Disruption of a dynamin homologue affects endocytosis, organelle morphology, and cytokinesis in Dictyostelium discoideum.
The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.
Wienke
D C
DC
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, D-69120 Heidelberg, Germany.
Knetsch
M L
ML
Neuhaus
E M
EM
Reedy
M C
MC
Manstein
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000502:physiology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure*; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D018390:Gene Targeting; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D015388:Organelles / Q000648:ultrastructure; D015800:Protozoan Proteins / Q000235:genetics / Q000502:physiology*; D014882:Water-Electrolyte Balance
D017931:DNA Primers; D015800:Protozoan Proteins; D019204:GTP-Binding Proteins; D034281:Dynamins; C117420:dynamin A protein, Dictyostelium discoideum