Centrosomal MPF triggers the mitotic and morphogenetic switches of fission yeast

PMCID: PMC3549529.-- et al.Activation of mitosis-promoting factor (MPF) drives mitotic commitment. In human cells active MPF appears first on centrosomes. We show that local activation of MPF on the equivalent organelle of fission yeast, the spindle pole body (SPB), promotes Polo kinase activity at the SPBs long before global MPF activation drives mitotic commitment. Artificially promoting MPF or Polo activity at various locations revealed that this local control of Plo1 activity on G2 phase SPBs dictates the timing of mitotic commitment. Cytokinesis of the rod-shaped fission yeast cell generates a naive, new, cell end. Growth is restricted to the experienced old end until a point in G2 phase called new end take off (NETO) when bipolar growth is triggered. NETO coincided with MPF activation of Plo1 on G2 phase SPBs (ref. 4). Both MPF and Polo activities were required for NETO and both induced NETO when ectopically activated at interphase SPBs. NETO promotion by MPF required polo. Thus, local MPF activation on G2 SPBs directs polo kinase to control at least two distinct and temporally separated, cell-cycle transitions at remote locations.This work was supported by Cancer Research UK (CRUK) grant number C147/A6058. V.S. and A.K. were supported by the Swiss National Science Foundation and EPFL.Peer reviewe

Extending the Schizosaccharomyces pombe molecular genetic toolbox

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.Swiss National Science Foundation (www.snf.ch). École polytechnique fédérale de Lausanne (www.epfl.ch). Cancer Research UK [CRUK] C147/A6058.Peer Reviewe

Polo-like kinases: a team that plays throughout mitosis

When the first mutant allele of the Drosophila genepolo was first characterized over 10 years ago, attention focused on the defects that centrosome behavior exhibited at various stages of development (Sunkel and Glover 1988). The subsequent realization that the serine-threonine kinase it encodes is highly conserved from yeasts to humans has provoked a flurry of investigation into the function of the enzyme. A role for the polo-like kinases (plks) in regulating centrosome behavior has been borne out in several organisms, and the enzymes have attracted further attention recently with the realization that they regulate multiple stages of mitotic progression. In this article we review the current status of our understanding of the functions of plks from the time of commitment to M phase in the activation of Cdc25, through the activation of the anaphase promoting complex (APC), to the regulation of late mitotic events essential for cytokinesis. We discuss how to reconcile the sometimes apparently disparate observations made upon plk function in different organisms

The S. pombe mitotic regulator Cut12 promotes spindle pole body activation and integration into the nuclear envelope

The fission yeast spindle pole body (SPB) comprises a cytoplasmic structure that is separated from an ill-defined nuclear component by the nuclear envelope. Upon mitotic commitment, the nuclear envelope separating these domains disperses as the two SPBs integrate into a hole that forms in the nuclear envelope. The SPB component Cut12 is linked to cell cycle control, as dominant cut12.s11 mutations suppress the mitotic commitment defect of cdc25.22 cells and elevated Cdc25 levels suppress the monopolar spindle phenotype of cut12.1 loss of function mutations. We show that the cut12.1 monopolar phenotype arises from a failure to activate and integrate the new SPB into the nuclear envelope. The activation of the old SPB was frequently delayed, and its integration into the nuclear envelope was defective, resulting in leakage of the nucleoplasm into the cytoplasm through large gaps in the nuclear envelope. We propose that these activation/integration defects arise from a local deficiency in mitosis-promoting factor activation at the new SPB

Polo-like kinases: a team that plays throughout mitosis

When the first mutant allele of the Drosophila genepolo was first characterized over 10 years ago, attention focused on the defects that centrosome behavior exhibited at various stages of development (Sunkel and Glover 1988). The subsequent realization that the serine-threonine kinase it encodes is highly conserved from yeasts to humans has provoked a flurry of investigation into the function of the enzyme. A role for the polo-like kinases (plks) in regulating centrosome behavior has been borne out in several organisms, and the enzymes have attracted further attention recently with the realization that they regulate multiple stages of mitotic progression. In this article we review the current status of our understanding of the functions of plks from the time of commitment to M phase in the activation of Cdc25, through the activation of the anaphase promoting complex (APC), to the regulation of late mitotic events essential for cytokinesis. We discuss how to reconcile the sometimes apparently disparate observations made upon plk function in different organisms

Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

Insertion into and release of the cytoplasmic domain of the Schizosaccharomyces pombe spindle pole body from a nuclear envelope fenestra during mitosis requires Brr6

Immunofluorescence Microscopy of Schizosaccharomyces pombe Using Chemical Fixation

Establishing the subcellular distribution of molecules of interest and the dynamics of their spatial control underpins all areas of cell and developmental biology. Although the ability to monitor the distribution of fluorescent fusion proteins has revolutionized cell and developmental biology, indirect immunofluorescence microscopy of fixed samples remains an essential complement to this approach. Immunofluorescence is often a more appropriate approach for the study of subcellular architecture. It avoids potential artifacts caused by studying fusion proteins, which might show altered function under stressful imaging conditions. Furthermore, the quantitative analysis of multiple cells in an unperturbed population by immunofluorescence invariably provides a more accurate assessment of the spatial and temporal control of a particular process than does the analysis of individual cells that is the hallmark of live-cell imaging. Parallel studies of living and fixed cells often provide complementary data sets, both of which can be considered necessary for a comprehensive understanding of molecular function. This protocol provides a method for the visualization of the Schizosaccharomyces pombe microtubule cytoskeleton by indirect immunofluorescence microscopy following chemical fixation with formaldehyde and glutaraldehyde. It includes discussion of common modifications used to monitor the distribution of other fission yeast antigens and forms a basis from which to develop protocols to localize new molecules of interest.</jats:p

Chromatin and Cell Wall Staining of Schizosaccharomyces pombe

Fission yeasts grow by tip extension, maintaining a constant width until they reach a critical size threshold and divide. Division by medial fission—which gives these yeast their name—generates a new end that arises from the site of cytokinesis. The old end, which was produced during the previous cell cycle, initiates progression of the new cell cycle, and in G2, the new end is activated in a process termed new-end takeoff (NETO). In this protocol, the fluorescent stains calcofluor and 4′,6-diamidino-2-phenylindole (DAPI) are used to give a rapid and informative assessment of morphogenesis and cell-cycle progression in the fission yeast Schizosaccharomyces pombe. Calcofluor reveals the timing of NETO because it stains the birth scars that are generated at new ends by cytokinesis less efficiently than the rest of the cell wall. Intense calcofluor staining of the septum and measurement of cell length are also widely used to identify dividing cells and to gauge the timing of mitotic commitment. Staining nuclei with DAPI identifies mono- and binucleated cells and complements the calcofluor staining procedure to evaluate the stages of the cell cycle and identify mitotic errors. Equally simple DAPI staining procedures reveal chromatin structure in higher resolution, facilitating more accurate staging of mitotic progression and characterization of mitotic errors.</jats:p