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.

Exploring the genetic background of the botulism neurotoxin BoNT/B2 in Spain.

To determine whether the neurotoxin BoNT/B2 causing botulism in Spain is clonal, the genetic diversity and phylogenetic relationships of Clostridium botulinum from food-borne episodes and infant cases of the condition were explored. The botulinum toxin gene (bont) subtype, the variable region of the flagellin gene (flaVR), and a seven-gene multi-locus sequence type were examined by sequencing 37 BoNT-positive cultures obtained over the period 2010 to 2022. Out of 37 botulism events, 16 food-borne episodes and 16 infant cases were associated with bont/b2. Eight bont/b2 alleles were detected [nucleotide distance range 0.0259-0.415%, Hunter and Gaston discrimination index (HGDI) 0.71]. The most common bont/b2 allele corresponded to that of strain Prevot 25 NCASE and its single and double locus variations (87.5%). Four known flaVR types were identified (HGDI 0.79), along with one previously unknown (flaVR-15). Sixteen sequence types (STs) (HGDI 0.89) were recorded including seven new STs (ST164-ST170; 10 new alleles) and five new STs (ST171-ST175; with new allele combinations) were also noted. Correlations among some STs and flaVR types were seen. Overall, the present results show that the combined analysis of bont/b2-flaVR-ST at the nucleotide level could be used to track botulism events in Spain. The neurotoxin BoNT/B2 has largely been responsible for human botulism in Spain. The polymorphism analysis of bont/b2, flaVR typing, and sequence type determinations, revealed a wide variety of clones to be responsible for human botulism, ruling out a common source of acquisition. IMPORTANCE Botulism, a potentially fatal disease, is classically characterized by a symmetrical descending flaccid paralysis, which if left untreated can lead to respiratory failure and death. Botulinum neurotoxin (BoNT), produced by certain species of Clostridium, is the most potent biological toxin known, and the direct cause of botulism. This study characterizes the acquisition in Spain of two forms of botulism, i.e., food-borne and infant botulism, which are largely caused by the main neurotoxin BoNT/B2. Polymorphism analysis of the bont/b2 gene, typing of the flagellin variable region sequence (flaVR), and multilocus sequence typing, were used to explore the genetic background of Clostridium botulinum group I. To our knowledge, this is the first phylogenetic and typing study of botulism undertaken in Spain.

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.

Recordar, Repetir y ( ) / Remember, Repeat and ( )

El eje central de este trabajo será pensar a la reelaboración freudiana como operación del final del análisis respecto de lo invariable e inasimilable de la exigencia pulsional y su articulación con la operación de escritura de la letra, que plantea J. Lacan como horizonte de la interpretación analítica. Por efecto de retroacción desde la última enseñanza de Lacan, es posible reconocer momentos excepcionales en la investigación freudiana donde se identifica lo que Lacan llamará "lo real imposible de simbolizar". El trauma, ubicado a partir del ombligo del sueño y de los fragmentos de vida real {stück realen lebens}, son el antecedente necesario para mi pregunta por el estatuto conceptual y ético de la reelaboración {durcharbeiten} de la resistencia. Desde esta lectura propuesta, la primera respuesta que imprime una discontinuidad al par inconciente-interpretación en la obra de Freud es la reelaboración {durcharbeiten} de la resistencia.

The central axis of this work will be to to think of the Freudian reworking as an operation of the end of the analysis regarding the invariable and unassimilable of the drive requirement and its articulation with the operation of letter writing, which J. Lacan poses as a horizon of interpretation analytics. As a result of feedback from the last teaching of Lacan, it is possible to recognize exceptional moments in Freudian research where what Lacan calls "the real impossible to symbolize" is identified. The trauma, located from the navel of the dream and the real life fragments (stück realen lebens), are the necessary antecedent to my question for the conceptual and ethical statute of the reworking (durcharbeiten) of the resistance. From this proposed reading, the first response that prints a discontinuity to the unconscious-interpretation pair in Freud's work is the reworking of the resistance.

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.

Los signos perceptivos como huellas freudianas de lo real / Perceptual signs as freudian traces of the real

Inconciente y real se leen topológicamente como una banda de Moebius, esta formulación es solidaria de los desarrollos epistémicos de la última enseñanza de J. Lacan. La hipótesis general que nos orienta en este trabajo es que aquel lo que en Lacan es el último giro de su enseñanza, tiene solidaridad con lo que en Freud encontramos al inicio de la suya. En particular nos centraremos en la construcción freudiana del primer modelo de aparato psíquico. En este los signos perceptivos nos orientan respecto de la conceptualización lacaniana de cuerpo y letra.

Unconscious and real is read topologically like a Moebius strip, this formulation is binding with the epistemic developments of the last teaching of Lacan. The general hypothesis who guides us in this work is what in Lacan the latest twist in his teaching, is solidary with what in Freud found in his beginning. In particular we will focus on the construction of the first Freudian model of psychic apparatus. The signs perceptive orientate us about the Lacanian conceptualization of body and letter.

Huellas freudianas en la última enseñanza de Lacan / Freudien traces in the last teaching of Lacan

A partir de nuestra investigación (UBACyT 2010-2012, “La aptitud de analista y la segregación contemporánea”. Facultad de Psicología, UBA. Código 20020090100300. Director Doctor Osvaldo Delgado), en los avances publicados, sostenemos que la “aptitud de analista” (tauglich) en Freud, es una respuesta inédita frente a la segregación que implica lo estereotipado de los mecanismos de defensa. Podemos pensar una dirección que va de la preocupación freudiana al final de su obra, acerca de la problemática de la pulsión y su ligadura (ligado, no ligado, insusceptible de ligadura) en la conclusión de los tratamientos; a la preocupación lacaniana en la última época de su enseñanza, por cómo se puede en un psicoanálisis acceder a un real más allá de los semblantes, pero contando con ellos. Nos interesa dar cuenta de una continuidad epistémica entre la segunda tópica que formula Freud y la última enseñanza de Lacan. Este recorrido permitiría pensar si se produce un segundo retorno de Lacan a Freud, segundo en relación con el que él mismo nombró como primero a la altura de su seminario Los cuatro conceptos fundamentales del psicoanálisis [Lacan: 1964].

Huellas freudianas en la última enseñanza de Lacan / Freudien traces in the last teaching of Lacan

A partir de nuestra investigación (UBACyT 2010-2012, ôLa aptitud de analista y la segregación contemporáneaö. Facultad de Psicología, UBA. Código 20020090100300. Director Doctor Osvaldo Delgado), en los avances publicados, sostenemos que la ôaptitud de analistaö (tauglich) en Freud, es una respuesta inédita frente a la segregación que implica lo estereotipado de los mecanismos de defensa. Podemos pensar una dirección que va de la preocupación freudiana al final de su obra, acerca de la problemática de la pulsión y su ligadura (ligado, no ligado, insusceptible de ligadura) en la conclusión de los tratamientos; a la preocupación lacaniana en la última época de su enseñanza, por cómo se puede en un psicoanálisis acceder a un real más allá de los semblantes, pero contando con ellos. Nos interesa dar cuenta de una continuidad epistémica entre la segunda tópica que formula Freud y la última enseñanza de Lacan. Este recorrido permitiría pensar si se produce un segundo retorno de Lacan a Freud, segundo en relación con el que él mismo nombró como primero a la altura de su seminario Los cuatro conceptos fundamentales del psicoanálisis [Lacan: 1964].(AU)

A genome-wide resource of cell cycle and cell shape genes of fission yeast.

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.

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.

La "aptitud de analista": una respuesta inédita frente a la segregación / The "analyst's aptitude": an unpublished response opposite to the segregation

El presente trabajo presenta desarrollos preliminares surgidos a partir del eje de investigación: las incidencias que los procesos de segregación tienen sobre la "aptitud de analista". Para abordarlo la referencia freudiana obligatoria es el capítulo VII de "Análisis terminable e interminable" [Freud: 1937], en el que Freud se pregunta por la aptitud -eignung (idoneidad)- y se responde por la aptitud -tauglich (saber hacer)-. Este avance de investigación está orientado por la preocupación acerca de las consecuencias posibles de los"saldos lamentables" de los análisis de los analistas, los mismos son manifestaciones de hostilidad, odio y partidismo, tanto en la producción doctrinaria como en los lazos con otros analistas y con la comunidad en general. Cómo pensar la enseñanza freudiana acerca de la "aptitud de analista" para pensar un colectivo social donde se pueda establecer con los otros un lazo que no rechace lo diferente.

The present work presents preliminary developments arisen from the axis of investigation: the incidents that the processes of segregation have on the " analyst's aptitude". To be approached, the Freudian obligatory reference it is the chapter VII of " Analysis terminable and endlessly ", where Freud asks himself about the aptitude-eignung (suitability) - and it is answered by the aptitude -tauglich (to be able to do)-. This advance of investigation is orientated by the worry brings over of the possible consequences of the " lamentable balances " of the analyses of the analysts, the same ones they are manifestations of hostility, hatred and partisanship, both in the doctrinaire production and in the bows with other analysts and with the community in general. How to think the Freudian bequest brings over of the " analyst's aptitude " for thinking a social group where it could establish with others a bow that does not reject the different thing.

Self-organization of microtubule bundles in anucleate fission yeast cells.

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.