Differential Effects of Caldesmon on the Intermediate Conformational States of Polymerizing Actin

Abstract

The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton.

The Conformational State of Actin Filaments Regulates Branching by Actin-related Protein 2/3 (Arp2/3) Complex

“…However, in our experiments, the increased branching occurs on actin filaments aged on a time scale significantly longer than that of phosphate release from actin (30). As has previously been shown, the effect induced by CaD does not involve ATP hydrolysis or phosphate release (9), meaning that virtually all Alexa Fluor 488 actin during the imaging assay was already in the ADP state. Interestingly, a transient post-phosphate release intermediate conformation of actin prior to mature ADP-Factin formation has been detected before (12); such a conformation may be stabilized by CaD.…”

“…Notably, CaD has been found to attenuate pyrene fluorescence enhancement of polymerizing actin without altering the rates of elongation and to prolong the early stage rough appearance of actin filaments under negative staining electron microscopy (9, 10). Because these effects were not seen when CaD was added to aged filaments, it was argued that they correspond to a conformational transition of F-actin occurring naturally during polymerization (9). It was further suggested that CaD blocks this transition, thus keeping the actin filament in the nascent state.…”

“…Pyrene-labeled actin was prepared as described previously (9). Alexa Fluor 488 actin was prepared by labeling surface lysine residues according to published procedures (16).…”

“…Although the muscle-specific isoform of CaD plays a regulatory role in smooth muscle contractility (3), nonmuscle CaD is more broadly implicated in cell motility (4), including processes such as cell migration (5), focal adhesion assembly (6), podosome dynamics (7), and neointimal formation (8). Recently, CaD was also suggested to control a key structural transition occurring during actin polymerization, known as "maturation" (9,10). We found that CaD or its C-terminal actin-binding fragment, when present before, but not after, the structural transition, inhibits the maturation process and arrests the filament at the pretransitional state (9).…”

“…Recently, CaD was also suggested to control a key structural transition occurring during actin polymerization, known as "maturation" (9,10). We found that CaD or its C-terminal actin-binding fragment, when present before, but not after, the structural transition, inhibits the maturation process and arrests the filament at the pretransitional state (9). Importantly, the same effect was also observed in solutions containing only ADP, indicating that this maturation differs from the conventional aging process of actin filaments that depends on nucleotide hydrolysis.…”