Complete microtubule–kinetochore occupancy favours the segregation of merotelic attachments

Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20 microtubules, even though single microtubules can generate sufficient force to move chromosomes. Here, we show that high microtubule occupancy at kinetochores ensures robust chromosome segregation by providing a strong mechanical force that favours segregation of merotelic attachments during anaphase. Using low doses of the microtubules-targeting agent BAL27862 we reduce microtubule occupancy and observe that spindle morphology is unaffected and bi-oriented kinetochores can still oscillate with normal intra-kinetochore distances. Inter-kinetochore stretching is, however, dramatically reduced. The reduction in microtubule occupancy and inter-kinetochore stretching does not delay satisfaction of the spindle assembly checkpoint or induce microtubule detachment via Aurora-B kinase, which was so far thought to release microtubules from kinetochores under low stretching. Rather, partial microtubule occupancy slows down anaphase A and increases incidences of lagging chromosomes due to merotelically attached kinetochores

Comparative genetic, proteomic and phosphoproteomic analysis of C. <i>elegans </i>embryos with a focus on <i>ham</i>-1/STOX and <i>pig</i>-1/MELK in dopaminergic neuron development

Asymmetric cell divisions are required for cellular diversity and defects can lead to altered daughter cell fates and numbers. In a genetic screen for C. elegans mutants with defects in dopaminergic head neuron specification or differentiation, we isolated a new allele of the transcription factor HAM-1 [HSN (Hermaphrodite-Specific Neurons) Abnormal Migration]. Loss of both HAM-1 and its target, the kinase PIG-1 [PAR-1(I)-like Gene], leads to abnormal dopaminergic head neuron numbers. We identified discrete genetic relationships between ham-1, pig-1 and apoptosis pathway genes in dopaminergic head neurons. We used an unbiased, quantitative mass spectrometry-based proteomics approach to characterise direct and indirect protein targets and pathways that mediate the effects of PIG-1 kinase loss in C. elegans embryos. Proteins showing changes in either abundance, or phosphorylation levels, between wild-type and pig-1 mutant embryos are predominantly connected with processes including cell cycle, asymmetric cell division, apoptosis and actomyosin-regulation. Several of these proteins play important roles in C. elegans development. Our data provide an in-depth characterisation of the C. elegans wild-type embryo proteome and phosphoproteome and can be explored via the Encyclopedia of Proteome Dynamics (EPD) - an open access, searchable online database

SPAT-1 acts with PLK-1 to regulate PAR polarity and cell cycle progression in the early C. elegans embryo

During cell division, cell cycle events have to be highly coordinated to happen in a correct order, with the proper timing and only once per division. Asymmetrically dividing cell must also coordinate polarity establishment and maintenance with other events of cell division. The presented work was pursued to study the link between cell cycle progression and cell polarity in asymmetrically dividing cells. An early C. elegans embryo enables simultaneous investigation and identification of proteins that control both these processes. The main players that have emerged from this work are: the mitotic kinase, PLK-1 and its regulator SPAT-1, the homologue of human Bora. As presented in the thesis, SPAT-1 together with PLK-1 controls both PAR polarity and cell cycle timing constituting a link between these events during asymmetric cell division

Cell polarity and asymmetric cell division: the C. elegans early embryo

Cell polarity is crucial for many functions including cell migration, tissue organization and asymmetric cell division. In animal cells, cell polarity is controlled by the highly conserved PAR (PARtitioning defective) proteins. par genes have been identified in Caenorhabditis elegans in screens for maternal lethal mutations that disrupt cytoplasmic partitioning and asymmetric division. Although PAR proteins were identified more than 20 years ago, our understanding on how they regulate polarity and how they are regulated is still incomplete. In this chapter we review our knowledge of the processes of cell polarity establishment and maintenance, and asymmetric cell division in the early C. elegans embryo. We discuss recent findings that highlight new players in cell polarity and/or reveal the molecular details on how PAR proteins regulate polarity processes

SPAT-1/Bora acts with Polo-like kinase 1 to regulate PAR polarity and cell cycle progression

During asymmetric cell division, cell polarity and cell cycle progression are tightly coordinated, yet mechanisms controlling both these events are poorly understood. Here we show that the Bora homologue SPAT-1 regulates both PAR polarity and cell cycle progression in C. elegans embryos. We find that, similarly to mammalian cells, SPAT-1 acts with PLK-1 and not with the mitotic kinase Aurora A (AIR-1), as shown in Drosophila. SPAT-1 binds to PLK-1, and depletion of SPAT-1 or PLK-1 leads to similar cell division defects in early embryos, which differ from the defects caused by depletion of AIR-1. Additionally, SPAT-1 and PLK-1 depletion causes impaired polarity with abnormal length of the anterior and posterior PAR domains, and partial plk-1(RNAi) or spat-1(RNAi), but not air-1(RNAi), can rescue the lethality of a par-2 mutant. SPAT-1 is enriched in posterior cells, and this enrichment depends on PAR polarity and PLK-1. Taken together, our data suggest a model in which SPAT-1 promotes the activity of PLK-1 to regulate both cell polarity and cell cycle timing during asymmetric cell division, providing a link between these two processes

Scattering of Exciting Light by Live Cells in Fluorescence Confocal Imaging: Phototoxic Effects and Relevance for FRAP Studies

As exciting light in a scanning confocal microscope encounters a cell and its subcellular components, it is refracted and scattered. A question arises as to what proportion of the exciting light is scattered by subcellular structures and whether cells in the vicinity of the imaged area, i.e., cells that are not directly illuminated by the laser beam, can be affected by either an exposure to scattered light and ensuing phototoxic reactions, or by the products of photoactivated reactions diffusing out of the directly illuminated area. We have designed a technique, which allows us to detect subtle cell photodamage and estimate the extent and range of phototoxic effects inflicted by interaction between scattered exciting light and fluorescent probes in the vicinity of the illuminated area. The technique is based on detecting an increased influx of acridine orange into photodamaged cells, which is manifested by a change of color. We demonstrate that phototoxic effects can be exerted not only on the illuminated cell, but also on fluorescently labeled neighboring cells. The damage inflicted on neighbors is due to exposure to light scattered by the imaged (i.e., directly illuminated) cell, but not phototoxic products diffusing out of the directly illuminated area. When light encounters a cell nucleus, scattering is so intense that photodamage can be inflicted even on fluorescently labeled cells located within a radius of ∼90 μm, i.e., several cell diameters away. This range of scattering is comparable with that caused by the glass bead resting on a coverslip (up to 120 μm). The intense scattering of exciting light imposes limits on FRAP, FLIP, and other techniques employing high intensity laser beams

Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms?

Spatio-temporal coordination of events during cell division is crucial for animal development. In recent years, emerging data have strengthened the notion that tight coupling of cell cycle progression and cell polarity in dividing cells is crucial for asymmetric cell division and ultimately for metazoan development. Although it is acknowledged that such coupling exists, the molecular mechanisms linking the cell cycle and cell polarity machineries are still under investigation. Key cell cycle regulators control cell polarity, and thus influence cell fate determination and/or differentiation, whereas some factors involved in cell polarity regulate cell cycle timing and proliferation potential. The scope of this review is to discuss the data linking cell polarity and cell cycle progression, and the importance of such coupling for asymmetric cell division. Because studies in model organisms such as Caenorhabditis elegans and Drosophila melanogaster have started to reveal the molecular mechanisms of this coordination, we will concentrate on these two systems. We review examples of molecular mechanisms suggesting a coupling between cell polarity and cell cycle progression