EXAMINATION OF THE ENDOSOMAL AND LYSOSOMAL PATHWAYS IN DICTYOSTELIUM-DISCOIDEUM MYOSIN-I MUTANTS

The role of myosin Is in endosomal trafficking and the lysosomal syste m was investigated in a Dictyostelium discoideum myosin I double mutan t myoB(-)/C-, that has been previously shown to exhibit defects in flu id-phase endocytosis during growth in suspension culture (Novak et al. , 1995). Various properties of the endosomal pathway in the myoB(-)/C- double mutant as well as in the myoB(-) and myoC(-) single mutants, i ncluding intravesicular pH, and intracellular retention time and exocy tosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contrac tile vacuole complex and the endocytic pathway in Dictyostelium, and t he localization of a myosin I to the contractile vacuole in Acanthamoe ba, led us to also examine the structure and function of this organell e in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB(-), myoC(-) or myoB(-)/ C- cell lines. The transport, processing, and localization of a lysoso mal enzyme, alpha-mannosidase, were also unaltered in all three mutant s. However, the myoB(-) and myoB(-)/C- cell lines, hut not the myoC(-) cell line, were found to oversecrete the lysosomal enzymes alpha-mann osidase and acid phosphatase, during growth and starvation. None of th e mutants oversecreted proteins following the constitutive secretory p athway. Two additional myosin I mutants, myoA(-) and myoA(-)/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mann osidase and acid phosphatase. Taken together, these results suggest th at these myosins do not play a role in the intracellular movement of v esicles, but that they may participate in controlling events that occu r at the actin-rich cortical region of the cell. While no direct evide nce has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fid elity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface.