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Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis

Nature Cell Biology volume 20, pages 175–185 (2018)Cite this article

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Abstract

Primary cilia play essential roles in signal transduction and development. The docking of preciliary vesicles at the distal appendages of a mother centriole is an initial/critical step of ciliogenesis, but the mechanisms are unclear. Here, we demonstrate that myosin-Va mediates the transportation of preciliary vesicles to the mother centriole and reveal the underlying mechanism. We also show that the myosin-Va-mediated transportation of preciliary vesicles is the earliest event that defines the onset of ciliogenesis. Depletion of myosin-Va significantly inhibits the attachment of preciliary vesicles to the distal appendages of the mother centriole and decreases cilia assembly. Myosin-Va functions upstream of EHD1- and Rab11-mediated ciliary vesicle formation. Importantly, dynein mediates myosin-Va-associated preciliary vesicle transportation to the pericentrosomal region along microtubules, while myosin-Va mediates preciliary vesicle transportation from the pericentrosomal region to the distal appendages of the mother centriole via the Arp2/3-associated branched actin network.

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Fig. 1: Myo-Va localizes to the preciliary vesicle, ciliary vesicle and ciliary sheath during ciliogenesis.
Fig. 2: Spatial-temporal localization of Myo-Va in RPE1 and IMCD3 cells, which use the intracellular and extracellular pathways, respectively.
Fig. 3: Myo-Va is required for cilia assembly and the trafficking of PCVs to the DA of the M-centriole.
Fig. 4: Myo-Va functions upstream of Rab11- and EHD1-mediated CV formation.
Fig. 5: Transportation of Myo-Va-associated PCVs to the M-centriole occurs via a dynein- and MT-dependent pathway.
Fig. 6: Low dose of cytochalasin D (CytoD) treatment stabilizes the Arp2-associated branched actin network and promotes ciliogenesis.
Fig. 7: Trafficking of Myo-Va-associated PCVs to the M-centriole requires the Arp2-associated branched actin network.

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Acknowledgements

The authors acknowledge support from the sequencing core facility (IBMS), the confocal imaging core facilities (IBMS, IMB, NPAS) and the EM core facilities (IMB, ICOB) of Academia Sinica. This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 105-2321-B001-016) and the Academia Sinica Investigator Award.

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Authors and Affiliations

  1. Taiwan International Graduate Program in Interdisciplinary Neuroscience, National Yang-Ming University and Academia Sinica, Taipei, Taiwan

    Chien-Ting Wu & Tang K. Tang

  2. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

    Chien-Ting Wu, Hsin-Yi Chen & Tang K. Tang

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Contributions

C.-T.W., a PhD student at the Taiwan International Graduate Program in Interdisciplinary Neuroscience, National Yang-Ming University and Academia Sinica, performed most of the experiments, designed the study, interpreted data and wrote the initial draft of the manuscript. H.-Y.C. performed experiments. T.K.T. conceived and designed the study, interpreted the data and wrote the manuscript.

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Correspondence to Tang K. Tang.

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Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary legends.

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Supplementary Table 1

List of antibodies and dilutions used in this study.

Supplementary Table 2

List of siRNA sequences used in this study.

Videos

Supplementary Video 1

Related to Supplementary Fig. 2b. RPE1-based inducible cells expressing GFP-Myo-Va-GTD and mCherry-Arl13b were treated with DOX for 24 h and then serum-starved. Live cell images were taken using a LSM780 Carl Zeiss confocal system under controlled CO2 (5%) and temperature (37 °C).

Supplementary Video 2

Related to Supplementary Fig. 2d. NIH3T3-based inducible cells expressing GFP-Myo-Va-GTD and mCherry-Arl13b were treated with DOX for 24 h and then serum-starved. Live cell images were taken using a LSM780 Carl Zeiss confocal system under controlled CO2 (5%) and temperature (37 °C).

Supplementary Video 3

Related to Fig. 2f. IMCD3-based inducible cells expressing GFP-Myo-Va-GTD and mCherry-Arl13b were treated with DOX for 24 h and then serum-starved. Live cell images were taken using a LSM780 Carl Zeiss confocal system under controlled CO2 (5%) and temperature (37 °C).

Supplementary Video 4

Related to Supplementary Fig. 1e. RPE1-based inducible cells expressing GFP-EHD1 and mCherry-Myo-Va-GTD were treated with DOX for 24 h and then serum-starved. Live cell images were taken using a LSM780 Carl Zeiss confocal system under controlled CO2 (5%) and temperature (37 °C).

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Wu, CT., Chen, HY. & Tang, T.K. Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis. Nat Cell Biol 20, 175–185 (2018). https://doi.org/10.1038/s41556-017-0018-7

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