Novel Actin Regulatory Activities Of The Iqgap-Apc-Dia1 Complex Revealed By Single-Molecule Imaging

Cell motility and morphogenesis depend on the precise spatiotemporal control of actin assembly at the cell periphery. IQGAP is a downstream effector of Cdc42 and Rac1 and a key factor linking signaling to cytoskeletal rearrangements at the leading edge. IQGAP binds to F-actin and scaffolds several actin regulatory proteins, including Adenomatous polyposis coli (APC) and the formin Dia1. However, the specific mechanisms by which IQGAP regulates actin dynamics remain poorly understood. Our previous work showed that a large C-terminal domain of APC (APC-C) nucleates actin polymerization and works collaboratively with Dia1 to promote actin assembly by a ‘Rocket Launcher’ mechanism (Breitsprecher et al., 2012). Here, we used single molecule imaging to characterize the effects of IQGAP and an N-terminal IQGAP-binding fragment of APC on actin dynamics. We find that IQGAP alone binds tightly to actin filament sides (dwell time > 15 min) and potently bundles F-actin. Further, IQGAP interacts transiently with barbed ends (dwell time ∼ 30 sec), blocking growth, thus defining it as a transient capper. As such, IQGAP attenuates actin assembly induced by APC-C and Dia1. Consistent with these in vitro observations, RNAi silencing of IQGAP in HeLa cells leads to a marked increase in APC-dependent F-actin accumulation at the leading edge. Thus, IQGAP restricts APC/Dia1-mediated actin assembly, possibly in order to make actin assembly signal-responsive. Analysis of the N-terminal armadillo (ARM) domain of APC revealed three novel functions: (1) APC-ARM inhibits APC-C-mediated actin assembly in trans, suggesting that APC is autoregulated; (2) APC-ARM inhibits IQGAP's barbed end capping effects, i.e., promoting actin assembly; (3) APC-ARM enhances Dia1's actin assembly-promoting activities. Together, these observations suggest a complex interplay among these three large, multi-domain proteins, and provide evidence of their mutual activation to promote actin assembly.