Cell Migration and Invadopodia Formation Require a Membrane-binding Domain of CARMIL2

Abstract

CARMILs regulate capping protein (CP), a critical determinant of actin assembly and actin-based cell motility. Vertebrates have three conserved CARMIL genes with distinct functions. In migrating cells, CARMIL2 is important for cell polarity, lamellipodial assembly, ruffling, and macropinocytosis. In cells, CARMIL2 localizes with a distinctive dual pattern to vimentin intermediate filaments and to membranes at leading edges and macropinosomes. The mechanism by which CARMIL2 localizes to membranes has not been defined. Here, we report that CARMIL2 has a conserved membrane-binding domain composed of basic and hydrophobic residues, which is necessary and sufficient for membrane localization, based on expression studies in cells and on direct binding of purified protein to lipids. Most important, we find that the membrane-binding domain is necessary for CARMIL2 to function in cells, based on rescue expression with a set of biochemically defined mutants. CARMIL1 and CARMIL3 contain similar membrane-binding domains, based on sequence analysis and on experiments, but other CPI motif proteins, such as CD2AP, do not. Based on these results, we propose a model in which the membrane-binding domain of CARMIL2 tethers this multidomain protein to the membrane, where it links dynamic vimentin filaments with regulation of actin assembly via CP.

CARMIL2 is a novel molecular connection between vimentin and actin essential for cell migration and invadopodia formation

“…A similar result was obtained for CARMIL1 ( Edwards et al ., 2013 ); therefore another function of CARMIL1 and CARMIL2 must be necessary for cell migration in wound healing. That function may be binding to membranes, which is mediated by a basic and hydrophobic membrane-binding domain, as described by us in a separate study ( Lanier et al ., 2015 ).…”

CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish

“…Our results provide new information about the function of CARMIL3, especially in the in vivo context of a whole vertebrate organism during the process of embryogenesis. Previous studies on CARMIL3 have used cell culture and mouse tumor models to uncover roles in neuronal synapse formation and cancer cell migration, based on actin assembly (Hsu et al, 2011;Lanier et al, 2016;Spence et al, 2019;Wang et al, 2020).…”

Comparative analysis of CPI-motif regulation of biochemical functions of actin capping protein

“…One cellular pool of CP with potentially great physiological significance is that found as a 1:1 complex with a CPI-motif protein that has been targeted to a membrane (11,14). Cell studies suggest that this pool of CP is part of the active fraction of CP, available to cap barbed ends of actin filaments that are near membranes (15,34). These barbed ends may have arrived at the membrane by growth of barbed ends that were nucleated by Arp2/3 complex or formins, whose activity can depend on membrane-associated signals and regulators.…”

“…This region enhances the ability of CARMILs to bind to and affect the activity of CP, relative to CPI motifs alone (19,35). CARMILs also possess a third region, downstream of (Cterminal to) the CSI motif, which also affects interaction with CP and which is responsible for binding to membranes (19,34,35). Thus, while proteins with CPI motifs were found to differ rather widely in their ability to bind and affect CP, based on the results here with CPI-motifcontaining peptides, we expect that CARMIL family proteins, with their CSI motifs and membrane-binding motifs, will differ from non-CARMIL CPI-motif proteins by an even larger degree.…”