ABSTRACT
We investigate the issue of end versus side branching of actin filaments by Arp2/3 complex, using a combination of analytic theory, polymerization assays, and quantitative modeling. The analytic theory shows that the effect of capping protein on the initial stages of actin polymerization in the presence of Arp2/3 complex depends strongly on whether new Arp2/3 complex-induced branches grow from the sides or ends of existing filaments. Motivated by these results, we measure and quantitatively model the kinetics of actin polymerization in the presence of activated Arp2/3 complex, for a range of concentrations of capping protein. Our model includes the most important types of events involving actin and actin-binding proteins, and can be adjusted to include end branching, side branching, or both. The side-branching model gives a better fit to the experimental data than the end-branching model. An end-plus-side model including both types of branching gives a moderate improvement in the quality of the fit. Another side-branching model, based on aging of subunits' capacity for branch formation, gives a significantly better fit than the end-plus-side model. We discuss implications for actin polymerization in cells.
Subject(s)
Actin Cytoskeleton/chemistry , Actins/chemistry , Cytoskeletal Proteins/chemistry , Microfilament Proteins/chemistry , Models, Chemical , Actin Depolymerizing Factors , Actin-Related Protein 2 , Actin-Related Protein 3 , Binding Sites , Computer Simulation , Destrin , Dimerization , Glutathione Transferase/chemistry , Kinetics , Protein Binding , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Several new papers report progress on the structure and function of Arp2/3 complex. A crystal structure, a cryo-EM structure, and a reconstitution of the complex from subunits have been reported. New results also address the nucleation mechanism and the role of bound nucleotide.
Subject(s)
Actins/chemistry , Actins/metabolism , Cytoskeletal Proteins , Actin-Related Protein 2 , Actin-Related Protein 3 , Animals , Models, Molecular , Protein Structure, Quaternary , Protein SubunitsABSTRACT
Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.
Subject(s)
Actins/physiology , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/physiology , Microfilament Proteins/metabolism , Microfilament Proteins/physiology , Phosphatidylinositol 4,5-Diphosphate/physiology , Saccharomyces cerevisiae Proteins , Yeasts/metabolism , Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors , Actins/metabolism , Adenosine Diphosphate/metabolism , Binding Sites , Destrin , Microfilament Proteins/genetics , Models, Biological , Mutation , Phosphatidylinositol 4,5-Diphosphate/metabolismABSTRACT
Assembly of branched actin filament networks at the leading edge of migrating cells requires stimulation of the Arp2/3 complex by WASp proteins, in concert with the WASp activators Cdc42, PIP(2) and profilin. Network disassembly and debranching appears to be linked to actin-bound ATP hydrolysis as filaments age.
Subject(s)
Actins/metabolism , Microfilament Proteins/metabolism , Protein Structure, Tertiary , Proteins/metabolism , Animals , Humans , Models, Biological , Polymers/metabolism , Proteins/genetics , Wiskott-Aldrich Syndrome ProteinABSTRACT
Survival and changes in mean body mass of whole-body irradiated mice were determined to examine the radioprotectant activity of 5-diethylsulfonamoylsalicylatocopper(II) [Cu(II) (5-DESS)]. One of four groups of 25 female C57BL/6 mice were treated subcutaneously (sc)with 0, 10, 20, 40, 60, 80, 100, or 120 mumol Cu(II)(5- DESS)/kg of body mass 3 hours before exposure to 8.0 Gy, gamma irradiation. In this paradigm, doses of Cu(II)(5- DESS) increased survival up to 92% above vehicle-treated control mice (P = 0.008). Mean body mass determinations revealed that mice treated with 80 to 120 mumol Cu(II)(5-DESS)/kg of body mass exhibited a smaller decrease in body mass than other complex-treated groups. These results support the hypothesis that Cu(II)(5-DESS) is an effective radioprotectant.
