Mechanisms of assembly and inheritance of yeast septin-containing structures

描述(申请人提供)：虽然其确切的分子功能尚不清楚，但在真核生物中广泛保守，并与多种细胞过程有关。到目前为止，在每个被研究的生物体中，都发现了多个Septin蛋白存在于杂寡体复合体中，它们的功能需要适当的组织。突变或错误调控扰乱了Septin杂寡体的化学计量比，是Septin相关人类疾病的常见特征，包括癌症和遗传性神经疾病。细胞分裂和分化过程中含有Septin的细胞结构是动态的，以时间和空间调节的方式经历组织的突变。最近的高分辨结构的一个人的间隔蛋白六聚体和一个萌芽酵母八聚体揭示了组成亚基和它们组装的界面惊人的相似性。目前尚不清楚细胞中如何构建具有适当排列的Septin亚单位的组件，或者它们在增殖和发育周期中是如何重组的。在这一点上，酵母分隔素代表了一个简单的模型系统，用来识别调节这些多亚单位大分子组装的结构组织的细胞机制。该奖项支持的实验将研究单个Septin蛋白如何在不同的组装中使用，以及共价修饰和Septin相关因素如何影响高阶Septin组装的动力学。这些研究采用SNAP-Tag技术标记酵母蛋白，允许用各种功能标签标记单个间隔蛋白，并通过细胞分裂和分化进行跟踪。酵母间隔蛋白被认为在母芽颈部形成细丝，但还没有直接测试间隔蛋白是否以及如何在这些结构中组织。直接测试基于Septin八聚体结构的模型的实验旨在解决这个长期存在的问题。最后，用于研究Septin动力学的相同SNAP-Tag方法将被应用于另一种动态组装(基于肌球蛋白II的细丝)，以确定拟议研究揭示的新机制见解的普遍性。总而言之，这项工作将为作为独立调查员的长期研究方向奠定基础。 相关性：虽然它们的分子功能还不清楚，但似乎不同的Septin蛋白正确地组织成多亚基组装是至关重要的。事实上，许多疾病(从神经元障碍到癌症)涉及Septin基因的突变或调控不当，其方式破坏了Septin组装的平衡。这项工作将确定酵母细胞构建和维持其Septin复合体的方式。

DESCRIPTION (provided by applicant): Septins are widely conserved among eukaryotes and implicated in a variety of cellular processes, although their precise molecular functions remain unknown. In every organism so far examined, multiple septin proteins are found in hetero-oligomeric complexes, the proper organization of which is required for their function. Mutation or misregulation that upsets the stoichiometry of septin hetero-oligomers is a common feature of septin-associated human diseases, which include cancer and hereditary neuropathies. Septin-containing cellular structures in dividing and differentiating cells are dynamic, undergoing abrupt changes in organization in a temporally and spatially regulated manner. The recent high-resolution structures of a human septin hexamer and a budding yeast octamer reveal the striking similarity of the constituent subunits and the interfaces by which they assemble. It is not known how assemblies with the proper arrangement of septin subunits are built in the cell, or how they are reorganized during cycles of proliferation and development. In this regard, the yeast septins represent a simple model system with which to identify cellular mechanisms regulating the structural organization of these multi- subunit macromolecular assemblies. Experiments supported by this award will examine how individual septin proteins are used in different assemblies, and how covalent modifications and septin-associated factors influence the kinetics of higher-order septin assembly. These studies adapt SNAP-tag technology for labeling of yeast proteins, allowing individual septins to be marked with a variety of functional tags and tracked through cell division and differentiation. Yeast septins are thought to form filaments at the mother-bud neck, but there has been no direct test of whether and how septins are organized in these structures. Experiments directly testing a model based on the structure of septin octamers aim to resolve this long-standing question. Finally, the same SNAP-Tag approach used to study septin dynamics will be applied to another dynamic assembly (myosin II-based filaments), to determine the generality of the new mechanistic insights revealed by the proposed research. Together, this work will lay the foundation for a long- term direction of study as an independent investigator. RELEVANCE: Although their molecular functions are not understood, it appears that the proper organization of different septin proteinss into multisubunit assemblies is critical. Indeed, a number of diseases (ranging from neuronal disorders to cancer) involve mutations in or misregulation of septin genes in ways that upset the balance of septin assembly. This work will identify the ways that yeast cells build and maintain their septin complexes.