Direct Evidence for a Critical Role of Myosin II in Budding Yeast Cytokinesis and the Evolvability of New Cytokinetic Mechanisms in the Absence of Myosin II

In the budding yeast Saccharomyces cerevisiae, an actomyosin-based contractile ring is present during cytokinesis, as occurs in animal cells. However, the precise requirement for this structure during budding yeast cytokinesis has been controversial. Here we show that deletion of MYO1, the single myosin II gene, is lethal in a commonly used strain background. The terminal phenotype of myo1Δ is interconnected chains of cells, suggestive of a cytokinesis defect. To further investigate the role of Myo1p in cytokinesis, we conditionally disrupted Myo1 function by using either a dominant negative Myo1p construct or a strain where expression of Myo1p can be shut-off. Both ways of disruption of Myo1 function result in a failure in cytokinesis. Additionally, we show that a myo1Δ strain previously reported to grow nearly as well as the wild type contains a single genetic suppressor that alleviates the severe cytokinesis defects of myo1Δ. Using fluorescence time-lapse imaging and electron microscopy techniques, we show that cytokinesis in this strain is achieved through formation of multiple aberrant septa. Taken together, these results strongly suggest that the actomyosin ring is crucial for successful cytokinesis in budding yeast, but new cytokinetic mechanisms can evolve through genetic changes when myosin II function is impaired

Zwilch, a New Component of the ZW10/ROD Complex Required for Kinetochore Functions

The Zeste-White 10 (ZW10) and Rough Deal (ROD) proteins are part of a complex necessary for accurate chromosome segregation. This complex recruits cytoplasmic dynein to the kinetochore and participates in the spindle checkpoint. We used immunoaffinity chromatography and mass spectroscopy to identify the Drosophila proteins in this complex. We found that the complex contains an additional protein we name Zwilch. Zwilch localizes to kinetochores and kinetochore microtubules in a manner identical to ZW10 and ROD. We have also isolated a zwilch mutant, which exhibits the same mitotic phenotypes associated with zw10 and rod mutations: lagging chromosomes at anaphase and precocious sister chromatid separation upon activation of the spindle checkpoint. Zwilch's role within the context of this complex is evolutionarily conserved. The human Zwilch protein (hZwilch) coimmunoprecipitates with hZW10 and hROD from HeLa cell extracts and localizes to the kinetochores at prometaphase. Finally, we discuss immunoaffinity chromatography results that suggest the existence of a weak interaction between the ZW10/ROD/Zwilch complex and the kinesin-like kinetochore component CENP-meta

Molecular Dissection of Cytokinesis by RNA Interference in Drosophila Cultured Cells

We have used double-stranded RNA-mediated interference (RNAi) to study Drosophila cytokinesis. We show that double-stranded RNAs for anillin, acGAP, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. Our phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and lead us to propose that the central spindle and the contractile ring are interdependent structures. Finally, our results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis

The Third P-loop Domain in Cytoplasmic Dynein Heavy Chain Is Essential for Dynein Motor Function and ATP-sensitive Microtubule Binding

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice