Disruption of a dynamin homologue affects endocytosis, organelle morphology, and cytokinesis in Dictyostelium discoideum

The identification and functional characterization of Dictyostelium discoid eum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynami n A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal a nd postlysosomal vacuoles. Overexpression of the protein has no adverse eff ect on the cells, whereas depletion of dynamin A by gene-targeting techniqu es leads to multiple and complex phenotypic changes. Cells lacking a functi onal copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucle ated due to a failure to complete normal cytokinesis. These pleiotropic eff ects of dynamin A depletion can be rescued by complementation with the clon ed gene. Morphological studies using cells producing green fluorescent prot ein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compar tment in D. discoideum, showed an almost complete colocalization of vacuoli n and dynamin A. Our results suggest that that dynamin A is likely to funct ion in membrane trafficking processes along the endo-lysosomal pathway of D , discoideum but not at the plasma membrane.