Michael J. Greenberg, PhD - US grants

FMRFamide (Phe-Met-Arg-Phe-NH2) is a neuropeptide isolated from clam ganglia; it is stored in granules, and released upon depolarization. FMRFamide is a potent molluscan pharmacon: it is cardioexcitatory or inhibitory depending on species; it causes contractures of various muscles and affects neuronal activity. FMRFamide is only one example of a set of heterogenous, but structurally and functionally homologous, molluscan neuropeptides. Moreover, it is hypothesized that FMRFamide-like peptides are widely distributed among animals, including vertebrates, and may have had a common origin with the opioid peptides in protein evolution. This project focuses on the phyletic distribution and mechanisms of action of FMRFamide. First, three peptides, already identified in the brains of the gastropods, Busycon contrarium and Helix aspersa, will be further purified by high performance liquid chromatography, and their amino acid compositions and sequences determined. The extraction and purification steps will be monitored by bioassays specific for FMRFamide (Busycon radula protactor muscle) or of the opioid peptides (guinea-pig ileum and mouse vas deferens). FMRFamide-like peptides will also be sought in other tissues: porcine adrenal medulla and sympathetic ganglia; and the central ganglia from other invertebrate phyla. As these peptides are characterized, their subcellular localization will be determined, and their release by potassium depolarization will be demonstrated. A particle-bound peptidase, coverting N-terminal extended precursors to FMRFamide, has been proposed; an attempt will be made to demonstrate it. FMRFamide receptors will be characterized by structure-activity studies on particular molluscan hearts and muscles and on rabbit vascular smooth muscle; the selected preparations are sensitive to the peptide; and their responses exemplify the range of its effects. The inotropic effects of FMRFamide will be correlated with its actions on cyclic nucleotide levels. The sucrose gap technique will be used to correlate electrical and mechanical responses.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

This award will partially support a symposium on neuropeptides at the Second International Congress of Comparative Physiology and Biochemistry.

The Whitney Marine Laboratory is a research institute of the University of Florida, established in 1974, and dedicated to the use of marine organisms for solving basic problems in experimental biology. The primary areas of study at the Laboratory are cell and neurobiology, but within those fields the scope is broad, including a variety of problems and approaches. The Whitney Laboratory has three strengths: it has broad technical expertise, and is particularly well instrumented for research in modern biochemistry, cell biology and physiology, an advantage maintained by support from the University and extramural agencies; a diverse fauna is available locally; and the rich animal resources of the tropics are easily accessible. Moreover, these organisms, as well as those from cold habitats, can be kept easily by virtue of a seawater supply that is unlimited, pristine and (in a new experimental aquarium facility) temperature controlled. This project will provide support for new construction and renovation to maintain and enhance the Laboratory's strengths. A new marine bio-resource facility, with work space, a collector's office and storage areas, will be built. A larger and more efficient tissue culture facility, with an adjoining room for image analysis, will be constructed. These activities will also free up space for molecular genetics, a common instrument room, and a laboratory for visiting scientists. These improvements will enhance the ability of the Whitney Laboratory to collect, maintain, and use marine animals, as well as maintaining its technological advantage in cell biology.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

This Research Experiences for Undergraduates award gives continuing support to The Whitney Laboratory, a research institute of the University of Florida, located on the Atlantic Coast, to provide research training for undergraduates in cell and neurobiology using marine animals. Eight faculty members will train students throughout the year in modern, experimental techniques of anatomy, biochemistry, pharmacology, toxicology and electrophysiology for which the facility is exceptionally well equipped. Students are recruited nationwide, and special efforts are made to attract women and minority students, and students in humanities departments who may be encouraged to try science as a career. The students will live on the campus of the laboratory, have access to all facilities, and will become full partners in the ongoing research efforts of their faculty advisors. Their independence will be encouraged, and they will receive full credit for their work in publications. In addition to their practical research training, the students' intellectual growth will be stimulated through participation in the Laboratory's regular seminar series, a special student seminar series, and research discussions provided by each member of the faculty.

Funds for a ultracentrifuge and gel analyzer for Whitney Lab, University of Florida, are requested. All the research efforts will be directed toward aquatic systems and vary over a broad range of subjects. Recently, molecular genetics has been added to the research portfolio at the lab. Among the species studied will be clams, shrimps, and jellyfish.

A one-half day Symposium, entitled "Signaling Systems, Venoms and Adhesives: Recurring Themes and Variations", is proposed for the Second International Marine Biotechnology Conference (IMBC' 91), October 13-16, 1991. The proposed Symposium assembles scientists whose studies with marine organisms make major contributions to our understanding of: (1) receptor systems for exogenous chemostimulants, neurotransmitters and hormones, (2) substances inhibiting specific receptors, and (3) the biosynthesis of waterproof adhesives. These seemingaly disparate subjects are integrated by recurring themes apparent in receptor structure, mechanisms of transduction, and the synthesis and employment of similar molecules for diverse functions. The following topics will be discussed: receptor-mediated larval settlement, the recurrence of a defined receptor type on echinoderm spermatozoa and mammalian cells, the evolution of families of venoms blocking specific receptors and ion channels, the role of peptides, steroids and prostaglandins as both endogenous hormones and exogenous pheromones and the incorporation of the catechol moiety into both neurotransmitters and adhesives. The Symposium contents will be summarized and integrated by the organizers for publication in the Biological Bulletin.//

This award provides funds to continue a successful Research Experiences for Undergraduates program at The Whitney Laboratory, a research institute of the University of Florida, located on the Atlantic Coast. The program provides research training for undergraduates in cell and neurobiology using marine animals. Ten faculty members will train students throughout the year in modern, experimental techniques of anatomy, biochemistry, molecular biology, pharmacology, toxicology and electrophysiology, for which the facility is exceptionally well equipped. Students are recruited nationwide and special efforts are made to attract women and minority students. The students live on the campus of the Laboratory, have access to all facilities, and become full partners in the ongoing research efforts of their faculty advisors. Student independence will be encouraged, and they will receive full credit for their work in publications. In addition to their practical research training, the students' intellectual growth will be stimulated through participation in the Laboratory's regular seminar series, a special student seminar series and research tutorials and discussions provided by each member of the faculty.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

This award supports summer research by undergraduates at the Whitney Laboratory of the University of Florida. Students pursue research related to ongoing faculty research programs that study the physiology, anatomy, biochemistry and molecular biology of various marine invertebrates and fishes. Students also participate in weekly seminars and informal research presentations, as well as discussions of scientific ethics and lectures and field trips that expose students to the ecology of nearby marine ecosystems.

DESCRIPTION (provided by applicant): The human genome contains 8 different isoforms of myosin I, making it the largest family of unconventional myosins expressed in humans. Myosin Ic is perhaps the best studied myosin I isoform due to its proposed roles in dynamic adaptation in hair cells, insulin stimulated GLUT-4 transport, compensatory excocytosis, and nuclear transcription. Despite its association with these cellular processes and disease such as congenital deafness, the molecular role of myo1c in the cell is unknown. It has been proposed that myosin Ic may act as a transporter, a force generator, or a strain-sensing tether. The ability of myosin Ic to function in these roles depends on its abilities to generate force and to modulate its biochemistry in response to load, with each of these roles placing very different and specific mechanical requirements on the myosin. Thus, by understanding the mechanics of myosin Ic, the molecular role of myosin Ic can be deduced. Despite the fact that myosin mechanics are at the heart of its cellular function, very little is known about the mechanics and load dependent mechanochemistry of myosin Ic. Furthermore, it has been proposed that calcium binding to calmodulins on the myosin Ic regulatory domain may play a central role in modulating its mechanics and response to load; however this notion has never been tested experimentally. Also, it is unknown whether calcium induced changes in myosin Ic mechanics and kinetics are relevant to cellular function since the response of myosin Ic to transient increases in calcium has never been studied. This research will address these gaps in our knowledge. Using a battery of single molecule techniques, the specific aims of this research are: 1. Determine the mechanical properties of the myosin Ic working stroke 2. Determine the load sensitivity of myosin Ic 3. Directly measure the effects of transient calcium on myosin Ic mechanics and kinetics All of these experiments will be conducted in the presence and the absence of calcium to test how calcium affects myosin kinetics and mechanics. Besides elucidating the molecular role that myosin Ic plays in the cell and how this role is regulated by calcium and force, this research will also shed light on the diversity within the myosin I superfamily. PUBLIC HEALTH RELEVANCE: Molecular motors within the human body are responsible for generating and responding to forces with malfunction of these motors leading to a wide array of diseases including deafness, hypertension, cardiac failure, and developmental defects. While the ability to respond to forces is central to the function of these motors, little is known about how this is accomplished. Using cutting-edge single molecule techniques, this research will examine force generation and tension sensing in of one of these motors, myosin Ic, helping us to understand both its cellular function and the role that tension sensing plays in molecular motors in both health and disease.

Project Summary/Abstract Familial cardiomyopathies are the leading cause of sudden death in people under 30. Two closely related cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are characterized by remodeling of the tissue in the ventricular wall, often accompanied by fibrosis, and myocyte disarray. These diseases are primarily caused by mutation of the proteins involved in generating or regulating power output by the heart. It has been proposed that HCM is caused by hypercontractility at the molecular level while DCM is caused by hypocontractility at the molecular level; however, it is not clear how mutations at the molecular level lead to alterations in the contractility and structure of heart cells and tissues. The goal of this research is to better understand the connections between molecular-based changes and the changes in contractility and structure at the cellular and tissue levels. To do this, we will harness biophysical, biochemical, cell biological, and tissue engineering techniques to measure contractility across these scales of organization, and then use mathematical modeling to connect these observations. We will examine two model mutations in troponin-T that cause either HCM or DCM. In Aim 1, we will dissect the molecular mechanism of two mutations in cardiac troponin-T, R92Q and ?K210, that cause hypercontractility and hypocontractility at the molecular level, respectively. In Aim 2, we will dissect the effects of these mutations on the structural and contractile properties of stem cell derived cardiomyocytes and engineered tissues. In Aim 3, we will examine whether these mutations affect the ability of cells and tissues to sense and respond to their mechanical environment. This bottom up approach will give unprecedented insights into the mechanisms of cardiac force production, mechanosensing by the heart, and the disease pathogenesis of familial cardiomyopathies.