Role of myosin II tail sequences in its function and localization at the cleavage furrow in Dictyostelium.

Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: Mydelta824-941, Mydelta943-1464, Mydelta943-1194 and Mydelta1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, except for Mydelta943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggest that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow.

Functional analysis of a cardiac myosin rod in Dictyostelium discoideum.

Manipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament-forming, or light meromyosin (LMM) domain of the myosin molecule in cytokinesis, development, and in the capping of cell surface receptors (see Spudich: Cell Regulation 1:1-11, 1989; Egelhoff et al.: Journal of Cell Biology, 112:677-688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat beta cardiac LMM was transfected into both wild-type and Dd mhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild-type and Dd mhc null cells. Although null cells expressing chimeric mhc at approximately 10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility.

Twenty-one polymorphic markers from human chromosome 12 for integration of genetic and physical maps.

Twenty-one physically mapped, polymorphic markers have been developed from a chromosome 12-specific cosmid library. The markers consist of CA repeat-containing sequence-tagged sites (STSs) derived from cosmid clones mapped by fluorescence in situ hybridization (FISH). Three methods for determining the sequence flanking CA microsatellites were used, including one using degenerate primer sets for direct sequence analysis. Oligonucleotide primer pairs suitable for use in polymerase chain reaction (PCR) were selected from the sequences flanking the CA microsatellite and were tested for their ability to generate unique PCR products. The informativeness of these STSs as genetic markers was determined by typing 10 unrelated individuals who are part of the Centre d'Etude du Polymorphisme Humaine (EPH) pedigrees. Eleven of the 21 FISH-mapped, polymorphic STSs are heterozygous in 7 or more of the individuals tested. Since these markers are derived from physically mapped cosmids, genetic linkage analysis with them will facilitate the integration of the developing physical and genetic maps of chromosome 12.