Molecular Basis for CARMIL Function in Lamellipodia

Abstract

Coordinated intracellular rearrangement of the actin cytoskeleton in response to extracellular cues is an important theme in cell motility. CARMIL is emerging as a crucial regulator of lamellipodia-based motility, linking GTPase signaling to actin filament dynamics at the leading edge. CARMIL's role in regulating lamellipodium formation and maintenance makes it an important player in tumor metastasis, neuronal development, and cell motility. However, the molecular basis for CARMIL's role in these cellular processes is unclear. The objective of this study is to determine the molecular mechanism by which CARMIL affects lamellipodia formation. We primarily used the methods of x-ray crystallography and small-angle x-ray scattering (SAXS), as well as biochemical and biophysical approaches for this objective. The main results of this study are: (1) we demonstrate for the first time that CARMIL can bind specific types of lipids in vitro. (2) Using site-directed mutagenesis, we identify several residues involved in membrane interaction. (3) Maximum lipid-binding activity requires a dimerization domain, which does not directly contribute to membrane binding. (4) The 2.9 A crystal structure of a large portion of CARMIL addresses the structural basis for membrane binding. Our main conclusions are that CARMIL is capable of directly binding the plasma membrane, leading to co-localization at the lamellipodium leading edge with its known binding partners Capping Protein (CP) and Myosin I to regulate actin dynamics. CARMIL's lipid-binding specificity provides a possible mechanism for further spatiotemporal regulation of lamellipodia. CARMIL dimerization may play a role in signal transduction by CARMIL from the membrane to the actin cytoskeleton, in addition to enhancing membrane interaction. Our results addressing CARMIL's mechanism of action in lamellipodial dynamics will likely also be relevant to its role in neuronal development and tumor metastasis.