Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.
Correct alignment of the mitotic spindle during cell division is crucial for cell fate determination, tissue organization, and development. Mutations causing brain diseases and cancer in humans and mice have been associated with spindle orientation defects. These defects are thought to lead to an imbalance between symmetric and asymmetric divisions, causing reduced or excessive cell proliferation. However, most of these disease-linked genes encode proteins that carry out multiple cellular functions. Here, we discuss whether spindle orientation defects are the direct cause for these diseases, or just a correlative side effect.
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
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 approximately 90 microm, 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 microm). The intense scattering of exciting light imposes limits on FRAP, FLIP, and other techniques employing high intensity laser beams.
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