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. Immunofluorescence studies with these antibodies showed profilin I to be distributed throughout the Acanthamoeba cytoplasm, except for lamellipodia, with the highest fluorescence intensity in cortical regions in which monomeric actin also was present, as shown by labeling with fluorescent DNase. In contrast, profilin II appeared to be uniformly associated with the plasma membrane except at sites of pseudopod extension, where the concentration was frequently decreased, in addition to cortical regions. Immunofluorescence studies using a monoclonal antibody specific for phosphatidylinositol-4,5-bisphosphate (PIP2) suggested that its distribution is mostly limited to the plasma membrane. In contrast to the distribution of profilin II, PIP2 immunofluorescence was prominent at the leading edge of cells, including the plasma membrane of lamellipodia. Quantitative immunoelectron microscopy showed that profilin II was approximately 36 times more likely to localize to the plasma membrane than profilin I. Immunofluorescence and confocal microscopy localized actobindin to the base of lamellipodia. The differential localization of the three actin monomer-binding proteins suggests that they have different biologic functions in Acanthamoeba and is consistent with the hypotheses that (1) profilin I functions predominantly as an actin monomer-binding protein; (2) profilin II regulates, or is regulated by, PIP2; and (3) actobindin inhibits nucleation of new filaments and facilitates elongation of existing polarized filaments in actively motile regions.
Bubb
M R
MR
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0301, USA.
Baines
I C
IC
Korn
E D
ED
D000048:Acanthamoeba / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002352:Carrier Proteins / Q000032:analysis*; D003285:Contractile Proteins; D019084:Fluorescent Antibody Technique, Indirect; D007150:Immunohistochemistry; D008840:Microfilament Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000032:analysis*; D051304:Profilins; D015800:Protozoan Proteins / Q000032:analysis*
D000913:Antibodies, Protozoan; D002352:Carrier Proteins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D051304:Profilins; D015800:Protozoan Proteins; C050869:actobindin protein, Acanthamoeba