Abstract. Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non-coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTPbinding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2-terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and a-, but not 3% adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over-expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.
The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.
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