THE EFFECTS OF VARYING ACTIN AND CAPPING PROTEIN CONCENTRATIONS ON ACTIN PATCH DYNAMICS IN FISSION YEAST

Abstract

Actin assembly into structures called endocytic actin patches is directly responsible for driving endocytic invagination and internalization. We tested the predictions made from the mathematical modeling of the Dendritic Nucleation Model of actin assembly at sites of clathrin-mediated endocytosis in fission yeast (Berro et al.; Sirotkin et al., 2010). The model predicts that increasing the concentration of cytoplasmic actin or deleting capping protein will cause an increase in the extent and the rate of actin assembly in actin patches. Conversely, the model predicts that increasing the concentration of capping protein or decreasing the actin concentration will cause a decrease in the extent and the rate of actin assembly in actin patches. To test these predictions, we used the actin cross-linking protein, fimbrin Fim1 tagged with a fluorescent protein to measure actin patch dynamics in strains that over- or under-express actin, over-express capping protein, or have deletions of the two capping protein genes. In contrast to model predictions, we found that manipulating capping protein concentrations did not have a significant effect on the extent of actin patch assembly and affected the rates of assembly to a lesser degree than expected from the model, suggesting that capping protein is not the only factor that limits actin patch assembly. Surprisingly, changes in the concentration of actin resulted in changes in the number of patches in a cell, suggesting that the concentration of actin is more important in controlling the initiation of new patches rather than in patch assembly. Through studying the biochemical pathway of actin assembly directly in living cells, we were able to gain insights into previously under-appreciated aspects of the mechanism of actin assembly at the sites of clathrin-mediated endocytosis.