Janet Shaw - US grants

Mitochondria cannot be synthesized de novo; instead, newly-formed daughter cells must inherit their mitochondria from the mother cell during division. Although the metabolic functions of mitochondria have been extensively studied, very little is known about the molecular mechanisms controlling mitochondrial transmission. Recently, an important class of genes (termed MDM for "mitochondrial distribution and morphology") has been identified through the use of temperature-sensitive mutants that disrupt mitochondrial inheritance during yeast budding. The precise functions of these gene products are not yet understood. All but two of these MDM genes are represented by single mutant alleles suggesting that additional components of the mitochondrial partitioning apparatus remain to be identified. We have identified eight new genes that are required for mitochondrial transfer into newly formed daughter cells during east budding. We propose to study the molecular basis of mitochondrial partitioning in yeast via the genetic, molecular and biochemical characterization of these 8 new MDM genes and the proteins they encode. FIRST, we will use a combination of vital staining, indirect immunofluorescence, and electron microscopy to characterize the range of mitochondrial inheritance and morphology defects caused by mutations in MDM genes. SECOND, wild-type and mutant alleles of the 8 MDM genes will be cloned, sequenced and analyzed. This analysis will allow us to identify any important structural motifs and functional domains in the predicted proteins. THIRD, we will use indirect immunofluorescence and cell fractionation experiments to determine the subcellular location of Mdm protein. FOURTH, we will analyze mitochondrial partitioning during meiosis in mdm mutants. These studies will allow us to determine whether Mdm proteins mediate mitochondrial movements during both meiosis and mitosis and may reveal details of mitochondrial partitioning that cannot be easily visualized in mitotically dividing cells. The experiments we propose will lay the foundation for future molecular, biochemical and genetic analyses of Mdm protein function. In the long- term, these studies should contribute to our basic understanding of the molecular mechanisms underlying mitochondrial movements during cell division and during early development in other organisms.

The establishment and maintenance of proper mitochondrial morphology involves regulated mitochondrial fusion events. Although mitochondrial fusion is observed in a wide range of organisms and cell types, very little is known about the molecules or mechanisms that coordinate the fusion of the inner and outer mitochondrial membranes. An important observation is that a transmembrane GTPase called Fzo1p regulates mitochondrial fusion in the budding yeast, Saccharomyces cerevisiae. A complete understanding of the biochemical activity of Fzo1p as well as other proteins necessary for fusion will require in vitro assays for each of the distinct steps of fusion. The purpose of this project is to develop in vitro assays that measure mitochondrial docking and membrane fusion in a reconstituted system using components isolated from yeast cells. These assays will exploit novel fusion proteins which contain green fluorescent protein (GFP) and blue fluorescent protein (BFP) and which can be targeted to different mitochondrial compartments. These proteins will be used to develop (1) a light microscopic assay for mitochondrial docking and aggregation, and (2) a complementation assay for actual fusion. The complementation assay will employ a construct of BFP and GFP linked together, which exhibits fluorescence resonance energy transfer (FRET), and a specific protease, which when fusion occurs cleaves the BFP-GFP linkage and causes loss of FRET. The assays will be used to study the role of wild-type and mutant forms of Fzo1p. Once developed, these assays can also be employed to identify and study other proteins involved in each step of the fusion process. Ultimately, the assays may lead to an understanding of how the mitochondria coordinate the separate fusion of their outer and inner membranes.

DESCRIPTION (applicant's description): Changes in mitochondrial morphology and copy number are associated with a variety of human diseases including neurological disorders and some types of cancer. Although the metabolic functions of mitochondria have been extensively studied, the molecular mechanisms that regulate mitochondrial membrane dynamics are not understood. In the current funding period, we characterized two different GTPases in S. cerevisiae that regulate mitochondrial morphology and copy number. Dnm1p is a dynamin-related GTPase that acts on the outer mitochondrial membrane to regulate fission. Fzo1p (fuzzy onions) is a transmembrane GTPase that regulates mitochondrial docking and/or fusion. Both GTPases have human homologues that, when mutated, cause defects in mitochondrial morphology. These findings illustrate how effectively yeast can be used as a tool to study the molecular mechanisms that control mitochondrial dynamics in human cells. During the next funding period, we will continue to study mitochondrial fission and fusion in yeast using a combination of genetic, molecular and biochemical approaches. Specifically, we propose: 1) to identify and characterize SFZ genes/proteins required for mitochondrial fission, 2) to determine the role of Fzo1p self-interactions in mitochondrial fusion, 3) to screen for binding partners that interact with Fzo1p in its GTP-bound state, and 4) to develop an in vitro assay for mitochondrial fusion. The studies we propose will provide new information about the molecular and biochemical basis of mitochondrial fission and fusion in eukaryotic cells.

DESCRIPTION (provided by applicant): Changes in mitochondrial morphology and copy number are associated with a variety of human diseases including neurological disorders and some types of cancer. Although the metabolic functions of mitochondria have been extensively studied, the molecular mechanisms that regulate mitochondrial membrane dynamics are not understood. In the past few years, we characterized two different GTPases in S. cerevisiae that act in opposition to regulate mitochondrial morphology and copy number. Dnm1p is a dynamin-related GTPase that acts on the outer mitochondrial membrane to regulate fission. Fzo1p (fuzzy onions) is a transmembrane GTPase that regulates mitochondrial docking and/or fusion. Both GTPases have human homologues that, when mutated, cause defects in mitochondrial morphology. These findings illustrate how effectively yeast can be used as a tool to study the molecular mechanisms that control mitochondrial dynamics in human cells. Most heterotypic membrane fusion events characterized to date require proteins on the donor membrane (called v-SNAREs) and proteins on the target membranes (called t-SNAREs) that allow proper recognition and docking of the two compartments prior to fusion. It is our hypothesis that the Fzo1 protein defines a new type of SNARE that utilizes GTP hydrolysis to regulate mitochondrial-mitochondrial membrane docking and/or homotypic fusion reactions. Although we are currently testing this hypothesis in living yeast cells, a complete understanding of the biochemical activity of Fzo1p will require an in vitro assay that reconstitutes mitochondrial fusion. The goal of the experiments described in this application is to develop an in vitro assay for mitochondrial fusion using components isolated from S. cerevisiae cells. The role of S. cerevisiae Fzo1p during fusion will then be studied using this assay. The studies we propose will ultimately provide new information about the molecular and biochemical basis of mitochondrial fusion in eukaryotic cells.

DESCRIPTION (provided by applicant): Mitochondria are dynamic organelles whose morphology and function are regulated by opposing fission and fusion. Using funds provided by this grant, my laboratory demonstrated that a dynamin-related GTPase called Dnm1 controls mitochondrial fission in budding yeast. Dnm1 defines a conserved family of mitochondrial GTPases with important but poorly understood functions in cell physiology and human health. The human homolog of yeast Dnm1 is called Drp1. Studies performed in mammals and cell lines suggest that Drp1 plays critical roles in maintaining mitochondrial function, mitochondrial fragmentation during apoptosis, and generation of mitochondrial fragments for autophagy. Based on these findings, molecules that regulate mitochondrial fission are potential targets for inhibitory drugs that reduce mitochondrial fragmentation and unwanted cell death, including the apoptotic death of cardiac cells after stroke or the neuronal cell death associated with a variety of neuropathies. This proposal focuses on the fission machinery of budding yeast, which serves as the prototype for mitochondrial fission machineries in all eukaryotes, including humans. This field has matured to the stage where the major questions are focused on how Dnm1 and its binding partners work together to elicit fission with the proper spatial and temporal control. To progress beyond this stage, it is critical to obtain a better mechanistic and structural understanding of how components of the Dnm1 fission complex interact and assemble, and how these interactions affect fission activity. The studies described here are designed to obtain this mechanistic and structural information. With this knowledge in hand, we will generate and test specific models for the regulation of Dnm1 assembly and fission complex activity. PUBLIC HEALTH RELEVANCE: The proposed studies will advance mechanistic and structural understanding of the molecules that regulate mitochondrial fission using yeast as a model system. This core mitochondrial fission machinery includes the Dnm1 GTPase, Mdv1/Caf4 adaptors, and the Fis1 membrane anchor. The results will be relevant to human development and health, as these proteins have human homologs including human Drp1 (yeast Dnm1 homolog) and its membrane receptor human Fis1 (yeast Fis1 homolog). Human Drp1 plays critical roles in maintaining mitochondrial function, mitochondrial fragmentation during apoptosis, and generation of mitochondrial fragments for autophagy. A mutation in Drp1 was recently linked to the death of an infant 37 days after birth. Experiments in this application take advantage of the sophisticated tools available in yeast to probe the mechanism of fission complex assembly and activity. What is learned from these studies will allow scientists and clinicians to manipulate the activities of these molecules for the benefit of human health

DESCRIPTION (provided by applicant): Membrane-anchored Miro GTPases and their adaptor proteins attach mitochondria to cytoskeletal motors that distribute the organelle throughout the cell. This process is particularly important in neurons, where mitochondria are moved long distances from the cell body to the synapse and back again. Aberrant mitochondrial movement and distribution is observed in a large number of human neurodegenerative disorders including Spastic paraplegias, Alzheimer's, Huntington's and Parkinson's diseases. A popular model proposes that energy (ATP) deprivation and changes in calcium buffering caused by mitochondrial distribution defects causes the neuronal degeneration associated with these diseases. However, whether mitochondrial motility defects are a primary cause or a secondary consequence of disease progression in these cases is not clear. The research proposed in this application will directly test this model. Using a conditional (floxed) allele, we generated two different Miro1 mutant mouse models. The first is a Miro1 neuron-specific KO that allows mice to survive postnatally, but causes a progressive neuropathy characterized by tremors, hind limb stiffness, kyphosis (spinal curvature) movement defects and death several weeks after birth. These phenotypes are hallmarks of neurodegenerative disorders. The second is a whole animal knockout (KO), which completes embryogenesis but fails to breathe and dies at birth. Preliminary studies indicate that defects in a specific neuronal circuit are responsible for this neonatal breathing defect. Using these mice as well as tissues and primary cell cultures from these animals, we will determine the effects of Miro1 loss on mitochondrial distribution and function. We will also determine whether/how any mitochondrial defects lead to neuronal degeneration and death. These studies will provide the first physiological analysis of Miro1 function and specific mitochondrial movement defects in mammals. Because our studies are based on mouse models with demonstrated neurological dysfunction, what we learn will advance understanding of the role of mitochondrial movement in the development and maintenance of neuronal health.

Project Summary/Abstract ? Project 2 Pelvic organ prolapse (POP), descent of the pelvic organs (bowel, bladder, uterus) into the vagina, and pelvic floor symptoms such as urinary incontinence, are common and costly conditions that impact the health and well being of millions of women world-wide. For many women, the stage for future pelvic floor disorders is set with the first vaginal delivery, yet we know little about how to maximize recovery following this seminal event. The long-term goal of this research is to develop evidence-based interventions that mitigate the impact of vaginal delivery on key indices of pelvic floor health. In this project, we plan to study how physical activity, sedentary time, measures of muscular strength and body habitus impact two primary outcomes: pelvic floor support and pelvic floor symptoms. Young women demonstrate a range of vaginal support and pelvic symptomatology during the first postpartum year, but little is known about why they differ from each other. By drawing parallels between vaginal delivery and other acute soft tissue injuries, this project will explore biologically plausible factors known about muscle and connective tissue healing that may be related to postpartum recovery and thereby pelvic floor health: timing, dose and type of physical activities, muscular strength and body habitus. We hypothesize that the early postpartum period (first 8 weeks) reflects acute healing and early recovery when moderate to vigorous physical activity (MVPA, measured using accelerometry) may impair healing and result in worse pelvic floor support and greater symptoms one year postpartum. Conversely, the remaining first postpartum year reflects a period to strengthen and improve supporting structures; we hypothesize that greater MVPA and less sedentary time will improve pelvic floor support and reduce symptoms. The scope of this project is relevant to NICHD's described mission, to ensure that ?women suffer no harmful effects from reproductive processes, and?to ensure the health, productivity, independence, and well-being of all people through optimal rehabilitation?, and specifically addresses pelvic floor disorders, as emphasized in the portfolio of the Gynecologic Health and Disease Branch. This prospective cohort study will recruit 1530 nulliparous women in the third trimester of pregnancy and follow those that deliver vaginally for 1 year postpartum. The aims of this project are to study the effect of 1) physical activity and inactivity, 2) muscular strength, and 3) body habitus, all measured in the early and later postpartum periods, on pelvic floor support and symptoms one year postpartum. We will also explore whether the presence of a high-risk delivery variable (forceps, prolonged 2nd stage of labor, shoulder dystocia, anal sphincter laceration) modifies the association between MVPA in the early postpartum period on pelvic floor support and symptoms at 1 year.