Quantitative analysis of the mechanism of endocytic actin patch assembly and disassembly in fission yeast.
We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30-50% between individual patches. The pathway begins with accumulation of 30-40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (approximately 120 End4p and approximately 230 Pan1p), activators of Arp2/3 complex (approximately 200 Wsp1p and approximately 340 Myo1p) and approximately 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (approximately 7000 actins, approximately 200 capping proteins, and approximately 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.
Sirotkin
Vladimir
V
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.
Berro
Julien
J
Macmillan
Keely
K
Zhao
Lindsey
L
Pollard
Thomas D
TD
D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D011485:Protein Binding; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D002966:Clathrin; C506561:End4 protein, S pombe; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D029702:Schizosaccharomyces pombe Proteins