The Structural Basis of Molecular Mechanisms in Cell Guidance and Adhesion.

The cells of a multicellular organism, such as a human, must be subject to an exquisite choreography during development. Each cell must achieve the particular balance between adhesion and motility appropriate to its role at a specific time and place in the developing organism. Much of the information to direct each cell must be garnered through interaction with its immediate environment including neighbouring cells. These interactions are mediated by various types of receptor molecules embedded in the cell surface. We wish to understand how one family of molecules, the semaphorins, work together with their receptors, the plexins, to control the ability of a cell to stick (adhere) or to move in a specific direction. Using the techniques of structural biology we aim to uncover, in atomic detail, the mechanisms by which the semaphorins and plexins control cell adhesion and guidance, for example in directing the wiring of the brain. These mechanisms must integrate receptor interactions occurring between cells and on the same cell surface, as well as spanning from the extracellular to the intracellular environment. To generate insight into such systems we will need to use state of the art techniques to produce suitable samples of the semaphorins, the plexins and their complexes, and to combine in vitro structural studies on the isolated molecules with in situ analyses in the functionally relevant context of the cell surface. This is basic research into the mechanisms by which biology works to build the nervous system and the blood vessels, to maintain bones and to activate the immune system. By understanding these mechanisms we will also be better equipped to explore molecular factors which may contribute to the failure of severed nerves to regenerate following spinal cord injury, to bone-related disorders, for example osteoporosis, to neurodevelopmental disorders such as autism disorder spectrum, and to cancer. Ultimately this knowledge can be used by the biotechnology and pharmaceutical industries, to inform and guide the design of novel therapeutics.

The semaphorins were first characterized as guidance cues exerting repulsive effects on axon growth cones during neural development. The number of tissues and biological processes for which we now know that the semaphorins and their type 1 single membrane spanning receptors, the plexins, play key roles has hugely expanded. Examples in the nervous system include neuronal migration, axon and dendrite guidance, branching, and synaptogenesis. Semaphorin function or dysfunction is also implicated in neurogenetic disorders, nerve regeneration, bone maintenance, immune cell differentiation and function, heart development, and cancer. Our knowledge of the molecular level mechanisms which deliver these myriad biological processes is still sparse. In this research programme we will apply an integrated structural and cellular biology approach to investigate the molecular mechanisms responsible for the biological activities of the class 1, 2, 5 and 6 semaphorins and their cognate plexin receptors. We will address three cross-cutting project areas:
1. Extracellular interactions governing signalling complex assembly
2. Linkage of the extracellular and cytoplasmic segments for signal transduction
3. Sub-cellular localisation and the interface with the cytoskeleton
We will generate atomic level detail using protein crystallography, and complement it with studies by electron microscopy (negative stain, and cryo). We will evaluate the functional hypotheses we generate in these molecular studies by using cellular imaging methods including electron cryo tomography as well as advanced fluorescence microscopy techniques such as localization microscopy (dSTORM) and FRET-FLIM. Our studies will also link through directly to in vivo and in vitro studies of cellular morphology and neuronal connectivity in collaborators' laboratories. Ultimately our results will inform and guide investigations into the semaphorin-plexin system as a point for therapeutic intervention (see Impact Summary).

As discussed in the Academic Beneficiaries section this research will be of interest and benefit to the structural biology community because it will generate new methods, protocols and reagents. Likewise it will provide information on molecular mechanisms which will underpin the design and interpretation of studies on biological function and clinical pathologies in a broad range of tissues including the nervous system, bone, the vasculature and the immune system.

The proposed programme is basic research of potential value to the commercial private sector, specifically the biotechnology and pharmaceutical industries, to inform and guide the design of novel therapeutics. Because the molecular mechanisms of semaphorin-plexin function targeted in this application are increasingly being found to play fundamental roles in the morphogenesis and homeostasis of many human tissues these molecules are emerging as attractive points for therapeutic intervention. Virtually all studies to date are at the pre-clinical stage, however, Vaccinex Inc is currently conducting two Phase I clinical trials for an anti-semaphorin (Sema4D) monoclonal antibody (VX15/2503): in adult patients with Multiple Sclerosis (ClinicalTrials.gov Identifier: NCT01764737), and in patients with advanced solid tumours (ClinicalTrials.gov Identifier: NCT01313065). Although the causality is not established, a growing number of studies link semaphorin expression levels, interactions and signalling pathways to a range of psychiatric and neurodevelopmental disorders, in particular those with delayed onset such as autism disorder spectrum. Semaphorins are also numbered among the molecules implicated in the failure of severed axons to regenerate following spinal cord injury. More definitively, recent results have highlighted the semaphorins as major regulatory molecules of bone cell activity and consequent targets for the treatment of bone-related disorders such as osteoporosis, osteopetrosis, and osteopenia. Molecular reagents can be of direct use for the development of therapeutics and diagnostic assays whilst structure-based design of therapeutics is well established in all major pharmaceutical companies.

The third area of impact for this research programme will be in the training and career development of a cohort of young research scientists. I have been very fortunate in the high level of talent and enthusiasm shown by the young scientists who have worked with me and I believe my laboratory has provided them with an excellent training environment. Past pre- and post-doctoral members of my laboratory include current MRC Senior Non-Clinical Fellowship, MRC Career Development Award, Cancer Research UK Senior Fellowship, Cancer Research UK Career Establishment Award, Netherlands Organisation for Scientific Research VIDI Grant and Human Frontiers Science Programme Fellowship holders whilst two recent doctoral students are now, respectively, a Staff Scientist at a UK biotechnology company, and a Team Leader at Roche Diagnostics GmbH, Germany.