My research focuses upon the regulation of membrane trafficking in mammalian cells. We work on two experimental systems, which are introduced briefly below. Follow the links for more detailed information.
GLUT4 trafficking in fat and muscle cells
Insulin stimulates glucose transport into fat and muscle by regulating delivery of the facilitative glucose transporter GLUT4 from an intracellular store to the cell surface. Upon binding its receptor, insulin initiates a signalling cascade that culminates in changes in the trafficking itinerary of GLUT4, releasing it from its intracellular store and delivering it to the cell surface. Individuals with insulin resistance and Type-2 diabetes exhibit defective insulin-stimulated GLUT4 translocation and consequently much effort has gone into defining the trafficking of GLUT4 in adipocytes and muscle cells. Our work in this area seek to define the mechanisms which control GLUT4 sorting to its unique intracellular store, and the mechanism by which GLUT4 is delivered to the cell surface.
Insulin stimulated glucose transport
The acute stimulation of glucose transport in adipocyte and muscle cells is a key action of insulin. Increased glucose transport is achieved by the delivery of glucose transporters (Glut4) from specialised intracellular stores (Glut4 storage vesicles; GSVs) to the plasma membrane (Figure 1), a process referred to as ‘translocation’. Insulin-stimulated Glut4 trafficking is also useful as a model of regulated exocytosis, a process that underpins many other physiologically relevant events such as mast cell degranulation, hormone secretion and antigen presentation. Research using Glut4 trafficking as a paradigm provides novel insight into how cells translate extracellular signals into membrane trafficking processes and is thus widely applicable across many research fields.
Type 2 diabetes
Blunted insulin-stimulated glucose transport is a hallmark of insulin resistance/Type 2 Diabetes mellitus, diseases with considerable health implications that are estimated to cost the UK in excess of £10 billion annually. Insulin resistance also accompanies ageing; e.g. over a quarter of Americans aged over 65 have diabetes and in the UK the incidence of Type 2 diabetes among the elderly is sharply increasing, suggesting that the costs of treatment of insulin resistance and Type 2 diabetes will rise still further as our population ages. Such observations clearly reveal a need to understand the cell biology of glucose transport as a means to identify potentially novel therapeutic targets.
Intracellular Glut4 trafficking into GSVs
Glut4 intracellular trafficking involves two inter-twined endosomal cycles. A rapid recycling pathway efficiently internalises Glut4 from the plasma membrane into early and recycling endosomes; Glut4 is then sorted via the trans Golgi network into GSVs via a slow trafficking pathway (Figure 2); GSVs are subsequently mobilised to the cell surface upon insulin stimulation. Insulin increases exocytosis of GSVs (and also modestly inhibits Glut4 endocytosis) thus redistributing Glut4 such that levels at the cell surface increase between 10- and 20-fold in isolated adipocytes. Upon removal of insulin, Glut4 is internalised into endosomes (cycle-1) and sorted back into GSVs (cycle-2). Thus, Glut4 populates and transits between multiple distinct intracellular compartments.
Detailed analysis of GLUT4 sorting and trafficking in peripheral tissues from patients with T2DM are consistent with the notion that impaired insulin-stimulated glucose transport can arise as a consequence of defective intracellular sequestration of GLUT4 into insulin-sensitive stores. For example, Garvey et al demonstrated that GLUT4 is sorted into a denser membrane fraction in muscles from patients with T2DM compared to control, insulin-sensitive individuals, and that insulin does not stimulate mobilisation of GLUT4 from this fraction to the cell surface. Similarly, defective GLUT4 sorting has been reported in adipocytes from patients with T2DM, and individuals with gestational diabetes. Altered GLUT4 trafficking and sorting has also been reported in cellular models of insulin resistance. Such studies underscore the need to define both the trafficking pathway(s) followed by GLUT4, and the mechanisms involved in GLUT4 sequestration into insulin-sensitive stores. Our group studies these trafficking events using a range of model systems.
SNAREs and GSV fusion
Like all membrane trafficking events, the fusion of GSVs with the plasma membrane involves SNARE proteins. Specifically, the fusion machinery thought to mediate this fusion event is Synaxtin-4/SNAP23 at the plasma membrane (the tSNAREs) and VAMP2 (the vSNARE) on the GSVs. Impaired fusion of GSVs to the cell surface in response to insulin may underpin defective glucose transport in T2DM, hence another major focus of our group is to study the regulation of GSV fusion with the cell surface, in particular understanding how SNARE proteins are regulated by insulin. Understanding which SNARE proteins interact with each other or with regulatory molecules, and whether their associations change (e.g. during different phases of exocytosis) is an important objective. Such knowledge can offer new potential therapeutic targets. This is challenging, as co-IP and epitope-tags may not reveal bona fide biological interactions and is ideally achieved by analysis of proteins at close to endogenous levels. To this end, we used Proximity Ligation Assays (PLA). This approach requires antibodies against the two proteins in question, raised in different species, which are used to quantify endogenous protein associations using a fluorescent PCR-amplification based approach. We identified two populations of plasma membrane t-SNARE Sx4 that function in distinct trafficking events, and showed that one of these is selectively engaged by the insulin signalling machinery via the SM protein Munc18c. These analyses and the insight they provide were made possible by the ability to study associations between SNARE components using endogenous proteins expressed at normal levels. Note that PLA also allowed us to demonstrate that SNARE complex formation in vivo is stimulated in response to an extracellular signal. This remains the first demonstration of this phenomenon in any cell type. Further work is aimed at defining the signalling mechanisms and the biochemical interactions they regulate using both in vitro and in vivo approaches.
This work is performed with our long standing collaborator, Professor Nia Bryant at University of York.
Membrane trafficking in cytokinesis
All cells divide by cytokinesis. After replication of the genetic material and formation of two nuclei, the mother cell divides by the formation of a furrow that constricts the cytoplasm, leaving two daughter cells connected by an intercellular bridge. Resolution of this bridge (abscission) then separates the daughter cells. Recent studies suggest that the formation of binucleate cells as a result of cytokinesis failure is an early event in tumour formation and underlies the subsequent development of genomic instability. Moreover, many key cytokinesis genes are proto-oncogenes and loss of tumour suppressors may promote carcinogenesis by disrupting cytokinesis. Such observations reinforce the idea that a detailed molecular description of cytokinesis is important to allow a full understanding of the development of cancer.
Cytokinesis is an intricate process that requires the integration of multiple cellular components into a contractile and membrane-reorganizing unit. Membrane traffic to the furrow is an essential facet of cytokinesis, reflecting a requirement for increased surface area during furrowing and for the delivery of intracellular signalling or membrane re-modelling activities to the correct spatial co-ordinates during cleavage. Understanding how this membrane traffic is controlled in space and time is the central objective of my lab.
Further detail is provided here.