An actin-nucleating activity in polymorphonuclear leukocytes is modulated by chemotactic peptides.

Abstract

Abstract. We examined the actin-nucleating activity in polymorphonuclear leukocyte lysates prepared at various times after chemotactic peptide addition. The actin nucleation increases two-to threefold within 15 s after peptide addition, decays to basal levels within 90 s, and is largely independent of cytoplasmic calcium fluxes. The peptide-induced nucleation sites behave as free barbed ends and therefore may increase the level of polymerized actin in vivo. The new nucleation sites may also determine the cellular sites of actin polymerization. This localization of actin polymerization could be important for the directional extension of lamellipodia during chemotaxis.D1r~G chemotactic peptides to polymorphonuclear leukocytes (PMNs) ~ induces actin polymerization (F-actin) (9,11,12,23,27,35). Concomitantly, PMNs spread and extend ruffles over their surface. Both responses peak within 30 s. At later times some of the ruffles withdraw and the amount of F-actin decreases to an intermediate level. Cytochalasin D (CD), which blocks the peptide-induced actin polymerization, inhibits the cell shape change (35,38). Consequently, actin polymerization appears to be required for the protrusion of surface ruffles in PMNs. The extension of filopodia on platelets, of the acrosomal process on thyone sperm, and of microvilli on fertilized sea urchin eggs have been previously shown to correlate with actin polymerization in similar studies (8,10,32).The cell must have mechanisms for preventing complete actin polymerization. Actin, in the ionic conditions and at the concentrations present within a cell, would always be 99 % polymerized in the absence of regulation (13, 30). However, by a number of criteria (actin sedimentation, inhibition of DNAase activity, nitrobenzoxadiazole phallacidin staining), 50-70% of the actin is unpolymerized in an unstimulated PMN (9, 23). This state could be maintained by regulatory proteins that bind free actin monomers with high affinity, thus preventing their polymerization, and by proteins that cap filament ends, thus blocking polymerization.Proteins with these properties, able to regulate actin polymerization, have been isolated from PMNs (13, 30). Profilin binds monomeric actin, preventing monomer addition to the nonpreferred (pointed) end of actin filaments but not to the preferred (barbed) end (5, 33, 34). Profilin could maintain a large pool of unpolymerized actin in PMNs in conjunction with a capping protein such as gelsolin, which blocks polymerization on the barbed end of an actin filament, (15, 39). Chemotactic peptides, therefore, could increase the fraction of actin polymerized by releasing actin monomers, by uncapping pre-existing filaments, or by creating new barbed end polymerization sites.Studies of isolated actin regulatory proteins suggest that none of the receptor-generated signals would yield the appropriate regulation of actin polymerization. The binding of chemotactic peptide to its receptor causes a transient decrease in the intracellular concentration of phosphafidylinositol 4...

Sphingosine‐1‐phosphate inhibits actin nucleation and pseudopodium formation to control cell motility of mouse melanoma cells

“…To test whether Sph-I-P inhibits in vitro actin nucleation, we used a fluorescence assay with pyrene-labeled actin [24,25]. In this assay, fluorescence intensity increases as actin nuclea- tion proceeds.…”

“…The nucleation assay was performed by the addition of buffer (0.4 mM ATP, 25 mM imidazole, pH 7.0, final concentrations) and pyrenelabeled actin to the sample. Fluorescence was measured with an LS-2B filter fluorometer (Perkin-Elmer Corp., Pomona, CA) at wavelengths of 366/407 nm [24,25]. Final concentration of ethanol was <0.25%, which did not affect actin nucleation.…”

“…Pyrene.labeled actin was prepared according to the method of M, Canon et al, [24] with slight modifications, Briefly, actin from rabbit muscle cell (Sigma, St, Louis, MO) in buffer (0,1 mM CaCl2, 0.2 mM ATP, I mM NaHCO~, i mM dithiothreitol, 0,02% NaN,~, pH 7,6) was polymerized by addition of MgCla to 2 mM and KCI to 0,1 M, A 7,$ molar excess of N-(I-pyret~yl)-iodoacetamide (Molecular Probes Inc,, Eugene, OR) in dimcthylformamide was added immediately after addition of KCL The reaction n~ixture was gently rotated in the dark for 24 h at room temperature, The F actin was pelleted by centrifugation at 180000×g for 2 h at 4°C, The pellet was homo£~nized in 0.2 mM CaCI2, 0,4 mM ATP, I mM dithiothreitol, 0,02°/o NaNa, 2 mM Tris-HCI, pH 8.0, and dialyzed against the same buffer, The homogenate was centrifuged at 180000×g for 30 min at 4°C, and the labeled actin was obzained in the supernatant.…”

Gelsolin mediates calcium-dependent disassembly ofListeriaactin tails

“…These observations raised the possibility that calcium might play a critical role in chemoattractant-induced actin assembly and disassembly. However, subsequently, chelation of intracellular calcium was found to minimally affect chemoattractantassociated changes in polymorphonuclear leukocyte actin filament content (9,10). Similarly, the changes in actin filament content associated with phagocytic stimuli proved to be minimally affected by reductions in intracellular calcium (11).…”

Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

“…Actin nucleating activity in PMNs was quantified with a rabbit muscle pyrenyl actin (Kouiyama and Mihashi, 1981) indicator system similar to that described by Carson et al (1986) but modified by cell fixation to allow 2-s time resolution of nucleating activity. Rabbit muscle actin was purified by polymerization/depolymerization cycles (Spudich and Watt, 1971) and column purified over Sephadex G150 (Pharmacia, Piscataway, NJ) to remove endogenous nucleating activity (Cassella and Maack, 1987).…”