MAPK-dependent control of mitotic progression in S. pombe.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress. RESULTS: We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit. CONCLUSIONS: Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.

Formation of Transient Protein Aggregate-like Centers Is a General Strategy Postponing Degradation of Misfolded Intermediates.

When misfolded intermediates accumulate during heat shock, the protein quality control system promotes cellular adaptation strategies. In Schizosaccharomyces pombe, thermo-sensitive proteins assemble upon stress into protein aggregate-like centers, PACs, to escape from degradation. The role of this protein deposition strategy has been elusive due to the use of different model systems and reporters, and to the addition of artificial inhibitors, which made interpretation of the results difficult. Here, we compare fission and budding yeast model systems, expressing the same misfolding reporters in experiments lacking proteasome or translation inhibitors. We demonstrate that mild heat shock triggers reversible PAC formation, with the collapse of both reporters and chaperones in a process largely mediated by chaperones. This assembly postpones proteasomal degradation of the misfolding reporters, and their Hsp104-dependent disassembly occurs during stress recovery. Severe heat shock induces formation of cytosolic PACs, but also of nuclear structures resembling nucleolar rings, NuRs, presumably to halt nuclear functions. Our study demonstrates that these distantly related yeasts use very similar strategies to adapt and survive to mild and severe heat shock and that aggregate-like formation is a general cellular scheme to postpone protein degradation and facilitate exit from stress.

Reversible protein aggregation as cytoprotective mechanism against heat stress.

Temperature fluctuation is one of the most frequent threats to which organisms are exposed in nature. The activation of gene expression programs that trigger the transcription of heat stress-protective genes is the main cellular response to resist high temperatures. In addition, reversible accumulation and compartmentalization of thermosensitive proteins in high-order molecular assemblies are emerging as critical mechanisms to ensure cellular protection upon heat stress. Here, we summarize representative examples of membrane-less intracellular bodies formed upon heat stress in yeasts and human cells and highlight how protein aggregation can be turned into a cytoprotective mechanism.

Acute Heat Stress Leads to Reversible Aggregation of Nuclear Proteins into Nucleolar Rings in Fission Yeast.

Upon acute heat stress (HS), overall mRNA transcription, processing, and export are inhibited, leading to cell growth arrest. However, how cells turn off mRNA metabolism is not fully understood. Here, we show that acute HS results in the segregation and aggregation of multiple nuclear and nucleolar proteins into ring-like structures located at the nucleolar periphery (nucleolar rings [NuRs]). NuRs sequester essential factors required for nuclear mRNA metabolism and nuclear pore complex function, as well as cell-cycle regulators. When cells are switched back to growing temperatures, NuRs disaggregate, and their components relocate to their functional environments in an Hsf1- and Hsp104-dependent manner, and concomitantly with the reinitiation of cell growth. These findings highlight the contribution of reversible protein aggregation to the inhibition of overall RNA-related activities in the nucleus and its functional relevance in the maintenance of cellular homeostasis during acute HS.

Selective Nuclear Pore Complex Removal Drives Nuclear Envelope Division in Fission Yeast.

An important question in cell biology is how cellular organelles partition during cell division. In organisms undergoing closed mitosis, the elongation of an intranuclear spindle drives nuclear division, generating two identically sized nuclei [1, 2]. However, how the site of nuclear division is determined and the underlying mechanism driving nuclear envelope (NE) fission remain largely unknown. Here, using the fission yeast, we show that the microtubule bundler Ase1/PRC1 at the spindle midzone is required for the local concentration of nuclear pore complexes (NPCs) in the region of the NE in contact with the central spindle. As the spindle elongates during anaphase B, components of these NPCs are sequentially eliminated, and this is accompanied by the local remodeling of the NE. These two events lead to the eventual removal of NPCs and nuclear division. In the absence of importin α, NPCs remain stable in this region and no event of NE remodeling is observed. Consequently, cells fail to undergo nuclear division. Thus, our results highlight a new role of the central spindle as a spatial cue that determines the site of nuclear division and point to NPC removal as the triggering event.

Nuclear Mechanics in the Fission Yeast.

In eukaryotic cells, the organization of the genome within the nucleus requires the nuclear envelope (NE) and its associated proteins. The nucleus is subjected to mechanical forces produced by the cytoskeleton. The physical properties of the NE and the linkage of chromatin in compacted conformation at sites of cytoskeleton contacts seem to be key for withstanding nuclear mechanical stress. Mechanical perturbations of the nucleus normally occur during nuclear positioning and migration. In addition, cell contraction or expansion occurring for instance during cell migration or upon changes in osmotic conditions also result innuclear mechanical stress. Recent studies in Schizosaccharomyces pombe (fission yeast) have revealed unexpected functions of cytoplasmic microtubules in nuclear architecture and chromosome behavior, and have pointed to NE-chromatin tethers as protective elements during nuclear mechanics. Here, we review and discuss how fission yeast cells can be used to understand principles underlying the dynamic interplay between genome organization and function and the effect of forces applied to the nucleus by the microtubule cytoskeleton.

Effects of the microtubule nucleator Mto1 on chromosomal movement, DNA repair, and sister chromatid cohesion in fission yeast.

Although the function of microtubules (MTs) in chromosomal segregation during mitosis is well characterized, much less is known about the role of MTs in chromosomal functions during interphase. In the fission yeast Schizosaccharomyces pombe, dynamic cytoplasmic MT bundles move chromosomes in an oscillatory manner during interphase via linkages through the nuclear envelope (NE) at the spindle pole body (SPB) and other sites. Mto1 is a cytoplasmic factor that mediates the nucleation and attachment of cytoplasmic MTs to the nucleus. Here, we test the function of these cytoplasmic MTs and Mto1 on DNA repair and recombination during interphase. We find that mto1Δ cells exhibit defects in DNA repair and homologous recombination (HR) and abnormal DNA repair factory dynamics. In these cells, sister chromatids are not properly paired, and binding of Rad21 cohesin subunit along chromosomal arms is reduced. Our findings suggest a model in which cytoplasmic MTs and Mto1 facilitate efficient DNA repair and HR by promoting dynamic chromosomal organization and cohesion in the nucleus.

Spatiotemporal control of spindle disassembly in fission yeast.

Maintenance of genomic stability during cell division is one of the most important cellular tasks, and it critically depends on the faithful replication of the genetic material and its equal partitioning into daughter cells, gametes, or spores in the case of yeasts. Defective mitotic spindle assembly and disassembly both result in changes in cellular ploidy that ultimately impinge proliferation fitness and might increase tumor malignancy. Although a great progress has been made in understanding how spindles are assembled to orchestrate chromosome segregation, much less is known about how they are disassembled once completed their function. Here, we review two recently uncovered mechanisms of spindle disassembly that operate at different stages of the fission yeast life cycle.

Importin α and vNEBD Control Meiotic Spindle Disassembly in Fission Yeast.

In metazoans, the nuclear envelope (NE) breakdown (NEBD) occurs during "open" mitosis and meiosis. In the fission yeast Schizosaccharomyces pombe, the mitosis and the first meiotic division (MI) are "closed," during which the NE is maintained. Intriguingly, during the second meiotic division (MII), the NE is also maintained, but nuclear and cytoplasmic molecules are mixed similarly to open mitosis, a phenomenon of unknown biological significance called "virtual" NEBD (vNEBD). Here, we show that importin-α-dependent nucleocytoplasmic transport regulates spindle disassembly late in anaphase B at MI, as previously reported for mitosis. At MII, however, spindle dissolution is triggered by vNEBD early in anaphase B, a mechanism that short-circuits the nucleocytoplasmic transport system. We demonstrate that the sequential action of these two spindle disassembly systems regulates the spatiotemporal order and ploidy of the meiotic products.

A new role for the nuclear basket network.

Our view of the nuclear pore complexes (NPCs) as gateways between the nuclear and cytoplasmic compartments has been largely expanded in recent years. NPCs have now demonstrated roles in genome regulation and maintenance from single cells to multicellular organisms. Both NPC proteins as well as components of the NPC basket act as dynamic scaffolds for silencing factors, and chromatin and cell cycle regulators. Components of the NPC basket also couple mRNA production and export, and prevent the exit of unprocessed mRNAs from the nucleus. Our recent work describes a novel function of the fission yeast nuclear basket component - the translocated promoter region (TPR) nucleoporin Alm1 - in proper localization of the proteasome to the nuclear envelope. Here we discuss how regulation of proteasome localization to the nuclear envelope by Alm1 is key to maintain kinetochores homeostasis and proper chromosome segregation.

The fission yeast nucleoporin Alm1 is required for proteasomal degradation of kinetochore components.

Kinetochores (KTs) are large multiprotein complexes that constitute the interface between centromeric chromatin and the mitotic spindle during chromosome segregation. In spite of their essential role, little is known about how centromeres and KTs are assembled and how their precise stoichiometry is regulated. In this study, we show that the nuclear pore basket component Alm1 is required to maintain both the proteasome and its anchor, Cut8, at the nuclear envelope, which in turn regulates proteostasis of certain inner KT components. Consistently, alm1-deleted cells show increased levels of KT proteins, including CENP-CCnp3, spindle assembly checkpoint activation, and chromosome segregation defects. Our data demonstrate a novel function of the nucleoporin Alm1 in proteasome localization required for KT homeostasis.

Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly.

During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.

Proteome-wide search for PP2A substrates in fission yeast.

PP2A (protein phosphatase 2A) is a major phosphatase in eukaryotic cells that plays an essential role in many processes. PP2A mutations in Schizosaccharomyces pombe result in defects of cell cycle control, cytokinesis and morphogenesis. Which PP2A substrates are responsible for these changes is not known. In this work, we searched for PP2A substrates in S. pombe using two approaches, 2D-DIGE analysis of PP2A complex mutants and identification of PP2A interacting proteins. In both cases, we used MS to identify proteins of interest. In the DIGE experiment, we compared proteomes of wild-type S. pombe, deletion of pta2, the phosphoactivator of the PP2A catalytic subunit, and pab1-4, a mutant of B-type PP2A regulatory subunit. A total of 1742 protein spots were reproducibly resolved by 2D-DIGE and 51 spots demonstrated significant changes between PP2A mutants and the wild-type control. MS analysis of these spots identified 27 proteins that include key regulators of glycerol synthesis, carbon metabolism, amino acid biosyntesis, vitamin production, and protein folding. Importantly, we independently identified a subset of these proteins as PP2A binding partners by affinity precipitation, suggesting they may be direct targets of PP2A. We have validated our approach by demonstrating that phosphorylation of Gpd1, a key enzyme in glycerol biogenesis, is regulated by PP2A and that ability of cells to respond to osmotic stress by synthesizing glycerol is compromised in the PP2A mutants. Our work contributes to a better understanding of PP2A function and identifies potential PP2A substrates.

Feedback regulation of SIN by Etd1 and Rho1 in fission yeast.

In fission yeast, the septation initiation network (SIN) is thought to promote cytokinesis by downstream activation of Rho1, a conserved GTPase that controls cell growth and division. Here we show that Etd1 and PP2A-Pab1, antagonistic regulators of SIN, are Rho1 regulators. Our genetic and biochemical studies indicate that a C-terminal region of Etd1 may activate Rho1 by directly binding it, whereas an N-terminal domain confers its ability to localize at the growing tips and the division site where Rho1 functions. In opposition to Etd1, our results indicate that PP2A-Pab1 inhibits Rho1. The SIN cascade is upstream-regulated by the Spg1 GTPase. In the absence of Etd1, activity of Spg1 drops down prematurely, thereby inactivating SIN. Interestingly, we find that ectopic activation of Rho1 restores Spg1 activity in Etd1-depleted cells. By using a cytokinesis block strategy, we show that Rho1 is essential to feedback-activate Spg1 during actomyosin ring constriction. Therefore, activation of Spg1 by Rho1, which in turn is regulated by Etd1, uncovers a novel feedback loop mechanism that ensures SIN activity while cytokinesis is progressing.

A Lallzyme MMX-based rapid method for fission yeast protoplast preparation.

Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi.

Cell migration and division in amoeboid-like fission yeast.

Yeast cells are non-motile and are encased in a cell wall that supports high internal turgor pressure. The cell wall is also essential for cellular morphogenesis and cell division. Here, we report unexpected morphogenetic changes in a Schizosaccharomyces pombe mutant defective in cell wall biogenesis. These cells form dynamic cytoplasmic protrusions caused by internal turgor pressure and also exhibit amoeboid-like cell migration resulting from repeated protrusive cycles. The cytokinetic ring responsible for cell division in wild-type yeast often fails in these cells; however, they were still able to divide using a ring-independent alternative mechanism relying on extrusion of the cell body through a hole in the cell wall. This mechanism of cell division may resemble an ancestral mode of division in the absence of cytokinetic machinery. Our findings highlight how a single gene change can lead to the emergence of different modes of cell growth, migration and division.

Yeast as a biosensor of detoxification: a tool for identifying new compounds that revert multidrug resistance.

During tumour progression, cells accumulate secondary mutations and/or chromosomal aberrations that generate genetic diversity within the tumour cell population. This may result in the acquisition of new properties that increase tumour malignancy, such as invasiveness or resistance to chemotherapy. One of the important mechanisms of chemotherapy resistance is overexpression or biochemical activation of ABC family transporters. ABC transporters remove anti tumour drugs from the cell, reducing their intracellular concentration and producing resistance against a wide range of chemically unrelated drugs, known as multidrug resistant phenotype (MDR). During recent decades, much effort has been devoted to the isolation of compounds able to inhibit the activity of these transporters. However, few such compounds have reached clinical practice and MDR remains a serious complication in cancer therapy. In an innovative approach to finding new ABC inhibitors, we propose using fission yeast Schizosaccharomyces pombe as a biosensor of detoxification that would enable cost-efficient screening of natural compounds and chemical libraries for molecules that revert the MDR phenotype. Existing fission yeast tools provide genetic, biochemical and cell biological analysis, thereby facilitating identification of drug targets. Putative inhibitors and modulators of ABC transporters could be used in combination with chemotherapeutic drugs for the treatment of multidrug resistant tumours.

Regulation of fission yeast morphogenesis by PP2A activator pta2.

Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity.

Antagonistic roles of PP2A-Pab1 and Etd1 in the control of cytokinesis in fission yeast.

In Schizosaccharomyces pombe, Etd1 is a positive regulator of the septation initiation network (SIN), a conserved GTPase-regulated kinase cascade that triggers cytokinesis. Here we show that a mutation in the pab1 gene, which encodes the B-regulatory subunit of the protein phosphatase 2A (PP2A), suppresses mutations in the etd1 gene. Etd1 is required for the function of the GTPase Spg1, a key regulator of SIN signaling. Interestingly, the loss of Pab1 function restored the activity of Spg1 in Etd1-deficient cells. This result suggests that PP2A-Pab1-mediated dephosphorylation inhibits Spg1, thus antagonizing Etd1 function. The loss of pab1 function also rescues the lethality of mutants of other genes in the SIN cascade such as mob1, sid1, and cdc11. Two-hybrid assays indicate that Pab1 physically interacts with Mob1, Sid1, Sid2, and Cdc11, suggesting that the phosphatase 2A B-subunit is a component of the SIN complex. Together, our results indicate that PP2A-Pab1 plays a novel role in cytokinesis, regulating SIN activity at different levels. Pab1 is also required to activate polarized cell growth. Thus, PP2A-Pab1 may be involved in coordinating polar growth and cytokinesis.

Interphase microtubule bundles use global cell shape to guide spindle alignment in fission yeast.

Correct spindle alignment requires a cell to detect and interpret its global geometry and to communicate this information to the mitotic spindle. In the fission yeast, Schizosaccharomyces pombe, the mitotic spindle is aligned with the longitudinal axis of the rod-shaped cell. Here, using wild-type and cell-shape mutants we investigate the mechanism of initial spindle alignment and show that attachment of interphase microtubules to the spindle pole bodies (SPB), the yeast equivalent of the centrosome, is required to align duplicated SPBs, and thus the mitotic spindle, with the long axis of the cell. In the absence of interphase microtubules or attachment between the microtubules and the SPB, newly formed spindles are randomly oriented. We show that the axis of the mitotic spindle correlates with the axis along which the SPB, as a consequence of interphase microtubule dynamics, oscillates just before mitosis. We propose that cell geometry guides cytoplasmic microtubule alignment, which in turn, determines initial spindle alignment, and demonstrate that a failure of the spindle pre-alignment mechanism results in unequal chromosome segregation when spindle length is reduced.