Structural and mechanistic studies of mitochondrial fission

描述（由申请人提供）：线粒体是动态细胞器，其形状和功能由连续的融合和裂变循环控制。特别是，线粒体裂变在细胞生理学中起着重要作用。在许多形式的细胞凋亡中，细胞的线粒体在细胞死亡之前经历了增加的线粒体裂变。抑制这种受调节的线粒体裂变可以降低细胞死亡水平，表明裂变是一个重要的组成部分。我们提出的结构研究将极大地推进我们对线粒体裂变的机制理解，并导致操纵这一过程的方法。线粒体分裂依赖于动力蛋白相关蛋白Dnm1在线粒体表面的聚集，在那里它组装成一个多聚体复合物，介导线粒体收缩。Dnm1的募集需要线粒体膜蛋白Fis1，衔接蛋白Mdv1和Caf4作为Fis1和Dnm1之间的分子桥梁。为了了解Fis1如何招募裂变机制，我们开发了一个共表达系统，以产生与Mdv1或Caf4片段结合的Fis1复合物。利用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，结合低温电子显微镜，了解Mdv1或Caf4与Dnm1的结合如何激活线粒体裂变。这些结构研究将为了解Fis1如何与Mdv1和Caf4物理相互作用以及裂变复合体的组装如何发生提供丰富的见解。我们将通过对Fis1、Mdv1和Caf4突变体进行广泛的结构/功能分析来评估这些结构发现的生理学相关性。我们的结构工作也确定了Fis1的二聚体形式，我们将使用生化和细胞生物学方法来了解线粒体裂变过程中Fis1的寡聚化。综上所述，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。提出的工作将导致对线粒体裂变的结构理解，这是细胞生理学中重要的基本细胞过程。线粒体分裂是细胞凋亡的重要组成部分，细胞凋亡是细胞程序性死亡的一种形式，在细胞发育、组织形态发生和癌症预防中起着重要作用。此外，线粒体分裂对神经细胞的功能很重要，并与衰老有关。这些研究将带来结构上的洞见，使研究人员能够操纵线粒体裂变，从而潜在地调节细胞凋亡、癌症和神经功能等过程。

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.