Regulation of mitotic spindles by IP3 receptors

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Every animal begins life as a single fertilized egg, which then divides repeatedly by mitosis to produce an animal with, in humans, some 30,000,000,000,000 cells. Mitosis continues throughout life to replenish lost and damaged cells. During mitosis, a diamond-shaped web forms (the spindle) and the replicated chromosomes align at its centre. The chromosomes are then drawn apart towards the spindle poles so that genetic material is evenly distributed between the two daughter cells. The orientation of the spindle - aligned with the surface to which the cell adheres or perpendicular to it - is important because it determines where the cell divides, and that influences how each cell will subsequently develop. Mitosis is tightly regulated, in large part by families of kinases that attach phosphate groups to other proteins, to ensure that each step proceeds only when preceding steps have been properly completed. Many cellular activities, including some steps in mitosis, are regulated by increases in intracellular calcium concentration. These calcium signals are generated when channels open and allow calcium to flow into the cell down a steep concentration gradient. One of the most important of these channels is the IP3 receptor, which allows controlled release of calcium from an intracellular store, the ER. In addition to distributing chromosomes to daughter cells, mitosis must also ensure that each cell gets its share of intracellular organelles, including the ER. There is, therefore, a massive rearrangement of intracellular structures, including the ER, during mitosis. Our recent work, using microscopes to report the movement of cellular components made visible by attachment of coloured proteins, has shown that during mitosis IP3 receptors accumulate around the spindle poles and that in cells without IP3 receptors the spindle does not align properly. We suggest that local calcium signals generated by these repositioned IP3 receptors control spindle orientation by regulating the tethers (astral microtubules) that hold the spindle poles to the plasma membrane that surrounds the cell. Immediately beneath the plasma membrane and at the spindle poles, there is an accumulation of actin, which, amongst many other functions, will later contribute to dividing the cell. We recently showed that IP3 receptors are 'licensed' to respond only when they associate with actin, and published work suggests that IP3 receptors may be stimulated after phosphorylation by one of the mitotic kinases (plk1) that accumulates at spindle poles. We suggest that accumulation of IP3 receptors at the spindle poles exposes them to signals (actin and plk1) that increase their activity, and that IP3 receptors then generate local calcium signals that control spindle orientation by regulating astral microtubules. In the proposed work, we will use advanced optical microscopy methods and newly developed tools that allow rapid repositioning of IP3 receptors to establish the mechanisms that move IP3 receptors to the spindle poles and to unravel how their activities at the poles control spindle orientation.