Viesturs Simanis

The septation initiation network (SIN) is a conserved signal transduction network, which is important for cytokinesis in Schizosaccharomyces pombe. The SIN component Etd1p is required for association of some SIN proteins with the spindle pole body (SPB) during anaphase and for contractile ring formation. We show that tethering of Cdc7p or Sid1p to the SIN scaffold Cdc11p at the SPB, rescues etd1-Δ. Analysis of a suppressor of the mutant etd1-M9 revealed that SIN signalling is influenced by the carbon source of the cell. Growth on a non-fermentable carbon source glycerol reduces the requirement for SIN signalling but does not bypass it. The decreased need for SIN signalling is mediated largely by reduction of protein kinase A activity, and it is phenocopied by deletion of pka1 on glucose medium. We conclude that protein kinase A is an important regulator of the SIN, and that SIN signalling is regulated by the carbon source of the cell.

Schizosaccharomyces pombe cells are rod shaped, and they grow by tip elongation. Growth ceases during mitosis and cell division; therefore, the length of a septated cell is a direct measure of the timing of mitotic commitment, and the length of a wild-type cell is an indicator of its position in the cell cycle. A large number of documented stage-specific changes can be used as landmarks to characterize cell cycle progression under specific experimental conditions. Conditional mutations can permanently or transiently block the cell cycle at almost any stage. Large, synchronously dividing cell populations, essential for the biochemical analysis of cell cycle events, can be generated by induction synchrony (arrest-release of a cell cycle mutant) or selection synchrony (centrifugal elutriation or lactose-gradient centrifugation). Schizosaccharomyces pombe cell cycle studies routinely combine particular markers, mutants, and synchronization procedures to manipulate the cycle. We describe these techniques and list key landmarks in the fission yeast mitotic cell division cycle.

Size selection of small cells from an asynchronous Schizosaccharomyces pombe culture offers a simple way to generate cultures in which progression through the mitotic cell division cycle is synchronized throughout the population. Here, we describe how density centrifugation of cells from asynchronous cultures through lactose gradients selects small G₂ cells to generate synchronized cultures as large as 500 mL. The ease and simplicity of this approach makes it an accessible and attractive method for generating synchronous cultures.

Cytokinesis in fission yeast is controlled by the Septation Initiation Network (SIN), a protein kinase signaling network using the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this paper, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.

The septation initiation network (SIN) regulates aspects of cell growth and division in Schizosaccharomyces pombe and is essential for cytokinesis. Insufficient signalling results in improper assembly of the contractile ring and failure of cytokinesis, generating multinucleated cells, whereas too much SIN signalling uncouples cytokinesis from the rest of the cell cycle. SIN signalling is therefore tightly controlled to coordinate cytokinesis with chromosome segregation. Signalling originates from the cytoplasmic face of the spindle pole body (SPB), and asymmetric localisation of some SIN proteins to one of the two SPBs during mitosis is important for regulation of the SIN. Recent studies have identified in vivo substrates of the SIN, which include components involved in mitotic control, those of the contractile ring and elements of the signalling pathway regulating polarised growth. The SIN is also required for spore formation following meiosis. This has provided insights into how the SIN performs its diverse functions in the cell cycle and shed new light on its regulation.

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.

The S. pombe Septation Initiation Network (SIN) regulates cytokinesis, and asymmetric association of SIN proteins with the mitotic spindle poles (SPBs) is important for its regulation. We have used semi-automated image analysis to study SIN proteins in large numbers of wild-type and mutant cells. Our principal conclusions are; First, the association of Cdc7p with the SPBs in early mitosis is asymmetric, with a bias in favour of the new SPB. Second, the early SPB association of Cdc7p-GFP depends on Plo1p but not Spg1p, and is unaffected by mutations that influence its asymmetry in anaphase. Third Cdc7p asymmetry in anaphase B is delayed by Pom1p and by activation of the spindle assembly checkpoint, and is promoted by Rad24p. Fourth, the length of the spindle, expressed as a fraction of the length of the cell, at which Cdc7p becomes asymmetric is similar in cells dividing at different sizes. These data reveal that multiple regulatory mechanisms control the SIN in mitosis and lead us to propose a two-state model to describe the SIN.

The Schizosaccharomyces pombe septation initiation network (SIN) is required for cytokinesis during vegetative growth and spore formation during meiosis. Regulation of the SIN during mitosis has been studied extensively, but less is known about its meiotic regulation. Here, we show that several aspects of the SIN regulation differ between mitosis and meiosis. First, the presence of GTP-bound spg1p is not the main determinant of the timing of cdc7p and sid1p association with the SPB during meiosis. Second, the localisation dependencies of SIN proteins differ from those in mitotic cells, suggesting a modified functional organisation of the SIN during meiosis. Third, there is stage-specific degradation of SIN components in meiosis; byr4p is degraded after meiosis I, while the degradation of cdc7p, cdc11p and sid4p occurs after the second meiotic division and depends upon the ubiquitin ligase dma1p. Finally, dma1p-dependent degradation is not restricted to the SIN, for we show that dma1p is needed for the degradation of mcp6p/hrs1p in meiosis I. Together, these data suggest that stage-specific targetted proteolysis will play an important role in regulating meiotic progression.

The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called “RodCellJ”, allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. (Continued on next page) (Continued from previous page) “RodCellJ” is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html.