LOCALIZATION OF ACTOBINDIN, PROFILIN-I, PROFILIN-II, AND PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE (PIP2) IN ACANTHAMOEBA-CASTELLANII

Specific polyclonal antisera were raised against purified Acanthamoeba actobindin and synthetic peptides corresponding to regions of maximum charge differences in Acanthamoeba profilin I and profilin II. Immuno fluorescence studies with these antibodies showed profilin I to be dis tributed throughout the Acanthamoeba cytoplasm, except for lamellipodi a, with the highest fluorescence intensity in cortical regions in whic h monomeric actin also was present, as shown by labeling with fluoresc ent DNase. In contrast, profilin II appeared to be uniformly associate d with the plasma membrane except at sites of pseudopod extension, whe re the concentration was frequently decreased, in addition to cortical regions. Immunofluorescence studies using a monoclonal antibody speci fic for phosphatidylinositol-4,5-bisphosphate (PIP2) suggested that it s distribution is mostly limited to the plasma membrane, In contrast t o the distribution of profilin IT, PIP2 immunofluorescence was promine nt at the leading edge of cells, including the plasma membrane of lame llipodia. Quantitative immunoelectron microscopy showed that profilin II was approximately 36 times more Likely to localize to the plasma me mbrane than profilin I. Immunofluorescence and confocal microscopy loc alized actobindin to the base of lamellipodia. The differential locali zation of the three actin monomer-binding proteins suggests that they have different biologic functions in Acanthamoeba and is consistent wi th the hypotheses that (1) profilin I functions predominantly as an ac tin monomer-binding protein; (2) profilin II regulates, or is regulate d by, PIP2; and (3) actobindin inhibits nucleation of new filaments an d facilitates elongation of existing polarized filaments in actively m otile regions. (C) 1998 Wiley-Liss, Inc.