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Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP

Nature Cell Biology volume 3, pages 927–932 (2001)Cite this article

Abstract

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles1,2. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains1,3,4,5,6,7. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

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Figure 1: Intersectin-l has guanine-nucleotide exchange factor (GEF) activity towards Cdc42.
Figure 2: Intersectin-l causes a Cdc42 activation phenotype in Swiss 3T3 fibroblasts and N1E-115 neuroblastoma cells.
Figure 3: Intersectin-l stimulates actin nucleation through N-WASP-dependent activation of Arp2/3.
Figure 4: N-WASP interactions with intersectin-l.
Figure 5: Intersectin-l guanine-nucleotide exchange factor (GEF) activity is upregulated by N-WASP binding.

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Acknowledgements

We would like to thank H. Miki, and M. Olson for important reagents used in this study. We also thank J. Bergeron and D. A. Schafer for discussion and J. Presley for assistance with confocal microscopy. This research was supported by Canadian Institutes of Health Research (CIHR) grants to P.S.M. and N.L.-V., and by a National Cancer Institute of Canada Grant to N.L.-V. P.S.M. and N.L.-V. acknowledge support from the Canadian Foundation for Innovation. T.P.S. is supported by a USPHS grant and by the Edwin S. Webster Foundation. C.C.Q. is supported by a NINDS grant to S. Hockfield of Yale University. N.K.H. and S.W. are supported by studentships from the Fonds De La Recherche En Santé Du Québec and the Natural Sciences and Engineering Research Council, respectively. S.J. is a CIHR postdoctoral fellow. N.L.-V. is a Junior Scholar of the Fonds De La Recherche En Santé Du Québec. P.S.M. is a CIHR Investigator.

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Authors and Affiliations

  1. Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Quebec, Canada

    Natasha K. Hussain, Sylwia Wasiak & Peter S. McPherson

  2. Department of Anatomy and Cell Biology, McGill University, Montreal, H3A 2B2, Quebec, Canada

    Sarah Jenna, Nathalie Lamarche-Vane & Peter S. McPherson

  3. Hematology Division, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Michael Glogauer & Thomas P. Stossel

  4. Section of Neurobiology, Yale University School of Medicine, New Haven, 06510, Connecticut, USA

    Christopher C. Quinn

  5. Division of Medical Genetics, University of Geneva Medical School, 1211 Geneva 4, Switzerland

    Michel Guipponi & Stylianos E. Antonarakis

  6. Department of Pharmacology, University of Wisconsin, Madison, 53706, Wisconsin, USA

    Brian K. Kay

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Correspondence to Peter S. McPherson.

Supplementary information

Supplementary methods and figures

Method S1 Production of cDNA constructs. (PDF 471 kb)

Figure S1 Myc-tagged Cdc42 binds to the DH domain of intersectin-l.

Figure S2 Intersectin-l tail constructs cause a Cdc42 activation phenotype in Swiss 3T3 fibroblasts.

Figure S3 Intersectin-l tail constructs stimulate neurite outgrowth in N1E-115 cells.

Figure S4 Overlay of N-WASP with intersectin SH3 domains.

Figure S5 Co-immunoprecipitation of intersectin-l and N-WASP from co-transfected cells.

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Hussain, N., Jenna, S., Glogauer, M. et al. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat Cell Biol 3, 927–932 (2001). https://doi.org/10.1038/ncb1001-927

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