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.

Sequences in the myosin II tail required for self-association.

The assembly of myosin II molecules into a filament requires the electrostatic interaction of a domain localized toward the carboxyl-terminus of the myosin II tail. However, the precise sequences involved in this interaction are not known. Here we show that the smallest Dictyostelium myosin II fragment that is necessary and sufficient for self-association is a fragment of 294 amino acids that contains four clusters of positively charged and negatively charged residues. Fragments of the same length but which lack one of these positively or negatively charged clusters are incapable of self-assembly. We postulate that this assembly domain is also found in myosin II from other species. Such charged clusters are found in a similar location in rabbit myosin II and are also essential for filament formation.