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

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.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness BFU2011-15216-E, P09-CTS-4697, and PGC2018-099849-B-100 to R.R.D.; National Institutes of Health (NIH) R01, GM067690, and GM115185 to F.C.; and NIH grants R01-GM085145 and R35-GM126910 to S.J

Cell migration and division in amoeboid-like fission yeast

Summary 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

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 10min. 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. © 2013 John Wiley & Sons, Ltd.This work was supported by grants to RRD from the Spanish Ministerio de Ciencia e Inovación (Grant No. BFU2010-21310) and the Junta de Andalucia (Grant No. P09-CTS-4697, Proyecto de Excelencia). I.F.-P. was supported by the Spanish Ministerio de Educación (Juan de la Cierva Programme).Peer Reviewe

La regulación del Spindle Assembly Checkpoint en Schizosaccharomyces pombe

Resumen del trabajo presentado a la 2ª Jornada de los másteres de biotecnología de la UPO, celebrada en Sevilla el 26 de abril de 2013.[Motivación]: Durante la mitosis, el huso mitótico formado por microtúbulos y moléculas asociadas, se encarga de la correcta segregación de los cromosomas. La pérdida o ganancia de cromosomas durante la mitosis puede promover cambios genéticos que predispongan al cáncer. Las células eucariotas tienen mecanismos que proporcionan a la célula un tiempo extra cuando existen problemas para capturar los cromosomas de forma adecuada. Este mecanismo, conservado de levaduras a humanos, se denomina Checkpoint del huso mitótico o Spindle Assembly Checkpoint (SAC). Una observación inicial mostró que las rutas de estrés son esenciales para el funcionamiento del Checkpoint del huso. Consistente con esta observación se comprobó que Bub1, una quinasa esencial para el funcionamiento del SAC, presenta 5 sitios consenso de fosforilación por las MAPKs. En este proyecto se estudió la relevancia funcional de los sitios de fosforilación por MAPK de Bub1 en la levadura S. pombe. [Métodos]: Para estudiar la regulación del SAC, se usó el análisis genético y microscópico de la división celular en mutantes defectivos de este sistema de control en mitosis. Técnicas como la transformación y la clonación también fueron utilizadas. [Resultados]: Hemos demostrado que el SAC está regulado por la ruta de estrés de la MAPK pmk1 en S. pombe. La inactivación de la ruta MAPK a través de la deleción de pmk1, hace que las células sean sensibles a una droga que induce despolimerización en los microtúbulos del spindle, indicativo de defectos en el SAC. Mediante análisis genético, encontramos que este mecanismo es dependiente de bub1, un regulador central del SAC. Además, se ha estudiado la división celular en un mutante de Bub1 en el que se han eliminado 5 sitios putativos de fosforilación por MAPKs observándose, que dicho mutante es incapaz de llevar a cabo una segregación cromosómica correcta. [Conclusiones]: Se ha determinado que la ruta de la MAPK pmk1 se requiere para la supervivencia de S. pombe en condiciones en las que se compromete la función del huso mitótico así como la importancia de los sitios de fosforilación consenso de MAPK presentes en la quinasa Bub1. Nuestro estudio revela una conexión entre las rutas de MAPKs y el correcto funcionamiento del SAC. La pérdida total o parcial de esta regulación da lugar a eventos de aneuploidía. Este estudio puede ser relevante para entender cómo las células eucariotas evitan la aneuploidía, una de las características más comunes en tumores y que suelen modificar la progresión tumoral hacia diferentes estados de malignidad.Peer reviewe