Dictyostelium has seven class-I myosins: three long-tailed (MIB, MIC, MID), three short-tailed (MIA, MIE, MIF) and one without a tail (MIK). The single heavy chains of these myosins have a motor domain, a neck and a non-helical tail. The tail of the long-tailed myosins contains a basic region with a basic-hydrophobic membrane-binding site (BH-site), a Gly-Pro-Gln (GPQ) region that binds F-actin, and a Src-homology 3 (SH3). The BH-site binds to acidic phospholipids. The GPQ region binds F-actin in the presence or absence of ATP, unlike the ATP-sensitive actin-binding site in the motor domain. The SH3 region binds CARMIL, and perhaps other proteins. The short-tailed myosins contain the basic region but not the GPQ region or SH3 domain. We have shown previously that the cellular localization of long-tailed MIB is very dynamic, paralleling the rapidly changing cell morphology, and determined the regions of the heavy chain responsible for targeting MIB to different cell compartments. MIB localizes uniformly to the plasma membrane of freshly plated, non-motile cells. During random movement of the amoebae, MIB relocates to other structures such as pseudopods, actin waves (dynamic structures associated with the basal plasma membrane), endocytic protrusions, cell-cell contacts, and the front of polarized, chemotaxing cells. The BH site is required for MIB localization to the plasma membrane, both the BH site and the GPQ region are required for MIB localization to actin waves and to macropinocytic protrusions, and the motor domain is required and sufficient for localization of MIB at the front of polarized cells. This year we have studied the molecular bases of the localizations of short-tailed MIA, MIE and MIF, determining their similarities and differences to each other and to long-tailed MIB. We found that the dynamic localizations of expressed short-tailed MIA were consistent with the previously determined roles of the motor domain, BH region and GPQ region in the localization of MIB. MIA localized to the regions of the cell that require only the motor domain or the BH site for localization of MIB, and MIA did not localize to the regions where localization of long-tailed MIB requires the GPQ region, which short-tailed MIA does not have. As expected, MIE and MIF, like MIA, localized uniformly to the plasma membrane, to cell-cell contacts, and the mouths of chemotaxing cells, and diffusely in pseudopods and the front of chemotaxing cells. Unexpectedly, however, unlike short-tailed MIA and like long-tailed MIB, short-tailed MIE and MIF localized to macropinocytic cups, macropinocytic protrusions and actin waves, but differently than long-tailed MIB. A manuscript reporting these new observations is in preparation.
