Caldesmon Stabilizes Nascent Actin Filaments and Promotes Branching by Arp2/3 Complex

Abstract

Caldesmon is an actin-binding protein found in nearly all vertebrate cells. The heavy caldesmon isoform, which is specific to smooth muscle cells, is known to inhibit actomyosin interactions in vitro in a phosphorylation-dependent manner. However, possible roles of caldesmon as a regulator of actin mechanics and turnover are being explored, and the effects of caldesmon on actin dynamics and structure are not well understood.We have recently demonstrated that polymerizing actin undergoes an irreversible structural transition (termed “maturation”), and that the caldesmon C-terminal fragment, H32K, if added during the early stage of actin polymerization, prevents this maturation process (Huang et al., 2010 J Biol Chem 285(1):71-79). Actin filaments stabilized in this “nascent” state by H32K appear rough under electron microscopy and exhibits attenuated pyrene fluorescence enhancement compared to normal, mature F-actin, but H32K does not otherwise affect the polymerization kinetics of actin (Collins et al., 2011 Bioarchitechture 1(3):127-133).Both phosphorylated caldesmon and the actin-branching protein complex Arp2/3 are present at the leading edge of motile cells, where actin assembly provides the force needed to protrude the plasma membrane. Here, we study the interaction of H32K-stabilized nascent F-actin with Arp2/3 complex. By direct visualization of actin assembly in the presence of Arp2/3 complex using confocal fluorescence microscopy, we show that actin polymerized in the presence of H32K exhibits increased branching activity. We propose that the observed change is a result of the structural state of the young actin filament, which is stabilized by the early added H32K. This effect suggests a role of caldesmon as a regulator of actin structure, in turn dictating the interaction of other actin-binding proteins with actin.