Antisense RNA Inactivation of Myosin Heavy Chain Gene Expression in Dictyostelium discoideum

Abstract

The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.

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.…”

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility

“…If the gene product is not essential for viability, the resulting variant can be scrutinized for phenotypic abnormalities. This approach has been successfully employed for the myosin I1 heavy chain [De Lozanne and Spudich, 1987;Knecht and Loomis, 1987;Manstein et al, 19891, a-actinin [Noegel et al, 19891 and ABP 120 [Brink et al, 1990;Knecht et al, 19901 genes of Dictyostelium. However, applying this approach to myosin I heavy chain function may be more complicated since there are at least five myosin I heavy chain genes in Dictyostelium Jung and Hammer, 19901, and the possibility therefore exists that disruption of one gene will be compensated for by the product of another in the gene family.…”

“…Disruption of the myosin I1 gene by homologous recombination [De Lozanne and Spudich, 1987;Manstein et al, 19891 resulted in a viable cell defective in cytokinesis, multicellular morphogenesis, and cellular translocation [De Lozanne and Spudich, 1987;Knecht and Loomis, 1987;Wessels et al, 19881. Computer-assisted analysis of intracellular and cellular motility, and immunofluorescent studies of the localization of F-actin, demonstrated that the absence of myosin I1 affected 1) cell shape, 2) cell velocity, 3) the dynamics of pseudopod extension, 4) the polarity of pseudopod extension, 5 ) intracellular particle velocity and directionality, and 6) the rapid inhibition of intracellular particle movement by 1Op6M cAMP Wessels and Soll, 1990;Soll et a]., 1990;Fukui et al, 19901. In contrast to the single copy myosin I1 heavy chain gene, disruption of any one myosin I heavy chain gene may have no significant effect on phenotype if the products of the other myosin I genes can functionally substitute for each other. Indeed, disruption of the myosin IB gene resulted in a viable cell capable of cytokinesis, chemotactic aggregation, and multicellular morphogenesis [Jung and Hammer, 19901.…”

Regulated Expression of the Centrosomal Protein DdCP224 Affects Microtubule Dynamics and Reveals Mechanisms for the Control of Supernumerary Centrosome Number

“…Promoter activity is high when cells are grown in axenic medium and low if they are grown at low density in bacterial suspension (Knecht and Loomis, 1987;Liu et al, 2002). Two independent clones with the desired promoter replacement were selected; one of them (named A3) was used for all experiments.…”