N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits.
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.
Benesch
Stefanie
S
Cytoskeleton Dynamics Group, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany.
Polo
Simona
S
Lai
Frank P L
FP
Anderson
Kurt I
KI
Stradal
Theresia E B
TE
Wehland
Juergen
J
Rottner
Klemens
K
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D018345:Mice, Knockout; D015398:Signal Transduction; D014162:Transfection; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000172:deficiency*
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D002966:Clathrin; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D004815:Epidermal Growth Factor; D066246:ErbB Receptors