Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin.

Abstract

Thyone sperm were demembranated with Triton X-IO0 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCI~. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22°C. Actin filaments rapidly assembled and aggregated into bundles when KCI was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (,-,10 ~tM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.

Acrosomal Reaction of theThyone SpermIII: The Relationship between Actin Assembly and Water Influx during the Extension of the Acrosomal Process

“…These observations are consistent with a model in which actin monomers, when released from their bound state adjacent to the nucleus, diffuse to the tip of the elongating acrosomal process where they assemble on the growing filaments, a model that is now confirmed by recent in vitro observations (17).…”

Profilin is required for the normal timing of actin polymerization in response to thermal stress

“…A possible explanation for this observation is that profilin normally serves to stimulate actin polymerization, perhaps by enhancing the rate of nucleotide exchange on actin monomers [22,23], or by shuttling actin monomers to the barbed/plus ends of growing filaments [16,24]. Alternatively, profilin may enhance the stability of existing actin filaments, countering their apparent thermal instability.…”

Role of the actin-binding protein profilin1 in radial migration and glial cell adhesion of granule neurons in the cerebellum

“…6,7 Profilins catalyze the nucleotide exchange of monomeric actin (G-actin) and funnel ATP-actin to the barbed ends of actin filaments (F-actin). 8,9 Thus, profilins can promote actin polymerization by recharging G-actin and by adding it to the filaments' growing ends. Apart from binding to actin, profilins can interact with phosphoinositides and proteins that contain poly-L-prolinerich domains.…”