Education & Certifications
B.S., The University of Tokyo
Ph.D, The University of Tokyo
Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses.
View details for DOI 10.1534/genetics.115.184663
View details for PubMedID 26715672
Rho-type small GTPases are involved in cytokinesis in various organisms, but their precise roles and regulation remain unclear. Rho proteins function as molecular switches by cycling between the active GTP-bound and inactive GDP-bound states; the GTP-bound proteins in turn interact with their downstream effectors to transmit the signal. Biochemical assays using Rho-binding domains of effector proteins have been used to specifically pull down GTP-bound Rho proteins from cell extracts. Here, we describe the application of such a method in combination with cell-cycle synchronization in the budding yeast Saccharomyces cerevisiae; this approach allows dissection of the activity of Rho1 at different stages of cytokinesis. We also present data showing the importance of caution in interpreting such biochemical data and of comparing to the results obtained with other approaches where possible. The principle of this protocol is also applicable to analyses of other Rho-type GTPases and cell-cycle events.
View details for DOI 10.1007/978-1-4939-3145-3_15
View details for PubMedID 26519315
Dicentric chromosomes are unstable products of erroneous DNA repair events that can lead to further genome rearrangements and extended gene copy number variations. During mitosis, they form anaphase bridges, resulting in chromosome breakage by an unknown mechanism. In budding yeast, dicentrics generated by telomere fusion break at the fusion, a process that restores the parental karyotype and protects cells from rare accidental telomere fusion. Here, we observed that dicentrics lacking telomere fusion preferentially break within a 25- to 30-kb-long region next to the centromeres. In all cases, dicentric breakage requires anaphase exit, ruling out stretching by the elongated mitotic spindle as the cause of breakage. Instead, breakage requires cytokinesis. In the presence of dicentrics, the cytokinetic septa pinch the nucleus, suggesting that dicentrics are severed after actomyosin ring contraction. At this time, centromeres and spindle pole bodies relocate to the bud neck, explaining how cytokinesis can sever dicentrics near centromeres.
View details for DOI 10.1101/gad.254664.114
View details for PubMedID 25644606
In yeast and animal cytokinesis, the small guanosine triphosphatase (GTPase) Rho1/RhoA has an established role in formation of the contractile actomyosin ring, but its role, if any, during cleavage-furrow ingression and abscission is poorly understood. Through genetic screens in yeast, we found that either activation of Rho1 or inactivation of another small GTPase, Cdc42, promoted secondary septum (SS) formation, which appeared to be responsible for abscission. Consistent with this hypothesis, a dominant-negative Rho1 inhibited SS formation but not cleavage-furrow ingression or the concomitant actomyosin ring constriction. Moreover, Rho1 is temporarily inactivated during cleavage-furrow ingression; this inactivation requires the protein Cyk3, which binds Rho1-guanosine diphosphate via its catalytically inactive transglutaminase-like domain. Thus, unlike the active transglutaminases that activate RhoA, the multidomain protein Cyk3 appears to inhibit activation of Rho1 (and thus SS formation), while simultaneously promoting cleavage-furrow ingression through primary septum formation. This work suggests a general role for the catalytically inactive transglutaminases of fungi and animals, some of which have previously been implicated in cytokinesis.
View details for DOI 10.1083/jcb.201302001
View details for Web of Science ID 000322062300014
View details for PubMedID 23878277
Cell morphogenesis is a complex process that relies on a diverse array of proteins and pathways. We have identified a transglutaminase-like protein (Cyk3p) that functions in fission yeast morphogenesis. The phenotype of a cyk3 knockout strain indicates a primary role for Cyk3p in cytokinesis. Correspondingly, Cyk3p localizes both to the actomyosin contractile ring and the division septum, promoting ring constriction, septation, and subsequent cell separation following ring disassembly. In addition, Cyk3p localizes to polarized growth sites and plays a role in cell shape determination, and it also appears to contribute to cell integrity during stationary phase, given its accumulation as dynamic puncta at the cortex of such cells. Our results and the conservation of Cyk3p across fungi point to a role in cell wall synthesis and remodeling. Cyk3p possesses a transglutaminase domain that is essential for function, even though it lacks the catalytic active site. In a wider sense, our work illustrates the physiological importance of inactive members of the transglutaminase family, which are found throughout eukaryotes. We suggest that the proposed evolution of animal transglutaminase cross-linking activity from ancestral bacterial thiol proteases was accompanied by the emergence of a subclass whose function does not depend on enzymatic activity.
View details for DOI 10.1091/mbc.E11-07-0656
View details for Web of Science ID 000306287400005
View details for PubMedID 22573890
Until recently, it had appeared that the septin family of proteins was restricted to the opisthokont eukaryotes (the fungi and animals and their close relatives the microsporidia and choanoflagellates). It has now become apparent that septins are also present in several other widely divergent eukaryotic lineages (chlorophyte algae, brown algae, and ciliates). This distribution and the details of the non-opisthokont septin sequences appear to require major revisions to hypotheses about the origins and early evolution of the septins.
View details for DOI 10.1515/BC.2011.086
View details for Web of Science ID 000293736800002
View details for PubMedID 21824002
Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.
View details for DOI 10.1515/BC.2011.083
View details for Web of Science ID 000293736800016
View details for PubMedID 21824009
During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.
View details for DOI 10.1128/MCB.01529-09
View details for Web of Science ID 000275980900017
View details for PubMedID 20123972
Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Delta cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow.
View details for DOI 10.1083/jcb.200903125
View details for Web of Science ID 000267134000009
View details for PubMedID 19528296
Sporulation of the fission yeast Schizosaccharomyces pombe is a developmental process that generates gametes and that includes the formation of spore envelope precursors called the forespore membranes. Assembly and development of forespore membranes require vesicular trafficking from other intracellular membrane compartments. We have shown that phosphatidylinositol 3-kinase (PtdIns 3-kinase) is required for efficient and proper development of forespore membranes. The role of a FYVE domain protein, Sst4p, a homolog of Vps27p/Hrs, as a downstream factor for PtdIns 3-kinase in sporulation was investigated. sst4Delta asci formed spores with oval-shaped morphology and with reduced viability compared to that of the wild-type spores. The extension of forespore membranes was inefficient, and bubble-like structures emerged from the leading edges of the forespore membranes. Sst4p localization was examined using fluorescent protein fusions and was found to be adjacent to the forespore membranes during sporulation. The localization and function of Sst4p were dependent on its FYVE domain and on PtdIns 3-kinase. Sst4p colocalized and interacted with Hse1p, a homolog of Saccharomyces cerevisiae Hse1p and of mammalian STAM. Mutations in all three UIM domains of the Sst4p/Hse1p complex resulted in formation of spores with abnormal morphology. These results suggest that Sst4p is a downstream factor of PtdIns 3-kinase and functions in forespore membrane formation.
View details for DOI 10.1128/EC.00211-07
View details for Web of Science ID 000251896100019
View details for PubMedID 17951524
The multivesicular body (MVB) sorting pathway is required for a number of biological processes, including downregulation of cell-surface proteins and protein sorting into the vacuolar lumen. The function of this pathway requires endosomal sorting complexes required for transport (ESCRT) composed of class E vacuolar protein sorting (Vps) proteins in Saccharomyces cerevisiae, many of which are conserved in Schizosaccharomyces pombe. Of these, sst4/vps27 (homologous to VPS27) and sst6 (similar to VPS23) have been identified as suppressors of sterility in ste12Delta (sst), although their functions have not been uncovered to date. In this report, these two sst genes are shown to be required for vacuolar sorting of carboxypeptidase Y (CPY) and an MVB marker, the ubiquitin-GFP-carboxypeptidase S (Ub-GFP-CPS) fusion protein, despite the lack of the ubiquitin E2 variant domain in Sst6p. Disruption mutants of a variety of other class E vps homologues also had defects in sorting of CPY and Ub-GFP-CPS. Sch. pombe has a mammalian AMSH homologue, sst2. Phenotypic analyses suggested that Sst2p is a class E Vps protein. Taken together, these results suggest that sorting into multivesicular bodies is dependent on class E Vps proteins, including Sst2p, in Sch. pombe.
View details for DOI 10.1099/mic.0.2007/006072-0
View details for Web of Science ID 000249044900038
View details for PubMedID 17660439
Schizosaccharomyces pombe defective in phosphatidylinositol (PtdIns) 3-kinase shows various defects in forespore membrane formation, including onset, growth orientation, and closure. Downstream factors of PtdIns 3-kinase in this system were explored. Among various phox homology (PX) domain-containing proteins, Vps5p and Vps17p, homologues of sorting nexins, were found to be required for efficient sporulation. Cells defective in these proteins showed a disordered growth orientation of the forespore membrane, as is the case with Deltapik3 cells. Vps5p and Vps17p with mutations in the PX domains failed to suppress the defects of their relevant disruptants. Vps5p and Vps17p migrated toward the the forespore membrane in a pik3+-dependent manner, suggesting that these proteins may interact with PtdIns(3)P. Electron-microscopic analysis revealed that the forespore membrane fails to engulf the nucleus in some of these cells, accumulating vesicle-like bodies similar to those seen in Deltaspo3 cells. These results suggest that Vps5p and Vps17p are the targets of PtdIns(3)P in vesicle transport required for onset of the forespore membrane formation.
View details for DOI 10.1111/j.1365-2443.2004.00744.x
View details for Web of Science ID 000222300100006
View details for PubMedID 15189449
The ste12+ gene of Schizosaccharomyces pombe codes for a phosphatidylinositol (PI) 3-phosphate 5'-kinase, which is required for efficient mating. Suppressor mutants for sterility of ste12Delta cells were screened for. Most of the mutant genes turned out to be recessive. Six genes were cloned and the open reading frames responsible for the suppressor activity were identified. They included genes coding for proteins with domains homologous to calcium transporters, casein kinase II, UBC13, AMSH, Vps23p, and Vps27p of Saccharomyces cerevisiae. Disruption of these genes resulted in suppression of the defects of the ste12Delta cells, including low mating efficiency and formation of large vacuoles. Since many of these gene products are homologous to the proteins involved in vesicle transport, sterility caused by inactivation of ste12 may be due to a disordered vesicle transport system.
View details for Web of Science ID 000185193700021
View details for PubMedID 12951513
From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.
View details for DOI 10.1002/yea.1011
View details for Web of Science ID 000184466200001
View details for PubMedID 12868054
In Schizosaccharomyces pombe, Pik3p phosphorylates phosphatidylinositol (PI) to produce PI 3-P, which is further phosphorylated by Ste12p to yield PI 3,5-P2. Pik3p is required for both conjugation and sporulation. To test which of PI 3-P and PI 3,5-P2 is required for sporulation, diploid cells defective in production of PI 3,5-P2 were used. They underwent sporulation almost normally provided that the osmotic pressure of the medium was controlled, suggesting that not PI 3,5-P2 but PI 3-P was important. Electron microscopic analysis confirmed normal sporulation in the absence of PI 3,5-P2 although the forespore membrane was found to be less dense in these cells.
View details for Web of Science ID 000183266100042
View details for PubMedID 12834310
Phosphatidylinositol (PI) 3-kinase (encoded by the pik3(+) gene) in Schizosaccharomyces pombe has been identified as a homologue of VPS34p, a protein required for proper vesicular protein sorting. The clone defective in this protein carries enlarged vacuoles and exhibits sensitivity to high temperature or high ion concentration. The effect of disruption of pik3(+) on sporulation of Sz. pombe was examined. The diploid cells underwent G(1) arrest and meiosis. However, the spores formed by the deltapik3 cells were not viable. Electron-microscopic analysis revealed that the growth of the forespore membrane of deltapik3 cells was not correctly orientated, failing to engulf the nucleus or forming extremely small spores, as was confirmed by the use of Spo3p-GFP and GFP-Psy1p, which are markers of the forespore membrane. The coating materials found along the forespore membrane of the wild-type were greatly reduced in these cells. PI 3-P, the product of Pik3p, was detected on the forespore membrane, suggesting that PI 3-P-dependent vesicle transport may take place in formation of the forespore membrane. Misshaped forespore membrane, accumulation of vesicles, formation of small non-viable spores, and suppression by over expression of Psy1p were the phenotypes commonly seen in deltapik3 and deltaspo3 cells, suggesting a relationship between the functions of Pik3p and Spo3p in formation of the forespore membrane in Sz. pombe.
View details for DOI 10.1002/yea.953
View details for Web of Science ID 000181059400001
View details for PubMedID 12557273