Jodi Marie Nunnari - US grants

9724143 Nunnari Accurate transmission of subcellular organelles during eukaryotic cell division is an essential and regulated process. In the case of the semi-autonomous respiratory organelle, the mitochondrion, this process is more complicated because, in order for progeny cells to be respiratory competent, both the organelle and its genome must be accurately transmitted. Although it is widely accepted that during cell division the partitioning of mitochondria is an active process dependent on the cytoskeleton, one fundamental question that remains unanswered is what additional mechanisms are required for the partitioning and distribution of mitochondrial DNA (mtDNA) both within the organelle and to daughter cells. The long term goal of the proposed work is to determine the cellular and molecular mechanisms responsible for mtDNA inheritance. These questions will be addressed using the simple eukaryote, Saccharomyces cerevisiae (budding yeast). From a wealth of genetic data obtained using this organism, it is clear that mtDNA inheritance is a non-random process. The ability to visualize both the organelle and mtDNA in vivo, and the opportunity to combine both genetic and biochemical approaches to understanding function, make S. cerevisiae an excellent model system for examining the cellular and molecular mechanisms involved in mitochondrial inheritance. It has recently been shown that when S. cerevisiae haploid cells fuse, mitochondrial fusion also occurs and a single, continuous dynamic mitochondrial reticulum is created in the zygote. While mitochondrial matrix proteins are able to freely diffuse within this reticulum, the diffusion of inner membrane-associated mtDNA is severely restricted. Although the movement of mtDNA within the organelle is restricted, mtDNA does enter emerging buds with the mitochondria. Elucidating the molecular mechanisms underlying mtDNA limited diffusion is critical to understanding the exact mechanisms for mtDNA inheri tance. The specific aims of this proposal are directed at further characterizing this mechanism and at identifying and determining the mechanisms of action of the proteins in S. cerevisiae that mediate mtDNA inheritance. Specifically, mtDNA movement within cells will be characterized by time lapsed imaging of a fluorescently tagged mtDNA-binding protein. The molecular components responsible for the limited diffusion and segregation of mtDNA will be identified using both genetic and biochemical approaches. Conditional mutants that are unable to stably maintain the mitochondrial genome during mitosis will be isolated. The mtDNA-protein complexes will be purified and characterized biochemically. Associated proteins will be identified using tandem mass spectrometry. The role of these proteins in mtDNA segregation will be determined using genetic, biochemical, and microscopic approaches. When cells divide, the various essential components (genes, cytoplasm, organelles) of the cell must be apportioned among the two daughter cells. In the case of the mitochondrion, the organelle possesses its own genome (in the form of multiple copies of mitochondrial DNA), and this must also be apportioned. Prior work has shown that the mtDNA distributes non-randomly, suggesting that mechanisms exist to somehow tether the DNA so that the copies do not intermix spatially within the organelle. This project will shed light on just how this occurs. ***

Mitochondria are semiautonomous intracellular organelles evolutionarily derived from endosymbiont prokaryotes. They are the primary site in eukaryotic cells where the energy of chemical bonds is oxidatively converted to a metabolically useful form. Mitochondria are therefore often referred to as the "powerhouses" of the eukaryotic cell. Like chloroplasts, mitochondria are separated from the cytoplasm of the cell by a double membrane system. The long term goal of this research project is to understand in molecular detail how the structure of the double-membraned mitochondrion is controlled. This is a complex problem because the chemical and physical structures of the outer membrane and inner membrane are different. In contrast to the outer membrane, the mitochondrial inner membrane is typically convoluted. These convolutions, referred to as cristae, form as a consequence of the greater surface area of the inner membrane, and presumably function to increase the organelle's ability to make energy. Interestingly, despite their distinct structures, current data suggest that fission and fusion of both membranes occur in tandem and that the molecular components that regulate these events are exclusively associated with the outer membrane. However, the range of cristae morphologies observed in cells suggests that the inner membrane is dynamic and that dedicated mechanisms and components exist to maintain its unique structure and perhaps to coordinate the behaviors of the two membranes. Virtually nothing is known about the mechanisms that regulate inner membrane structure in mitochondria. In addition to the outer membrane-associated dynamin-related GTPase, Dnm1p, analyses from Dr. Nunnari's laboratory indicate that a second dynamin-related GTPase, Mgm1p, is localized to the mitochondrial inner membrane and that inner membrane structure in mgm1 mutant cells is specifically aberrant. Members of the dynamin GTPase family share the common function to regulate the structure of various biological membranes. Thus, based on these findings, Dr. Nunnari hypothesizes that Mgm1p regulates the structure of mitochondria in cells by functioning to remodel the inner membranes. The experiments that will be done with support from this award will test this hypothesis utilizing a combination of genetic, biochemical, and cytological techniques.

DESCRIPTION:(provided by applicant) Mitochondria perform essential cellular functions that are influenced by the morphology and dynamics of the organelle. The goal of the proposed work is to understand the mechanisms involved in the fundamental and topologically complex process of mitochondrial fission, using the model eukaryote, S. cerevisiae. In yeast, we have shown that mitochondrial fission is mediated by the dynamin-related GTPase, Dnmlp, which is concentrated in punctate structures at sites where mitochondrial membrane constriction and fission occur. Human and C. elegans Dnm1p homologs also have been shown to control mitochondrial fission. Thus, elucidating the mechanism of mitochondrial fission in yeast will be relevant to fission in human cells and will ultimately help us to understand how changes in mitochondrial morphology and copy number contribute to the development of diseases. To date, additional components of the mitochondrial fission machinery have not been reported. We have isolated three novel nuclear genes required for mitochondrial fission, MDV1-MDV3 for Mitochondrial Division. Mdvlp is a predicted soluble cytosolic protein, containing at least three distinct regions: a novel NH2-terminal region; a middle region predicted to form a coiled-coil structure; and a C-terminal region containing 7 WD repeats. Four sets of data suggest that Mdvlp interacts with Dnm1p to mediate mitochondrial fission: 1)mdvl and dnm1 alleles genetically interact, 2) two-hybrid analysis reveals a strong interaction between Mdvlp and Dmnlp, 3) Mdvlp localizes to punctate structures associated with mitochondria, a pattern similar to Dnmlp's, and 4) localization of Mdvlp to punctate structures, but not to the mitochondrial membrane, requires Dnm1p function. In dmdv1 cells, Dnm1p is associated with punctate structures on mitochondrial membranes, but these structures are not able to mediate fission. Our data, therefore, suggest that Mdvlp functions relatively late in the fission process to stimulate Dnmlp-dependent mitochondrial membrane constriction and/or fission. The experiments proposed in this grant will test this hypothesis and determine the role of additional fission components using a combination of cytological, genetic and biochemical approaches. Specifically, we will characterize the step at which Mdvlp acts in the fission process, determine the structural features of Mdv1p required for mitochondrial fission, Mdv1p's interaction with Dnm1p, and the mitochondrial localization of Mdv1p in delta dnm1 cells, identify and characterize Mdvlp binding partners and, clone and characterize novel fission components. In the long term, our goal is to determine the molecular mechanism of mitochondrial fission by reconstituting this process in vitro.

DESCRIPTION (provided by applicant): Mitochondrial shape and size are governed by frequent fission and fusion events. Recently, defects in mitochondrial morphological caused by aberrant mitochondrial fission and fusion in mitochondria have been found to lead to optic neurodegeneration associated with autosomal dominant optic atrophy (adOA), and in apoptosis. These findings underscore the physiological importance of mitochondrial fission and fusion in cells. We propose a chemical genetic approach to investigate both the molecular mechanisms and physiological implications of mitochondrial membrane dynamics. We have identified small molecule inhibitors of these processes through screens conducted at the Institute of Chemistry and Cell Biology at Harvard. The most potent to date is a mitochondrial fission inhibitor that targets the mitochondrial fission dynamin-related GTPase and acts with equal efficacy in yeast and mammalian cells. We will exploit this inhibitor to determine the mechanistic role of dynamin-related GTPases in mitochondrial fission. We will also characterize our other candidate inhibitors from our screen and identify their targets to examine the molecular mechanisms of both mitochondrial fission and fusion. We exploit our inhibitors further in mammalian cell culture model systems to examine the physiological role of mitochondrial membrane dynamics in apoptosis and to test the theraputic effects of small molecule fission inhibitors on optic atrophies that result from mitochondrial dysfunction.

DESCRIPTION (provided by applicant): We propose to purchase a Leica TCS SP2 AOBS laser scanning confocal microscope equipped with special 405 nm diode and 594 nm orange HeNe lasers to be housed in our existing light microscopy Imaging Facility. This facility was used by 77 different researchers in calendar year 2002, including 56 using our fiveyear- old Leica SP1 UV confocal. The growth and increasing sophistication of our users have revealed two significant shortcomings of the existing microscopes. First is the inability to perform key experiments, including 1) fluorescence recovery after photobleaching (FRAP), 2) photo-activation of fluorophores in defined regions of interest, and 3) time-resolved imaging of more than one component at rates faster than one two-component image every 1-2 seconds. Second is a debilitating lack of sensitivity from our existing SP1 confocal for even moderately bright samples. This leads many experiments better suited for a confocal to be done on our deconvolution microscope, despite the optical flaws, simply to get adequate sensitivity. A straightforward way of addressing all of these concerns is the replacement of our existing confocal with one of contemporary design. Of contemporary confocals, the Leica TCS SP2 AOBS and Zeiss LSM 510 META are best suited to meet the needs of our most sophisticated users while still allowing a multi-user facility. We have selected the Leica TCS SP2 AOBS because 1) the Leica can meet our experimental needs and has superior sensitivity as evidenced during demos by Leica and Zeiss; 2) the superior Leica "filter-free" optical design; 3) the continuity of user interface for our large user group, making extensive retraining unnecessary; 4) continuity of our strong relationship with our Leica field service engineer, and 5) the trade in value of our existing Leica SP1. Major NIH-funded users will be Jodi M. Nunnari ("Regulation of Mitochondrial Fission"), Jonathon M. Scholey ("Roles of Microtubule-Based Motility in Mitosis and Intracellular Transport"), and Carol A. Erickson ("Direct Observation of Neural Crest Cell Emigration in a Living Embryo"). These users will account for approximately 50% of the instrument time, with the balance being offered to our large imaging community. This use will continue to be under the direction of one PhD and one MS scientist and administered under the current policies of an existing departmental Imaging Committee. A letter showing strong institutional support for maintenance and continued operation of the confocal is included.

DESCRIPTION (provided by applicant): Mitochondrial are complex organelles whose cellular roles extend beyond the well-characterized metabolic pathways of respiration, fatty-and amino-acid metabolism and ion homeostasis. Their central role in the cell is reflected by the fact that they function as regulators of apoptosis. Mitochondria are also dynamic organelles, which continually undergo fission and fusion. Using the yeast S. cerevisiae as our experimental system, we have identified proteins directly required for mitochondrial fission and fusion, whose orthologs in mammalian cells also regulate apoptosis. Among them are 3 dynamin-related proteins (DRPs), which are large self-assembling GTPases that regulate membrane dynamics in a variety of cellular processes. To date, our work has placed these key players into a framework of molecular events that occur during fission and fusion. We propose to build on these studies to understand in more detail the mechanism of how these proteins collaborate to carry out the mechanics of membrane fission and fusion. Towards this goal, we have established in vitro assays that recapitulate mitochondrial fusion and mitochondrial fission events. We will use these assays combined with genetic and cytological approaches to determine the molecular mechanism of mitochondrial fission and fusion. Mutations in 2 conserved human mitochondrial fusion proteins have been linked to the neurodegenerative diseases, Charcot-Marie-Tooth and dominant optic atrophy. Resolving the mechanisms of mitochondrial fission and fusion will provide insight into the etiology of these fusion protein-linked diseases and will help to understand how mitochondrial dynamics regulates apoptosis.

[unreadable] DESCRIPTION (provided by applicant): We propose to conduct a high through put screen in the Molecular Libraries Screening Centers Network identical to the one that, on a smaller screening scale, has already successfully identified mitochondrial fission and fusion inhibitors. Specifically, we will employ straightforward growth-based assays in S. cerevisiae strains that monitor mitochondrial fission and fusion activity to identify additional small molecule inhibitors. Given our success, we are confident that many novel compounds that will be found. The medical importance of events regulated by mitochondrial membrane dynamics, such as apoptosis, indicates that the chemical genetic approach may also lead to the identification and development of novel therapeutic agents for stroke, myocardial infarction, neurodegenerative diseases, and cancer. We believe that the novel therapeutic potential of these compounds, our expertise in finding their targets, and our unique ability to exploit them to more fully understand mechanism, justifies our request to expand our screen under this funding mechanism. We propose to conduct a high through put screen in the Molecular Libraries Screening Centers Network identical to the one that, on a smaller screening scale, has already successfully identified mitochondrial fission and fusion inhibitors. Specifically, we will employ straightforward growth-based assays in S. cerevisiae strains that monitor mitochondrial fission and fusion activity to identify additional small molecule inhibitors. Given our success, we are confident that many novel compounds will be found. The medical importance of events regulated by mitochondrial membrane dynamics, such as apoptosis, indicates that the chemical genetic approach may also lead to the identification and development of novel therapeutic agents for stroke, myocardial infarction, neurodegenerative diseases, and cancer. We believe that the novel therapeutic potential of these compounds, our expertise in finding their targets, and our unique ability to exploit them to more fully understand mechanism, justifies our request to expand our screen under this funding mechanism. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A user group including nine NIH funded investigators requests $498,113.89 to purchase an integrated TIRF/live cell confocal/fast widefield fluorescence microscope from Intelligent Imaging Innovations (3i) to be housed in the Section of MCB/College of Biological Sciences Imaging Facility. This instrument provides important new live cell imaging capabilities not possible with the two existing "turn key" instruments currently in the facility. This instrument will also free time on the existing point scanning confocal, which will be saturated in the next year. The proposed instrument builds from a base 3i Marianas Real Time Confocal SDC workstation by sharing the laser excitation sources (required for TIRF and multiple-aperture confocal) and sensitive EM-CCD cameras. The system is configured with two separate emission ports that encompass three imaging modalities. One is shared for the TIRF and rapid widefield applications and the other dedicated to the multiple-aperture confocal. Changing between TIRF/widefield and confocal is accomplished using a motorized fiber optic switch which is easily operated by the user. We have demonstrated that this instrument design works well for all three imaging modalities during a weeklong demonstration in our facility by 3i in December 2006. The MCB/CBS Imaging Facility currently serves a very large number of UC-D researchers (64 researchers in 17 departments in fiscal year 2005-2006). The sophisticated and versatile instrument requested here meets the needs of advanced researchers whose research problems and imaging needs have outgrown the capabilities of our existing instrumentation. Three of the major users in this proposal obtained significant new results during our instrument demonstrations that could not be obtained with the existing instrumentation in the Facility. In recognition of the important contribution this instrument would make to the UC-Davis research enterprise, the university administration has pledged $107,691 towards the purchase price. Management of the proposed instrument will be integrated into the existing, successful management scheme (2119 user instrument hours and $96,402 of recharges in fiscal year 2005-2006), which is supervised by a PhD-level scientist and guided by a faculty departmental imaging committee. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a Competitive Revision Application (notice number NOT-OD-09-058 and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications), which is focused on broadening the scope of the parent grant, GM062942 entitled "Mechanism of Mitochondrial Fusion" to encompass the structural analysis of the fusion DRP, Mgm1. Mitochondria are dynamic, essential, double-membraned organelles that perform a myriad of tasks within cells. Unlike their bacterial ancestors, they are not discrete entities. Isolated mitochondria are transient and in communication via fusion to form both localized and widespread mitochondrial syncytia within cells. Mitochondrial division antagonizes fusion and together these events function to create a compartment that is connected, with access to mtDNA, and thus functional, yet able to be distributed to distant cellular destinations via transport on actin or microtubule networks. We are focused on understanding the molecular mechanism of mitochondrial fusion to gain insight into the roles and regulation of fusion in cells and disease. In addition to its fundamental cellular role, mitochondrial fusion also regulates intrinsic apoptosis in cells and conversely, the pro-apoptotic Bcl-2 proteins regulate mitochondrial fusion in healthy cells. The important cellular roles of fusion are underscored by the fact that mitochondrial fusion is required for embryonic development and mutations in fusion proteins cause neurodegenerative diseases and stroke. Mitochondrial fusion is unique in that it is mediated by the action of highly conserved dynamin-related proteins (DRPs). DRPs are large GTPases that, through their ability to self-assemble and hydrolyze GTP, control membrane remodeling events. Our powerful yeast in vitro assay has revealed a wealth of information regarding the fusion mechanism. We have demonstrated that DRPs mediate both membrane tethering and lipid-mixing steps in fusion at the mitochondrial outer and inner membranes. Our recent work also indicates that a non-DRP outer membrane fusion protein is required post-membrane tethering, at the lipid-mixing step of outer and inner membrane fusion. Information from the structural analysis of Mgm1 will provide novel insight into the molecular events of inner membrane fusion and guide the biochemical experiments proposed in the original grant. In addition, the structural analysis of assembled Mgm1 will provide invaluable insight in the general function, mechanism and properties of DRP family members. The funds provided by this grant will address the goals of the Recovery act by providing for the salary and training of a junior post-doctoral scientist. PUBLIC HEALTH RELEVANCE: Mitochondria perform many important roles in cells, including the production of energy. This critical mitochondrial function and others depend on mitochondrial fusion and defects in mitochondrial fusion in humans cause neurodegenerative diseases and stroke.

DESCRIPTION (provided by applicant): Mitochondria are dynamic, essential, double-membraned organelles that perform a myriad of tasks within cells. Unlike their bacterial ancestors, they are not discrete entities. Isolated mitochondria are transient and in communication via fusion to form both localized and widespread mitochondrial syncytia within cells. Mitochondrial division antagonizes fusion and together these events function to create a compartment that is connected, with access to mtDNA, and thus functional, yet able to be distributed to distant cellular destinations via transport on actin or microtubule networks. We are focused on understanding the molecular mechanism of mitochondrial fusion to gain insight into the roles and regulation of fusion in cells and disease. In addition to its fundamental cellular role, mitochondrial fusion also regulates intrinsic apoptosis in cells and conversely, the pro-apoptotic Bcl-2 proteins regulate mitochondrial fusion in healthy cells. The important cellular roles of fusion are underscored by the fact that mitochondrial fusion is required for embryonic development and mutations in fusion proteins cause neurodegenerative diseases and stroke. Mitochondrial fusion is unique in that it is mediated by the action of highly conserved dynamin-related proteins (DRPs). DRPs are large GTPases that, through their ability to self-assemble and hydrolyze GTP, control membrane remodeling events. Our powerful yeast in vitro assay has revealed a wealth of information regarding the fusion mechanism. We have demonstrated that DRPs mediate both membrane tethering and lipid-mixing steps in fusion at the mitochondrial outer and inner membranes. Our recent work also indicates that a non-DRP outer membrane fusion protein is required post-membrane tethering, at the lipid-mixing step of outer and inner membrane fusion. Building on our success, we have now reconstituted the analogous mammalian mitochondrial fusion reaction in vitro. Using our yeast and mammalian systems, we will determine the fundamental mechanism of mitochondrial fusion, explore the mechanistic significance of the unique features of the mammalian mitochondrial fusion machines, and determine the mechanistic basis of the regulation of mitochondrial fusion by Bcl-2 proteins. Data from the proposed experiments will provide a foundation to understand the physiological roles mitochondrial fusion plays in cells. In addition, they will directly provide information that illuminates the basis of the diseases linked to mutations fusion components. Finally, by probing the mechanistic link between mitochondrial fusion and apoptosis, our experiments will provide fundamental information regarding the function and regulation of the Bcl-2 family of proteins, which will impact how scientists view the regulation of apoptosis.

DESCRIPTION (provided by applicant): Mitochondrial division, through the action of a conserved DRP, mediates the intracellular distribution of mitochondria in concert with transport and tethering pathways. Regulation of mitochondrial division is critical; excessive mitochondrial division is linked to numerous diseases, including neurodegeneration. Our long-term goal is to understand the mechanism and regulation of mitochondrial division and how division proteins collaborate with other pathways to distribute mitochondria and contribute to cellular homeostasis. Using a combination of structural, biochemical, genetic, and cytological approaches in yeast and mammalian cells, we will address the outstanding question of how on the molecular level division DRPs harness the GTPase cycle to divide mitochondria. Although DRPs can function as minimal machines in vitro, in cells all require additional proteins, whose mechanisms of action are not well understood. We will determine how mitochondrial division DRP effector proteins mechanistically function in yeast and mammalian cells. This will provide new insight into how they are used to integrate mitochondrial functions with cellular signaling pathways and are co-opted to regulate non-traditional DRP cellular events, such as apoptosis. We will determine how DRPs are harnessed for different activities through the analysis of the Dnm1 interacting protein, Num1, which is a cortical protein that mediates mitochondrial tethering. Our focus on Num1 will also serve to fill the gap in our understanding of the molecular basis of tethering-based distribution, which is common in cell types, such as neurons that have a functionally important population of stationary mitochondria. The basic mechanisms of mitochondrial division and distribution and their regulation are directly relevant to our understanding of the molecular basis of an increasing number of diseases, such as Parkinson's disease and diabetes and also acute pathological conditions, such as stroke and heart attack. As such, this work will pave the way for new and better therapeutic strategies for these diseases and conditions in humans. )

DESCRIPTION (provided by applicant): Mitochondria are required for a large number of cellular functions, including ion homeostasis, respiration, and programmed cell death. Consequently, defects in mitochondrial function have emerged as causative or contributing factors in a growing number of diverse human diseases such as cancer, cardiomyopathies, metabolic syndrome, and various neurodegenerative disorders. These diseases affect more than 50 million adults in the United States. Ad hoc proteomic and genetic studies in mammalian system have uncovered new mitochondrial protein (MP) complexes or pathways, but yet there has been no systematic experimental study of the mitochondrial interactome in mammalian system describing how these proteins function together in networks of pathways and complexes. Furthermore, it is also difficult to pinpoint the role of mitochondrial dysfunction in human disease because the interactions of MPs, both within and outside the mitochondria, are extensive and can be difficult to detect. This proposal seeks to begin addressing this deficit by creating a detailed physical (protein-protein) and genetic (gene-gene) interaction maps among MPs, which will help determine how mitochondrial protein complexes, within the framework of higher order networks, regulate and execute the associated processes. Using an optimized lentivirus-delivered tagging system coupled with mass spectrometry, a mammalian mitochondrial physical interactome map will be created using both native and mutant MPs with known association with human diseases. The resulting interaction networks will be compared to identify protein candidates that are relevant to disease onset and progression, and assess for variation in putative posttranslational modification sites involved in the progression of mitochondrial diseases (Aim1). Because genetic interaction (GI) is critical for revealing pathway-level relationships, optimized high precision quantitative pooled shRNAi coupled with deep sequencing approach, pioneered by the Weissman laboratory, will be used to query genes encoding for MP function by comparing the growth of pooled shRNAi treated cells on glucose relative to galactose as carbon sources in the media (Aim 2). This GI screening procedure will uncover new candidates that can toggle the glycolysis/mitochondrial respiration switch, which can be harnessed for therapeutic intervention. Finally, the GI data resulting from Aim 2 will be integrated with the proteomics data from Aim 1 (Aim 3) to investigate the functional relatedness and overall pathway architecture of the MP complexes to understand the fundamental mitochondrial biology and the role of mitochondrial dysfunction in disease. Collectively, these objectives are designed to provide new insights into the complex etiologies of diseases and have the potential to identify novel therapeutic avenues.

DESCRIPTION (provided by applicant): Mitochondria are required for a large number of cellular functions, including ion homeostasis, respiration, and programmed cell death. Consequently, defects in mitochondrial function have emerged as causative or contributing factors in a growing number of diverse human diseases such as cancer, cardiomyopathies, metabolic syndrome, and various neurodegenerative disorders. These diseases affect more than 50 million adults in the United States. Ad hoc proteomic and genetic studies in mammalian system have uncovered new mitochondrial protein (MP) complexes or pathways, but yet there has been no systematic experimental study of the mitochondrial interactome in mammalian system describing how these proteins function together in networks of pathways and complexes. Furthermore, it is also difficult to pinpoint the role of mitochondrial dysfunction in human disease because the interactions of MPs, both within and outside the mitochondria, are extensive and can be difficult to detect. This proposal seeks to begin addressing this deficit by creating a detailed physical (protein-protein) and genetic (gene-gene) interaction maps among MPs, which will help determine how mitochondrial protein complexes, within the framework of higher order networks, regulate and execute the associated processes. Using an optimized lentivirus-delivered tagging system coupled with mass spectrometry, a mammalian mitochondrial physical interactome map will be created using both native and mutant MPs with known association with human diseases. The resulting interaction networks will be compared to identify protein candidates that are relevant to disease onset and progression, and assess for variation in putative posttranslational modification sites involved in the progression of mitochondrial diseases (Aim1). Because genetic interaction (GI) is critical for revealing pathway-level relationships, optimized high precision quantitative pooled shRNAi coupled with deep sequencing approach, pioneered by the Weissman laboratory, will be used to query genes encoding for MP function by comparing the growth of pooled shRNAi treated cells on glucose relative to galactose as carbon sources in the media (Aim 2). This GI screening procedure will uncover new candidates that can toggle the glycolysis/mitochondrial respiration switch, which can be harnessed for therapeutic intervention. Finally, the GI data resulting from Aim 2 will be integrated with the proteomics data from Aim 1 (Aim 3) to investigate the functional relatedness and overall pathway architecture of the MP complexes to understand the fundamental mitochondrial biology and the role of mitochondrial dysfunction in disease. Collectively, these objectives are designed to provide new insights into the complex etiologies of diseases and have the potential to identify novel therapeutic avenues.

? DESCRIPTION (provided by applicant): Mitochondria perform fundamental functions in eukaryotic cells, including ATP production via respiration and cellular ion and phospholipid homeostasis. They also serve as platforms to integrate signaling pathways such as cell death and innate immunity. Mitochondrial functions are tightly linked to mitochondrial form, established through separate, but somehow coordinated machines that control dynamics, positioning, motility and mitochondrial DNA (mtDNA) transmission. The endoplasmic reticulum (ER) has emerged as an integral and pervasive player in the regulation of mitochondrial form and function. The ER exerts its role through contacts with mitochondria, which we hypothesize create specialized microdomains, which can recruit and/or modulate effectors that control and integrate mitochondrial physiology with other organelles and signaling pathways. In most cases, however, the molecular composition of ER-mitochondria contacts is poorly defined and their exact mechanisms of action, their functional scope and modes of communication are poorly understood. In the aims of this grant, we will address these deficits by exploring the molecular basis and functions of different types of ER-mitochondria contact sites in yeast and we will extend our findings to mammalian cells using comparative and forward strategies. New information in this area of cell biology will provide insight into the general architecture and rols of ER contacts and their regulation of mitochondrial function and cellular homeostasis to more accurately reveal role of mitochondria in human diseases that result from mitochondrial and ER dysfunction.

PROJECT SUMMARY Mitochondria are endosymbiotic organelles that possess a residual genome (mtDNA) encoding a handful of proteins and ribosomal and transfer RNAs essential for their functions. Human cells possess 100-1000s of mtDNAs, actively condensed into nucleoids - protein-DNA structures that are the cellular unit of mtDNA inheritance ? distributed within dynamic mitochondria ?syncytia?. Although the molecular players involved in mtDNA replication and packaging have been described, much less is understood about how at the cellular level nucleoids are distributed within mammalian cells to meet the needs for mitochondrial function, for example, how they are selected for mtDNA replication and how the cellular copy number of mtDNA is controlled. We discovered that in human cells, nucleoids engaged in mtDNA replication are spatially linked to a small subset of ER-mitochondria contact sites destined for mitochondrial division and motility. We found that the successive events of mtDNA replication, mitochondrial division and mitochondrial motility function together in a pathway that preferentially distributes nascent mtDNA in cells, which we term ER-linked mtDNA transmission. In this grant, we explore the underlying mechanisms of this ER-linked mtDNA transmission pathway by addressing the cell biology and behavior of the mammalian nucleoid. New information in this understudied area of cell biology will more accurately reveal the etiology of human metabolic diseases caused by mutations in mtDNA and in nuclear genes that affect mtDNA maintenance and in aging and neurodegenerative disorders, which are also linked to defective mtDNA maintenance and mitochondrial and ER dysfunction.

PROJECT(SUMMARY/ABSTRACT( (( Increasing evidence indicates that contacts formed between two major eukaryotic cellular organelles ? the endoplasmic reticulum (ER) and mitochondria ? are regulatory hubs essential for cell homeostasis. Although their pervasive significance for cell is established, they are poorly understood on both the molecular and functional levels ? a deficit that is directly addressed by this grant. Insight into the mechanisms of action, functional scope and modes of communication of ER-mitochondria contact sites will pave the way for a better understanding of the etiology of human diseases associated with mitochondrial and ER dysfunction and could reveal new targets for therapeutics.(

Summary Mitochondria are endosymbiotic organelles and have retained a reduced genome packaged into 100-1000 copies of nucleoids per cell, distributed within dynamic mitochondria ?syncytia?. Mitochondria are the metabolic hubs of eukaryotic cells, producing ATP via oxidative phosphorylation and other critical building blocks. They have evolved to behave dynamically and to be an integrating platform for the assembly and regulation of signaling pathways, such as cell death and innate immunity. Given their central roles, it is not surprising that that mitochondrial dysfunction is associated with an increasingly large proportion of human inherited disorders and with common diseases, such as neurodegenerative disorders, metabolic syndromes, and cancer. To gain insight into the roles of mitochondria in sickness and in health, we are focused on how mitochondrial behavior is controlled within cells and how this behavior is intertwined with metabolism and cell behavior. Our current research addresses the outstanding questions of how mitochondrial DNA copy number and transmission is controlled in cells, how the mitochondrial inner membrane is differentiated into distinct domains and how mitochondrial behavior, metabolism and cell behavior are integrated. Addressing these questions will illuminate how mitochondria contribute to cellular homeostasis and pathogenesis.