When DNA replication is challenged, cells activate a DNA synthesis checkpoint blocking cell cycle progression until they are able to overcome the replication defects. In fission yeast, Cds1 is the effector kinase of this checkpoint, inhibiting M phase entry, stabilizing stalled replication forks and triggering transcriptional activation of S-phase genes; the molecular basis of this last effect remains largely unknown. The MBF complex controls the transcription of S-phase genes. We have purified novel interactors of the MBF complex and among them we have identified the repressor Max1. When the DNA synthesis checkpoint is activated, Max1 is phosphorylated by Cds1 resulting in the abrogation of its binding to MBF. As a consequence, MBF-dependent transcription is maintained active until cells are able to overcome this challenge. / Cuando la replicación del DNA se ve alterada, las células activan un mecanismo de control bloqueando la progresión del ciclo celular hasta que son capaces de superar el daño. En la levadura de fisión, Cds1 es la proteína kinasa efectora de dicha respuesta, mediante inhibición de la entrada en fase M, estabilización las horquillas de replicación bloqueadas, e inducción de la activación de la transcripción de los genes de fase S; siendo la base molecular de este último proceso poco conocida. El factor de transcripción MBF controla la transcripción de los genes de fase S. Hemos purificado proteínas que interaccionan con MBF, y entre ellas, hemos identificado al represor Max1. Cuando el checkpoint de síntesis de DNA es activado, Max1 es fosforilado por la kinasa Cds1, y esto se traduce en la disociación de Max1 del complejo MBF. Como consecuencia, la transcripción MBF-dependiente se mantiene activa hasta que las células son capaces de superar el daño.

La cohésion des chromatides sœurs est assurée par un complexe protéique en forme d’anneau assurant leur capture topologique. Ce complexe est constitué par des protéines conservées de la levure à l’Homme regroupées sous le terme « cohésine » : Smc1, Smc3 et la phosphoprotéine Scc1 fermant l’anneau (respectivement Psm1, Psm3 et Rad21 chez Schizosaccharomyces pombe). Les protéines régulatrices Rad61-Wapl, Pds5 et Scc3 (Wpl1,Pds5 et Psc3 respectivement chez S. pombe) interagissent avec l’anneau via Scc1. Il a été proposé que la capture de l’ADN par les cohésines nécessite l’ouverture transitoire de l’interface Smc1/Smc3. La réaction de dissociation fait quant à elle intervenir le sous-complexe Wapl/Pds5/Scc3 entraînant vraisemblablement l’ouverture de l’interface Scc1/Smc3. Le mécanisme par lequel la cohésion est créée et celui par lequel Wapl promeut la dissociation des cohésines des chromosomes, sont encore inconnus. Parmi les mutants de cohésion chez Saccharomyces cerevisiae, la mutation thermosensible eco1-1 affecte le gène ECO1 codant une acétyl-transférase, essentielle à la viabilité cellulaire, conservée de la levure à l’Homme (Eco1 « Establishment of Cohesion » chez S. cerevisiae, Eso1 chez S.pombe, ESCO1-2 chez l’Homme) et ayant Smc3 pour substrat. Il a été montré que l’acétyl-transférase s’oppose à l’action de dissociation de Wapl. C’est un crible génétique réalisé par plusieurs équipes, visant à trouver des mutants suppresseurs d’eco1-1, qui a permis d’identifier les gènes codant les protéines Wapl, Pds5, Scc3 et Smc3 comme composants du mécanisme d’ouverture de l’anneau de cohésine. Un crible similaire a été réalisé chez S.pombe dans notre laboratoire, dans le but de trouver des suppresseurs de la mutation thermosensible eso1-H17. Ce crible a identifié les gènes orthologues à ceux trouvés chez la levure : wpl1, pds5, psc3 et psm3 mais aussi le gène codant la sous-unité catalytique du complexe sérine/thréonine phosphatase de type IV (PP4), noté pp4c. Nous avons alors mis en œuvre des expériences pour caractériser PP4c ainsi que sa sous-unité régulatrice Psy2 qui s’est révélée être également impliquée dans la cohésion des chromatides soeurs. Nous avons également identifié la protéine Rad21 comme substrat du complexe PP4, puis identifié les phosphosites potentiellement cibles de PP4, pour ensuite cribler et analyser des phosphomutants de Rad21 récapitulant l’effet suppresseur de la délétion de PP4. / Sister-chromatid cohesion is ensured by a ring shape protein complex which is in charge of their topological embrace. This complex consists of proteins which are conserved from yeast to human and grouped under the term “cohesin”: Smc1, Smc3 and the phosphoprotein Scc1 which closes the ring (respectively Psm1, Psm3 and Rad21 in Schizosaccharomyces pombe). The regulatory proteins Rad61-Wapl, Pds5 and Scc3 (Wpl1,Pds5 and Psc3 respectively in S. pombe) interact with the ring via Scc1. It has been suggested that DNA capture by the cohesin complex involves the transient opening of the Smc1/Smc3 interface. The dissociation reaction involves the sub-complex Wapl/Pds5/Scc3 which likely causes the opening of the Scc1/Smc3 interface. The mechanisms by which cohesion is created and by which Wapl promotes the cohesin dissociation from chromosomes are still unknown. Among the cohesion mutants in Saccharomyces cerevisiae the thermosensitive eco1-1 mutation affects the ECO1 gene encoding an acetyl-transferase essential for cell viability and conserved from yeast to human (Eco1 « Establishment of Cohesion » in S.cerevisiae, Eso1 in S. pombe and ESCO1-2 in human) and whose substrate is Smc3. It has been shown that the acetyl-transferase counteracts the dissociation action of Wapl. A genetic screen carried out by several teams in order to find suppressors of the eco1-1 mutation has led to the identification of the genes encoding the Wapl, Pds5, Scc3 and Smc3 proteins as components of the opening mechanism of the cohesin ring. A similar screen was carried out in S. pombe in our lab to find suppressors of the thermosensitive mutation eso1-H17. This screen identified the orthologous genes to those found in the budding yeast: wpl1,pds5, psc3 and psm3 and also the gene encoding the catalytic subunit of the type 4 serine/threonine phosphatase complex (PP4) named pp4c. We have therefore carried out experiments to characterize PP4c and its regulatory subunit Psy2 which has also been found to be involved in sister-chromatid cohesion. We have likewise identified the Rad21 subunit as a PP4 substrate and identified phosphosites as potential targets of PP4. We have then screened and analyzed Rad21 phosphomutants which were able to mimic the suppressor effect of the deletion of pp4c.

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Cbf11 and Cbf12 proteins, the members of the CSL transcription factors family, are involved in a wide range of cellular processes in the fission yeast Schizosaccharomyces pombe - among other things they regulate cell adhesion and they have also been implicated in maintenance of genome integrity. At the level of the whole genome we previously identified target loci bound by CSL proteins in vivo. Many of them do not contain any consensus CSL-binding element. There are probably different DNA binding modes of the Cbf11/12 proteins and it has not been known what specific biological function is associated with the particular way of DNA binding. For the purpose of studying CSL DNA binding modes we have worked in this project on the implementation of the DNA binding mutation (DBM), which prevents direct DNA binding of CSL proteins to canonical motif in vitro, into the chromosomal locus of the cbf11 and cbf12 genes. Using the "ura4 selection system" we have successfully constructed the scar-less Cbf12-TAP and Cbf12DBM-TAP knock-ins, i.e. the strains without/with DBM in the open reading frame of Cbf12 where Cbf12 is C- terminally TAP-tagged and contains the intact 3'UTR. In our laboratory we have established the CRISPR/Cas9 system by which we have been able to prepare the Cbf11- TAP strain. We have failed to...

As the effector component of the Notch signaling pathway the transcription factors of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/Lag-1) are essential for many developmental processes in metazoan organisms, but they can function also independently of Notch. Recently, their presence was proved in fungal organisms lacking the Notch pathway as well as most of the known metazoan interacting partners. Cbf11 and Cbf12, the CSL proteins of the unicellular yeast Schizosaccharomyces pombe, were determined experimentally as non-essential nuclear transcription factors, which regulate cell adhesion, extracellular material production, colony morphology, septation and daughter cell separation, coordination of nuclear and cell division, and ploidy maintenance in an antagonistic way. The responsive genes of these factors are not known yet. In this study, genes of S. pombe, whose promoter regions represent potential direct targets for the Cbf proteins binding, were predicted. The binding of the Cbf11 and Cbf12 proteins, and of a truncated version Cbf12∆N to CSL response elements contained in the regulatory regions of selected S. pombe genes was tested in vitro by EMSA, and consequently, in the case of the Cbf11 protein, also in vivo by ChIP. Cbf11 and Cbf12∆N recognize specifically the response elements in...

Cells need to complete DNA replication in a timely and error-free manner. To ensure that replication is completed efficiently and in a finite amount of time, cells regulate origin firing. To prevent any errors from being transmitted to the next generation, cells have the checkpoint mechanism. The S-phase DNA damage slows replication to allow the cell to repair the damage. The mechanism of replication slowing by the checkpoint was not clear in fission yeast, Schizosaccharomyces pombe, at the start of my thesis. The downstream targets of the DNA damage checkpoint in fission yeast were also unclear. I worked on identifying the downstream targets for the checkpoint by studying if Cdc25, a phosphatase, is a target of the checkpoint. Work from our lab has shown that origin firing is stochastic in fission yeast. Origins are also known to be inefficient. Inefficient origins firing stochastically would lead to large stretches of chromosome where no origins may fire randomly leading to long replication times, an issue called the random gap problem. However, cells do not take a long time to complete replication and the process of replication itself is efficient. I focused on understanding the mechanism by which cells complete replication and avoid the random gap problem by attempting to measure the firing efficiency of late origins. Genome-wide origin studies in fission yeast have identified several hundred origins. However, the resolution of these studies can be improved upon. I began a genome-wide origin mapping study using deep sequencing to identify origins at a greater resolution compared to the previous studies. We have extended our origin search to two other Schizosaccharomyces species- S. octosporus and S. japonicus.There have been no origin mapping studies on these fission yeasts and identifying origins in these species will advance the field of replication. My thesis research shows that Cdc25 is not a target of the S-phase DNA damage checkpoint. I showed that DNA damage checkpoint does not target Cdc2-Y15 to slow replication. Based on my preliminary observation, origin firing might be inhibited by the DNA damage checkpoint as a way to slow replication. My efforts to measure the firing efficiency of a late replicating sequence were hindered by potentially unidentified inefficient origins firing at a low rate and replicating the region being studied. Studying the origin efficiency was maybe further complicated by neighboring origins being able to passively replicate the region. To identify origins in recently sequenced Schizosaccharomyces species, we initiated the genome-wide origin mapping. The mapping was also done on S. pombe to identify inefficient origins not mapped by other mapping studies. My work shows that deep sequencing can be used to map origins in other species and provides a powerful tool for origin studies.

No description available.

It is known that fission yeast Schizosaccharomyces pombe maintains its nuclear envelope during mitosis and it undergoes an interesting shape change during cell division - from a spherical via an ellipsoidal and a peanut-like to a dumb-bell shape. However, the biomechanical system behind this amazing transformation is still not understood. What we know is, that the shape must change due to forces acting on the membrane surrounding the nucleus and the microtubule based mitotic spindle is thought to play a key role. To estimate the locations and directions of the forces, the shape of the nucleus was recorded by confocal light microscopy. But such data is often inhomogeneously labeled with gaps in the boundary, making classical segmentation impractical. In order to accurately determine the shape we developed a global parametric shape description method, based on a Fourier coordinate expansion. The method implicitly assumes a closed and smooth surface. We will calculate the geometrical properties of the 2-dimensional shape and extend it to 3-dimensional properties, assuming rotational symmetry. Using a mechanical model for the lipid bilayer and the so called Helfrich-Canham free energy we want to calculate the minimum energy shape while respecting system-specific constraints to the surface and the enclosed volume. Comparing it with the observed shape leads to the forces. This provides the needed research tools to study forces based on images.

It is known that fission yeast Schizosaccharomyces pombe maintains its nuclear envelope during mitosis and it undergoes an interesting shape change during cell division - from a spherical via an ellipsoidal and a peanut-like to a dumb-bell shape. However, the biomechanical system behind this amazing transformation is still not understood. What we know is, that the shape must change due to forces acting on the membrane surrounding the nucleus and the microtubule based mitotic spindle is thought to play a key role. To estimate the locations and directions of the forces, the shape of the nucleus was recorded by confocal light microscopy. But such data is often inhomogeneously labeled with gaps in the boundary, making classical segmentation impractical. In order to accurately determine the shape we developed a global parametric shape description method, based on a Fourier coordinate expansion. The method implicitly assumes a closed and smooth surface. We will calculate the geometrical properties of the 2-dimensional shape and extend it to 3-dimensional properties, assuming rotational symmetry. Using a mechanical model for the lipid bilayer and the so called Helfrich-Canham free energy we want to calculate the minimum energy shape while respecting system-specific constraints to the surface and the enclosed volume. Comparing it with the observed shape leads to the forces. This provides the needed research tools to study forces based on images.