1. Cell Biology

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

  1. Tomoharu Kanie  Is a corresponding author
  2. Roy Ng
  3. Keene L Abbott
  4. Niaj Mohammad Tanvir
  5. Esben Lorentzen
  6. Olaf Pongs
  7. Peter K Jackson  Is a corresponding author
  1. University of Oklahoma Health Sciences Center, United States
  2. Stanford University, United States
  3. Aarhus University, Denmark
  4. Saarland University, Germany
https://doi.org/10.7554/eLife.85998
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  1. Tomoharu Kanie
  2. Roy Ng
  3. Keene L Abbott
  4. Niaj Mohammad Tanvir
  5. Esben Lorentzen
  6. Olaf Pongs
  7. Peter K Jackson
(2025)
Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages
eLife 14:e85998.
https://doi.org/10.7554/eLife.85998
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Abstract

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 ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary 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 a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary 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 ciliary vesicles.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for 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. Roy Ng

    Department of Microbiology and Immunology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Keene L Abbott

    Department of Microbiology and Immunology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6166-704X

  4. Niaj Mohammad Tanvir

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
    Competing interests
    The authors declare that no competing interests exist.

  5. Esben Lorentzen

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6493-7220

  6. Olaf Pongs

    Institute for Physiology, Saarland University, Saarbrücken, Germany
    Competing interests
    The authors declare that no competing interests exist.

  7. 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 (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.

Ethics

Animal experimentation: All mice were maintained under specific pathogen-free conditions at the Stanford animal care facility. All experiments were approved by Administrative Panel on Laboratory Animal Care at Stanford University (Institutional Animal Care and Use Committee protocol number: 28556) and were performed in strict accordance with their guidelines.

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. Roy Ng
  3. Keene L Abbott
  4. Niaj Mohammad Tanvir
  5. Esben Lorentzen
  6. Olaf Pongs
  7. Peter K Jackson
(2025)
Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages
eLife 14:e85998.
https://doi.org/10.7554/eLife.85998

Share this article

https://doi.org/10.7554/eLife.85998

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Further reading

    1. Cell Biology

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

    Tomoharu Kanie, Beibei Liu ... Peter K Jackson
    Research Article Updated

    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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    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.

    Conclusions:

    While PASMCs and PAAFs retain their core cellular identities, they acquire distinct disease-associated states. These findings provide new insights into the dynamic interplay of pulmonary vascular mesenchymal cells in disease pathogenesis.

    Funding:

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