Dynamics and function of early endsomes

The behaviour of cells within a tissue is controlled by their environment. Amongst the most important signals that cells receive are in the form of circulating small proteins called growth factors. These bind to specific receptors that are found on the surface of cells. Binding of growth factors causes the receptors to alter their pattern of interactions with many molecules inside the cell that control cell growth. In this way growth factor receptors act as essential bridges between the cell exterior and interior to stimulate cell growth, cell division and cell migration, so-called mitogenic responses. To prevent uncontrolled mitogenic activation, which would be harmful to the tissue, the cell must make sure the strength of the response is carefully adjusted, and that the response is switched off after a short time. Such control is achieved by removing the activated receptor from the cell surface and sending it through a series of specialised compartments within the cell, where its activity can be closely regulated and where it can eventually be degraded. Transfer of the receptor through these compartments is accompanied by highly complicated movements of these compartments, as they set about transferring the receptor between each other. All of these movements are determined by specialised cellular proteins called motor proteins, each of which are designed to move particular compartments around the cell with characteristic directions and speeds. This project seeks to unravel which motor proteins move which compartments, and how movement is coordinated with the transfer of growth factor receptor. Understanding such complex behaviour will be important, since defects in this movement and transfer are linked to a range of diseases.

Mitogenic growth factors act by triggering the activation of signalling cascades, and early endosomes play a crucial role in this process by providing spatially discrete platforms for the assembly of signalling protein networks. The duration of signalling is controlled by the progress of activated receptors through early endosomes, and this in turn is linked to the maturation of the endosomes themselves. We have shown that early endosomes, identified by the presence of Rab5, are highly motile structures that exhibit very complex dynamics, moving both towards and away from the cell centre, and switching from moving to stationary phases and back. Since we have previously demonstrated that the dynein-driven movement of endosomes at a later stage in the endocytic pathway contributes to their maturation and their ability to sort cargo, we now wish to test the hypothesis that microtubule- and actin-based motility make fundamental contributions to the function and maturation of endosomes shortly after receptor internalisation. The key questions we will address are: 1. Do the dynamics of these early endosomes alter during the passage of cargo and during their maturation? 2. Does endosome motility control endosome maturation, morphology and cargo transit? 3. Does interfering with endosome dynamics influence cell signalling?

Our approach will be to image living cells expressing low levels of fluorescent proteins that mark different populations of signalling endosomes, cargo molecules, and the phosphoinositide-modifying proteins that are vital for endosome maturation. We will analyse and compare the motility of these different markers quantitatively using newly-developed algorithms that allow us to automatically track all moving endosomes within living cells and define their motile properties. A key advance in this proposal will be to image two colours simultaneously at ~30 fps, enabling us to analyse the behaviour of pairs of endosomal proteins. These studies will establish whether steps in the signalling and maturation pathways correlate with alterations in motility, such as switching from moving to stationary, or from short range to long range motion. We will determine the contribution that motors make to endosomal maturation and signalling by disrupting the function of key microtubule- and actin-based motors. In addition, we will test whether changes in endosome motility are linked to the flux in phosphoinositides that occurs as endosomes mature. Our extensive background work tracking and analysing the motility of Rab5-positive endosomes makes us uniquely qualified to address these important questions and contribute to the understanding of mitogenic signalling.