Mika Toya

|[gamma]|-Tubulin complex-mediated anchoring of spindle microtubules to spindle-pole bodies requires Msd1 in fission yeast

The anchoring of microtubules to subcellular structures is critical for cell polarity and motility. Although the process of anchoring cytoplasmic microtubules to the centrosome has been studied in some detail, it is not known how spindle microtubules are anchored to the mitotic centrosome and, particularly, whether anchoring and nucleation of mitotic spindles are functionally separate. Here, we show that a fission yeast coiled-coil protein, Msd1, is required for anchoring the minus end of spindle microtubules to the centrosome equivalent, the spindle-pole body (SPB). msd1 deletion causes spindle microtubules to abnormally extend beyond SPBs, which results in chromosome missegregation. Importantly, this protruding spindle is phenocopied by the amino-terminal deletion mutant of Alp4, a component of the γ-tubulin complex (γ-TuC), which lacks the potential Msd1-interacting domain. We propose that Msd1 interacts with γ-TuC, thereby specifically anchoring the minus end of microtubules to SPBs without affecting microtubule nucleation.

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

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.

Mal3, the fission yeast EB1 homologue, cooperates with Bub1 spindle checkpoint to prevent monopolar attachment.

Bipolar microtubule attachment is central to genome stability. Here, we investigate the mitotic role of the fission yeast EB1 homologue Mal3. Mal3 shows dynamic inward movement along the spindle, initial emergence at the spindle pole body (SPB) and translocation towards the equatorial plane, followed by sudden disappearance. Deletion of Mal3 results in early mitotic delay, which is dependent on the Bub1, but not the Mad2, spindle checkpoint. Consistently, Bub1, but not Mad2, shows prolonged kinetochore localization. Double mutants between mal3 and a subset of checkpoint mutants, including bub1, bub3, mad3 and mph1, but not mad1 or mad2, show massive chromosome mis‐segregation defects. In mal3bub1 mutants, both sister centromeres tend to remain in close proximity to one of the separating SPBs. Further analysis indicates that mis‐segregated centromeres are exclusively associated with the mother SPB. Mal3, therefore, has a role in preventing monopolar attachment in cooperation with the Bub1/Bub3/Mad3/Mph1‐dependent checkpoint.

Pob1 ensures cylindrical cell shape by coupling two distinct rho signaling events during secretory vesicle targeting.

Proper cell morphogenesis requires the co‐ordination of cell polarity, cytoskeletal organization and vesicle trafficking. The Schizosaccharomyces pombe mutant pob1‐664 has a curious lemon‐like shape, the basis of which is not understood. Here, we found abundant vesicle accumulation in these cells, suggesting that Pob1 plays a role in vesicle trafficking. We identified Rho3 as a multicopy suppressor of this phenotype. Because Rho3 function is related to For3, an actin‐polymerizing protein, and Sec8, a component of the exocyst complex, we analyzed their functional relationship with Pob1. Pob1 was essential for the formation of actin cables (by interacting with For3) and for the polarized localization of Sec8. Although neither For3 nor Sec8 is essential for polarized growth, their simultaneous disruption prevented tip growth and yielded a lemon‐like cell morphology similar to pob1‐664. Thus, Pob1 may ensure cylindrical cell shape of S. pombe by coupling actin‐mediated vesicle transport and exocyst‐mediated vesicle tethering during secretory vesicle targeting.

The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos

Through an RNAi-based modifier screen, we identified the nucleoporin Nup205/NPP-3 as a negative regulator of mitotic onset in Caenorhabditis elegans. Strikingly, NPP-3 is lost from the nuclear envelope at mitotic onset in an AIR-1– and centrosome-dependent manner. We propose a model whereby centrosomes and AIR-1 promote timely mitosis by locally removing NPP-3.

Imaging of mitotic spindle dynamics in Caenorhabditis elegans embryos.

Development of the nematode Caenorhabditis elegans is highly reproducible, and the cell division patterns are virtually invariant. Transparency of the eggshell and cells enables the observation of intracellular events with a high temporal and spatial resolution. These unique features, along with the sophisticated genetic techniques, make this organism one of the most attractive model systems for dissecting regulatory mechanisms of dynamic cellular behaviors, such as mitosis, at an organismal level. In this chapter, we describe immunofluorescence and live imaging methods for analyzing mitotic spindle regulation. In particular, we present the use of double- or triple-labeled fluorescent strains for high-resolution two-dimensional and three-dimensional live imaging to analyze dynamic behaviors of mitotic spindles.

The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A

UBXN-2, a substrate adaptor of the AAA ATPase CDC-48/p97, is required to coordinate centrosome maturation timing with mitosis.

Caenorhabditis elegans ortholog of the p24/p22 subunit, DNC-3, is essential for the formation of the dynactin complex by bridging DNC-1/p150Glued and DNC-2/dynamitin

Dynactin is a multisubunit protein complex required for the activity of cytoplasmic dynein. In Caenorhabditis elegans, although 10 of the 11 dynactin subunits were identified based on the sequence similarities to their orthologs, the p24/p22 subunit has not been detected in the genome. Here, we demonstrate that DNC‐3 (W10G11.20) is the functional counterpart of the p24/p22 subunit in C. elegans. RNAi phenotypes and subcellular localization of DNC‐3 in early C. elegans embryos were nearly identical to those of the known dynactin components. All other dynactin subunits were co‐immunoprecipitated with DNC‐3, indicating that DNC‐3 is a core component of dynactin. Furthermore, the overall secondary structure of DNC‐3 resembles to those of the mammalian and yeast p24/p22. We found that DNC‐3 is required for the localization of the DNC‐1/p150Glued and DNC‐2/dynamitin, the two components of the projection arm of dynactin, to the nuclear envelope of meiotic nuclei in the adult gonad. Moreover, DNC‐3 physically interacted with DNC‐1 and DNC‐2 and significantly enhanced the binding ability between DNC‐1 and DNC‐2 in vitro. These results suggest that DNC‐3 is essential for the formation of the projection arm subcomplex of dynactin.