Characterization of Arp2/3 complex in chicken tissues

Abstract

Arp2/3 protein complex consists of seven subunits (Arp2, Arp3, p41-Arc, p34-Arc, p21-Arc, p20-Arc and p16-Arc) in apparent 1:1 stoichiometry. This complex has been shown to promote the formation of Y-branch structures of F-actin in cultured cells. We generated specific antibodies against chicken Arp2, Arp3, and p34-Arc to analyze the distribution of these subunits in chicken tissues. In whole samples of brain and gizzard, antibodies against each recombinant protein reacted with single bands of predicted molecular mass based on their cDNA sequences of the antigens. Anti-p34-Arc antibody detected at least two neighboring spots in 2D-PAGE, which might suggest the existence of isoforms or modified forms. Arp2/3 complex bound to an F-actin affinity column from gizzard extract. However, Arp2/3 complex did not tightly bind major actin cytoskeleton because the complex was extracted easily when gizzard smooth muscle was homogenized in PBS. Immunoblot analysis of various tissues revealed that the amounts of Arp2/3 subunits were lower in striated muscle than in non-muscle and smooth muscle tissues. Amounts and ratio of the three subunits varied in tissues, as estimated by quantitative immunoblotting. With immunofluorescence microscopy, we also observed localization of Arp3 and p34-Arc in frozen sections of gizzard with different staining patterns around blood vessels. These results suggest that the Arp2/3 complex exists also in places where rapid actin polymerization does not occur, and that a part of the subunits may exist in different forms from the complex containing the seven subunits in some tissues.