Regulation of mitotic spindles by IP3 receptors

每一种动物都是以单个受精卵的形式开始生命的，然后通过有丝分裂反复分裂，产生一个在人类身上有大约3000亿个细胞的动物。有丝分裂在整个生命中持续，以补充丢失和受损的细胞。在有丝分裂过程中，形成一个菱形的网状结构(纺锤体)，复制的染色体排列在纺锤体的中心。然后，染色体朝着纺锤体两极分开，这样遗传物质就会均匀地分布在两个子细胞之间。纺锤体的方向--与细胞附着的表面对齐或垂直--很重要，因为它决定了细胞分裂的位置，并影响了每个细胞随后的发育方式。有丝分裂受到严格的调控，很大程度上是由将磷酸基团连接到其他蛋白质上的激酶家族来控制的，以确保只有当前面的步骤正确完成时，每一步才能进行。许多细胞活动，包括有丝分裂的一些步骤，都受到细胞内钙浓度增加的调节。这些钙信号是在通道打开时产生的，并允许钙沿陡峭的浓度梯度流入细胞。其中最重要的通道之一是IP3受体，它允许从细胞内的内质网中控制钙的释放。除了将染色体分配给子细胞外，有丝分裂还必须确保每个细胞获得包括内质网在内的细胞内细胞器的份额。因此，在有丝分裂期间，包括内质网在内的细胞内结构有大量的重排。我们最近的工作，使用显微镜来报告通过附着有色蛋白而可见的细胞组件的运动，表明在有丝分裂期间，IP3受体在纺锤体极周围积累，并且在没有IP3受体的细胞中，纺锤体不能正确排列。我们认为，这些重新定位的IP3受体产生的局部钙信号通过调节将纺锤体极固定在细胞周围质膜上的纽带(星形微管)来控制纺锤体的方向。在质膜的正下方和纺锤体的两极，有肌动蛋白的积聚，肌动蛋白在许多其他功能中，将有助于细胞分裂。我们最近发现，IP3受体只有在与肌动蛋白相关的时候才能做出反应，发表的工作表明，IP3受体可能在被聚集在纺锤体极点的一种有丝分裂酶(PLK1)磷酸化后被刺激。我们认为，IP3受体在纺锤体极点的积累使它们暴露在增加其活性的信号(肌动蛋白和PLK1)中，然后IP3受体产生局部钙信号，通过调节星形微管来控制纺锤体的方向。在拟议的工作中，我们将使用先进的光学显微镜方法和新开发的工具，允许IP3受体快速重新定位，以建立将IP3受体移动到纺锤体极点的机制，并揭示它们在极点的活动如何控制纺锤体的方向。

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.