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

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.

Bi-directional motility of membranes is a fundamental feature of eukaryotic cells. We recently discovered the first adapter for kinesin-3 and dynein on early endosomes (EEs), using an unbiased genetic screen using a fungal model system. This adapter consists of a hook protein (Hok1), a homologue of human FTS (Fts1) and FHIP (Fhp1). This complex was first described in human cells, but its role in motor binding and membrane trafficking was not known. We found evidence for a conserved function of Hook proteins in motor binding and that kinesin-3 and Hook are always paired (choanoflagellates to humans), while absent from plants and yeasts. Thus, Kinesin-3 and its adapter likely form a functional pair, essential for membrane trafficking in most eukaryotes.
How this complex is targeted to the organelle and binds and coordinates kinesin-3 (Kin3) and dynein remains elusive. We will address this by complementing our biochemical experiments with forward genetic screening and live-cell imaging. Our preliminary data indicate a central role for Fhp1 and hint towards a role of small GTPase Rab5a. We will investigate this by quantitative live-cell imaging of Kin3, Hok1, Fts1 and Fhp1 on EEs in various rab5a mutants. We also identified putative interactors of Fhp1, Fts1 or both. We will test localization of these on EEs, their role in EE motility and subsequently will investigate their role in adapter and motor anchorage to the organelles. The same strategy promises to identify factors that bridge between Hok1 and motors. Preliminary data suggests that Fts1 is pivotal for motor binding. We will investigate proteins, which interact with Hok1, Kin-3 and Fts1 in this role. Finally, we will invoke use of our genetic screen, analyse 10 more mutant strains, and will identify further candidates. This will deepen our understanding of the regulation and molecular requirement for bi-directional EE motility.

This project addresses the molecular mechanism of coordinating kinesin-3 and dynein activity during bi-directional motility of early endosomes (EEs). It is based on our most recent discovery of an early endosome-associated adaptor complex in a fungal model system, consisting of conserved proteins that are also present and described in humans. We will use our unique fungal model system and exploit its technical advantages to address this question in a comprehensive and unbiased way.

Academia:
We are at the beginning of an understanding of motor cooperation in eukaryotes. This work addresses the most common and important membrane transport kinesin-3 and its Hook adaptor complex on Rab5-carrying endosomes. Kinesin-3, Hook and motile early endosomes are implicated in numerous human diseases (e.g. Pal et al., 2006, J. Cell Biol., 172:605; Nath et al., 2015, Hum Mol Genet. 24:450; Riviere et al., 2011, Am. J. Hum. Genet. 89:219; Herrmann et al., 2015, Plos One 10:e0119423), but also play essential roles in fungal pathogenicity in plants (Bielska et al., 2014, Nat. Commun. 5: 5097). Thus, this work benefits academic research in three ways:
(1) It will provide fundamental insight into the mechanism of motor coordination in general.
(2) It will impact on our understanding of human diseases
(3) It will inform infection-related research in plant and animal pathogenic fungi

Society:
Democratic policy is rooted in public understanding of the challenges facing society. Therefore, the PI will use opportunities to disseminate scientific knowledge about fungal pathogenesis and general principles of motor coordination in eukaryotic cells in a series of public presentations. These are detailed in the pathways to impact document.

Industry:
This project covers an area of fundamental research, pivotal to further our understanding of defects, underlying human diseases. Thus, insight into the regulation of membrane trafficking will inform research in the pharmaceutical industry. In addition, deeper understanding of the process of EE motility in fungi may lead to the discovery of new anti-fungal target sites, which is of high interest to a AgriTech industry.