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 (similar to 120 End4p and similar to 230 Pan1p), activators of Arp2/3 complex (similar to 200 Wsp1p and similar to 340 Myo1p) and similar to 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (similar to 7000 actins, similar to 200 capping proteins, and similar to 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over similar to 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.