The microtubule-associated proteins CKAP2 and its paralog CKAP2-Like control ciliogenesis in human cells
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eLife Assessment
In this valuable study, the authors build on previous work demonstrating that the microtubule-associated protein CKAP2 functions at the mitotic spindle to regulate microtubule dynamics for proper chromosome segregation. Here, the authors show that CKAP2 and its paralog CKAP2L also function at the cilium to regulate cilium formation and Hedgehog signaling. The writing is clear, the experiments and data are generally convincing, and the proper data analysis is carried out; however, the evidence supporting the proposal that CKAP2 and CKAP2L compensate for each other is incomplete. Overall, the findings advance the field in our knowledge of microtubule-associated proteins with roles in both the mitotic spindle and the cilium.
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Abstract
Ciliogenesis is an evolutionarily conserved process that leads to the assembly of cilia. This process relies on microtubule-associated proteins (MAPs) to regulate axonemal microtubule dynamics. Misregulation of MAPs often leads to changes in ciliary homeostasis, contributing to numerous ciliopathies. Although CKAP2 and CKAP2-Like are best known for regulating microtubule dynamics during cell division, they are also components of ciliary organelles. Notably, CKAP2 overexpression promotes chromosomal instability and cancer, whereas CKAP2-Like deficiency causes Filippi syndrome, a developmental disorder with clinical features overlapping those of ciliopathies. How both MAPs operate at primary cilia remains poorly understood. Here, we identify both MAPs as axonemal components of motile and primary cilia in human cells. We find that CKAP2-positive cilia are linked to cell cycle progression and that the conserved intrinsically disordered microtubule-binding domain of CKAP2, which is essential for microtubule polymerization and stabilization, is required for its ciliary localization. Unexpectedly, CRISPR-mediated loss of either paralog results in elongated cilia without affecting ciliogenesis, accompanied by increased ciliary enrichment of the remaining paralog and impaired Gli2 accumulation at the ciliary tip. In contrast, simultaneous deletion of both MAPs suppresses ciliogenesis and has minimal effect on the ciliary length as compared to wild-type levels. These findings uncover that CKAP2 and CKAP2-Like are axonemal MAPs that contribute to ciliogenesis, partially compensating for each other in human cells.
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eLife Assessment
In this valuable study, the authors build on previous work demonstrating that the microtubule-associated protein CKAP2 functions at the mitotic spindle to regulate microtubule dynamics for proper chromosome segregation. Here, the authors show that CKAP2 and its paralog CKAP2L also function at the cilium to regulate cilium formation and Hedgehog signaling. The writing is clear, the experiments and data are generally convincing, and the proper data analysis is carried out; however, the evidence supporting the proposal that CKAP2 and CKAP2L compensate for each other is incomplete. Overall, the findings advance the field in our knowledge of microtubule-associated proteins with roles in both the mitotic spindle and the cilium.
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Reviewer #1 (Public review):
Summary:
The authors examine the roles of CKAP2 and CKAP2-Like in ciliogenesis. They assess the localization of both proteins in primary and motile cilia of the human male reproductive tract and use hTERT-RPE1 cells to investigate CKAP2 function. They report that CKAP2 localization is associated with cell-cycle progression and that its intrinsically disordered region is required for ciliary localization. Loss of either CKAP2 or CKAP2-Like results in elongated cilia, whereas loss of both proteins reduces ciliogenesis and restores ciliary length to approximately wild-type levels. Based on these findings, the authors propose compensatory interactions between the two paralogs.
Strengths:
The study combines tissue localization, knockout cell lines, quantitative analysis of ciliogenesis and ciliary length, and …
Reviewer #1 (Public review):
Summary:
The authors examine the roles of CKAP2 and CKAP2-Like in ciliogenesis. They assess the localization of both proteins in primary and motile cilia of the human male reproductive tract and use hTERT-RPE1 cells to investigate CKAP2 function. They report that CKAP2 localization is associated with cell-cycle progression and that its intrinsically disordered region is required for ciliary localization. Loss of either CKAP2 or CKAP2-Like results in elongated cilia, whereas loss of both proteins reduces ciliogenesis and restores ciliary length to approximately wild-type levels. Based on these findings, the authors propose compensatory interactions between the two paralogs.
Strengths:
The study combines tissue localization, knockout cell lines, quantitative analysis of ciliogenesis and ciliary length, and live imaging of CKAP2 dynamics. The findings extend previous work implicating CKAP2 and CKAP2-Like in microtubule regulation and support roles for both proteins in ciliogenesis and cell-cycle-associated ciliary dynamics. Overall, the study is convincing and should be of interest to readers.
Weaknesses:
The proposed compensation model requires further support. Because cilia are longer in the single-knockout cells, the ciliary fluorescence signals of CKAP2 and CKAP2-Like should be normalized to ciliary length. The same issue applies to the acetylated-tubulin fluorescence measurements. Furthermore, the ciliary-length phenotypes and increased localization of the remaining paralog do not by themselves establish functional compensation. Additional experiments are needed to determine whether acute depletion of the remaining paralog in each single-knockout background reproduces the double-knockout phenotype and whether expression of either paralog can rescue the double-knockout cells.
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Reviewer #2 (Public review):
Summary:
The authors build on previous work demonstrating that the microtubule-associated protein CKAP2 functions at the mitotic spindle to regulate microtubule dynamics for proper chromosome segregation. Here they show that CKAP2 and its paralog CKAP2L also function at the cilium to regulate cilium formation and Hedgehog signaling.
Strengths:
A major strength of the manuscript is the use of GFP-CKAP2 knockin cells and the examination of single and double CKAP2 and CKAP2L knockout cells. The authors use live imaging of the knockin cells to show that CKAP2 localizes to the primary cilium of RPE1 cells after assembly.
The authors use the knockout cells to show that loss of either CKAP2 or CKAP2L results in longer cilia and a dampened Hedgehog response, whereas loss of both CKAP2 and CKAP2L results in shorter …
Reviewer #2 (Public review):
Summary:
The authors build on previous work demonstrating that the microtubule-associated protein CKAP2 functions at the mitotic spindle to regulate microtubule dynamics for proper chromosome segregation. Here they show that CKAP2 and its paralog CKAP2L also function at the cilium to regulate cilium formation and Hedgehog signaling.
Strengths:
A major strength of the manuscript is the use of GFP-CKAP2 knockin cells and the examination of single and double CKAP2 and CKAP2L knockout cells. The authors use live imaging of the knockin cells to show that CKAP2 localizes to the primary cilium of RPE1 cells after assembly.
The authors use the knockout cells to show that loss of either CKAP2 or CKAP2L results in longer cilia and a dampened Hedgehog response, whereas loss of both CKAP2 and CKAP2L results in shorter cilia and an even more dampened Hedgehog response.
Weaknesses:
The data are compelling and support the conclusions. However, the findings would be significantly strengthened by using additional markers of the cilium, not just acetylated tubulin.
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The microtubule-associated proteins CKAP2 and its paralog CKAP2-Like control ciliogenesis in human cells https://www.biorxiv.org/content/10.64898/2026.09.03.749104v1
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The microtubule-associated proteins CKAP2 and its paralog CKAP2-Like control ciliogenesis in human cells https://www.biorxiv.org/content/10.64898/2026.09.03.749104v1
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