Nucleation and elongation of actin filaments in the presence of high speed supernate from neutrophil lysates: modulating effects of Ca2+ and phosphatidylinositol-4,5-bisphosphate.
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 (PIP2) 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 PIP2 micelles and micromolar [Ca2+] 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 [Ca2+]. Under these conditions, PIP2 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 [Ca2+] and totally abolished by immunoprecipitation of gelsolin from HSS; incubation of HSS with PIP2 at micromolar [Ca2+] slightly decreased the number of calcium-induced nucleation sites in the supernate. Incubation of HSS with PIP2 before the addition of calcium led to a greater reduction in Ca2+-inducible nucleation sites. HSS possessed more nucleation sites after simultaneous exposure to PIP2 and Ca2+, followed by chelation of Ca2+ with EGTA, than HSS preincubated at micromolar [Ca2+] without PIP2. At submicromolar [Ca2+], PIP2 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 PIP2, presumably by preventing capping by capping protein-beta2 in the supernate. Pointed end nucleating sites in HSS, attributable to gelsolin, could be created by adding micromolar [Ca2+]. The preincubation of HSS with PIP2 in the absence of micromolar [Ca2+] decreased the number of Ca2+-inducible nucleation sites in the HSS. Under conditions mimicking the sequential rise and fall of cytosolic [Ca2+] after stimulation, PIP2 accelerated actin polymerization despite the inhibitory action of HSS by maintaining Ca2+-activated nucleation sites. These observations suggest that a possible role for PIP2 in modulating cytoskeletal growth in vivo may be to regulate nucleation sites activated by sequential changes in cytosolic [Ca2+].
DiNubile
M J
MJ
Division of Infectious Diseases, Department of Medicine, Cooper Health System, UMDNJ/Robert Wood Johnson Medical School, Camden, NJ, USA. dinubile@umdnj.edu
D000199:Actins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002465:Cell Movement / Q000502:physiology; D002474:Cell-Free System; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D011817:Rabbits
D000199:Actins; D001704:Biopolymers; D002614:Chelating Agents; D019269:Phosphatidylinositol 4,5-Diphosphate; D004533:Egtazic Acid; D002118:Calcium