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First published online 28 June 2005
doi: 10.1242/jcs.02444
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Research Article |
1 Cytoskeleton Dynamics Group, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany
2 IFOM, the FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, 20139, Milan, Italy
3 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
4 Department of Cell Biology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany
Author for correspondence (e-mail: klemens.rottner{at}gbf.de)
Accepted 19 April 2005
WASP and WAVE family proteins promote actin polymerization by stimulating Arp2/3-complex-dependent filament nucleation. Unlike WAVE proteins, which are known to drive the formation of protrusions such as lamellipodia and membrane ruffles, vertebrate cell functions of WASP or N-WASP are less well established. Recent work demonstrated that clathrin-coated pit invagination can coincide with assembly of actin filaments and with accumulation of N-WASP and Arp2/3 complex, but the relevance of their recruitment has remained poorly defined. We employed two-colour total internal reflection microscopy to study the recruitment and dynamics of various components of the actin polymerization machinery and the epidermal growth factor receptor signalling machinery during clathrin-coated pit internalization in control cells and cells genetically deficient for functional N-WASP. We found that clathrin-coated pit endocytosis coincides with the recruitment of N-WASP, Arp2/3 complex and associated proteins, but not of WAVE family members. Actin accumulation at clathrin-coated pits requires the Arp2/3 complex, since Arp2/3 complex sequestration in the cytosol abolished any detectable actin assembly. The absence of N-WASP caused a significant reduction in the frequencies of actin and Arp2/3 complex accumulations at sites of clathrin-coated pit invagination and vesicle departure. Although N-WASP was not essential for Arp2/3-complex-mediated actin assembly at these sites or for EGF receptor-mediated endocytosis, N-WASP deficiency caused a marked reduction of EGF internalization.
We conclude that the assembly of WASP subfamily proteins and associated factors at sites of clathrin-coated pit invagination amplifies actin accumulations at these sites promoting efficient internalization of ligands via clathrin-mediated endocytosis.
Key words: N-WASP, Arp2/3 complex, Actin, EGF receptor, Clathrin, Endocytosis
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