Localization of Dictyostelium myoB and myoD to filopodia and cell-cell contact sites using isoform-specific antibodies.

To date, five myosin I heavy chain genes (myoA-E) have been sequenced in Dictyostelium. Among them, myoB, myoC and myoD possess tail domains that contain a putative membrane-binding domain, a nucleotide-insensitive actin-binding site, and an src homology (SH)-3 domain. In this study, we have determined the intracellular localizations of myoB and myoD by immunofluorescence using isoform-specific antibodies raised against bacterially expressed fusion proteins. MyoB is concentrated at the leading edges of lamellipodia and at sites of cell-cell contact in both stationary and aggregation stage cells. Based on its distinctive appearance, we have named the myosin I-rich, interdigitating cell-cell contact structure in the stationary stage cells "zipper". To analyze the staining of filopodia, we employed the ratio imaging technique. We find that myoB is present in filopodia in either a uniform or an apical staining pattern. Like myoB, myoD is concentrated in leading edges of lamellipodia and at sites of cell-cell contact in stationary stage cells. MyoD is also present in filopodia, although the intensity is weaker than that of myoB staining. Despite persistence of myoD protein in the cells, myoD largely disappears from lamellipodia and cell-cell contact regions in aggregation stage cells, suggesting the occurrence of a developmentally regulated relocalization to the cytoplasm. While these results, along with the striking similarity in their tail domain structures, suggest that myoB and myoD have overlapping functions, differences in their localization in developing cells indicate that they have unique functions as well.