geneticists exploring ciliary pathology.
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Abstract
Distal appendages are ninefold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for the formation of the primary cilium, by regulating at least four critical steps: preciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here, we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in the RAB34 + vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
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eLife assessment
This important and detailed study presents the most comprehensive view of the functional organization and requirements for a mother centriole's distal appendage in primary cilia assembly published to date. Crispr-knockouts and super-resolution microscopy analysis of the distal appendage proteins provides convincing evidence to support the claims of the authors. This work will be of high value to cell biologists and biophysicists working on the structure and function of the centrosome as well as human geneticists exploring ciliary pathology.
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Reviewer #1 (Public Review):
In this work, Kanie and colleagues explored the composition, structure, and assembly hierarchy of distal appendage proteins. The microscopy was well executed and appropriately quantified. Importantly, the quality of individual antibodies was documented with a discussion of how this might complicate results. The hierarchy of assembly was established by careful quantification of assembly in an extensive set of knockout cell lines. This work will be of interest to cell biologists exploring organelle assembly as well as human geneticists trying to understand the clinical implications of mutations.
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Reviewer #2 (Public Review):
Kanie et all have carried out a tour-de-force effort to further understand the hierarchy and function of centriole distal appendages in ciliogenesis. They made a thorough effort to understand the localization of all the known distal appendage proteins. To examine the distal appendage hierarchy, they used an automated analysis of centrosomal localization. It is not clear how this was quantified and pictures are not shown. They used CEP170, a marker for subdistal appendages, to define a mask around centrioles. It is not clear how the experiment was analyzed and normalized. The techniques used in this study cannot be compared with those commonly used in the field which normally include storm and other super-resolution techniques (which are less prone to artifacts) and correlated electron microscopy. Thus, it is …
Reviewer #2 (Public Review):
Kanie et all have carried out a tour-de-force effort to further understand the hierarchy and function of centriole distal appendages in ciliogenesis. They made a thorough effort to understand the localization of all the known distal appendage proteins. To examine the distal appendage hierarchy, they used an automated analysis of centrosomal localization. It is not clear how this was quantified and pictures are not shown. They used CEP170, a marker for subdistal appendages, to define a mask around centrioles. It is not clear how the experiment was analyzed and normalized. The techniques used in this study cannot be compared with those commonly used in the field which normally include storm and other super-resolution techniques (which are less prone to artifacts) and correlated electron microscopy. Thus, it is not possible to make a head-to-head comparison. The lack of rescue experiments further weakens the conclusions of this paper.
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Reviewer #3 (Public Review):
Distal appendages are multiprotein complexes that are only present on the mother centriole as a 9-fold symmetric structure that functions in ciliogenesis. How distal appendage proteins are organized and assembled still remains poorly understood. In this manuscript, Kanie et al. comprehensively analyzed the localizations of known and newly described distal appendage proteins using super-resolution microscopy. They investigated mechanisms associated with distal appendage assembly and their roles in the early stages of ciliogenesis in CRISPR-Cas9 knockout cells, which enabled a clearer investigation of these structures compared to previous RNAi depletion studies. These studies confirm previous findings for distal appendage protein ciliogenesis function and demonstrate the CEP83-SCLT1-CEP164-TTBK2 module is …
Reviewer #3 (Public Review):
Distal appendages are multiprotein complexes that are only present on the mother centriole as a 9-fold symmetric structure that functions in ciliogenesis. How distal appendage proteins are organized and assembled still remains poorly understood. In this manuscript, Kanie et al. comprehensively analyzed the localizations of known and newly described distal appendage proteins using super-resolution microscopy. They investigated mechanisms associated with distal appendage assembly and their roles in the early stages of ciliogenesis in CRISPR-Cas9 knockout cells, which enabled a clearer investigation of these structures compared to previous RNAi depletion studies. These studies confirm previous findings for distal appendage protein ciliogenesis function and demonstrate the CEP83-SCLT1-CEP164-TTBK2 module is critical for both distal appendage assembly and the initiation of ciliogenesis. Notably, they find that CEP89 is dispensable for distal appendage assembly, but is needed for the recruitment of RAB34-positive ciliary vesicles to the mother centriole for ciliogenesis. Finally, this work introduces the application of single-molecule 3D super-resolution microscopy as a tool for interrogating the relationship between membranes and distal appendages. Overall this work extends our fundamental understanding of distal appendage structure/function in ciliogenesis.
An interesting observation from this work is that CEP83 is found localized both at the innermost region and the outermost region of the distal appendages when detected by antibodies that recognize a different epitope of CEP83 (Figure 1A), suggesting a helical structure that could serve as a platform for distal appendage assembly. A previous study using STORM imaging also showed that another distal appendage protein CEP164 occupies a wider region of the distal appendages when using an antibody recognizing the N-terminal residues of Cep164 (M Bowler et al. 2019). Together these studies show the importance of evaluating the structure of distal appendage proteins and the challenges of using antibody detection to reveal distal appendage hierarchy.
This work also highlights the potential differences in functional conclusions that can be drawn when comparing RNAi and CRISPR knockout depletion approaches. The latter which expectedly can lead to a more precise functional analysis of these small distal appendage structures, albeit with the potential for knockout cells to display compensatory regulation. Although not directly addressed in the text, the authors find that RPE-1 MYO5A knockout cells could ciliate which differs from a report by Wu et al. (2018). Furthermore, in the case of RAB34 knockout cells, the authors find CP110 removal from the mother centriole, while in previously published RAB34 KO studies this was not observed. In the case of the. RAB34 data a plausible explanation for the results given by the authors is that different assay conditions were used as was noted by the authors.
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