Characterization of actin filament severing by actophorin from Acanthamoeba castellanii.

Abstract

Abstract. Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem . 261 :477-485) . Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1 :1 covalent complex of actin and actophorin . Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steadystate actin polymer concentration gave a Kd of 0.2 fiM T o understand the mechanisms that regulate the assembly and dynamics of the actin cytoskeleton, a catalog of more than 30 types of actin-binding proteins has been compiled in the hope that understanding the parts will give insight into the whole (Stossel et al., 1985;Pollard and Cooper, 1986) . The redundancy of the system is striking at the biochemical level where a variety of proteins can have similar activities. In a given cell type more than one protein can sequester actin monomers or nucleate actin polymerization or cap actin filaments or crosslink actin filaments . Furthermore, an individual protein can have two or more of these activities. The actin filament severing proteins illustrate this redundancy.The best characterized severing proteins are the -90-kD gelsolin group (Yin and Stossel, 1979) and the -42-kD fragmin/severin group (Hasegawa et al., 1980 ;Brown et al ., 1982) . Gelsolin requires Cam to sever actin filaments and is inhibited by phosphoinositides (Janmey and Stossel, 1987) . It also caps the barbed end of actin filaments and forms nuclei for elongation by binding two actin monomers. These proteins consist of multiple functionally specialized domains sharing a common sequence motif. It is generally believed that both groups arose from a precursor of -125 amino acids by a series of gene duplications resulting in fragmin/severin with three of these domains (Ampe and Vandekerckhove, 1987;Andre et al ., 1988) and gelsolin/villin with six domains (Kwaitkowski et al., 1986;Way and Weeds, 1988;Bazari et al ., 1988). The current models for severing by both groups of proteins involve the interaction of multiple domains with actin filaments. for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration . The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate .

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

“…Actin interacting protein 1 (Aip1p) and cofilin are two actin binding proteins that function in concert to promote the rapid disassembly of actin filaments. Biochemical assays show that cofilin promotes depolymerization of actin filaments by accelerating pointed-end filament disassembly (Carlier et al, 1997;Lappalainen and Drubin, 1997) and by a severing activity that is very weak at physiological pH (Maciver et al, 1991;Ichetovkin et al, 2000). Aip1p dramatically enhances the depolymerization activity of cofilin-decorated actin filaments and is suspected to do so by assisting cofilin-induced severing and/or by capping the barbed ends of actin filaments (Okada et al, 1999;Rodal et al, 1999;Okada et al, 2002;Balcer et al, 2003;Mohri et al, 2004;Ono et al, 2004).…”

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

“…9 and 20) interact with both monomeric and filamentous actin, in vitro, and sever filaments (9,17,(21)(22)(23). The members of the ADF group show a high conservation of primary structure and function.…”

Relationships between Actin Regulatory Mechanisms and Measurable State Variables

“…The only known influences on these rates are proteins of the ADF/cofilin family, which are present at significant concentrations in cells, $20 lM. 1,12,14,44 Cofilins bind preferentially to ADP-bound subunits in actin filaments and accelerate depolymerization of these filaments by increasing the off-rate of ADP-actin subunits, 9 severing filaments to create more ends, 31 and increasing the Pi release rate on ADPAEPi-bound subunits. 5 However, it is not clear what the relative contributions of these three effects are and how they relate to the concentration of cofilin.…”

“…These proteins bind the ADP-bound subunits of filaments found most prominently near pointed ends. 9 Cofilins destablize filament structure leading to both filament severing 31 and enhanced depolymerization. 9 Also regulating the actin cycle in cells are proteins that cap the barbed and pointed ends.…”