Mitotic cell mechanics in a tissue context

Both symmetric and asymmetric cell divisions require a complex set of molecular processes to ensure the proper orientation of the microtubule-based spindle with respect to cortical cues. In asymmetric divisions, cells first establish an axis of polarity in response to internal or external cues. This is then read by astral microtubules to orient the spindle to segregate cell fate determinants asymmetrically in the two daughter cells. In symmetric divisions, the spindle typically reads apical-basal polarity cues and mitotic cell shape in order to align in such a way as to divide to release tissue strain. In both cases, spindle orientation is likely to be important for high fidelity chromosome segregation. Understanding the role of mechanics in mitosis is therefore a fundamental problem in cell biology. In addition, it is currently thought that controlled ratio of symmetric versus asymmetric cell division plays a critical role in stem cell homeostasis, a phenomenon thought to be misregulated in cancer.

While much progress has been made in identifying biochemical signalling pathways that regulate cell division in general, little is known about the mechanisms by which normal epithelial cells and stem cells sense, respond to and resist forces. Only recently the role of force has been studied in spindle orientation of individual adherent cells, and mechanical stress is known to be a major external signal involved in epithelial cell regulation. Our preliminary data now show that forces also play a significant role in spindle orientation in tissues. Building on this work we plan to test how spindle orientation responds to external force during symmetric and asymmetric divisions and to identify the molecular machinery involved.

To do so we will explore the molecular and cellular mechanisms by which mitotic cells sense, respond to and resist mechanical forces using two complementary experimental systems developed in the Baum and Charras labs: i) a device that enables the mechanical perturbation of MDCK epithelial monolayers, and ii) the Drosophila notum, a tissue where genetics, mechanical perturbations and live imaging can be readily combined. In this way, we expect to identify conserved molecular mechanisms that ensure that cell division occurs with high fidelity in the context of an epithelium subject to
changes in mechanics. We will further test whether force also a play a role in asymmetric divisions (as a simple model of stem cell divisions).

We expect this work to have a significant impact on our understanding of fundamental questions in the fields of cell division, tissue homeostasis, stem cell biology and regenerative medicine. There are several lines of evidence that make clear the importance of bridging this gap in our understanding of the role of mechanics in cell division. First, passage through mitosis involves dramatic active changes in cell shape and cortical rigidity, which when perturbed may lead to cell division failure and chromosome mis-segregation; potentially contributing to cancer development. Second, the ability of a cell to divide and form a colony in a mechanically soft medium is a key test of cellular transformation and malignancy, implying a role for mechanics in cell division. Third, in cell culture the mitotic spindle typically aligns parallel to a substrate plane along the axis of greatest tensile force. Fourth, it is possible that symmetrical divisions which tend to increase the number of stem cells will be triggered by tensile stress. In this way tissues may respond directly to the need for more cells by increasing the number of cells via tensile-controlled proliferation combined to oriented cell division ensuring relieve of the tension across the tissue.

The project aims at exploring the role of external forces in spindle orientation in epithelial mitotic cells in a tissue context. To address this question we will use a combination of methods using the fly notum and a device to apply tension on MDCK cells as complementary experimental systems.

MDCK monolayers can be grown for few hours under external force, fixed, immunostained and imaged for markers of spindle orientation in normal and perturbed conditions. Using this device we will reveal the role of applied tensile and compressive forces on spindle orientation. We will stain for key proteins involved in spindle orientation including NuMA, Dynein and E-Cad to get at the molecular processes involved and will then image these events live using the MDCK monolayers expressing tagged versions of NuMA/EB1. In parallel, we will use similar GFP-tagged markers to precisely correlate analogous changes in cell shape, spindle assembly and orientation in the fly notum. Importantly, in the fly we can use our understanding of force differences across the tissue together with laser ablation to determine the relative effects of force and cell shape on the pattern of cell division, which preliminary work shows can be separated.

Using both systems we expect to come to general about how forces (tension and compression) affect mitotic cell shape, spindle morphology, assembly, and orientation. To determine the molecules involved in force-dependent cell behaviour in fly and MDCK cells (sensitivity and robustness), we will begin with NuMA, which we showed polarises in response to tension prior to spindle alignment. Next we will test the involvement of potential upstream regulators (e.g. Galphai), the actomyosin cortex and of osmo-regulators identified in the RNAi screen carried out with the Piel lab.

Finally, we will test the role of force in asymmetric P1 divisions in the notum and on apical-basal oriented spindles in MDCK monolayers as a model for stem cell divisions.

The main beneficiaries of this research are likely to be the scientific communities working on questions related to cell division, spindle orientation, cellular mechanics, epithelium homeostasis and stem cell biology. We also expect these data to have an impact in the field of cancer research, in relation to high fidelity cell division and the dysregulation of stem cells. This research also has the potential to contribute to nation health through its exploration of the fundamental biology underlying cell division in a tissue context, a process that is relevant to development, homeostasis, regeneration and disease. In the longer term this research may also be of relevance to tissue engineers, since there is a great interest in the role of mechanics in tissue growth. Through its potential impact on our understanding of stem cell biology and our development of tools for the application of force to entire tissues, this work is also likely to benefit the stem cell research community.

To ensure this work has an impact in these areas we aim to present our results, technological developments and new ideas at conferences that cover different relevant topics including cell division, role of force in tissue homeostasis, engineering, development, cell biology and at the BSCB (British Society for Cell Biology) and Cell Mechanics meetings and the ASCB. We also aim to publish the main biological findings in 2 papers in high impact journals. Where possible we will publish in open access journals. We expect the first of these papers to be submitted in 2014.

Through our involvement in HFSP, the EMBO YIP forum, EU and Weizmann-UK networks, we will ensure that this work reaches the global scientific community and leads to the development of new international research collaborations. Most significantly it will strengthen our collaboration with the Piel lab at the Curie Institute, another leader in the field.

Importantly, we will also make use of our MeDiCI network to make this work known to our commercial (Cytokinetics, Pharmatest; JPK; CYTOO and Cellastix) and clinical partners (G. Williams and O. Carpen). Moreover, in our discussions with them we will explore how best to ensure the exploitation and commercialisation of our research findings and tools.

Tools that will be developed through the project will be made available to the community. To ensure impact we will carry out workshops to train researchers in the novel methods refined during the course of this analysis. We expect this type of approach to make a contribution to the emerging field of cell and tissue mechanics, where such tools are sorely lacking.

A large number of graduate students will benefit from involvement in this interdisciplinary systems level research through rotation projects in the lab and through MRes and tutorial activities associated with these programmes. Similarly, undergraduates will be exposed to this work through internships and short projects. Short research training projects will be offered in 2013-15. Through this experience, we expect students to gain an understanding of the way productive interdisciplinary collaborations work.

We would hope to publicise the implications of our research by writing a review of the field in 2015 targeted to reach a general audience. In the long-term, this research is likely to have an impact on lifelong human health and well being. UK Plc will directly benefit from this high profile research as technological developments will be commercialised through UCL and will be made available to UK companies working in tissue engineering.