Abstract
Quantitative microscopy is a valuable tool for inferring molecular mechanisms of cellular processes such as clathrin-mediated endocytosis, but, for quantitative microscopy to reach its potential, both data collection and analysis needed improvement. We introduce new tools to track and count endocytic patches in fission yeast to increase the quality of the data extracted from quantitative microscopy movies. We present a universal method to achieve "temporal superresolution" by aligning temporal data sets with higher temporal resolution than the measurement intervals. These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP. We show that the time course of actin assembly and disassembly varies <600 ms between patches. Actin polymerizes during vesicle formation, but we show that polymerization does not participate in vesicle movement other than to limit the complex diffusive motions of newly formed endocytic vesicles, which move faster as the surrounding actin meshwork decreases in size over time. Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated.