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Functional genomic screen for modulators of ciliogenesis and cilium length

Nature volume 464, pages 1048–1051 (2010)Cite this article

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Abstract

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways1,2. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities3. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance4. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell–cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

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Figure 1: Regulators of actin dynamics modulate cilium assembly.
Figure 2: Endocytic recycling pathway is linked to ciliogenesis.
Figure 3: Pharmacological rescue of ciliary defect on IFT88 mutant cells.

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Acknowledgements

The screen was performed at the Functional Genomics Core (LJINCC grant P30 NS057096 to S. Lipton) with technical assistance from A. Cortez and E. Sisman and with imaging support from the UCSD Microscopy Core (P30 NS047101 and P30 CA23100). We thank P. A. Beachy for the Smo–EGFP plasmid and J. F. Reiter for IFT88 orpk/orpk MEFs. We thank C. Kintner, V. M. Fowler, S. J. Field and S. Ferro-Novick for discussion. This work was supported by grants from NARSAD (Young Investigator Award to J.K.), NINDS (RO1 NS052455 to J.G.G.), the NIH (GM070743 to T.I. and K.L.), the Korea Food and Drug Administration (10182KFDA992 to K.L.) and the Brain Research Center of the 21st Century Frontier Research Program (M103KV010008-07K2201-00810 to K.L.). J.G.G. is an investigator of the Howard Hughes Medical Institute.

Author Contributions J.G.G. conceived and directed the project. J.K., S.H.-G. and P.A.-B. designed the screen and J.K., S.H.-G. and E.S. performed the screen. J.K., K.O., K.L. and T.I. analysed the screen data. J.K. designed the follow-up experiments and J.K. and J.E.L. performed the experiments. J.K. interpreted the results and J.K. and J.G.G. wrote the paper with contributions from S.H.-G. and P.A.-B.

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Authors and Affiliations

  1. Departments of Neurosciences and Pediatrics, Institute for Genomic Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla, California 92093, USA,

    Joon Kim, Ji Eun Lee & Joseph G. Gleeson

  2. High Content Screening and Functional Genomics Core, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA ,

    Susanne Heynen-Genel, Eigo Suyama & Pedro Aza-Blanc

  3. Departments of Medicine and Bioengineering, University of California San Diego, La Jolla, California 92093, USA,

    Keiichiro Ono, KiYoung Lee & Trey Ideker

  4. Department of Biomedical Informatics, Ajou University School of Medicine, Suwon 443-749, Korea

    KiYoung Lee

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Corresponding author

Correspondence to Joseph G. Gleeson.

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Competing interests

The authors have applied for a patent to develop treatments for ciliopathy diseases on the basis of the findings described in this paper.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1- 16 with legends and legends for Supplementary Tables 1 - 4 (see separate files for tables). (PDF 1750 kb)

Supplementary Table 1

This file contains primary screen data (see Supplementary Information file for full legend). (XLS 5474 kb)

Supplementary Table 2

This file contains confirmation screen data (see Supplementary Information file for full legend). (XLS 374 kb)

Supplementary Table 3

This file summarizes hit classification study (see Supplementary Information file for full legend). (XLS 44 kb)

Supplementary Table 4

This file contains the sequences of siRNAs for selected hits. (XLS 30 kb)

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Kim, J., Lee, J., Heynen-Genel, S. et al. Functional genomic screen for modulators of ciliogenesis and cilium length. Nature 464, 1048–1051 (2010). https://doi.org/10.1038/nature08895

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