Mechanism of interaction of Dictyostelium severin with actin filaments.

Abstract

Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2÷-dependent manner, was purified 500-fold to >99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29/k and consists of a single polypeptide chain. !t contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluorescence-labeled severin shows that this protein changes its conformation on binding Ca 2÷. Actin filaments are rapidly fragmented on addition of severin and Ca 2÷, but severin does not interact with actin filaments in the absence of Ca 2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (tl/2 = 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments wth the disassembled actin pool (tl/2 -~ 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin:severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.One of the central problems in cell motility is to understand the molecular basis for the transient nature of the nonmuscle contractile system. For instance, it is clear that cytokinesis, pseudopod formation, phagocytosis, and substrate adherence are dynamic events which demand both a spatial and temporal control of actin assembly and disassembly. Consequently, a pressing question has been to determine to what extent dynamic control over actin disassembly and subunit exchange is an inherent property of the filaments themselves. Extensive subunit exchange has been observed with purified actin filaments under some conditions (5 l, 52). However, recent experiments (39) indicate this may not occur under physiologically related ionic conditions. Under such conditions Pardee et al. (39) have shown by fluorescence energy transfer, 35S-actin monomer exchange, and steady state nucleotide incorporation into filaments that highly purified actin filaments from both muscle and Dictyostelium amoebae undergo only limited subunit exchange. This observation poses a further question. Are there accessory proteins which can function to cause filament disassembly and enhance filament subunit exchange in response to metabolic signals?Recent studies ...

Influx of extracellular Ca2+ is necessary for electrotaxis inDictyostelium

“…In support for this, focal Ca 2+ release induces biased motility of growth cones (Zheng, 2000;Hong et al, 2000). In addition, actin-binding proteins, such as ␣-actinin and severin are sensitive to [Ca 2+ ] i (Witke et al, 1993;Yamamoto et al, 1982). Surprisingly, genetic analysis shows that Ca 2+ signalling is not required for chemotaxis of Dictyostelium cells.…”

Proteins Exposed at the Adult Schistosome Surface Revealed by Biotinylation

“…Paracrystalline actin is a major constituent of tegumental spines (23), whereas severin and fimbrin are likely determinants of spine structure (Fig. 5), the former cross-linking actin (24) and the latter capping growing filaments (25). The tight apposition of the plasma membrane to the underlying spines (26) would require minimal penetration of reagent to label the three proteins.…”

Mechanism of regulation of actin polymerization by Physarum profilin.

“…Thus, free G-actin has to be in equilibrium not only with F-actin but also with complex ofprofilin-G-actin at the steady state. Profilin reduces the concentration of F-actin unaffecting the length distribution of filaments unlike the effects of end-blocking proteins ; for example, fragmin, villin, and severin sever F-actin filaments and block the association of the fragments by capping their ends (3,11,37).…”