Cell migration and division in amoeboid-like fission yeast

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.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.This work was supported by grants to R.R.D. from the Spanish Ministerio de Ciencia e Inovación BFU2010-21310 and P09-CTS-4697 (Proyecto de Excelencia) from La Junta de Andalucia. I.F.-P. was supported by the Spanish Ministerio de Educación (Juan de la Cierva Program). M.B. was supported by the Spanish Ministerio de Educación (FPI Fellowship, BES-2008-004018). J.Z. was supported by grant P09-CTS-4697.Peer Reviewe

Phosphorylation-mediated regulation of the gamma tubulin complex.

Motivation: Microtubules perform vital functions such as chromosome segregation, nuclear positioning, vesicular transport and determination of cell polarity and morphogenesis. The gamma tubulin complex is an essential element responsible for microtubule nucleation and is highly conserved from yeast to human and the core of the complex is basically composed of three esential proteins (Alp4/GCP2, Alp6/GCP3 and Gtb1/GTB). Its function is subject to a multiple regulation processes both temporally and spatially, and phosphorilation is one of the main postranslational modifications to regulate protein function. The fission yeast Schizosaccharomyces pombe is a potent model for studying microtubules (MT) nucleation because of its simple MT cytoskeleton, genetic tractability, limited number of regulators, and amenability to assays of individual nucleation events. In this work, we use S. pombe to determine the role of different putative phosphorylation sites to understand the regulation of this complex in different stages of the cell cycle. Methods: In our laboratory, a collection of mutants of Alp4 and Alp6 genes was generated. These mutants consisted of the modification of the phosphorylation sites described in massive phosphoproteomes. Usind directed mutagenesis we generated non-phosphorylatable and phosphomimetic alleles (changins Serines to Alanine or Aspartic acid residues respectively. In this project, a phenotypic analysis of the different mutants from this collection has been performed using different approaches such as spot tests, western blots, fluorescence microscopy with live cells, and genetic interaction analysis techniques. Results: We found that different mutants display phenotypic differences under the conditions analysed. The most representative phenotypes are related to delayed and aberrant mitotic spindle formation, no mitotic spindle formation and the appearance of stress granules at high temperatures. This establishes a connection between the phosphorylation state of Alp4 and Alp6 with microtubule formation, and its implication in the biology of microtubules in the cell. Conclusions: Our data suggest that phosphorylation of components of the gamma tubulin complex is involved in the regulation of microtubule nucleation in different stages of the cell cycle and cell phisiology. This shed light in new molecular mechanisms not been previously described

Analysis and optimization of the efficiency of CRISPR/Cas systems in gene editing and expression depletion in the fission yeast S. Pombe.

The CRISPR/Cas system is a gene editing tool that was discovered as a protection mechanism in bacteria. Thissystem has the ability to perform alterations in DNA and RNA sequences in a precise way, through the action of anRNA molecule that acts as a guide and a Cas protein that interacts with the guide and cuts the target strand [1]. Inthis project, we aim for a double goal. First, we implement a previously published CRISPR/Cas9 editing system in ourbiological model to enable a versatile tool for genome editing in our lab. We have used a replicative plasmid for Cas9expression together with corresponding gRNAs to perform and analysing the tagging efficiency of mex67 gene(nuclear protein involved in mRNA transport) with a fluorescent protein; in comparison to the traditional method.Second, we aim to develope a novel, highly efficient, method to deplete gene expression by targeting the Cas13dRNA nuclease to specific RNAm molecules. We are developing a fusion of Cas13d with Mex67 to maximize thephysical interaction between target RNA molecules and the RNA nuclease in obtaining RNA knockdowns [1,2,3].Fluorescence microscopy in living cells results show that it is possible to perform fluorescent labeling of mex67 bythe CRISPR/Cas9 system, although not at the reported efficiency in the literature for other genes. Nevertheless, up toour knowledge, this is the first Mex67 fluorescently tagged version under its own promoter in the field. We show thatthe intracellular distribution of this protein is nuclear; cells are viable and they proliferate as wild type controls, whichmake this protein a good candidate to acomplish our second goal

Decoding the Role of a Microtubule-related protein in Meiosis

Several meiosis-specific proteins of Schizosaccharomyces pombe play essential roles in meiotic progression. In this study we report that a conserved microtubule-related protein is required for proper spore formation. Sporulation in fission yeast represents a unique model to study gametogenesis in sexual reproducing organisms. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores [1]. After the two sequential meiotic divisions, FSMs expand and encapsulate the haploid nuclei, producing the membrane-surrounded spore precursors [2]. During anaphase II, two FSM sacs expand from the poles encasing the nuclei, and these elongates with the intranuclear spindle to divide into two. Finally, these sacs enclose four newborn nuclei individually. The leading edge of the elongating FSM forms a ring and comprises some associated proteins [3]. The leading edge ring is a crucial structure in meiosis that remains poorly understood.  Our research investigates its role and dynamics, focusing on the localization and function of this microtubule-related protein during spore formation in fission yeast. Transgenic S. pombe strains were generated by tagging the gene of interest with GFP along with either mCherry-labeled microtubules or mCherry-Psy1 to observe simultaneous dynamics of these markers throughout meiosis in vivo. Fluorescence microscopy was used to capture images at different Z-plane levels for 3D reconstruction. These experiments reveal that this protein is localizes in the leading-edge ring. In addition, we studied the lack-of-function by deletion of this gene, revealing a phenotype characterized by malformed spindles and delayed assembly/disassembly during meiosis II. This could lead to issues in chromosome segregation and result in mutants with altered chromosomal content

From Yeast to Fly: A Study of the Evolutionary Conservation of Cohesin in Schizosaccharomyces pombe and Drosophila melanogaster

Motivation: Cohesin is a crucial protein complex composed of Psm1, Psm3 and Rad21, arranged in a like-ring conformation. This complex plays a key role in maintaining chromosome structure, facilitating accurate chromosome segregation, regulating DNA repair, and controlling gene expression by influencing chromatin architecture and transcriptional processes. Defects in cohesin can lead to severe cellular consequences, including chromosome missegregation, genomic instability, and developmental disorders. Schizosaccharomyces pombe is an wide-spread eukaryotic model organism for genetic studies due to its simplicity, ease of genetic manipulation, and well-characterized genetic structure, making it an ideal tool for research in fundamental cellular processes. The evolutionary gap between Schizosaccaromyces pombe and Drosophila melanogaster (approximately 1.2 billion years) and between D. melanogaster and humans (around 600 million years) provides a unique opportunity to investigate the conservation and divergence [3] of cohesin function across a broad evolutionary spectrum. This study aims to explore the conservation and functional roles of cohesin in these species, providing valuable insights into its molecular mechanisms and the potential impact of its dysfunction across different eukaryotic organisms. Methods: Each of the three S. pombe cohesin subunits, Psm1, Psm3 and Rad21, were replaced by their D. melanogaster orthologues. This was achieved by transforming a diploid strain with a linear fragment containing the Drosophila gene, a selection marker and homology sequences with the upstream and downstream regions of the gene to be replaced. The correct insertion of the fragment was verified by PCR. Viability of the transgenic yeast in haploidy was studied by sporulating the heterozygous mutants and segregating their four spores using a micromanipulator. Results: Psm1 and Psm3 genes were replaced by their fly orthologues, SMC1 and SMC3, in a diploid strain resulting in a double heterozygous transgenic mutant. Upon tetrad segregation, it was observed that drosophila´s version can not substitute yeast´s natives genes

The ceramide synthase subunit lac1 regulates cell growth and size in fission yeast

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.Junta de Andalucía P18-FRJ1132Universidad de Sevilla VIPPIT-2020-I.5Japan Society for the Promotion of Science JP19H02922, JP21K19088Ministerio de Ciencia, Innovación y Universidades BFU2017-89700-

Measurement of asthma control according to global initiative for asthma guidelines : a comparison with the asthma control questionnaire

Introduction: Asthma Control Questionnaire (ACQ) is a validated tool to measure asthma control. Cut-off points that best discriminate " well-controlled" or " not well-controlled" asthma have been suggested from the analysis of a large randomized clinical trial but they may not be adequate for daily clinical practice.Aims: To establish cut-off points of the ACQ that best discriminate the level of control according to Global Initiative for Asthma (GINA) 2006 guidelines in patients with asthma managed at Allergology and Pulmonology Departments as well as Primary Care Centers in Spain.Patients and methods: An epidemiological descriptive study, with prospective data collection. Asthma control following GINA-2006 classification and 7-item ACQ was assessed. The study population was split in two parts: 2/3 for finding the cut-off points (development population) and 1/3 for validating the results (validation population).Results: A total of 1,363 stable asthmatic patients were included (mean age 38 ± 14 years, 60.3% women; 69.1% non-smokers). Patient classification according to GINA-defined asthma control was: controlled 13.6%, partially controlled 34.2%, and uncontrolled 52.3%. The ACQ cut-off points that better agreed with GINA-defined asthma control categories were calculated using receiver operating curves (ROC). The analysis showed that ACQ < 0.5 was the optimal cut-off point for " controlled asthma" (sensitivity 74.1%, specificity 77.5%) and 1.00 for " uncontrolled asthma" (sensitivity 73%, specificity 88.2%). Kappa index between GINA categories and ACQ was 0.62 (p < 0.001).Conclusion: The ACQ cut-off points associated with GINA-defined asthma control in a real-life setting were <0.5 for controlled asthma and ≥1 for uncontrolled asthma. © 2012 Olaguibel et al.; licensee BioMed Central Ltd

Anti-Spike antibodies 3 months after SARS-CoV-2 mRNA vaccine booster dose in patients on hemodialysis: the prospective SENCOVAC study

Background: Patients on hemodialysis are at high-risk for complications derived from coronavirus disease 2019 (COVID-19). The present analysis evaluated the impact of a booster vaccine dose and breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on humoral immunity 3 months after the booster dose. Methods: This is a multicentric and prospective study assessing immunoglobulin G anti-Spike antibodies 6 and 9 months after initial SARS-CoV-2 vaccination in patients on hemodialysis that had also received a booster dose before the 6-month assessment (early booster) or between the 6- and 9-month assessments (late booster). The impact of breakthrough infections, type of vaccine, time from the booster and clinical variables were assessed. Results: A total of 711 patients [67% male, median age (range) 67 (20-89) years] were included. Of these, 545 (77%) received an early booster and the rest a late booster. At 6 months, 64 (9%) patients had negative anti-Spike antibody titers (3% of early booster and 29% of late booster patients, P =. 001). At 9 months, 91% of patients with 6-month negative response had seroconverted and there were no differences in residual prevalence of negative humoral response between early and late booster patients (0.9% vs 0.6%, P =. 693). During follow-up, 35 patients (5%) developed breakthrough SARS-CoV-2 infection. Antibody titers at 9 months were independently associated with mRNA-1273 booster (P =. 001), lower time from booster (P =. 043) and past breakthrough SARS-CoV-2 infection (P <. 001). Conclusions: In hemodialysis patients, higher titers of anti-Spike antibodies at 9 months were associated with mRNA-1273 booster, lower time from booster and past breakthrough SARS-CoV-2 infectionThe present project has been supported by Fresenius Medical Care, Diaverum, Vifor Pharma, Vircell, Fundación Renal Iñigo Álvarez de Toledo and ISCIII FEDER funds RICORS2040 (RD21/0005

The transcription factor rbf1 is the master regulator for b-mating type controlled pathogenic development in Ustilago maydis

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.We would like to thank R. Kahmann and the Max-Planck Institute for terrestrial Microbiology, Marburg, for generous support.Peer Reviewe