The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.
The eukaryotic slime mold Dictyostelium discoideum has a single conventional myosin heavy chain gene (mhcA). The elimination of the mhcA gene was achieved by homologous recombination. Two gene replacement plasmids were constructed, each carrying the G418 resistance gene as a selective marker and flanked by either 0.7 kb of 5′ coding sequence and 0.9 kb of 3′ coding sequence or 1.5 kb of 5′ flanking sequence and 1.1 kb of 3′ flanking sequence. Myosin null mutants (mhcA‐ cells) were obtained after transformation with either of these plasmids. The mhcA‐ cells are genetically stable and are capable of a variety of motile processes. Our results provide genetic proof that in Dictyostelium the conventional myosin gene is required for growth in suspension, normal cell division and sporogenesis, and illustrate how gene targeting can be used as a tool in Dictyostelium.
Abstract. To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A.
Abstract. Several members of the rho/rac family of small GTP-binding proteins are known to regulate the distribution of the actin cytoskeleton in various subcellular processes. We describe here a novel rac protein, racE, which is specifically required for cytokinesis, an actomyosin-mediated process. The racE gene was isolated in a molecular genetic screen devised to isolate genes required for cytokinesis in Dictyostelium. Phenotypic characterization of racE mutants revealed that racE is not essential for any other cell motility event, including phagocytosis, chemotaxis, capping, or development. Our data provide the first genetic evidence for the essential requirement of a rho-like protein, specifically in cytokinesis, and suggest a role for these proteins in coordinating cytokinesis with the mitotic events of the cell cycle.T HE intimate association between mitosis and cytokinesis requires a means of coordination between these two processes to insure that the newly duplicated nuclei segregate properly with half of the cytoplasm into the daughter cells. Although much is known about these processes, the mechanism(s) by which they are coordinated remains unknown. The regulation of the mitotic cell cycle has been intensively studied over the last several years. Biochemical and genetic approaches have combined to identify many of the key proteins that control different aspects of the cell cycle. In addition, many of the structural proteins that compose the mitotic apparatus have been characterized. Similarly, much is understood about how cells achieve proper cytoplasmic division. In animal cells, this involves the formation of an equatorial contractile ring that consists largely of actin and myosin and constricts to divide the cell into two . However, it is not understood how these proteins localize to the equator of the cell at the appropriate time and in the correct orientation. From the work of Rappaport (1990), it is clear that the astral microtubules of the mitotic apparatus are intimately involved in determining the placement of the contractile ring. What is not clear is what kind of signals may be involved or how they may be transmitted by the mitotic apparatus to the cell cortex.The rho family of ras-related small GTP-binding proteins (including rho, cdc42, and rac proteins) are known to have profound effects on the actin cytoskeleton (Hall, 1994). Rho proteins have been implicated in the regulation of cytokinesis in both sand dollar (Mabuchi et al., 1993)
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