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

细胞的组织和功能需要沿着细胞骨架的蛋白质纤维进行细胞内运输。就像“高速公路”一样，这些纤维为蛋白质机器提供了“轨道”，即所谓的分子马达，它们利用化学能在细胞内移动它们的“货物”。其中一种“货物”是早期核内体（EE）。这些膜状容器沿着细胞骨架纤维的一个亚类——微管移动。它们的转运发生在相反的方向（双向），并由抵消马达运动蛋白-3和动力蛋白介导。这一过程对我们体内的细胞非常重要，而EE运动、动力蛋白-3或动力蛋白功能的缺陷被认为是严重人类疾病的基础。致病真菌入侵我们的作物的能力也需要同样的马达和EEs，这再次强调了这一过程在细胞中的普遍重要性。丝状真菌具有主要的技术优势，我们最近已经利用这些优势来深入了解控制病菌黑穗病菌EE运动和分布的因素。利用这一模型系统，我们最近发现了一种蛋白质复合物，可以使马达驱动蛋白-3和动力蛋白结合EEs。这种复合体（命名为Hok1/Fts1/Fhp1复合体）也在人类中发现，我们提供了将马达与其“货物”连接起来的类似功能的证据。在本项目中，我们将阐明Hok1/Fts1/Fhp1靶向“货物”的分子环境和详细机制，以及该复合体如何协调对立马达kinesin-3和dynein的结合。我们发现了初步证据，这两种机制（锚定到“货物”和结合马达）涉及额外的，但尚未确定的蛋白质因素。在准备这一应用程序时，我们进行了扩展的生化实验，得出了一系列假定的相互作用蛋白质。我们将测试这些蛋白是否与EEs结合，以及当从细胞中移除时，EE的运动性、运动结合或Hok1/Fts1/Fhp1锚定是否受损。我们还将通过对随机产生的突变体、EE分布和运输中的缺陷进行遗传筛选，以完全公正的方式解决双向EE运动的过程。先前进行了这项筛选，并鉴定了美国maydis中的Hok1/Fts1/Fhp1复合体（见上文）。我们将遵循相同的实验策略，并将在其他突变株中测试更多可能的因素，以了解EE运动和分布的作用。我们已经从这个筛选中确认了3个蛋白附着在EEs上，并将阐明它们在运动结合和运动调节或Hok1/Fts1/Fhp1募集到“货物”膜上的假设作用。此外，这种方法有可能揭示完全意想不到的方式，通过这种方式，细胞可以实现EEs的双向运输和均匀分布。总之，本提案以公正和全面的方式解决了在动物，人类和丝状真菌中发现的基本过程。我们广泛的初步结果以一种非常结构化的方式指导我们阐明马达在细胞中移动EEs的分子机制。了解这一过程将为医学研究提供信息，但也为开发抗真菌药物提供了新的途径。

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.