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Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a "leaky"(More)
Polymorphonuclear leukocyte (PMN) chemotaxis has been examined under conditions which allow phase microscope observations of cells responding to controlled gradients of chemotactic factors. With this visual assay, PMNs can be seen to orient rapidly and reversibly to gradients of N-formylmethionyl peptides. The level of orientation depends upon the mean(More)
We have established a cell-free system to investigate pathways that regulate actin polymerization. Addition of GTP ␥ S to lysates of polymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba induced formation of filamentous actin. The GTP ␥ S appeared to act via a small G-protein, since it was active in lysates of D. discoideum mutants missing(More)
We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at(More)
The rate of filamentous actin (F-actin) de-polymerization is proportional to the number of filaments depolymerizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymor-phonuclear leukocytes and the change in filament number and length that occurs during the increase in(More)
We have found that hypertonic medium inhibited the receptor-mediated uptake of the chemotactic peptide N-formylnorleucylleucylphenylalanine without affecting fluid-phase endocytosis by polymorphonuclear leukocytes (PMNs). Morphological and biochemical evidence demonstrated that cells in hypertonic medium did not accumulate peptide in a receptor-mediated(More)
To exhibit chemotaxis, the orientation of locomotion along a chemical gradient cells sense differences in concentrations of a chemotactic factor by detecting some difference in the occupancy of their chemotactic receptors. Thus chemotaxis is sensitive to the number of receptors present and might be used to evaluate the consequences of receptor(More)
The dynamics of the chemotactic peptide receptor on rabbit peritoneal polymor-phonuclear leucocytes were followed using the tritiated peptide N-formylnorleucylleucylphen-ylalanine (FNLLP). We have used a kinetic analysis to examine the possible interrelationships between receptor loss (down-regulation), receptor-mediated peptide uptake, and receptor(More)
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(More)