DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium

We have identified a novel gene, dwwA, which is required for cytokinesis of Dictyostelium cells on solid surfaces. Its product, Dd WW domain containing protein A (DWWA), contains several motifs, including two WW domains, an IQ motif, a C2 domain, and a proline-rich region. On substrates, cells lacking dwwA were multinucleated and larger and flatter than wild-type cells due to their frequent inability to sever the cytoplasmic bridge connecting daughter cells after mitosis. When cultured in suspension, however, dwwA-null cells seemed to carry out cytokinesis normally via a process not driven by the shearing force arising from agitation of the culture. GFP-DWWA localized to the cell cortex and nucleus; analysis of the distributions of various truncation mutants revealed that the N-terminal half of the protein, which contains the C2 domain, is required for the cortical localization of DWWA. The IQ motif of DWWA binds calmodulin in vitro. Given that the scission process is also defective in calmodulin knockdown cells cultured on substrates (Liu et al., 1992), we propose that DWWA's multiple binding domains enable it to function as an adaptor protein, facilitating the scission process through the regulation of Ca(2+)/calmodulin-mediated remodeling of the actin cytoskeleton and/or modulation of membrane dynamics

Living microtransporter by uni-directional gliding of Mycoplasma along microtracks

The gliding bacterium Mycoplasma mobile adheres to plastic surfaces, and moves around vigorously. However, it has not been possible to control the direction of movements on plain surfaces. Here we report that, on patterned lithographic substrates, M. mobile cells are unable to climb tall walls, and move along the bottom edge of the walls. This property to move persistently along walls enabled us to design patterns that control direction of movements, resulting in uni-directional circling or one-way gating between two areas. Furthermore, cells loaded with streptavidin beads following biotinylation of surface proteins moved at normal speeds. These bacteria could be useful as living microtransporters, carrying cargo around within micrometer-scale spaces

Adhesion-dependent and Contractile Ring-independent Equatorial Furrowing during Cytokinesis in Mammalian Cells

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 μM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 μM), which inhibits Rho-kinase, was similar to 30 μM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 μM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 μM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis

Role of Myosin II Tail Sequences in its Function and Localization at the Cleavage Furrow in \u3ci\u3eDictyostelium\u3c/i\u3e

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: My∆824-941, My∆943-1464, My∆943-1194 and My∆1156- 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 My∆943- 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

Multiple Myosin II Heavy Chain Kinases: Roles in Filament Assembly Control and Proper Cytokinesis in Dictyostelium

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B(-)/C(-) triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified