1. Cell Biology

A hierarchical pathway for assembly of the distal appendages that organize primary cilia

  1. Tomoharu Kanie  Is a corresponding author
  2. Beibei Liu
  3. Julia F Love
  4. Saxton D Fisher
  5. Anna-Karin Gustavsson
  6. Peter K Jackson  Is a corresponding author
  1. University of Oklahoma Health Sciences Center, United States
  2. Rice University, United States
  3. Stanford University, United States
https://doi.org/10.7554/eLife.85999
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  1. Tomoharu Kanie
  2. Beibei Liu
  3. Julia F Love
  4. Saxton D Fisher
  5. Anna-Karin Gustavsson
  6. Peter K Jackson
(2025)
A hierarchical pathway for assembly of the distal appendages that organize primary cilia
eLife 14:e85999.
https://doi.org/10.7554/eLife.85999
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Abstract

Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary 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 RAB34+ ciliary 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.

Data availability

All data generated or analysed during this study including Macro for automated fluorescent intensity measurement are included in the manuscript and supporting file; Source Data files have been provided for all the corresponding figures.

Article and author information

Author details

  1. Tomoharu Kanie

    Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, United States
    For correspondence
    Tomoharu-Kanie@ouhsc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2084-1451

  2. Beibei Liu

    Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Julia F Love

    Department of Chemistry, Rice University, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6642-6301

  4. Saxton D Fisher

    Department of Chemistry, Rice University, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Anna-Karin Gustavsson

    Department of Chemistry, Rice University, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0980-1168

  6. Peter K Jackson

    Department of Microbiology and Immunology, Stanford University, Stanford, United States
    For correspondence
    pjackson@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institute of General Medical Sciences (P20GM103447)

  • Tomoharu Kanie

National Institute of General Medical Sciences (1R35GM151013)

  • Tomoharu Kanie

National Institute of General Medical Sciences (R00GM134187)

  • Anna-Karin Gustavsson

Welch Foundation (C-2064-20210327)

  • Anna-Karin Gustavsson

Cancer Prevention and Research Institute of Texas (RR200025)

  • Anna-Karin Gustavsson

National Institute of General Medical Sciences (R01GM114276)

  • Peter K Jackson

National Institute of General Medical Sciences (R01GM121565)

  • Peter K Jackson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2025, Kanie et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Tomoharu Kanie
  2. Beibei Liu
  3. Julia F Love
  4. Saxton D Fisher
  5. Anna-Karin Gustavsson
  6. Peter K Jackson
(2025)
A hierarchical pathway for assembly of the distal appendages that organize primary cilia
eLife 14:e85999.
https://doi.org/10.7554/eLife.85999

Share this article

https://doi.org/10.7554/eLife.85999

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Further reading

    1. Cell Biology

    Myristoylated Neuronal Calcium Sensor-1 captures the preciliary vesicle at distal appendages

    Tomoharu Kanie, Roy Ng ... Peter K Jackson
    Research Article Updated

    The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of preciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures preciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the preciliary vesicle recruitment, but not for other steps of cilium formation (Kanie et al., 2025). The lack of a membrane-binding motif in CEP89 suggests that it may indirectly recruit preciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and the centriole-associated vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similar to CEP89 knockouts, preciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the preciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the preciliary vesicles.

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    Pulmonary vascular remodeling is a progressive pathological process characterized by functional alterations within pulmonary artery smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs). Mechanisms driving the transition to a diseased phenotype remain elusive.

    Methods:

    We combined transcriptomic and proteomic profiling with phenotypic characterization of source-matched cells from healthy controls and individuals with idiopathic pulmonary arterial hypertension (IPAH). Bidirectional cellular crosstalk was examined using direct and indirect co-culture models, and phenotypic responses were assessed via transcriptome analysis.

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    PASMC and PAAF undergo distinct phenotypic shifts during pulmonary vascular remodeling, with limited shared features, such as reduced mitochondrial content and hyperpolarization. IPAH-PASMC exhibit increased glycosaminoglycan production and downregulation of contractile machinery, while IPAH-PAAF display a hyperproliferative phenotype. We identified alterations in extracellular matrix components, including laminin and collagen, alongside pentraxin-3 and hepatocyte growth factor, as potential regulators of PASMC phenotypic transitions mediated by PAAF.

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    This work was supported by Cardio-Pulmonary Institute EXC 2026 390649896 (GK) and Austrian Science Fund (FWF) grant I 4651-B (SC).