Rab proteins, microtubule motors and the organisation of the endocytic pathway

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 proliferative, or mitogenic responses. In order to prevent such mitogenic responses continuing endlessly, which would lead to uncontrolled cell division, the growth factor receptor must be inactivated shortly after the growth factor binds. This is achieved by removing the activated receptor from the cell surface and sending it to a specialised compartment within the cell, where it can be degraded. This compartment is called the lysosome. Movement of the receptor from the cell surface to the lysosome involves the receptor being sequestered into regions of the cell surface membrane that invaginate and pinch off to form spherical packages, or vesicles, within the cell interior. These vesicles move in a directed fashion to the lysosome, via a number of intermediate compartments. The situation becomes more complicated when it is realised that the route that growth factor receptors take to the lysosome is also followed part of the way by other types of receptor, which are engaged in taking up nutrients. These receptors are returned to the cell surface from the intermediate compartments so that they can participate in further rounds of nutrient uptake. Hence, at a critical point along the pathway towards the lysosome, these receptors are selected away. The aim of this project is to understand how this diversion takes place.

Cell surface receptors that have been internalised are sorted within the early endosome, either entering a recycling pathway back to the cell surface or being transported to the lysosome in order to be degraded. It is essential that this separation occur efficiently, to minimise the loss of recycling receptors to the degradative pathway. Transport to the lysosome is characterised by the maturation of the vacuolar regions of early endosomes into late endosomes, which are able to fuse directly with the lysosome. This project will explore the possibility that a sensing mechanism helps delay this maturation until all recycling receptors have been delivered from the vacuolar endosome into associated recycling tubules, thus ensuring that none of these receptors enter the lysosome. We will focus on two key aspects of this potential regulatory step.
Firstly, since Rab GTPases are fundamental to organising functional domains within the endocytic pathway, we will examine whether there is co-ordination between Rab proteins participating in lysosomal transport and those involved in recycling. Specifically, we will examine whether vacuolar endosomes lose Rab5 and acquire Rab7, an event linked to their maturation to late endosomes, only after Rab4 and Rab11, which specialise in recycling, have been released into neighbouring membranes. Assuming this is the case, we will test whether Rab4 and Rab11 either directly or indirectly control the activation and membrane recruitment of Rab7.
Secondly, we will investigate the ability of microtubule motors to maintain close spatial links between the recycling and degradative pathways and thereby maximise the efficiency of cargo sorting. To this end, we will investigate which combination of plus and minus end-directed microtubule motors are responsible for the movement and localisation of each of the endosomal Rab domains. Based on our preliminary findings that cytoplasmic dynein moves Rab5-positive endosomal vacuoles to the perinuclear region, we will investigate whether dynein is also responsible for localising Rab4 and Rab11-positive membranes to this region. We will then examine which plus end-directed microtubule motor is responsible for moving Rab4 and Rab11-positive membranes to the periphery, concentrating in the first place on the activities of kinesin 1 and kinesin 2.
These collaborative studies, based on preliminary investigations and our extensive experience of endosomal trafficking and microtubule motors, will rely heavily on our proven ability to image endocytic trafficking in live cultured cells at very high temporal resolution in multiple fluorescence channels.