Microfilaments in the yeast Saccharomyces cerevisiae

描述(申请人提供)：真核细胞的内部组织和动力学在很大程度上由细胞骨架元件和分子马达的框架决定，这些分子马达将细胞器和大分子复合体运送到细胞中的特定目的地。长距离运输一般由基于微管的马达调节，短程运输和捕获由以肌动蛋白为基础的马达肌球蛋白介导。肌球蛋白-V家族的分子马达在进化上在动物、植物和真菌之间高度保守，脊椎动物肌球蛋白-V的缺陷可以导致疾病。许多不同的细胞器是由肌球蛋白-V运输的，但它们如何识别特定的货物，运输并在目的地释放货物，人们知之甚少。为了解决这些问题，研究人员使用了萌芽酵母，其中由Myo2编码的基本肌球蛋白-V作为其主要货物运输生长所需的分泌小泡，但也运输过氧化体、分泌途径的隔间和细胞周期中用于分离的液泡，以及在有丝分裂之前用于核定位的微管末端。这些转运事件都是由细胞器特异性受体介导的，其中许多是已知的。在这里，研究人员探索了Myo2分泌囊交付周期的三个基本方面。在第一个目标中，他们使用定量的活细胞成像来量化每个分泌小泡的马达数量，并确定如何确定这一数量，以及递送周期的动力学，以及这如何与目的地的小泡拴系和融合相结合。他们还探索了Myo2的循环和调节机制。在第二个目标中，他们使用遗传和活细胞成像方法来探索两种蛋白质MMR1和Smy1的功能，这两种蛋白质结合在Myo2的尾巴上，似乎在其传递周期中发挥作用。在第三个目标中，他们首先提出利用遗传和生化方法来扩展他们对Myo2识别和绑定分泌小泡的受体的研究。他们还通过识别损害与单个受体相互作用的特定突变，以及探索与一个受体的结合如何影响与另一个受体的结合，来探索Myo2货物结合尾巴上结合位点之间的关系。最后，他们建议建立一种体外试验，根据在早期AIMS中收集的遗传、生化和细胞生物学信息，重建MyO2与分泌小泡的结合。总体而言，这项研究将为Myo2的传递周期提供前所未有的见解，这将具有广泛的相关性，因为真菌和脊椎动物的肌球蛋白-V之间高度保守，以及它们在格里斯塞利综合征等疾病中的功能障碍。

DESCRIPTION (provided by applicant): The internal organization and dynamics of eukaryotic cells is largely determined by the framework of cytoskeletal elements and molecular motors that transport organelles and macromolecular complexes to specific destinations in the cell. Long range transport is generally mediated by microtubule-based motors, and short range transport and capture by the actin-based motors, myosins. The myosin-V family of molecular motors is evolutionarily highly conserved between animals, plants and fungi, and defects in vertebrate myosin-Vs can cause disease. Many different organelles are transported by myosin-Vs, but how they recognize specific cargo, transport and release it at its destination is poorly understood. To address these questions, the researchers use budding yeast where the essential myosin-V, encoded by Myo2, transports secretory vesicles for growth as its major cargo, but also transports peroxisomes, compartments of the secretory pathway, and the vacuole for segregation during the cell cycle, and microtubule ends for nuclear orientation prior to mitosis. These transport events are all mediated by organelle-specific receptors, many of which are known. Here the researchers explore three fundamental aspects of the Myo2 secretory vesicle delivery cycle. In the first aim, they use quantitative live-cell imagining to quantify the number of motors per secretory vesicle and establish how this is determined, and the kinetics of the delivery cycle and how this is integrated with tethering and fusion of the vesicles at their destination. They also explore mechanisms for recycling and regulation of Myo2. In the second aim, they use genetic and live cell imaging methods to explore the function of two proteins, Mmr1 and Smy1, that bind the tail of Myo2 and seem to play a role in its delivery cycle. In the third aim, they first propose to exploit genetic and biochemical approaches to extend their studies on the receptor by which Myo2 identifies and binds secretory vesicles. They also explore the relationship between binding sites on the Myo2 cargo-binding tail by identifying specific mutations that compromise the interactions with individual receptors, and also exploring how binding to one receptor affects binding to another. Finally, they propose to set up an in vitro assay to reconstitute the binding of Myo2 to secretory vesicles based on the genetic, biochemical and cell biological information collected in the earlier aims. Overall, this study will provide unprecedented insights into the delivery cycle of Myo2, which will be of broad relevance due to the high conservation between fungal and vertebrate myosin-Vs as well as their dysfunction in diseases such as Griscelli's syndrome.