Regulation of mitotic spindles by IP3 receptors

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.

During mitosis, the spindle assembles to segregate chromatids to daughter cells. Intracellular organelles, including the endoplasmic reticulum (ER), are also reconfigured. The orientation of the spindle is important because it determines the plane of cell division, which ensures that cellular components are shared appropriately between daughter cells, and it influences cell fate. Our recent work shows that IP3 receptors (IP3R), which are widely expressed intracellular channels that release Ca2+ from the ER, are redistributed to metaphase centrosomes, and loss of IP3Rs perturbs spindle alignment. The latter is likely to be caused by perturbation of relationships between centrosomes, astral microtubule and cortical actin. IP3Rs are known substrates of some mitotic kinases, notably cyclin-dependent kinase 1 (Cdk1/Cyclin B), which orchestrates mitosis; and polo-like kinase 1 (plk1), which associates with, and controls assembly of, centrosomes. Our recent work has shown that association of IP3Rs with actin 'licenses' them to respond. We propose that regulated redistribution of IP3Rs to centrosomes exposes them to regulatory proteins (actin, plk1) that prime them to respond. The resulting local Ca2+ signals, we suggest, regulate astral microtubules and thereby spindle orientation. Using optical microscopy, re-expression of mutated IP3Rs in cells devoid of native IP3Rs, and newly developed nanobody tools, we will address the following aims:

1 How are IP3Rs accumulated at metaphase centrosomes?
a. What is the temporal relationship between IP3R translocation and spindle assembly?
b. Do microtubule motors or a diffusion-trap mechanism mediate IP3R accumulation at centrosomes?
c. Does phosphorylation of IP3R by Cdk1 contribute to IP3R translocation?

2 How do IP3Rs regulate spindle orientation?
a. Are local Ca2+ signals required for IP3R to align spindles, and when are such signals required?
b. Do plk1 and actin selectively regulate IP3R activity at spindle poles?

Our work will advance understanding of one of the most fundamental properties of all cells, their progression through the cell cycle. Our proposal, focused on how redistribution of IP3Rs controls spindle alignment, seeks to integrate several areas of fundamental cell biology. This fundamental question, allied with the importance of spindle alignment for normal cell division and fate determination, and the pathologies that result from aberrant spindle behaviour will ensure that our work delivers impact. Our expected impacts across diverse fields within the academic community are described in Academic Beneficiaries.

Training
Staff are encouraged to develop the skills and experience required for independence. They engage fully with every aspect of the project from developing proposals, managing budgets, reviewing and developing research programmes, to preparing publications and presenting work. Staff apply state-of-the art techniques equipping them for work in the best labs. Staff gain experience of teaching by supervising project/PhD students, teaching practical classes and in a lecture on advanced techniques to final year students. All staff contribute fully to weekly lab meetings, where they present and critically evaluate work. In my absence, lab meetings are chaired by postdocs. A major impact is our proven ability to train staff equipped to meet future needs of industry, the public sector and academia. Our contributions to providing the UK and beyond with well-prepared scientists comes also from our public engagement activities. These benefits will come at a particularly important time for Dr Lagos-Cabre, the named PDRA, as he prepares for the next step towards independence, but they apply also to the technical assistant and to the summer students that Dr Lagos-Cabre is likely to supervise during the tenure of his appointment.

Public understanding and schools
My lab organises the Young Pharmas scheme, which seeks to inspire year-12 students, ensure that they appreciate the importance of creativity and critical evaluation in science, and the economic impact of pharmacology. Parents and teachers also gain exposure to these activities through the final poster session/guest lecture evening. Our interests in mitotic spindles will be assimilated into Young Pharmas activities. We request funds (£500pa) to partially support these activities. Staff contribute to Cambridge Science Festival with a hands-on demonstration of the actions of common drugs on waterfleas. We provide occasional visits to schools, providing practical experience of, for example, insect biology and microscopy. We provide at least one placement for an undergraduate to gain research experience before deciding on postgraduate options. These activities encourage informed interest in science from students who have not yet finalised their career choices, and a more widespread appreciation of the importance of addressing fundamental questions in biology.

We work with press offices to maximize the impact of our work by bringing it to more diverse audiences than our primary publications can reach. Two press releases from BBSRC described our recent work.

Health
Our work addresses fundamental questions in cell biology, but it does have clear links with human medicine. Misaligned spindles can cause developmental defects, they may become more frequent with age, and they contribute to decisions relating to fate and stem cell niches. Our work is likely to provide a deeper understanding of how spindles may contribute to these changes. The impacts for clinical medicine are impossible to predict. We will actively engage with clinicians, stem cell biologists, and the pharmaceutical industry to ensure that our findings are presented at an early stage to communities with direct interests in clinical development.