Stimulation of a calcium-dependent actin nucleation activity by phorbol 12-myristate 13-acetate in rabbit macrophage cytoskeletons.
Cytoskeletons of detergent-extracted quiescent macrophages have nucleation sites that increase the rate of pyrene-labeled actin assembly in vitro. Cytochalasin D, which inhibits actin assembly at the fast-exchanging ends of filaments (barbed with respect to heavy meromyosin decorated filaments), only partially inhibits the increased assembly rate, demonstrating that pyrene-actin monomers add to both ends of filaments present in the cytoskeletons. Cytoskeletons prepared from macrophages treated with phorbol 12-myristate 13-acetate for 20-30 s before permeabilization, markedly stimulated (300% of control) the rate of actin assembly, and this increment was completely cytochalasin-sensitive, indicating that exposure to phorbol leads to formation of free barbed ends. Nucleation activity required more than 5 nM free calcium only in the assay and was maximal in the presence of 200 nM calcium. Concentrations of calcium of at least 30 nM dissociate the nucleation activity from the cytoskeleton, and it is recovered fully active in the calcium wash.
Hartwig
J H
JH
Hematology-Oncology Unit, Massachusetts General Hospital, Charlestown 02129.
Janmey
P A
PA
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D015638:Cytochalasin D; D003572:Cytochalasins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D015879:Myosin Subfragments / Q000378:metabolism; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000199:Actins; D003572:Cytochalasins; D015879:Myosin Subfragments; D015638:Cytochalasin D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium