Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis

  1. Peter Walentek  Is a corresponding author
  2. Ian K Quigley
  3. Dingyuan I Sun
  4. Umeet K Sajjan
  5. Christopher Kintner
  6. Richard M Harland  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Salk Institute for Biological Studies, United States

Abstract

Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and post-transcriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function.

Data availability

The following data sets were generated
    1. Quigley IK
    2. Kintner C
    (2015) RNAseq profiling of multiciliated cells
    Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE76342).
The following previously published data sets were used
    1. Ma L
    2. Quigley IK
    3. Kintner C
    (2014) Multicilin drives centriole biogenesis via E2f proteins
    Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE59309).

Article and author information

Author details

  1. Peter Walentek

    Division of Genetics, Genomics and Development, University of California, Berkeley, Berkeley, United States
    For correspondence
    walentek@berkeley.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2332-6068
  2. Ian K Quigley

    Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0075-8324
  3. Dingyuan I Sun

    Division of Genetics, Genomics and Development, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Umeet K Sajjan

    Division of Genetics, Genomics and Development, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Christopher Kintner

    Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Richard M Harland

    Division of Genetics, Genomics and Development, University of California, Berkeley, Berkeley, United States
    For correspondence
    harland@berkeley.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8247-4880

Funding

Deutsche Forschungsgemeinschaft (Wa 3365/1-1)

  • Peter Walentek

National Heart, Lung, and Blood Institute (K99HL127275)

  • Peter Walentek

National Institute of General Medical Sciences (GM42341)

  • Richard M Harland

National Institute of General Medical Sciences (GM076507)

  • Christopher Kintner

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

Ethics

Animal experimentation: This work was done with approval of University of California, Berkeley's Animal Care and Use Committee. University of California, Berkeley's assurance number is A3084-01, and is on file at the National Institutes of Health Office of Laboratory Animal Welfare.

Reviewing Editor

  1. Janet Rossant, University of Toronto, Canada

Publication history

  1. Received: May 5, 2016
  2. Accepted: September 12, 2016
  3. Accepted Manuscript published: September 13, 2016 (version 1)
  4. Version of Record published: September 30, 2016 (version 2)

Copyright

© 2016, Walentek et al.

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

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  1. Peter Walentek
  2. Ian K Quigley
  3. Dingyuan I Sun
  4. Umeet K Sajjan
  5. Christopher Kintner
  6. Richard M Harland
(2016)
Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis
eLife 5:e17557.
https://doi.org/10.7554/eLife.17557

Further reading

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    Background:

    Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.

    Methods:

    To test whether dystrophin mutations lead to defective cardiac NaV1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.

    Results:

    Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers.

    Conclusions:

    We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5–Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5–Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.

    Funding:

    Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.