A unique set of affinity-purified anti-profilin and anti-actin antibodies generated against a covalently coupled version of the profilin:actin complex was used to assess the distribution of profilin and non-filamentous actin in mouse melanoma cells. In agreement with the profilin:actin complex being the principal source of actin for filament formation, we observed extensive co-distribution of both antibody preparations with vasodilator-stimulated phosphoprotein (VASP) and the p34 subunit of the Arp2/3 complex, both of which are components of actin polymer-forming protein complexes in the cell. This suggests that the localization of profilin and actin revealed with these antibodies in fact reflects the distribution of the profilin:actin complex rather than the two proteins separately. Significantly, protruding lamellipodia and filopodia showed intensive labeling. The two antibody preparations were also used to stain HeLa cells infected with Listeria monocytogenes or vaccinia virus. In both cases, the pattern of antibody staining of the pathogen-induced microfilament arrangement differed, suggesting a varying accessibility for the antibody-binding epitopes.
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Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:
Nucleation-promoting proteins tightly regulate actin polymerization in cells. Whereas many of these proteins bind actin monomers directly, formins use the actin-binding protein profilin to dynamically load actin monomers onto their flexible Formin Homology 1 (FH1) domains. Following binding, FH1 domains deliver profilin-actin complexes to filament ends.
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Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455
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Department of Bioengineering, University of California, Berkeley, CA, USA.
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Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA.
Formins accelerate actin polymerization, assumed to occur through flexible Formin Homology 1 (FH1) domain-mediated transfer of profilin-actin to the barbed end. To study FH1 properties and address sequence effects, including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of a set of representative FH1 domains of formins: mouse mDia1 and mDia2, budding yeast Bni1 and Bnr1, and fission yeast Cdc12, For3, and Fus1. We find FH1 has flexible regions between high-propensity polyproline helix regions.
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