Arp2/3 Exhibits Higher Affinity for Young than for Mature Actin Filaments

Abstract

Actin undergoes a conformational transition during polymerization, known as maturation. Caldesmon (CaD) or its C-terminal fragment, H32K, when present before the transition, inhibits this process and arrests the actin filament at the pre-transitional “young” state, which is otherwise transient (Huang et al., J Biol Chem 285, 71-79, 2010). However, CaD in cells usually binds to stable actin filaments, except when it is phosphorylated and translocated to the cell leading edge where actin is being actively assembled (Kordowska et al., Exp Cell Res 312, 95-110, 2006); this may be the only occasion that CaD could interfere with actin maturation in vivo. We hypothesized that the young actin filaments stabilized by phosphorylated CaD are rendered more amenable to remodeling. Electron microscopic images indeed show that these young filaments have a rough and fuzzy morphology (Collins et al., BioArchitecture 1, 127-133, 2011). The question then arises: Could such an irregular appearance in fact provide more available docking sites for other actin-binding proteins, and thereby facilitate actin dynamics? To test this idea, we have performed binding experiments between Arp2/3 and actin filaments formed under the condition favoring either the young or the mature state. Actin filaments polymerized in the presence and absence of the CaD fragment H32K were incubated with the Arp2/3 complex and centrifuged; the amount of actin-bound Arp2/3 in the pellet was then analyzed by immuno-reactivity toward anti-Arp2 after serial dilutions. We found that the level of Arp2/3 cosedimented with actin filaments polymerized in the presence of H32K was higher than that bound to CaD-free F-actin, indicating stronger binding of Arp2/3 to the young actin filaments than to the mature filaments. These results appear to support our hypothesis. Supported by a grant from NIH.