The role of microtubule motor proteins in cargo sorting

The correct function of all cells in the human body depends on the fact that they are internally divided in to discrete compartments. This ensures that biochemical reactions can occur specifically and efficiently (such as the synthesis of new proteins, their modification and processing) . It also means that the cell requires a means to communicate between compartments and to transport material between them (for example for sequential processing of a newly synthesized hormone in to its final state as occurs for insulin). We study the process by which material is moved between compartments and the mechanisms by which these carriers moving between them are transported. Key questions are centred on the mechanisms that ensure that compartments remain distinct (?compartment identity?) as well as the way in which specific ?cargo? is selected for transport but other cargo excluded or removed if incorporated by mistake. Our hypothesis, which is built on very solid foundations from our own work as well as that of others, is that the sorting of cargo within these structures is inherently linked to the motors that drive their movement. Specific motors exist to move these carrier vesicles in one direction or another. We propose (and have evidence that) motors are coupled to distinct cargoes. Applying force to different cargos using motors of opposing polarity would segregate these cargoes within a structure in to discrete domains. Thus, we would generate a motor-dependent sortig of cargo to be directed in one direction versus another. We propose to use our experience of advanced microscopy to test these ideas in living cells and to correlate both the movement and morphology of carriers with these sorting events. These experiments should lead to a clear understanding of the mechanism by which cargo sorting occurs. Building on a very strong existing collaboration, we wish to integrate two membrane trafficking approaches to provide a comprehensive analysis and to use this synergistic approach to provide a fuller understanding of the general mechanisms at work. This approach is likely to have significant implications for the mechanisms of membrane trafficking in both normal and disease states.

The organization of all metazoan cells depends on the fidelity of membrane trafficking processes. Movement of vesicles and tubules between compartments is responsible for the correct sorting of proteins and lipids in to discrete compartments, the maintenance of compartment identity, and the secretion of newly synthesized proteins. All organelles are linked to the microtubule cytoskeleton and increasing evidence indicates that this membrane-microtubule coupling is responsible for the morphology and positioning of organelles within cells as well as the movement of both organelles and vesicles during membrane trafficking. Recent work from us and others has provided mechanistic insight in to the mechanisms by which motor proteins that drive vesicles along the microtubules couple to the membranes. This work also demonstrates that the force imparted by motors on membranes can drive formation of trafficking intermediates as well as dictating cargo selection in to these carriers. Motor proteins drive cargo in one direction along microtubules, dynein family motors to the minus end and most kinesin family members to the plus end. This proposal is built on the hypothesis the opposing forces imparted by these motors direct the sorting of cargo within organelles. During the transport of biosynthetic cargo from its site of synthesis in the ER to the Golgi apparatus, secretory cargo is sorted away from recycling cargo that is to be returned to the ER. Anterograde (secretory) cargo is driven by dynein towards the Golgi while recycling cargo is directed by kinesin back to the ER in the cell periphery. Similarly, in the endocytic pathway, cargo molecules destined for different destinations (e.g. the plasma membrane, lysosomes, or the TGN) are sorted from one another to maintain compartment identity and correct intracellular routing of receptors. Microtubules direct the organization and motility of transport carriers between these compartments and provide a highly related system to study the role of motors of opposing force in the biogenesis and maturation of transport intermediates. Our hypothesis is that opposing motors (dyneins and kinesins) drive bidirectional motility of these carriers in both the biosynthetic and endocytic systems, and that this motility is inherently linked to cargo sorting within these structures. Here, we propose to test this hypothesis applying high resolution light and electron microscopy to the study of cargo sorting within motile carriers. This work should establish fundamental principles for the way in which motor proteins direct membrane trafficking.