Structural and mechanistic studies of mitochondrial fission

描述（由申请人提供）：线粒体是动态细胞器，其形状和功能由融合和分裂的连续循环控制。特别是，线粒体分裂在细胞生理学中具有重要作用。在许多形式的细胞凋亡中，细胞的线粒体在执行细胞死亡之前经历增加的线粒体分裂。抑制这种受调节的线粒体分裂可以降低细胞死亡的水平，表明分裂是一个重要的组成部分。我们提出的结构研究，将大大推进我们的线粒体裂变的机械理解，并导致方法来操纵这个过程。线粒体分裂依赖于发动蛋白相关蛋白Dnm 1在线粒体表面的募集，在那里它组装成介导线粒体收缩的多聚体复合物。Dnm 1的募集需要线粒体膜蛋白Fis 1，而衔接蛋白Mdv 1和Caf 4作为Fis 1和Dnm 1之间的分子桥梁。为了理解Fis 1如何募集裂变机制，我们开发了一种共表达系统来产生与Mdv 1或Caf 4片段结合的Fis 1复合物。使用X射线晶体学，我们将解决这些配合物的原子结构。此外，我们将使用类似的方法，加上冷冻电子显微镜，以了解Mdv 1或Caf 4与Dnm 1的结合如何激活线粒体裂变。这些结构研究将为Fis 1如何与Mdv 1和Caf 4物理相互作用以及裂变复合物的组装如何发生提供丰富的见解。我们将使用广泛的结构/功能分析的突变体的Fis 1，Mdv 1，和Caf 4的这些结构研究结果的生理相关性进行评估。我们的结构工作还确定了Fis 1的二聚体形式，我们将使用生物化学和细胞生物学方法来了解线粒体分裂过程中Fis 1的寡聚化。总之，这些研究将为理解线粒体分裂提供结构和机制基础，线粒体分裂是一个与细胞凋亡，神经元功能和衰老有关的过程。拟议的工作将导致线粒体分裂的结构理解，这是一个基本的细胞过程，在细胞生理学中是重要的。线粒体分裂是细胞凋亡的重要组成部分，细胞凋亡是一种程序性细胞死亡，在发育、组织形态发生和癌症预防中起作用。此外，线粒体分裂对神经细胞的功能很重要，并与衰老有关。这些研究将导致结构的见解，使研究人员能够操纵线粒体分裂，从而潜在地调节过程，如细胞凋亡，癌症和神经功能。

DESCRIPTION (provided by applicant): Mitochondria are dynamic organelles whose shape and function are controlled by continual cycles of fusion and fission. In particular, mitochondrial fission has important roles in cell physiology. In many forms of apoptosis, the mitochondria of cells undergo increased mitochondrial fission prior to the execution of cell death. Inhibition of this regulated mitochondrial fission can reduce the level of cell death, indicating that fission is an important component. We propose structural studies that will greatly advance our mechanistic understanding of mitochondrial fission, and lead to methods to manipulate this process. Mitochondrial fission depends on recruitment of the dynamin-related protein, Dnm1, to the mitochondrial surface, where it assembles into a multimeric complex that mediates mitochondrial constriction. Dnm1 recruitment requires the mitochondrial membrane protein Fis1, with the adaptor proteins Mdv1 and Caf4 acting as molecular bridges between Fis1 and Dnm1. To understand how Fis1 recruits the fission machinery, we have developed a co- expression system to produce complexes of Fis1 bound to fragments of Mdv1 or Caf4. Using X-ray crystallography, we will solve the atomic structure of these complexes. In addition, we will use a similar approach, coupled with cryo-electron microscopy, to understand how binding of Mdv1 or Caf4 to Dnm1 activates mitochondrial fission. These structural studies will provide a wealth of insight into how Fis1 physically interacts with Mdv1 and Caf4, and how assembly of the fission complex occurs. We will evaluate the physiological relevance of these structural findings using extensive structure/function analysis of mutants of Fis1, Mdv1, and Caf4. Our structural work has also identified a dimeric form of Fis1, and we will use biochemical and cell biological approaches to understand the oligomerization of Fis1 during mitochondrial fission. Taken together, these studies will provide a structural and mechanistic basis for understanding mitochondrial fission, a process with links to apoptosis, neuronal function, and senescence. The proposed work will lead to a structural understanding of mitochondrial fission, a fundamental cellular process that is important in cell physiology. Mitochondrial fission is an important component of apoptosis, a form of programmed cell death that plays a role in development, tissue morphogenesis, and prevention of cancer. In addition, mitochondrial fission is important for the function of nerve cells and has been linked to aging. These studies will lead to structural insights that will enable investigators to manipulate mitochondrial fission, and thereby potentially modulate processes such as apoptosis, cancer, and nerve function.