MAARS Software for Automatic and Quantitative Analysis of Mitotic Progression.

In this chapter, we describe a software called MAARS (Mitotic Analysis And Recording System) that enables automatic and quantitative analysis of mitotic progression on an open-source platform. This computer-assisted analysis of cell division allows the unbiased acquisition of multiple parameters such as cell shape or size, metaphase or anaphase delays, as well as various mitotic abnormalities. This chapter describes the power of such an expert system to highlight the complexity of the mechanisms required to prevent mitotic chromosome segregation errors, leading to aneuploidy.

Condensin positioning at telomeres by shelterin proteins drives sister-telomere disjunction in anaphase.

The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here, we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome remodeler associated with shelterin.

Aurora B and condensin are dispensable for chromosome arm and telomere separation during meiosis II.

In mitosis, while the importance of kinetochore (KT)-microtubule (MT) attachment has been known for many years, increasing evidence suggests that telomere dysfunctions also perturb chromosome segregation by contributing to the formation of chromatin bridges at anaphase. Recent evidence suggests that Aurora B kinase ensures proper chromosome segregation during mitosis not only by controlling KT-MT attachment but also by regulating telomere and chromosome arm separation. However, whether and how Aurora B governs telomere separation during meiosis has remained unknown.

Nuclear envelope attachment of telomeres limits TERRA and telomeric rearrangements in quiescent fission yeast cells.

Telomere anchoring to nuclear envelope (NE) is a key feature of nuclear genome architecture. Peripheral localization of telomeres is important for chromatin silencing, telomere replication and for the control of inappropriate recombination. Here, we report that fission yeast quiescent cells harbor predominantly a single telomeric cluster anchored to the NE.

Splicing Defects of the Profilin Gene Alter Actin Dynamics in an S. pombe SMN Mutant.

Spinal muscular atrophy (SMA) is a devastating motor neuron disorder caused by mutations in the survival motor neuron (SMN) gene. It remains unclear how SMN deficiency leads to the loss of motor neurons. By screening Schizosaccharomyces pombe, we found that the growth defect of an SMN mutant can be alleviated by deletion of the actin-capping protein subunit gene acp1.

MAARS: a novel high-content acquisition software for the analysis of mitotic defects in fission yeast.

Faithful segregation of chromosomes during cell division relies on multiple processes such as chromosome attachment and correct spindle positioning. Yet mitotic progression is defined by multiple parameters, which need to be quantitatively evaluated. To study the spatiotemporal control of mitotic progression, we developed a high-content analysis (HCA) approach that combines automated fluorescence microscopy with real-time quantitative image analysis and allows the unbiased acquisition of multiparametric data at the single-cell level for hundreds of cells simultaneously.

Aurora B kinase controls the separation of centromeric and telomeric heterochromatin.

The segregation of chromosomes is coordinated at multiple levels to prevent chromosome loss, a phenotype frequently observed in cancers. We recently described an essential role for telomeres in the physical separation of chromosomes and identified Aurora B kinase as a double agent involved in the separation of centromeric and telomeric heterochromatin.

Cutting edge science: Laser surgery illuminates viscoelasticity of merotelic kinetochores.

Increasing evidence in eukaryotic cells suggests that mechanical forces are essential for building a robust mitotic apparatus and correcting inappropriate chromosome attachments. In this issue, Cojoc et al. (2016.

Fission yeast kinesin-8 controls chromosome congression independently of oscillations.

In higher eukaryotes, efficient chromosome congression relies, among other players, on the activity of chromokinesins. Here, we provide a quantitative analysis of kinetochore oscillations and positioning in Schizosaccharomyces pombe, a model organism lacking chromokinesins. In wild-type cells, chromosomes align during prophase and, while oscillating, maintain this alignment throughout metaphase.

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52.

Aurora B prevents chromosome arm separation defects by promoting telomere dispersion and disjunction.

The segregation of centromeres and telomeres at mitosis is coordinated at multiple levels to prevent the formation of aneuploid cells, a phenotype frequently observed in cancer. Mitotic instability arises from chromosome segregation defects, giving rise to chromatin bridges at anaphase. Most of these defects are corrected before anaphase onset by a mechanism involving Aurora B kinase, a key regulator of mitosis in a wide range of organisms.

A stochastic model of kinetochore-microtubule attachment accurately describes fission yeast chromosome segregation.

In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs.

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast.

The Dam1 complex is a kinetochore component that couples chromosomes to the dynamic ends of kinetochore microtubules (kMTs). Work in the budding yeast Saccharomyces cerevisiae has shown that the Dam1 complex forms a 16-unit ring encircling and tracking the tip of a MT in vitro, consistent with its cellular function as a coupler. Dam1 also forms smaller, nonring patches in vitro that track the dynamic ends of MTs.

Tip1/CLIP-170 protein is required for correct chromosome poleward movement in fission yeast.

The plus-end microtubule binding proteins (+TIPs) play an important role in the regulation of microtubule stability and cell polarity during interphase. In S. pombe, the CLIP-170 like protein Tip1, together with the kinesin Tea2, moves along the microtubules towards their plus ends.

Ase1/Prc1-dependent spindle elongation corrects merotely during anaphase in fission yeast.

Faithful segregation of sister chromatids requires the attachment of each kinetochore (Kt) to microtubules (MTs) that extend from opposite spindle poles. Merotelic Kt orientation is a Kt-MT misattachment in which a single Kt binds MTs from both spindle poles rather than just one. Genetic induction of merotelic Kt attachment during anaphase in fission yeast resulted in intra-Kt stretching followed by either correction or Kt disruption.

The dynamin related protein Dnm1 fragments mitochondria in a microtubule-dependent manner during the fission yeast cell cycle.

Mitochondria are dynamic organelles that undergo cycles of fission and fusion. In the fission yeast, Schizosaccharomyces pombe, mitochondria align with microtubules and mitochondrial integrity is dependent upon an intact microtubule cytoskeleton. Here we show that mitochondria re-organize during the cell cycle and that this process is both dynamin- and microtubule-dependent.

Sister kinetochore recapture in fission yeast occurs by two distinct mechanisms, both requiring Dam1 and Klp2.

In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. We have studied chromosome recapture in the fission yeast Schizosaccharomyces pombe. We show by live cell analysis that lost kinetochores interact laterally with intranuclear microtubules (INMs) and that both microtubule depolymerization (end-on pulling) and minus-end-directed movement (microtubule sliding) contribute to chromosome retrieval to the spindle pole body (SPB).

Lost your orientation? Find your way with PtdIns(3,4,5)P3!

Faithful chromosome segregation requires correct positioning of the spindle during mitosis. In this issue of Developmental Cell, Toyoshima et al. describe a new mechanism for spindle orientation involving phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3].

Dynein participates in chromosome segregation in fission yeast.

Background Information: In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. For faithful segregation of sister chromatids, each sister kinetochore must attach to microtubules that extend to opposite poles (chromosome bi-orientation). At the metaphase-anaphase transition, cohesion between sister chromatids is removed, and each sister chromatid is pulled to opposite poles of the cell by microtubule-dependent forces.

The fission yeast spindle orientation checkpoint: a model that generates tension?

In all eukaryotes, the alignment of the mitotic spindle with the axis of cell polarity is essential for accurate chromosome segregation as well as for the establishment of cell fate, and thus morphogenesis, during development. Studies in invertebrates, higher eukaryotes and yeast suggest that astral microtubules interact with the cell cortex to position the spindle. These microtubules are thought to impose pushing or pulling forces on the spindle poles to affect the rotation or movement of the spindle.

btn1, the Schizosaccharomyces pombe homologue of the human Batten disease gene CLN3, regulates vacuole homeostasis.

We have cloned the Schizosaccharomyces pombe homologue of the human Batten disease gene, CLN3. This gene, btn1, encodes a predicted transmembrane protein that is 30% identical and 48% similar to its human counterpart. Cells deleted for btn1 were viable but had enlarged and more alkaline vacuoles.

Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis.

In the fission yeast, Schizosaccharomyces pombe, uptake of the fluorescent styryl dye FM4-64 via the endocytic pathway to the vacuole was localised to the poles of growing, interphase cells and to the cell equator during cell division, regions of cell wall deposition that are rich in actin. When the pattern of growth or the plane of cytokinesis was altered, the relationship between the actin cytoskeleton and the site of endocytosis was maintained. Transfer of the label to the vacuolar membrane was dependent upon the Rab GTPase Ypt7 and, hence, vesicle fusion.

Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3-dependent checkpoint in fission yeast.

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton.

Mechanism controlling perpendicular alignment of the spindle to the axis of cell division in fission yeast.

In animal cells, the mitotic spindle is aligned perpendicular to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring (CAR). We show that, in fission yeast, spindle rotation is dependent on the interaction of astral microtubules with the cortical actin cytoskeleton.