Michael Frohman - US grants

This proposal focuses on investigation of the function of genes expressed during mammalian gastrulation. The ultimate goal of this laboratory is to integrate more than a century of experimental embryology with an understanding of gastrulation on a molecular level. Little is known in mammals about the genes that direct anteroposterior axis formation early in embryo genesis or of the signals that initiate their expression. Gastrulation, the period during which they act, is a critical time for development. A large number of poorly understood human genetic and epigenetic embryological abnormalities arise during this time, including renal, cardiac, CNS, and assorted midline malformations. Several immediate goals are addressed here. First, functional studies win be performed on two homeobox-containing genes that in preliminary experiments have been found to become expressed during gastrulation in an intriguing manner. These studies will include (i) isolating full length cDNAs and genomic clones, (ii) performing high resolution mapping of the position of one of the genes which is located near a known developmental mutation, (iii) characterizing expression of the genes in embryonic stem cells to develop an in vitro model for their expression, (iv) characterizing their expression in embryos homozygous for known developmental mutations, and (v) performing loss-of-function and gain-of-function experiments accompanied by morphological, histochemical, and molecular analyses. Subsequent experiments are designed to address the effort and the length of time required to perform loss-of-function experiments using homologous recombination to inactivate genes. The aim of this section of the proposal to develop a simple and rapid assay to determine if inactivation of a gene specifically during gastrulation would be likely to result in an informative phenotype. The first approach attempted win be to use modified antisense oligonucleotides to inhibit gene expression in embryos cultured transiently in vitro. The feasibility of inhibiting genes known or presumed to be required for gastrulation (cyclin, beta-actin, and T) will be determined. Finally, genes acquired from a variety of sources including this laboratory that are known to be expressed during gastrulation will be screened using this protocol, to identify ones critically required for normal development. The information gained from these studies will lead, ultimately, to an understanding of the functions of these genes in early development, and to new insights into gastrulation.

DESCRIPTION: The central hypothesis of the proposal is that PLDs are important regulators of cell biological processes. More specifically, the P.I. proposes that PLD1 regulates secretion in response to stimulation and that PLD2 plays a role in receptor-mediated recycling or secretion. The specific aims address the central hypothesis. Aims 1 and 3 deal mainly with the molecular biology, enzymology, and activation of the PLDs, while Aims 2 and 4 deal more directly with the functions of the PLDs.

During the past several years, a superfamily of phosphatidylcholine-hydrolyzing Phospholipase D (PLD) genes conserved from prokaryotes to mammals has been described. Mammalian PLDs are activated by G-protein-coupled receptor and tyrosine kinase receptor signal transduction pathways. The biochemical step mediated by PLD and the second messenger generated by it are fairly well characterized, but functional roles for PLDs although thought to be important are not well defined except in yeast, where intriguing results have been generated. These results, together with less definitive mammalian studies, suggest that PLD promotes specialized types of membrane biogenesis as it relates to vesicular trafficking from the Golgi and plasma membrane. This proposal addresses potential roles of PLD in Drosophila melanogaster embryogenesis in cellularization, which is a specialized form of membrane biogenesis originating from the Golgi. We have mapped and cloned a PLD gene from Drosophila (denoted dPLD). Similar to mammalian PLD, dPLD appears to be regulated by Protein Kinase C-stimulated pathways. Unlike mammalian PLD but similar to yeast PLD, dPLD is not stimulated by the small G-protein ARF. dPLD-specific antisera reveal that it is expressed in germ cells and in the periphery of the embryo during cellularization. We propose 1) to define the mechanisms that regulate dPLD; 2) to characterize dPLD spatial expression and subcellular localization at high resolution in wild type embryos and embryos with defects in cellularization; 3) to generate and characterize loss-of-function dPLD alleles; 4) to examine the consequence of overexpression of wild-type and mutant PLD during cellularization and germ cell migration. Ultimately, we seek to understand what functional roles dPLD mediates, to model the more complex cell biological and physiological roles undertaken by the mammalian PLDs, and to bridge the growing knowledge concerning PLD mechanism of action in yeast and mammals using technological approaches unique to Drosophila.

9909888
Frohman

This award supports a two year collaborative research project between Professor Michael Frohman of the State University of New York (SUNY), Stony Brook and Professor Yasunori Kanaho of the Tokyo Institute of Technology in Japan. The researchers will be undertaking a study of the physiological and cellular functions for the signalling enzyme phospholipase D (PLD). PLD is a recently identified mammalian signal transducer that is believed to play important roles in agonist-induced cellular responses including membrane vesicular trafficking (secretion and endocytosis), proliferation, and differentiation. Recent studies have demonstrated to some extent the mechanism through which PLD is regulated. However, the physiological and cellular functions of PLD remain to be clarified. The aims of this project are to determine the signaling pathway(s) in which PLD is involved and the consequence(s) of PLD activation, using biochemical, molecular biological, cell biological, and transgenic approaches. The researchers will identify effectors downstream of PLD activation and determine how their activity changes in the absence of PLD, or when wild type, dominant negative, and constitutively active PLD mutants are over-expressed.

The project brings together the efforts of two laboratories that have complementary expertise and research capabilities. The U.S. researchers' expertise is in the area of molecular biology and the Japanese have expertise in biochemical probes. Results of the research will provide a greater understanding of cellular stimulus response pathways. As these pathways are defined and their metabolic intermediates identified, therapeutic advances are being realized as specific new pharmaceutical agents are discovered. This research advances international human resources through the participation of postdocs and graduate students. Through the exchange of ideas and technology, this project will broaden our base of basic knowledge and promote international understanding and cooperation. The researchers plan to publish results of the research in scientific journals.

DESCRIPTION (provided by applicant): A Medical Scientist Training Program (MSTP) is offered by the State University of New York at Stony Brook in conjunction with Brookhaven National Laboratory and Cold Spring Harbor Laboratory. The purpose of the MSTP is to train academic medical scientists for both research and teaching in medical schools and research institutions. Graduates of this program (30 to date) will be equipped to study major medical problems at the basic level and at the same time, to recognize the clinical significance of their pursuits and discoveries. Of our 28 graduates since 1990, many are already in faculty positions at prestigious institutions throughout the U.S. Ten are Assistant Professors (one each at Brown, the U of South Dakota, Mt. Sinai, UVA, UT Houston, Penn, Yale, and Einstein, as well as two at Columbia). Three are instructors (one at UWash and two at Harvard). Several enjoy competitive research funding from the NIH including one active RO1, one active PO1, one active R29 and five active KO8 awards. At least two other graduates hold competitive NRSA Fellowships. Thirteen of our graduates all of whom graduated in 1995 or later are still in training and only two of the 28 are in private practice. Graduate Programs in which the Ph.D. may be earned include Anatomical Sciences, Biochemistry & Structural Biology, Biomedical Engineering, Genetics, Molecular & Cellular Biology (including Biochemistry and Pathology), Molecular & Cellular Pharmacology, Molecular Microbiology, Neurobiology & Behavior, Oral Biology & Pathology, and Physiology & Biophysics. Each program listed is an independent doctoral degree-granting unit. Several major new initiatives are also now underway at Stony Brook including ones in genomics, proteomics and computational biology/bioinformatics. Finally, our small MSTP has diversity and flexibility that permit the design of individualized educational opportunities to meet the needs of trainees with a variety of interests, backgrounds and career goals.

[unreadable] DESCRIPTION (provided by applicant): Insulin-stimulated glucose uptake in muscle and adipose tissue is primarily mediated by the insulin-responsive glucose transporter isoform, Glut-4. In the basal state, Glut-4 slowly cycles with the majority of the protein sequestered into intracellular storage sites. In response to insulin, the rate of Glut-4 exocytosis becomes markedly increased, resulting in a large accumulation of Glut-4 at the cell surface. Recent findings have suggested that this translocation process may be regulated by Phospholipase D (PLD), a membrane-associated enzyme that is also activated by insulin. PLD catalyzes the hydrolysis of phosphatidylcholine, the most abundant membrane phospholipid, to generate the signaling lipid phosphatidic acid (PA). PA has been proposed to have several cellular functions including the facilitation of membrane vesicle trafficking. There are two mammalian PLD genes, PLD1 and PLD2. We have recently shown that PLD1 facilitates the fusion of neuroendocrine secretory granules into the plasma membrane during regulated exocytosis and that both genes facilitate regulated exocytosis of mast cell histamine-containing granules. Importantly, the fusion mechanisms controlling these secretory granules have many features in common with the insulin-stimulated plasma membrane fusion of Glut-4 vesicles, and we have found recently that PLD1 and PLD2 activation or inhibition alters insulin-stimulated Glut-4 translocation to the plasma membrane. Based upon these novel data, we propose to examine systematically the function of PLD as a key regulatory component in the insulin-stimulated trafficking of Glut-4 by developing reconstitution assays and establishing the role of PLD in physiologically relevant contexts. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Regulated exocytosis of secretory membrane vesicles proceeds via recruitment of the vesicles to the plasma membrane at specific sites conducive for exocytosis, followed by staged fusion into the plasma membrane. Generalized defects in regulated exocytosis cause single or multi-system disease in humans, and the promotion (e.g in diabetes) or inhibition (e.g. in anaphylaxis) of regulated exocytosis underlies numerous therapeutic modalities. Our general hypothesis is that manipulation of the iipid environment is a key element in this process. Published reports and our preliminary evidence suggest that the signal-transducing enzyme Phospholipase D1 (PLD1) plays a role late in this process during fusion of the vesicles into the plasma membrane via production of phosphatidic acid (PA), the Iipid product of PLD action. Many avenues of investigation are now timely to explore to determine the mechanism through which it functions. We propose to carry out the following specific aims to address these questions: 1. What are the temporal and spatial relationships of PLD1 activation and PA generation to their facilitation of regulated exocytosis? We will examine whether PLD1 activation is required acutely at the time of secretion, which of PLDVs activators participate in the PLD1-facilitated process, and where PA is produced during the fusion event. 2. How is regulated exocytosis facilitated by increasing levels of PA? We will use electron microscopy to examine the morphology of vesicles hindered from completing fusion in cells lacking PLD1 to determine which step the fusion process is blocked at. We will also examine potential mechanisms though which PLD1 and its product PA may be functioning, including regulation of the production of PI4,5P2 by stimulation of PI4P5KI, the enzyme family that generates PI4,5P2;recruitment to exocytic fusion sites of CAPS, a PI4,5P2-binding protein that is required for fusion;and potential affects on the fusion process itself though promotion of fusion pore formation or expansion. The results from the proposed experiments will substantially further our understanding of the mechanisms through which PLD1 promotes secretion during regulated exocytosis.

This application requests continued support for the tri-institutional Medical Scientist Training Program (MSTP) at Stony Brook University, Cold Spring Harbor Laboratory and Brookhaven National Laboratory. Continuously supported by the NIGMS since 1992, our MSTP has over a 30-year history of training physician-scientists, many of whom have gone on to make important discoveries and become leaders in biomedical research at academic medical centers across the country. Of our 87 graduates thus far, 30 are still in training, almost all in strong academic medical programs. Of the remaining graduates, 83% have academic faculty positions, many of which are highly prestigious, or are affiliated with university hospitals, NIH, or biotechnology / pharmaceutical groups. The goal of the program is to provide a rigorous training path that creates and fosters a physician-scientist mentality and a culture that imbues trainees with the tools, motivation, identity, and drive to pursue careers in academic medicine. Students are exposed to both clinical and basic scientific activities throughout their training, reinforcing the special nature of this program. Core courses unique to our MSTP include monthly seminar / dinners with both internal and external physician-scientist speakers, and a monthly journal club ? clinical pathological correlation dinner-evening that combines basic science with clinical applications in an active-learning manner. Most recently, our students have been strongly encouraged to register for mini-courses in our new and internationally-unique Alan Alda Center for Communicating Science. PhD degrees are offered in a wide range of disciplines with the students being guided to undertake high-quality basic / translational science training to address problems relevant to human health. Since the last review, there have been many changes at SBU including (i) the recruitment of an extraordinarily dynamic physician-scientist Dean who in turn has recruited new MD and MD-PhD physician-scientists as Chairs of the major clinical departments and the Cancer Center, (ii) the founding of a new Biomedical Informatics department by an MSTP-trained physician-scientist, (iii) major new philanthropy, and (iv) the $423M construction in progress of a Cancer Center and Children's Hospital. The MSTP has grown from 40 students to 58, annual institutional support to the MSTP has risen by 60%, the applicant pool size has almost doubled, and both applicant and matriculant quality have increased substantially. In 2014/15, we are matriculating 7 students. Many of our students hold individual NRSAs, publish high-impact articles, and have received a variety of honors, both intra- and extramurally. Overall time-to-graduation has averaged 7.9 years over the past decade. Our program was recommended for 18 lines of support by the NIH study section and Council in the last review, but federal funding constraints resulted in the current level of support being held to 8 slots. In this application, we request the level of support approved in the prior cycle, i.e. 18 slots, in line with the recent growth of our institution. We are committed to the training of exceptional physician-scientists, which renewed funding will greatly facilitate.

Abstract Mitochondria are dynamic organelles that function autonomously in many respects to produce energy via glucose (pyruvate) metabolism, undergo morphological change through fusion and fission, and move about the cell. However, these processes are also responsive to signals delivered from the extracellular environment; for example, insulin signals cells to upregulate mitochondrial fusion and alter the production of energy. The regulation of fusion and fission is also key for moving mitochondria properly to synaptic terminals in neurons in response to neurotrophic stimuli. These fundamental processes, when abnormal, cause many types of human disorders including neurodegenerative disease and diabetes. The links between extracellular signaling and mitochondrial responses are understood only in part. We previously uncovered a new role for the signaling lipid Phosphatidic Acid (PA) in mitochondrial fusion [11]. Our more recent unpublished work has connected the production of this signaling lipid on the mitochondrial surface to the generation of an inter-related signaling lipid, Diacylglycerol (DAG). PA can be converted to DAG by the lipid phosphatase Lipin 1, which we have found translocates to mitochondria when surface PA levels increase there. Lipin 1 mutations in mice and humans have been shown to cause a form of lipodystrophy with similarities to Type II diabetes. Taken together, these and other findings suggest that the generation of lipid signals on the surface of the mitochondria may regulate mitochondrial fusion, fission, and energetics in the context of insulin signaling and other extracellular signaling pathways. In this application, we propose in Aim 1 to characterize the external face of the mitochondrial outer membrane as a platform for lipid signaling involving PA and DAG, including analysis of the recruitment of the key enzymes that control their production and elimination, and identification of the physiological signaling pathways that upregulate them. In Aim 2, we will investigate the roles of these signaling lipids in the regulation of mitochondrial fusion, fission, and energy production as a consequence of extracellular signaling. By the end of the proposed experiments, we will have firmly established connections between extracellular agonists, lipid signaling at the mitochondrial surface, and mitochondrial physiological responses in the context of diabetes. Since many of these signaling steps represent drugable targets, gaining insight into the control of these fundamental processes may provide leads to novel therapeutic approaches in diabetes and other disease settings.

DESCRIPTION (provided by applicant): Mitochondria, once viewed as the simple powerhouse of the cell, are increasingly recognized to be involved in many different cell biological processes that involve interaction with non-mitochondrial organelles such as the endoplasmic reticulum and plasma membrane. These interactions take place in part via machinery on the mitochondrial surface that generates and senses protein and lipid signals, which has been an area of focus for us in the context of mitochondrial dynamics (fusion) and changes in energetic in response to extracellular signaling events. Our recent work has uncovered a new role for mitochondria in a specialized form of RNA processing that appears to take place at the interface between the mitochondrial surface and juxtaposed RNA granules. More specifically, we have linked a signaling enzyme on the mitochondrial surface to the production of piRNAs, a third type of RNAi, in an electron-dense structure known as nuage that is similar to P-bodies. Using cell biological, in culture, and animal model approaches, we propose to study how lipid signaling at the mitochondrial surface promotes this newly identified physical and functional interaction between mitochondria and RNA-processing complexes via underlying mechanisms that potentially include kinesin-regulated trafficking of RNAs, processing granules and mitochondria on microtubules. Relevance: This initial story applies to a pathway that is critical for spermatogenesis, has relevance to male infertility, and offers opportunities fr a novel type of male contraceptive. However, other types of RNA recruitment to and processing at the mitochondrial surface are known and represent a broader context in which this newly identified pathway will likely be important, including in connection to mesenchymal stem cells, brain and cardiac function, and cancer. PUBLIC HEALTH RELEVANCE: Mobilization of transposons can cause genetic diseases including cancer. piRNAs, the third type of RNAi discovered, function to maintaining genome integrity by suppressing transposon mobilization and performing germ-line imprinting. We have found that mitochondria play a role in generation of piRNAs via the action of a signaling enzyme anchored on the mitochondrial surface. We propose to increase our understanding of how a signal generated on the mitochondrial surface regulates this process, which may involve controlling how mitochondria and the RNA structures traffic through the cell on microtubules. If successful, the proposed research could facilitate the development of tools to broadly manipulate the epigenetic structure of genes and thus control their expression, might identify some causes of male infertility, and will present a potential therapeutic opportunity for a male contraceptive. It may also be important more broadly, as piRNAs are now being reported in many tissues.

DESCRIPTION (provided by applicant): This proposal focuses on the hypothesis that a lipid signaling pathway on the surface of the mitochondria facilitates the process of mitochondrial fission. We have published evidence that the enzyme Lipin 1, which is recruited to the mitochondrial surface by the lipid phosphatidic acid (PA) via a PA-binding domain in the center of the protein, converts the PA to the related signaling lipid diacylglycerol (DAG), which then promotes mitochondrial fission. Unexpectedly, we also found that Lipin 1 harbors a second, cryptic mitochondrial targeting sequence in the catalytic domain that exhibits highly-specific subcellular localization to sites of future fission events. This led to a model that binding to PA triggers a conformational change to expose the second site which avidly targets fission sites on the mitochondrial tubule and robustly triggers fission in a collaborative but also partially independent manner with Drp1, the dynamic-like protein most widely studied as the physical mediator of fission. This topic has direct clinical significance. Manipulation of mitochondrial fission is being tested for therapeutic application in stroke, cardiac ischemia, and pulmonary hypertension; increased knowledge about mechanisms underlying the fission process will aid in development of these approaches. We propose to pursue areas of interest that have been developed in the context of the Lipin 1 - fission story based on exploration of how DAG triggers fission in collaboration with other components of the fission machinery. Such questions include defining the proteins Lipin 1 interacts with at fission sites or recruits to the fission sites throgh the production of DAG, whether these proteins have roles in the fission process, and whether their function or the fission process itself is driven by DAG production by Lipin 1. We will also examine roles for other members of the Lipin enzyme family in fission, explore how Lipin is recruited to fission sites, and its relationship to the endoplasmic reticulum (ER) and actin cytoskeletal reorganization, which play important roles in the fission process. Taken together, these studies will further our knowledge of the mechanisms underlying fission and be of utility in the development of therapies targeting diseases with connections to this intrinsic process.