Integrin specificity regulating chemotaxis in 3-dimensional matrix.

The movement of different types of cell within the body is vital for normal embryo development, immunity and wound healing in mammals. It is vital that the way in which cells move or 'migrate' and the proteins involved in this process are fully understood in order to allow the design of therapies to tackle diseases where cell movement is uncontrolled. Cell migration in humans is not a random process, but a highly complex, tightly regulated one. Initially, the cell must receive a signal from the environment to use as a cue to migrate. This provides the cell with a sense of direction and stimulates movement persistently in one direction, namely towards the source of the signal. For example, during wound healing, soluble factors are released into the wound itself by blood cells or invading bacteria. These factors trigger cells at the edge of the wound to move in and repopulate the wound space, and begin the healing process. The actual mechanism that cells use to co-ordinate these events is still not well understood. However, what is known is that cells must first attach to the surrounding tissue proteins, such as collagen, to be able to move. Attachment is achieved mainly using specialized membrane-bound (receptor) proteins called integrins. Integrins anchor the cells to the surrounding matrix and, in doing so, trigger changes to specific proteins inside the cell. A key early component of cell attachment is a protein called actin, which forms long structural polymers to give the cell a rigid shape. These actin polymers, known as the cytoskeleton, provide a crucial mechanical scaffold for the cell to use to propel itself forward. However, in order to allow the cell to move efficiently, the cytoskeleton is constantly being remodeled and reformed. This process is controlled initially from the cell membrane by integrins, although the way in which the cell does this is not well understood. The study outlined here aims to use a number of ways of imaging combined with biochemical analysis to characterise the role of integrins and associated proteins in cell migration in isolated human cells. It is important to understand exactly how and where in the cell the integrin is being controlled by binding proteins, and how this can alter the cell's response to external stimuli. It is possible to directly track the behaviour of these proteins in living cells. The protein of interest is tagged with a fluorescent dye, meaning it can be seen if excited by light of a specific colour. These molecules are then delivered into living cells. The protein can be seen using a highly sensitive camera attached to a microscope. By watching cells whilst they are moving using microscopy, and following protein movement inside the cells, we can work out the importance of these proteins in cell motility. This is a very exciting and important scientific field which allows us to study the fundamental questions of how human cells move and ultimately how they change to become, for example, cancer cells. This information has very important implications for developing new therapies to treat diseases. Once we understand the way in which a cell uses the integrins to move, we can begin to manipulate these proteins with a view to developing treatments to prevent disorders such as cancer, inflammatory asthma and developmental abnormalities.

Cell migration is a crucial process in the development of both normal mammalian tissues, and a number of pathological conditions. Integrin receptors control the spatial and temporal response to extracellular cues during adhesion and motility. Integrins also control a number of key intracellular signalling events upon matrix binding. Many cells use more than one integrin to adhere and migrate, and those integrins share some intracellular binding proteins. A number of studies have shown a dependence upon both b1 and b3 integrins for migration. Recent studies in this laboratory have made use of b1 integrin knockout and RNAi knockdown cells to investigate the role of this receptor in formation of adhesions. Preliminary data demonstrates altered in focal adhesion formation in b1 null cells, a change in b3 localisation, GTPase activity and migratory behaviour. This proposal aims to build on these observations. The specific aims are: 1) To establish the role of b1 integrin in focal adhesion stability by a) live analysis of focal adhesion turnover in b1 and b3 integrin null vs. wild-type cells b) analysis of the effects of b1 integrin deficiency upon b3 integrin activity and talin binding. 2) To quantify b1 integrin control of chemotaxis by a) analysis of chemotaxis in b1 null cells in 3-D matrices b) determining integrin binding and localisation of talin in the absence of b1 or b3 integrins. 3) To investigate the role of b1 integrin intracellular binding to talin in GTPase activation by a) Assessing GTPase activity in the presence and absence of b1 integrin in live cells b) directly competing integrin:talin association and assessing active GTPase localisation in chemotaxing cells.