Masamitsu Sato

New drug‐resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe

We describe new heterologous modules for PCR‐based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug‐resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999 ). Vector modules for gene disruption and C‐terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a–MX6–derived plasmids (Bähler et al., 1998 ; Wach et al., 1997 ). In combination with the existing systems that are based upon the G418‐resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one‐step integration of a monomeric RFP (mRFP) to the C‐terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post‐genomic molecular analysis of protein functions in fission yeast. Copyright

Phosphorylation of Mei2 and Ste11 by Pat1 Kinase Inhibits Sexual Differentiation via Ubiquitin Proteolysis and 14-3-3 Protein in Fission Yeast

Fission yeast Pat1 kinase inhibits sexual differentiation by phosphorylating the meiotic inducer Mei2 and the transcription factor Ste11. Here, we show how Pat1 downregulates these proteins. Mei2 is degraded via a ubiquitin-proteasome pathway in a phosphorylation-dependent fashion. The E2 Ubc2 and the E3 Ubr1 are required for this proteolysis. In addition, Pat1 negatively regulates Ste11 via Rad24/14-3-3, thereby repressing mei2+ transcription. The Pat1 phosphorylation sites of Ste11 match the consensus recognition sequence for 14-3-3. Rad24 binds preferentially to phosphorylated Ste11, and this binding results in inhibition of the transcriptional activation capacity of Ste11. Overall, therefore, these results show that Pat1 coordinates concerted molecular mechanisms that govern the sexual differentiation developmental decision.

Alp7/TACC is a crucial target in Ran-GTPase-dependent spindle formation in fission yeast.

Microtubules are essential intracellular structures involved in several cellular phenomena, including polarity establishment and chromosome segregation. Because the nuclear envelope persists during mitosis (closed mitosis) in fission yeast (Schizosaccharomyces pombe), cytoplasmic microtubules must be reorganized into the spindle in the compartmentalized nucleus on mitotic entry. An ideal mechanism might be to take advantage of an evolutionarily conserved microtubule formation system that uses the Ran-GTPase nuclear transport machinery, but no targets of Ran for spindle formation have been identified in yeast. Here we show that a microtubule-associated protein, Alp7, which forms a complex with Alp14, is a target of Ran in yeast for spindle formation. The Ran-deficient pim1 mutant (pim1-F201S) failed to show mitosis-specific nuclear accumulation of Alp7. Moreover, this mutant exhibited compromised spindle formation and early mitotic delay. Importantly, these defects were suppressed by Alp7 that was artificially targeted to the nucleus by a Ran-independent and importin-α-mediated system. Thus, Ran targets Alp7–Alp14 to achieve nuclear spindle formation, and might differentiate its targets depending on whether the organism undergoes closed or open mitosis.

|[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.

Fission yeast Pcp1 links polo kinase-mediated mitotic entry to γ-tubulin-dependent spindle formation

The centrosomal pericentrin‐related proteins play pivotal roles in various aspects of cell division; however their underlying mechanisms remain largely elusive. Here we show that fission‐yeast pericentrin‐like Pcp1 regulates multiple functions of the spindle pole body (SPB) through recruiting two critical factors, the γ‐tubulin complex (γ‐TuC) and polo kinase (Plo1). We isolated two pcp1 mutants (pcp1‐15 and pcp1‐18) that display similar abnormal spindles, but with remarkably different molecular defects. Both mutants exhibit defective monopolar spindle microtubules that emanate from the mother SPB. However, while pcp1‐15 fails to localise the γ‐TuC to the mitotic SPB, pcp1‐18 is specifically defective in recruiting Plo1. Consistently Pcp1 forms a complex with both γ‐TuC and Plo1 in the cell. pcp1‐18 is further defective in the mitotic‐specific reorganisation of the nuclear envelope (NE), leading to impairment of SPB insertion into the NE. Moreover pcp1‐18, but not pcp1‐15, is rescued by overproducing nuclear pore components or advancing mitotic onset. The central role for Pcp1 in orchestrating these processes provides mechanistic insight into how the centrosome regulates multiple cellular pathways.

Spatial segregation of polarity factors into distinct cortical clusters is required for cell polarity control

Cell polarity is regulated by evolutionarily conserved polarity factors whose precise higher-order organization at the cell cortex is largely unknown. Here we image frontally the cortex of live fission yeast cells using time-lapse and super-resolution microscopy. Interestingly, we find that polarity factors are organized in discrete cortical clusters resolvable to ~50–100 nm in size, which can form and become cortically enriched by oligomerization. We show that forced co-localization of the polarity factors Tea1 and Tea3 results in polarity defects, suggesting that the maintenance of both factors in distinct clusters is required for polarity. However, during mitosis, their co-localization increases, and Tea3 helps to retain the cortical localization of the Tea1 growth landmark in preparation for growth reactivation following mitosis. Thus, regulated spatial segregation of polarity factor clusters provides a means to spatio-temporally control cell polarity at the cell cortex. We observe similar clusters in Saccharomyces cerevisiae and Caenorhabditis elegans cells, indicating this could be a universal regulatory feature.

The fission yeast meiotic regulator Mei2p undergoes nucleocytoplasmic shuttling

Schizosaccharomyces pombe Mei2p is an RNA‐binding protein that switches the cell cycle from mitotic to meiotic. Mei2p forms a unique dot in the nucleus prior to meiosis I, aided by a non‐coding RNA molecule termed meiRNA. Here we show that Mei2p intrinsically undergoes nucleocytoplasmic shuttling. Artificial acceleration of nuclear migration of Mei2p advances nuclear dot formation, but meiRNA does not appear to promote the dot formation by modulating the migration rate of Mei2p into the nucleus. Rather, this RNA is likely to facilitate the assembly of Mei2p into a dot structure and trap the protein as such in the nucleus.

Spindle pole body components are reorganized during fission yeast meiosis

We show that spindle pole body (SPB) remodeling during meiosis in fission yeast is essential for meiosis. Many SPB components disappear during meiotic prophase and return to the SPBs at meiosis I onset. We found novel functions for Polo kinase/Plo1 and centrin/Cdc31 in the meiotic reorganization of SPB components.

Nuclear Compartmentalization Is Abolished during Fission Yeast Meiosis

In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells. It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle. Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.

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.