Nucleation and elongation of actin filaments in the presence of high speed supernate from neutrophil lysates: modulating effects of Ca 2+ and phosphatidylinositol-4,5-bisphosphate

Abstract

Cell motility depends on the rapid growth of cortical actin filaments whose barbed ends are capped in the resting cell. High speed supernates (HSS) of dilute neutrophil lysates contain actin monomers and/or oligomers that can be induced to polymerize by certain stimuli. We questioned whether some of the actin remaining in the supernate after high speed centrifugation exists as occult nucleation sites which can elongate when uncapped. Phosphatidylinositol-4,5-bisphosphate (PIP 2) may play a critical role as an intracellular messenger in cytoskeletal rearrangement after stimulation by removing cappers from barbed filament ends. The experiments reported here examine the separate and interactive effects of PIP 2 micelles and micromolar [Ca 2+] on the rates of nucleation and elongation of pyrenyl-G-actin in the presence of HSS. HSS slowed the nucleation and elongation rates of gel-filtered pyrenyl-G-actin polymerized at submicromolar [Ca 2+]. Under these conditions, PIP 2 only slightly increased the number of nucleation sites, but delayed the slowing of the elongation rate in the presence of HSS. Nucleating activity in HSS could be induced by the addition of micromolar [Ca 2+] and totally abolished by immunoprecipitation of gelsolin from HSS; incubation of HSS with PIP 2 at micromolar [Ca 2+] slightly decreased the number of calcium-induced nucleation sites in the supernate. Incubation of HSS with PIP 2 before the addition of calcium led to a greater reduction in Ca 2+-inducible nucleation sites. HSS possessed more nucleation sites after simultaneous exposure to PIP 2 and Ca 2+, followed by chelation of Ca 2+ with EGTA, than HSS preincubated at micromolar [Ca 2+] without PIP 2. At submicromolar [Ca 2+], PIP 2 only generated a few barbed end nucleation sites in the HSS, but lessened the gradual slowing of elongation seen with HSS in the absence of PIP 2, presumably by preventing capping by capping protein-β 2 in the supernate. Pointed end nucleating sites in HSS, attributable to gelsolin, could be created by adding micromolar [Ca 2+]. The preincubation of HSS with PIP 2 in the absence of micromolar [Ca 2+] decreased the number of Ca 2+-inducible nucleation sites in the HSS. Under conditions mimicking the sequential rise and fall of cytosolic [Ca 2+] after stimulation, PIP 2 accelerated actin polymerization despite the inhibitory action of HSS by maintaining Ca 2+-activated nucleation sites. These observations suggest that a possible role for PIP 2 in modulating cytoskeletal growth in vivo may be to regulate nucleation sites activated by sequential changes in cytosolic [Ca 2+].