Bridging the Gaps: Investigating Molecular Mechanisms That Coordinate Actin Filament Assembly

Abstract

Cell division, migration, and maintaining cell morphology are all essential and dynamic cell processes that require precise coordination of the actin cytoskeleton. Actin monomers assemble into polar actin filaments that have a faster-growing (barbed) end and a slower-growing (minus) end. Here we examine the role of IQ-motif containing GTPase Activating Protein 1 (IQGAP1) in regulating actin filament assembly. This 189 kDa actin- binding protein slows actin filament assembly by interacting with the barbed end. Using extensive truncation analysis and single molecule microscopy techniques, we determined that IQGAP1 interacts with actin filament ends via residues within its IQ motifs. The barbed ends of actin filaments are intricately and competitively regulated by specific proteins and complexes of proteins that promote (formins) or inhibit (capping proteins) actin filament growth. We next examined the role of IQGAP1 in competitive interactions with the prominent barbed end regulators including formin and capping protein. Using fluorescently tagged proteins, IQGAP1 can be directly visualized on filament ends with individual formins and capping proteins and with formin-capping protein complexes. Interactions between IQGAP1 and formin on the ends of filaments slows formin-mediated actin assembly from 22.68 ± 2.9 subunits s-1μM-1 to 6.13 ± 0.7 subunits s-1μM-1. Further, IQGAP1 interacts with decision complexes on filament ends, creating a more complex decision complex, which decreases the dwell time on the end by 18-fold. We next examined the relevance of IQGAP1-mediated capping in cells using readouts of actin assembly: cell morphology, actin filament structure, and cell migration. Cells lacking IQGAP1 displayed significant changes to cell morphology and actin filament structures Cells expressing IQGAP1 or a capping deficient IQGAP1(CD), unable to bind filament ends, on a plasmid did not display significant changes to cell morphology or actin filament structure compared to wildtype cells. However, cells expressing IQGAP1(CD) displayed significantly slower wound closure compared to cells with endogenous IQGAP1. These results suggest that IQGAP1-mediated capping is a physiologically relevant mechanism of regulating actin filament assembly. This study reveals a role for IQGAP1 as a transient capper that promotes protein exchange on filament ends, which may have implications in the regulation of actin filament lengths in cells.