We propose a detailed analysis of the mechanisms regulating mitochondrial division dynamics. The core mitochondrial fission regulator, Drp1 is a dynamin-related GTPase that also plays an important regulatory role in intrinsic apoptosis. Drp1 assembles on the mitochondrial membrane to execute division;however the mechanisms regulating these events remain largely unknown. We are working to identify and characterize Drp1 effectors to determine how these proteins mediate Drp1 recruitment and assembly for execution of mitochondrial division (Aim 1). The recent observation that the endoplasmic reticulum (ER) contacts mitochondria, and these contacts are sites of mitochondrial constriction and division, suggests that the ER strongly influences mitochondrial dynamics. However the mechanisms that coordinate this process are uncharacterized. We will identify components of the ER-mitochondrial tethering complexes using mass spectrometry and proteomics approaches, and identify how these sites influence mitochondrial division dynamics (Aim 2). We will use established microscopic and cell-based approaches, as well as siRNA methods to study how effectors function in a pathway of mitochondrial division in cells, and in vitro assays to specifically address the mechanism by which effectors influence Drp1 assembly and GTP hydrolysis, and determine the structure of assembled Drp1-effector complexes using negative-stain electron microscopy. We will use candidate proteins that localize to ER-mitochondrial contacts to identify new tethering components, and characterize their influence on mitochondrial division dynamics. Given the fact that the ER influences mitochondrial division, we will study the relationship between the Drp1 division effectors and ER-mitochondrial tethering complexes in order to understand the mechanisms that regulate the site and execution of mitochondrial division. Both ER and mitochondria undergo dynamic remodeling under stressed conditions;we will apply what we learn regarding the regulation of mitochondrial division under healthy conditions to learn about the specific mechanisms that influence the remodeling that occurs in response to stress. It is likely that different combinations of effector proteins, as well as post-translational modifications, influence the rates and sites of mitochondrial division dynamics. In the long term we wish to understand how ER and mitochondria coordinate their behavior in healthy and pathologic conditions to regulate ER and mitochondrial function, distribution, and the induction of apoptosis in order to understand the impact of these processes on cellular health. Understanding the fundamental mechanism of mitochondrial division and its regulation is directly relevant to understanding the basis of an increasing number of diseases associated with ER stress and dysregulated mitochondrial division, such as Alzheimer's, Parkinson's and diabetes, heart disease and stroke, and will potentially allow for the development of therapies targeting mitochondrial division to treat a wide variety of human diseases.
Our research will elucidate the mechanisms used by healthy cells to maintain cellular energy production and adapt to stressful conditions. We hope that by better understanding these processes we will able to create new therapies to treat or prevent a variety of diseases that results from pathologic stress, including heart disease, diabetes, and neurodegeration including Alzheimer's, Huntington's and Parkinson's diseases.
