Masamitsu Sato

• PhD (University of Tokyo)
• Professor at Waseda University

In mammalian epithelial cells, cytoplasmic microtubules are mainly non-centrosomal, through the functions of the minus-end binding proteins CAMSAP2 and CAMSAP3. When cells enter mitosis, cytoplasmic microtubules are reorganized into the spindle composed of both centrosomal and non-centrosomal microtubules. The function of the CAMSAP proteins upon s...

The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP21...

Non-coding RNAs (ncRNAs) ubiquitously exist in normal and cancer cells. Despite their prevalent distribution, the functions of most long ncRNAs remain uncharacterized. The fission yeast Schizosaccharomyces pombe expresses >1800 ncRNAs annotated to date, but most unconventional ncRNAs (excluding tRNA, rRNA, snRNA and snoRNA) remain uncharacterized....

Centromeres are established by nucleosomes containing the histone H3 variant CENP-A. CENP-A is recruited to centromeres by the Mis18–HJURP machinery. During mitosis, CENP-A recruitment ceases, implying the necessity of CENP-A maintenance at centromeres, although the exact underlying mechanism remains elusive. Herein, we show that the inner kinetoch...

The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP21...

Centromeres are established by nucleosomes containing the histone H3 variant CENP-A. CENP-A is recruited to centromeres by the Mis18-HJURP machinery. During mitosis, CENP-A recruitment ceases, implying the necessity of CENP-A maintenance at centromeres, although the exact underlying mechanism remains elusive. Herein, we show that the kinetochore pr...

Dormancy breaking is a common physiological phenomenon that is shared by eukaryotes. Germination of spores in fungi is one of the most representative cases of dormancy breaking. Understanding the mechanisms of spore germination is therefore fundamental to basic studies on the control of cell proliferation and differentiation, as well as agricultura...

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a ‘transition zone’. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how th...

The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microt...

Meiosis is a specialized style of cell division conserved in eukaryotes, particularly designed for the production of gametes. A huge number of studies to date have demonstrated how chromosomes behave and how meiotic events are controlled. Yeast substantially contributed to the understanding of the molecular mechanisms of meiosis in the past decades...

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were sev...

CRISPR/Cas9 is a powerful tool for genome editing. Several studies have been conducted to take the benefit of the versatile tool in the fission yeast Schizosaccharomyces pombe. However, the protocols for the CRISPR/Cas9 system proposed in previous studies are complicated in culture conditions compared to traditional genome editing methods. In this...

How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are l...

Microtubules in the mitotic spindle are organised by microtubule-associated proteins. In the late stage of mitosis, spindle microtubules are robustly organised through bundling by the antiparallel microtubule bundler Ase1/PRC1. In early mitosis, however, it is not well characterised whether spindle microtubules are actively bundled, as Ase1 does no...

Bipolar spindles are organized by motor proteins that generate microtubule-dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end directed motor Pkl1 (kinesin-14) generates the inward force. Balanced...

How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are l...

The fission yeast Schizosaccharomyces pombe is a powerful model organism for cell biology and molecular biology, as genetic manipulation is easily achieved. Introduction of exogenous genes cloned in episomal plasmids into yeast cells can be done through well-established transformation methods. For expression of genes in S. pombe cells, the multi-co...

A key goal of functional genomics is to elucidate how genes and proteins act together in space and time, wired as pathways, to control specific aspects of cell biological function. Here, we develop a method to quantitatively determine proteins' localization interdependencies at high throughput. We show that this method can be used to systematically...

Meiosis is a specialised cell division process for generating gametes. In contrast to mitosis, meiosis involves recombination followed by two consecutive rounds of cell division, meiosis I and II. A vast field of research has been devoted to understanding the differences between mitotic and meiotic cell divisions from the viewpoint of chromosome be...

Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase...

For proper partitioning of genomes in mitosis, all chromosomes must be aligned at the spindle equator before the onset of anaphase. The spindle assembly checkpoint (SAC) monitors this process, generating a 'wait anaphase' signal at unattached kinetochores of misaligned chromosomes. However, the link between SAC activation and chromosome alignment i...

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration pla...

Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in S. pombe, from anaphase to early septation, and throughout G2 phase...

Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulat...

BackgroundMeiotic cells undergo two rounds of nuclear division and generate gametes. Previous studies have indicated that a number of transcription factors modulate the transcriptome in successive waves during meiosis and spore formation in fission yeast. However, the mechanisms underlying the post-transcriptional regulation in meiosis are not full...

Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in th...

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid res...

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. Cytoplasmic microtubule arrays during interphase are reorganized into the spindle at mitotic onset. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7-Alp14 (TACC-TOG), enters the nucleus upon...

Faithful chromosome segregation during meiosis is indispensable to prevent birth defects and infertility. Canonical genetic manipulations have not been very useful for studying meiosis II, since mutations of genes involved in cell cycle regulation or chromosome segregation may affect meiosis I, making interpretations of any defects observed in meio...

Meiosis comprises two consecutive nuclear divisions, meiosis I and II. Despite this unique progression through the cell cycle, little is known about the mechanisms controlling the sequential divisions. In this study, we carried out a genetic screen to identify factors that regulate the initiation of meiosis II in the fission yeast Schizosaccharomyc...

Cell polarity—the intrinsic property of cells to take different shape, migration or growth patterns suited to their function—is one of those things intuitively we are all aware of. It is part of our collective imagination, and indeed if prompted all of us immediately think of cells as

Timing of cell division is coordinated by the Septation Initiation Network (SIN) in fission yeast. SIN activation is initiated at the two spindle pole bodies (SPB) of the cell in metaphase, but only one of these SPBs contains an active SIN in anaphase, while SIN is inactivated in the other by the Cdc16-Byr4 GAP complex. Most of the factors that are...

Tethering kinetochores at spindle poles facilitates their efficient capture and segregation by microtubules at mitotic onset in yeast. During meiotic prophase of fission yeast, however, kinetochores are detached from the poles, which facilitates meiotic recombination but may cause a risk of chromosome mis-segregation during meiosis. How cells circu...

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 cluste...

Supplementary Figures S1-S8, Supplementary Table S1, Supplementary Methods and Supplementary References

Tea3-3GFP node movement at the cell ends imaged by conventional microscopy. Images were taken every 1 s for 60 s and are shown at 7 frames per second. Movie created from maximum intensity projections of 3D-deconvolved, wide-field z-stacks.

A Tea1 packet is carried on a microtubule tip and deposited at the cell end as a single observable node. Tea1-GFP with mCh-Atb2 labelled microtubules. Movie is from images in Fig. 2a. Images were taken every 3 s and are shown at 7 frames per second. Movie created from maximum intensity projections of 3D-deconvolved, wide-field z-stacks.

In silico computer simulation of cluster formation of two polarity factors. Simulation of two polarity factors (red, green). In the first sequence the red polarity factor can oligomerize and is deposited to the cell cortex by microtubules in localized sites. In the second sequence red can oligomerize but is deposited uniformly everywhere onto the c...

Tea1-3mCh nodes visually co-localizing with Tea3-GFP nodes in time.Tea3-GFP and Tea1-3mCh cells frontally on the OMX widefield microscope in conventional mode. 2 μm thick stacks at 125 nm intervals were taken every 3 s for 30 s. Video is created from maximum intensity projections of 3D-deconvolved, wide-field z- stacks and shown at 3.5 frames per s...

Cell cycle stage determination using SPB separation. A single, unseparated SPB indicates that the cell is in interphase. The movie corresponds to the image sequence in Supplementary Fig. S7c top. It is of an 18 ?m stack taken at 0.125 ?m intervals of an Alp6-GFP Tea3-GFP Tea1-3mCh head-on cell in the GFP channel. Individual planes are 3-D deconvolv...

Cell cycle stage determination using SPB separation. Two separated SPBs within a 4 ?m radius of each other indicate that the cell is in mitosis. The movie corresponds to the image sequence in Supplementary Fig. S7c bottom. It is of an 18 ?m stack taken at 0.125 ?m intervals of an Alp6-GFP Tea3-GFP Tea1-3mCh head-on cell in the GFP channel. Individu...

Par6-GFP clusters in a two cell stage C. elegans embryo. Movie is of a 3-D deconvolved max-intensity projection of a 2 ?m stack taken at 0.125 ?m intervals. Time-steps are every 0.8 s for 8 s and are shown at 7 frames per second.

Tea1-3mCh nodes visually co-localizing with GFP-Mod5 nodes in time. Movie is from images in Fig. 2b. Images were taken every 3 s and are shown at 7 frames per second. Movie created from maximum intensity projections of 3D-deconvolved, wide-field z-stacks.

In Tea1-GBP-mCh Tea3-GFP cells, both node populations remained at the cell cortex over relatively long time periods and became gradually displaced from the cell end cortex to the cell sides. Movie is of a 6 ?m OAI stack in the GFP channel only, taken at 360 second time-steps and shown at 7 frames per second.

Kel1-GFP clusters in S. cerevisiae. Movie is of a 3-D deconvolved max-intensity projection of a 2 ?m stack taken at 0.125 ?m intervals. Time-steps are every 0.85 s for 8.5 s and are shown at 7 frames per second.

Tea1-3GFP node movement at the cell ends imaged by conventional microscopy. Stacks were taken every 1.05 s for 61 s and are shown at 7 frames per second. Movie created from maximum intensity projections of 3D-deconvolved, wide-field z-stacks.

Tea1-3mCh nodes visually co-localizing with GFP-Mod5 nodes in time. Images were taken every 3 s and are shown at 7 frames per second. Movie created from maximum intensity projections of 3D-deconvolved, wide-field z-stacks.

Spa2-GFP clusters in S. cerevisiae. Movie is of a 3-D deconvolved max-intensity projection of a 2 ?m stack taken at 0.125 ?m intervals. Time-steps are every 0.76 s for 7.6 s and are shown at 7 frames per second.

The number of nuclear divisions in meiosis is strictly limited to two. Although the precise mechanism remains unknown, this seems to be achieved by adjusting the anaphase-promoting complex/cyclosome (APC/C) activity to degrade cyclin. Here, we describe a fission yeast cuf2 mutant that enters into a third nuclear division cycle, represented by ectop...

Robustness analysis for the betweenness centrality ranking for polarity, cytokinesis and cell cycle networks in fission yeast. We analysed the robustness of ranking proteins by BC centrality in the presence of imperfect network interaction data. We added 10% extra edges at random to the network, calculated BC for every node after adding the edges,...

Functional modularity in the core networks. To calculate how much the functional modularity (the ratio of interactions between nodes with a shared GO category versus interactions between nodes with no GO category in common) observed for the core network of budding and fission yeast deviated from a random network, we kept all the category labels for...

Predicting essentiality by network measures in fission and budding yeast. (PDF)

Analysis of the neighbors of high linkerity proteins. (PDF)

Symmetric steady state solutions for SIN levels at the two SPBs in the minimal model of SIN asymmetry establishment show that asymmetry emerges through a pitchfork bifurcation. Stable (solid lines) and unstable (dashed) steady states of SIN activity at the old or new SPB. The two solutions totally overlap as the system is fully symmetrical. The cal...

Analysis of the genome-wide fission yeast network. See detailed description under Table S2. (XLS)

Analysis of the genome-wide budding yeast network. Tabulated file (in .xls format) containing network measures for all protein in the largest connected component of the genome-wide network of fission (S1) and budding (S2) yeast. Columns include: Common name: Common name. Systematic name: Systematic name (for fission yeast), GO database ID (for budd...

Analysis of the core budding yeast network. Tabulated file (in .xls format) containing network measures for all protein in the core network of fission (S3) and budding (S4) yeast. Columns include: Common name: Common name. Systematic name: Systematic name (for fission yeast), GO database ID (for budding yeast) Description: Brief description of know...

All members of the cliques identified on Figure 3B. List of all proteins belonging to the cliques described in Figure 3. Clique 1 corresponds to the top left clique in Figure 3B, with cliques increasing moving from left to right. (PDF)

The cell cycle + cytokinesis + polarity = core interaction network of budding yeast proteins. (A) Venn diagram showing the overlap among the different Gene Ontology functional groups in the proteins present in the core network of budding yeast. Proteins with multiple functional annotations have colours that are the sum of the colours of the individ...

Robustness analysis of linkerity of proteins in the fission yeast polarity network. We systematically analysed the robustness of linkerity in the presence of imperfect network interaction data. We added 10% edges preferentially to nodes with high degree (A) or removed 10% edges at random (B) to the core network. In the preferential attachment model...

Linkerity at various network confidences. (PDF)

Time-lapse analysis of Sts5 localization in fission yeast cells. Microtubules are visualized using mCherry labeled tubulin (Atb2) to identify cell cycle stage (A and B right column and Sts5-3GFP is visualized on the left). As the cell cycle progresses, Sts5 starts to accumulate into cytoplasmic dots, which then rapidly disappear upon septum formati...

Analysis of the core fission yeast network. See detailed description under Table S4. (XLS)

Correlation between linkerity and other network measures. (PDF)

The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify 'lin...

During meiosis, the centrosome/spindle pole body (SPB) must be regulated in a manner distinct from that of mitosis to achieve a specialized cell division that will produce gametes. In this paper, we demonstrate that several SPB components are localized to SPBs in a meiosis-specific manner in the fission yeast Schizosaccharomyces pombe. SPB componen...

The mitotic spindle consists of two types of microtubules. Dynamic kinetochore microtubules capture kinetochores, whereas stable interpolar microtubules serve as the structural backbone that connects the two spindle poles. Both have been believed to be indispensable for cell division in eukaryotes. Here we demonstrate that interpolar microtubules a...

The SCF (Skp1-Cul1-F-box) complex contributes to a variety of cellular events including meiotic cell cycle control, but its function during meiosis is not understood well. Here we describe a novel function of SCF/Skp1 in meiotic recombination and subsequent chromosome segregation. The skp1 temperature-sensitive mutant exhibited abnormal distributio...

The progression of meiosis is controlled by a number of gene-expression systems in the fission yeast Schizosaccharomyces pombe. A forkhead-type transcription factor Mei4 activates a number of genes essential for progression from the middle to late stages of meiosis, which include meiosis I, meiosis II and sporulation. The mei4-deletion mutant (mei4...

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...

Microtubules form a multifunctional filamentous structure essential for the cell. In interphase, microtubules form networks in the cytoplasm and play pivotal roles in cell polarity and intracellular transport of various biomolecules. In mitosis, microtubules dramatically change their morphology to assemble the mitotic spindle, thereby pulling the c...

Supplementary Information

Supplementary Movie S3

Review Process File

Supplementary Movie S1

Supplementary Movie S2

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 gamma-tubulin complex (gamma-T...

Ran GTPase activates several target molecules to induce microtubule formation around the chromosomes and centrosomes. In fission yeast, in which the nuclear envelope does not break down during mitosis, Ran targets the centrosomal transforming acidic coiled-coil (TACC) protein Alp7 for spindle formation. Alp7 accumulates in the nucleus only during m...

Supplementary information

Mitotic spindle microtubules pull chromosomes toward each pole to generate two daughter cells. Proper spindle formation and function are required to prevent tumorigenesis and cell death. The fission yeast Schizosaccharomyces pombe has been widely used as a model organism to understand the molecular mechanism of mitosis due to its convenience in gen...

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 mitot...

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 compartmenta...

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 result...

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...

The Ase1/Prc1 proteins constitute a conserved microtubule-associated protein family that is implicated in central spindle formation and cytokinesis. Here we characterize a role for fission yeast Ase1. Ase1 localizes to microtubule overlapping zones and displays dynamic alterations of localization during the cell cycle. In particular, its spindle lo...

Microtubules display dramatic morphological alterations from mitotic spindles to fibrous interphase structures upon exit from mitosis. In this issue of Developmental Cell, Zimmerman et al. shed a novel light on the molecular mechanism of microtubule structure reorganization during cytokinesis.