The molecular basis of crosstalk between integrin-mediated cell-matrix adhesions and the microtubule network.

All cells in the human body are held in the correct place via adhesion to neighbouring cells, and to a dense meshwork of proteins that surround cells called the extracellular matrix (ECM). Cell attachment to the ECM is important for cell proliferation, survival and migration during embryonic development, and in adult organisms for processes such as blood clotting and wound healing. Cell-matrix adhesion is mediated by a family of proteins, the integrins, that are found on the cells surface that act as receptors that grab hold of ECM proteins.

Integrins are organised into specialised structures called focal adhesions (FAs) - sticky feet - and cell migration requires the coordinated assembly and disassembly of FAs coupled to force exerted by the cells contractile machinery which enables the cell to pull itself forwards. While cell migration is essential for the development of multicellular organisms, it must be tightly controlled - cancer metastasis is a product of uncontrolled cell migration.

From our previous research and that of other laboratories, the assembly of FAs is now fairly well understood, and an intracellular protein called talin has been shown to be essential for FA assembly as it binds to integrins and couples them to the cells' contractile machinery. However, the mechanisms underlying the disassembly of FAs are less clear. It is now well recognised that recruitment of intracellular structures called microtubules to FAs enhances their turnover and increases the speed at which cells migrate, but what directs microtubules to FAs has remained elusive. Excitingly, we have discovered a novel link between talin and a protein called Kank1 that is involved in stabilising microtubules. The hypothesis that we would like to test is that the interaction between talin and Kank1 directs microtubules to adhesion sites and in doing so leads to FA disassembly and consequently increased cell migration.

Drugs that disrupt microtubules are commonly used to kill cancer cells, but have severe side effects because they also kill normal cells. However, if we can leave the microtubule network intact, but specifically prevent microtubule recruitment to FAs, this should inhibit FA turnover and suppress cell migration, and therefore offer a new way to treat cancer metastasis.

Until we characterise how the talin:Kank1 interaction works, and determine the precise atomic structure of the binding sites where this linkage is made, we cannot design drugs to prevent the talin-dependent recruitment of microtubules to FAs that enhances the invasiveness of cancer cells. The Aim of this project is to establish how talin and Kank1 interact so as to design mutations in the Kank1 binding site that prevent the interaction. We will use a combination of biochemical and structural approaches to obtain this information, approaches that are well established in the applicant's laboratory. The effect of such mutations on cell migration will then be tested in collaboration with Prof. Anna Akhmanova (Utrecht).

Ultimately this study could lead to the identification of new strategies for the design and development of drugs that inhibit microtubule recruitment to adhesion sites, and thus prevent cancer growth and spread in humans.

Cancer metastasis is a consequence of mis-regulated cell migration. Increasing evidence shows that crosstalk between microtubules and integrin-mediated adhesions plays a key role in controlling adhesion turnover and the rate of cell migration, although the mechanism by which microtubules target adhesions is unknown.

The aim of this study is to build on our recent finding of a connection between the cortical microtubule stabilization complex protein Kank1 and the mechanosensitive integrin-associated protein talin. We will use NMR and X-ray crystallography to determine the molecular basis of the linkage between talin and Kank1, and we will design and characterise mutations that sever this link. Such mutants will enable us to explore the role microtubules play in controlling adhesion turnover, the rate of cell migration and cell polarity (collaboration with Prof Akhmanova, Utrecht).

There are two human talin genes. Whilst the talin1 isoform is uniformly distributed throughout adhesions, using super-resolution microscopy, we find talin2 co-localises with Kank1 at the adhesion periphery. This raises the intriguing possibility that talin2 may preferentially bind Kank1, and we will characterise the interaction between Kank1 and both talin isoforms to explore their relative roles in modulating adhesion dynamics. The coordinated positioning of proteins within adhesions is crucial in dictating cell behaviour, and this work will allow us to begin to understand the mechanisms by which this spatial segregation occurs.

In summary, we aim to determine the molecular basis of the novel linkage we have identified that recruits microtubules to cell-matrix adhesions. Successful completion of this project will establish how adhesion and microtubule dynamics are coupled in migrating cells. Ultimately this study could open up new routes for the design of drugs that inhibit microtubule-dependent adhesion turnover and prevent tumour metastasis.

Our proposed study will have impact across a number of different areas. Within the academic arena, we will present our findings at national and international scientific meetings and invited talks at institutions. This work is particular relevant to scientists working in the fields of cell adhesion, microtubules, mechanobiology, cancer and metastasis. This impact will be immediate: i.e. within the lifetime of the grant and beyond. The research will provide a foundation governing the molecular mechanisms of how dynamic microtubules are stabilised at cell-matrix adhesion sites.

We are part of a new grouping, Mechanics of Dynamics of Cells and Proteins (MaDCaP), at the University of Kent that aims to improve the Impact of our extensive collective expertise in the study of the cell cytoskeleton. We will organise a meeting in Year 2 to promote this grouping and to discuss these new cytoskeletal connections identified in this project. This provides the perfect opportunity to host a meeting to discuss dynamic microtubules and the actin cytoskeleton and we will apply to the Royal Society to fund/host this. Such a meeting would be very timely as this is an exciting and fast-moving field, and bringing together mathematicians, engineers, cell biologists and physicists will likely have significant impact in driving new collaborative partnerships within the UK.

We will also promote and provide multidisciplinary training opportunities for the PDRA associated with this proposal both in house and in our collaborators laboratories. This will come in the form of direct interactions and time spent at the Mechanobiology Institute in Singapore and in our collaborators laboratory in Utrecht. We plan to interact regularly with our collaborators and inform them of progress, share reagents, and provide the opportunity for us to develop an iterative approach where our data feeds immediately into the biology, and the results obtained in the biology orient this research. This collaborative approach will enable us to maximise the Impact of this work.

We additionally plan to use data arising from this study as a means to communicate the importance of "mechanics in biology" to the general public. We will do so using Science Extravaganza and University Open Days as platforms to showcase these concepts as well as visits into Secondary Schools. We will use our findings, coupled with movies of live cells and interactive molecular models of how mechanosensitive proteins like talin, can undergo shape changes, and how this might help control cell function in disease processes. We will also continue our on-going outreach visits to primary schools to teach children about the power of microscopy in cell biology.

The linkage between cell-extracellular matrix and dynamic microtubules is emerging as an important regulator of cell migration, mis-regulation of which drives metastatic disease, and as such this new connection we have identified represents a potentially important therapeutic target. However, the lack of current information on precisely how microtubules are stabilised at adhesion sites has thwarted attempts to design drugs to target this process in cancer metastasis. The talin:Kank1 interaction therefore has the potential to be a major driver in cancer disease progression, and as such, we have already started to engage with the pharmaceutical and drug development sector about the potential for using information and reagents (such as peptide inhibitors) for future screening for therapeutic development programmes. These discussions have been met with considerable enthusiasm. We plan to re-visit these discussions in year 2 of the project (following structural analysis and identification of disrupting mutant) to share our emerging data and discuss potential future plans/funding for developmental projects.