Biochemistry, Department of


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Published in Journal of Cell Science 114 (2001), 2673-2683. Copyright © 2001 The Company of Biologists Ltd. Used by permission.


Scar, a member of the WASp protein family, was discovered in Dictyostelium discoideum during a genetic screen for second-site mutations that suppressed a developmental defect. Disruption of the scar gene reduced the levels of cellular F-actin by 50%. To investigate the role of Scar in endocytosis, phagocytosis and endocytic membrane trafficking, processes that depend on actin polymerization, we have analyzed a Dictyostelium cell line that is genetically null for Scar. Rates of fluid phase macropinocytosis and phagocytosis are significantly reduced in the scar- cell-line. In addition, exocytosis of fluid phase is delayed in these cells and movement of fluid phase from lysosomes to post-Iysosomes is also delayed. Inhibition of actin polymerization with cytochalasin A resulted in similar phenotypes, suggesting that Scar-mediated polymerization of the actin cytoskeleton was important in the regulation of these processes. Supporting this conclusion, fluorescence microscopy revealed that some endo-Iysosomes were ringed with F -actin in control cells but no F -actin was detected associated with endo-Iysosomes in Scar null cells. Disruption of the two genes encoding the actin monomer sequestering protein profilin in wild-type cells causes defects in the rate of pinocytosis and fluid phase effiux. Consistent with a predicted physical interaction between Scar and profilin, disrupting the scar gene in the profilin null background results in greater decreases in the rate of fluid phase internalization and fluid phase release compared to either mutant alone. Taken together, these data support a model in which Scar and profilin functionally interact to regulate internalization of fluid and particles and later steps in the endosomal pathway, probably through regulation of actin cytoskeleton polymerization.