Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell.

We characterized the novel Schizosaccharomyces pombe genes myo4(+) and myo5(+), both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

Identification and functional analysis of the gene for type I myosin in fission yeast.

BACKGROUND: Type I myosin is highly conserved among eukaryotes, and apparently plays important roles in a number of cellular processes. In the budding yeast, two myosin I species have been identified and their role in F-actin assembly has been inferred. RESULTS: We cloned the fission yeast myo1 gene, which apparently encoded a myosin I protein. Disruption of myo1 was not lethal, but it caused growth retardation at high and low temperatures, sensitivity to a high concentration of KCl, and aberrance in cell morphology associated with an abnormal distribution of F-actin patches. An abnormal deposition of cell wall materials was also seen. Homothallic myo1Delta cells could mate, but heterothallic myo1Delta cells were poor in conjugation. Myo1p was necessary for the encapsulation of spores. The tail domain of Myo1p was pivotal for its function. Calmodulin could bind to Myo1p through the IQ domain at the neck. CONCLUSIONS: Myo1p appears to control the redistribution of F-actin patches during the cell cycle. Loss of Myo1p function is likely to slow down the actin assembly/disassembly process, which results in a failure of the actin cycle to catch up with other events in both the mitotic and meiotic cell cycles, including extension of the conjugation tubes.

Contractile ring formation in Xenopus egg and fission yeast.

How actin filaments (F-actin) and myosin II (myosin) assemble to form the contractile ring was investigated with fission yeast and Xenopus egg. In fission yeast cells, an aster-like structure composed of F-actin cables is formed at the medial cortex of the cell during prophase to metaphase, and a single F-actin cable(s) extends from this structure, which seems to be a structural basis of the contractile ring. In early mitosis, myosin localizes as dots in the medial cortex independently of F-actin. Then they fuse with each other and are packed into a thin contractile ring. At the growing ends of the cleavage furrow of Xenopus eggs, F-actin at first assembles to form patches. Next they fuse with each other to form short F-actin bundles. The short bundles then form long bundles. Myosin seems to be transported by the cortical movement to the growing end and assembles there as spots earlier than F-actin. Actin polymerization into the patches is likely to occur after accumulation of myosin. The myosin spots and the F-actin patches are simultaneously reorganized to form the contractile ring bundles. The idea that a Ca signal triggers cleavage furrow formation was tested with Xenopus eggs during the first cleavage. We could not detect any Ca signals such as a Ca wave, Ca puffs or even Ca blips at the growing end of the cleavage furrow. Furthermore, cleavages are not affected by Ca-chelators injected into the eggs at concentrations sufficient to suppress the Ca waves. Thus we conclude that formation of the contractile ring is not induced by a Ca signal at the growing end of the cleavage furrow.

The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p.

In order to identify additional components important for cell division in the fission yeast Schizosaccharomyces pombe we have screened a bank of conditional cold-sensitive mutants for cytokinesis defects. One of these mutants showed a delay in cell cleavage, and strong genetic interactions with other genes implicated in medial ring formation. Cloning of the corresponding gene indicates that it encodes a protein with significant homology to the regulatory light chain of non-muscle myosins. We have named the gene rlc1 (regulatory light chain 1). The gene is not essential for division, but null mutants display a cell cleavage defect and form an aberrant F-actin ring. Two myosin-II heavy chains have been identified in fission yeast: Co-immunoprecipitation experiments indicate that rlc1p associates more strongly with myo3p than myo2p.

Molecular mechanism of myosin-II assembly at the division site in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells divide by virtue of the F-actin-based contractile ring (F-actin ring). Two myosin-II heavy chains, Myo2 and Myp2/Myo3, have been localized to the F-actin ring. Here, we investigated the mechanism of myosin-II assembly at the division site in S. pombe cells. First, we showed that Cdc4, an EF-hand protein, appears to be a common myosin light chain associated with both Myo2 and Myo3. Loss of function of both Myo2 and Myo3 caused a defect in F-actin assembly at the division site, like the phenotype of cdc4 null cells. It is suggested that Myo2, Myo3 and Cdc4 function in a cooperative manner in the formation of the F-actin ring during mitosis. Next, we investigated the dynamics of myosin-II during mitosis in S. pombe cells. In early mitosis when accumulation of F-actin cables in the medial region was not yet observed, Myo2 was detected primarily as dots widely located in the medial cortex. Myo2 fibers also became visible following the appearance of the dots. The Myo2 dots and fibers then fused with each other to form a medial cortical network. Some Myo2 dots appeared to be localized with F-actin cables which are also accumulated in the medial region. Finally these structures were packed into a thin contractile ring. In mutant cells that cannot form the F-actin ring such as cdc3(ts), cdc8(ts) and cdc12(ts), Myo2 was able to accumulate as dots in the medial cortex, whereas no accumulation of Myo2 dots was detected in cdc4(ts) cells. Moreover, disruption of F-actin in the cell by applying latrunculin-A did not affect the accumulation of Myo2 dots, suggesting that F-actin is not required for their accumulation. A truncated Myo2 which lacks putative Cdc4-binding sites (Myo2dIQs) was able to rescue myo2 null cells, myo3 null cells, cdc4(ts) mutant cells and cdc4 null cells. The Myo2dIQs could assemble into a normal-shaped ring in these cells. Therefore, its assembly at the division site does not require the function of either Cdc4 or Myo3.

Overproduction of elongation factor 1alpha, an essential translational component, causes aberrant cell morphology by affecting the control of growth polarity in fission yeast.

BACKGROUND: Elongation factor 1alpha (EF1alpha), an essential component of the eukaryotic translational machinery, has been shown to possess various biochemical and biological activities, including F-actin-binding and -bundling, microtubule- severing, and the activity of making fibroblasts highly susceptible to transformation. However, our understanding of the biological significance of EF1alpha with respect to these various biochemical or biological activities remains limited. Here we report the identification of EF1alpha-encoding genes as genes whose over-expression causes aberrant cell morphology in fission yeast. RESULTS: Overproduction of EF1alpha caused aberrant cell morphology-elliptic, curved or branched-and growth defects in yeast cells at high temperatures. EF1alpha-overproducing cells showed a supersensitivity to the actin inhibitor cytochalasin D and to the tubulin inhibitor thiabendazole. Genetic analyses using cdc mutants suggested that excess EF1alpha disturbed the establishment and the maintenance of growth polarity in the G1 phase by pre- venting the localization of F-actin to the polarized growing site and the organization of microtubules. Results from DNase I column chromatography indicated that EF1alpha was bound to G-actin. Indeed, the fission yeast actin was immunoprecipitated along with EF1alpha. Moreover, the temperature sensitivity caused by the overproduction of EF1alpha was restored by co-overproduction of actin. CONCLUSIONS: Fission yeast EF1alpha has the ability to alter the cell morphology of yeast by affecting the control of actin and microtubule cytoskeletons.

Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe.

We cloned the myo3+ gene of Schizosaccharomyces pombe which encodes a type-II myosin heavy chain. myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F-actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F-actin ring in S. pombe.