Isolation and characterization of a novel cytokinesis-deficient mutant in Dictyostelium discoideum.
Cytokinesis is a dramatic event in the life of any cell during which numerous mechanisms must coordinate the legitimate and complete mechanical separation into two daughter cells. We have used Dictyostelium discoideum as a model system to study this highly orchestrated event through genetic analysis. Transformants were generated using a method of insertional mutagenesis known as restriction enzyme-mediated integration (REMI) and subsequently screened for defects in cytokinesis. Mutants isolated in a similar screen suffered a disruption in the myosin II heavy chain gene, a protein known to be essential for cytokinesis and in a novel gene encoding a rho-like protein termed racE [Larochelle et al., 1996]. In the screen reported here we isolated a third type of mutant, called 10BH2, which also had a complete defect in cytokinesis. 10BH2 mutant cells are able to propagate on tissue culture plates by fragmenting into smaller cells by a process known as traction-mediated cytofission. However, when grown in suspension culture, 10BH2 cells fail to divide and become large and multinucleate. Phenotypic characterization of the mutant cells showed that other cytoskeletal functions are preserved. The distribution of myosin and actin is identical to wild type cells. The cells can chemotax, phagocytose, cap crosslinked receptors, and contract normally. However, the 10BH2 mutants are unable to complete the Dictyostelium developmental program beyond the finger stage. The mutant cells contain functional genes for myosin II heavy and light chains and the racE gene. Thus, based on these findings, we conclude that 10BH2 represents a novel cytokinesis-deficient mutant.
Vithalani
K K
KK
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Shoffner
J D
JD
De Lozanne
A
A
D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics*; D003001:Cloning, Molecular; D003208:Concanavalin A; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics*; D004023:Dictyostelium / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D019204:GTP-Binding Proteins / Q000032:analysis; D018628:Gene Dosage; D015321:Gene Rearrangement; D017125:Genes, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D009218:Myosins / Q000032:analysis / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D020744:rac GTP-Binding Proteins
D000199:Actins; D016054:DNA, Protozoan; C100414:RacE protein, Dictyostelium discoideum; D003208:Concanavalin A; D012343:RNA, Transfer; D019204:GTP-Binding Proteins; D009218:Myosins; D020744:rac GTP-Binding Proteins