IQGAP1 mediates the structure and dynamics of a novel multi-vesicular compartment
Samson, Edward B
Diehl, Michael R
Doctor of Philosophy
IQGAP1 is a master cytoskeletal regulatory protein that connects extracellular signaling to changes in cell polarity, motility, and adhesion with adjacent cells. IQGAP1 achieves these fundamental outcomes by acting as a scaffolding protein that coordinates a wide variety of signaling cascades in a highly spatially-dependent manner. This dissertation details the use of multiple imaging modalities to characterize localized, highly-dynamic IQGAP1-related processes in epithelial MCF-10A cells. This led to the discovery of a novel multi-vesicular compartment that is surrounded by an outer layer of IQGAP1-associated actin filaments. Further studies showed that this compartment shares many common identifiers with traditional multi-vesicular bodies and participates in the internalization of cell-cell adhesion proteins via endocytic and recycling pathways. Live-cell imaging studies were conducted to correlate local cytoskeletal remodeling of this outer layer to various dynamic behaviors of the multi-vesicular core. These studies showed that IQGAP1 localization negatively correlates with actin polymerization during compartment formation and stabilization. During this time, rapid actin assembly appears to be constrained by a negative feedback mechanism. In contrast, IQGAP1 dissociation from the compartment’s surface is followed by a rapid, non-linear increase in actin polymerization that coincides with compartment disassembly and the release of multiple, high-motile intraluminal vesicles. Taken together, these results suggest a potential regulatory role of IQGAP1 in the trafficking of cell-cell adhesion proteins by promoting the stabilization of a novel multi-vesicular sorting compartment.