Non-glycolytic roles for aldolase in actin-dependent cellular processes
Ritterson Lew, Carolyn
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The enzyme fructose-1,6-bisphosphate aldolase (aldolase) has well-defined roles in metabolism, and has recently been implicated in cellular functions unrelated to metabolism. These "moonlighting" functions include vesicle transport, signal transduction, and cell motility, which also involve the actin cytoskeleton. Aldolase is an actin-binding protein, however, the function of this interaction is not known. The hypothesis that aldolase is a "molecular adaptor" in cells, which localizes proteins to the cytoskeleton, is tested. Because of its importance in metabolism, aldolase cannot be completely ablated in cells. Therefore, an RNAi approach that reduces aldolase expression is employed. After siRNA transfection into mouse fibroblast cells, the following effects are seen: aberrant cell morphology, a disorganized cytoskeleton, and a decrease in cell proliferation. Moreover, several rapidly proliferating transformed cell lines show an 80-90% decrease in proliferation following aldolase knockdown. This reduction in cell proliferation is rescued by the expression of exogenous aldolase. Surprisingly, aldolase knockdown has no effect on the metabolic state of the cell. Instead, a multinucleation defect is observed, likely due to a cytokinesis defect that involves the interaction of aldolase with the cytoskeleton-regulating Wiskott-Aldrich syndrome protein (WASP). The function of the aldolase-WASP interaction is tested in vitro and in vivo. In vitro, aldolase inhibits WASP-stimulated actin polymerization through sequestering of WASP (IC50 =250 nM). In vivo, knockdown of aldolase inhibits two WASP-mediated processes - cell motility and cell spreading. Two aldolase variants are tested in rescue experiments. The R42A variant, which cannot bind actin well, fails to inhibit in vitro actin polymerization and cannot rescue cell motility defects due to aldolase knockdown. The D33S variant, which is catalytically inactive, inhibits in vitro actin polymerization and rescues the cell migration defect due to aldolase knockdown. These studies suggest that aldolase regulates the activity of WASP in cells through an actin-binding mechanism. A model is presented wherein aldolase binds activated WASP in cells but inhibits its activity until it is properly localized.
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