Identifying the molecular mechanism by which the conserved Hook/Fts/Fhip complex controls kinesin-3 and dynein attachment to early endosomes

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The organisation and function of cells requires intracellular transport along protein fibres of the cytoskeleton. Like "highways", these fibres provide the "tracks" for protein machines, the so-called molecular motors, which use chemical energy to move their "cargo" throughout the cell. One such "cargo" is the early endosomes (EE). These membranous containers move along a sub-class of cytoskeletal fibres, the microtubules. Their transport occurs in opposite direction (bi-directional) and is mediated by the counteracting motors kinesin-3 and dynein. This process is high importance for the cells in our body, and defects in EE motility, kinesin-3 or dynein function is thought to underlie severe human diseases. The same motors and EEs are required for the ability of pathogenic fungi to invade our crops, which, again, highlights the general importance of this process in cells.Filamentous fungi have major technical advantages that we have exploited recently to gain insight into the factors that control EE motility and distribution in the pathogen Ustilago maydis. Making use of this model system, which is also an important crop pathogen, we found recently a protein complex that enables the motors kinesin-3 and dynein to bind EEs. This complex (named Hok1/Fts1/Fhp1 complex) is also found in humans and we provided evidence for similar functions in linking motors to their "cargo". In this proposed project, we will elucidate the molecular environment and detailed mechanism by which Hok1/Fts1/Fhp1 is targeted to the "cargo", and how the complex coordinates the binding of the opposing motors kinesin-3 and dynein. We found preliminary evidence that both mechanisms (anchorage to "cargo" and binding motors) involves additional, yet unidentified protein factors. In preparation for this application, we have performed extended biochemical experiments that led to a list of putative interacting proteins. We will test if these proteins bind to EEs and if, when removed from the cell, EE motility, motor binding or Hok1/Fts1/Fhp1 anchorage is impaired. We will also address the process of bi-directional EE motility in an entirely unbiased way, by using genetic screening for randomly-generated mutants, defective in EE distribution and transport. This screen was undertaken previously and led to the identification of the Hok1/Fts1/Fhp1 complex in U. maydis (see above). We will follow the same experimental strategy and will test more putative factors in additional mutant strains for a role in EE motility and distribution. We have already confirmed the attachment of 3 proteins from this screen to EEs, and will elucidate their putative role in motor binding and motor regulation or Hok1/Fts1/Fhp1 recruitment to "cargo" membranes. In addition, this approach has the potential to reveal entirely unexpected ways by which the cell enables bi-directional transport and even distribution of EEs.In summary, this proposal addresses a fundamental process found in animals, humans and filamentous fungi in an unbiased and comprehensive way. Our extensive preliminary results are guiding us in a very structured way to elucidate the molecular mechanism by which motors move EEs in the cell. Understanding this process will inform medical research, but also provides new avenues towards the development of anti-fungal drugs.