Disruption of the Dictyostelium Myosin Heavy Chain Gene by Homologous Recombination

Abstract

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.

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum

“…This process occurred in the interphase state of the cell cycle (Fig. 6C, middle and right panels) and resembled the traction-mediated cytofission observed in other mutants of D. discoideum [2,13,14]. By traction-mediated cytofission, portions of a multi-nucleated cell are pinched off due to the active movement into different directions of multiple leading edges [15].…”

“…Mutants of D. discoideum known to be disturbed in cytokinesis fall into one of two categories. (1) Mutants defective in proteins like myosin II [13,14] and the actin-bundling proteins cortexillin I and II [2], that contribute to the contractile activities or the mechanical properties of the cell cortex. (2) Mutants altered in proteins that play a regulatory role in mitosis.…”

Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis.

“…Dictyostelium amoebae are haploid, and many genes, including calmodulin (Goldhagen and Clarke, 1986), that are present in multiple copies in mammalian cells are single-copy in Dictyostelium. Thus, for several Dictyostelium proteins, it has been possible to manipulate gene expression by deleting or altering the appropriate genomic sequences via homologous recombination (DeLozanne and Spudich, 1987;Witke et al, 1987;Manstein et al, 1989;Jung and Hammer, 1990;Doring et al, 1991;Kumagai et al, 1991;Sun and Devreotes, 1991;Cox et al, 1992). Reduction of mRNA levels by the introduction of vectors producing antisense RNA also has proved effective.…”

Loss of Cell Adhesion Causes Hydrocephalus in Nonmuscle Myosin II-B–ablated and Mutated Mice

“…Nonmuscle myosin (NM) II, one of the major cytoskeletal motor proteins, plays an important role in cell migration (Svitkina et al, 1997;Ma et al, 2004;Even-Ram et al, 2007;Vicente-Manzanares et al, 2007), cell-cell adhesion Shewan et al, 2005;Giannone et al, 2007), and cell division (De Lozanne and Spudich, 1987;Takeda et al, 2003;Bao et al, 2005). The molecular structure of NM II is a hexamer consisting of a pair of myosin heavy chains (200 kDa) and two pairs of light chains (20 and 17 kDa).…”

“…The defect may be accompanied by an obstructed aqueduct of Sylvius, the narrow channel connecting the third and fourth brain ventricles (noncommunicating hydrocephalus), or by a normal aqueduct (communicating hydrocephalus). The pathogenesis of most cases of communicating hydrocephalus is largely unknown (for reviews, see Perez-Figares et al, 2001;Crews et al, 2004).Nonmuscle myosin (NM) II, one of the major cytoskeletal motor proteins, plays an important role in cell migration (Svitkina et al, 1997;Ma et al, 2004;Even-Ram et al, 2007;Vicente-Manzanares et al, 2007), cell-cell adhesion Shewan et al, 2005;Giannone et al, 2007), and cell division (De Lozanne and Spudich, 1987;Takeda et al, 2003;Bao et al, 2005). The molecular structure of NM II is a hexamer consisting of a pair of myosin heavy chains (200 kDa) and two pairs of light chains (20 and 17 kDa).…”