Grant Abstracts: Dr. Sanford Bernstein's Lab Personnel Home Page

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Title: Research Education Core: SDSU/UCSD Cancer Center Comprehensive Partnership

Funding Source: National Cancer Institute Training Grant

Abstract:

The U54 Partnership Scholars Program led by San Diego State University (SDSU) and the University of California, San Diego (UCSD) will offer exceptional research and education experiences in cancer biology and health disparities to undergraduates. The program will: 1) Enhance success and retention of both underrepresented and non-underrepresented undergraduates by providing educational workshops, 2) Increase participation of undergraduate students from underrepresented groups in cancer research projects and broaden the range of disciplines these students pursue by providing research opportunities, and 3) Collaborate with ongoing research education programs at both partnership sites to enrich these ongoing programs in the areas of cancer research and disparities. Cancer-related research and education opportunities for first and second year students will be promoted through publicity and workshops. Five cohorts of 12 students each will be recruited during the end of their second year to participate in the Partnership Scholars Program. Students from SDSU (the institution serving underserved health disparity populations and underrepresented students) will comprise the majority of recruited students and most will be placed at UCSD's Moores Cancer Center for their research experiences. Other research opportunities with additional principal investigators at both SDSU and UCSD, including those serving in Partnership research projects and in the Outreach Core, will be available as well. Principal Investigators already recruited to the Partnership Scholars Program represent highly qualified faculty members with a track record of educating students in cancer-related research. The Scholars' research experience will begin during an intensive summer period following their second year that will include workshops in research ethics, cancer biology, cancer health disparities and professional development. Scholars will have onsite graduate or postdoctoral level supervisors to provide hands-on education in research activities. Scholars will continue participation in research during the academic year and take advantage of existing and new workshops that guide them toward involvement in a cancer research career and in developing their portfolio for graduate level education. Talented and motivated students may continue their program experience during the ensuing year. Dr. Sanford Bernstein (SDSU) and Dr. Sheila Crowe (UCSD), who have complementary expertise in running undergraduate and graduate NIH-funded education programs, will lead the program in conjunction with a core coordinator. Program outcomes will be monitored for success through assessment activities under the guidance of the Planning and Evaluation Core. The Internal Advisory Committee will use data gathered via assessment activities to implement program changes. The proposed program represents an innovative approach, not currently available to underrepresented students in San Diego, which will prime the “pipeline” for initiating successful careers in cancer research.

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Title: Mechanism of Myosin Chaperone UNC-45: Structural, Functional & Genetic Approaches

Funding Source: National Institutes of Health Research Grant

Abstract:

UNC-45 is a molecular chaperone that is required for myosin accumulation and myofibril assembly in striated muscle. Its C-terminal UCS domain interacts directly with myosin, while its N-terminal TPR domain binds the chaperone Hsp90. Although its mechanism of action is unknown, UNC-45 appears to be critical both for myosin folding in vivo and for protecting myosin from stress-induced denaturation. Further, changes in UNC-45 levels correlate with skeletal muscle inclusion body myopathy and cardiac failure, implicating UNC-45 in human disease. To begin to understand structure-function relationships in this enigmatic protein, we solved the first crystal structure of UNC-45. This proposal builds upon this Drosophila melanogaster structure to identify the molecular mechanisms and consequences of UNC-45 dimerization, UNC-45 interaction with myosin and UNC-45's relationship with yet to be identified partners. Aim 1 will map the structural and functional basis of our recent discovery that UNC-45 dimerizes. We will employ high-resolution electron microscopy, molecular modeling, cross-linking studies and functional analyses to test the hypothesis that dimerization of UNC-45 is a critical step in its mechanism of action. Aim 2a will be the first structure-function based mutagenesis of UNC-45 and will test the role of a highly-conserved surface groove that we defined in the UCS domain. Mutant versions of the protein will be analyzed in vitro through myosin-binding and aggregation assays, and in vivo by muscle structure and function analysis in transgenic Drosophila. This will test the hypothesis that the conserved cleft in the UCS domain of UNC-45 binds myosin, aids in myosin accumulation in muscle and/or protects myosin from stress-induced denaturation. Aim 2b will explore our observed differential localization of UNC-45 within sarcomeres of different muscle types along with our electron microscopy results showing that UNC-45 can bind to the neck region of myosin. We will use transgenic fly lines expressing alternative versions of the neck converter region along with confocal microscopy to test the hypothesis that UNC-45 binds specifically to the converter domain of the myosin neck and preferentially binds to specific versions of this myosin domain. Aim 3 will employ both genetic and biochemical approaches to define new partners for UNC-45 and test their importance in muscle structure and function. We will use flies with a depleted UNC-45 background in conjunction with the powerful genetic techniques of deficiency mapping and microRNA-enabled knockdown to define these partners. Further, we will use mass spectrometry to identify proteins isolated from developing and stressed muscles by UNC-45-based protein pull-down. We will examine the roles of these proteins during muscle development and stress by RNAi-based transient knockdown in vivo. This aim will test the hypothesis that UNC-45 has different binding partners and functions during myosin folding, during its occupancy of the muscle sarcomere and during muscle stress. Overall, our integrative analysis will provide important insights into the mechanism of action of UNC-45 and its role in muscle development, stasis and stress.

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Title: Genetics and Molecular Biology of Striated Muscle Myosin

Funding Source: National Institutes of Health Research Grant

Abstract:

This application seeks to understand mechanistic elements of the myosin motor of striated muscle and to determine how specific human mutations disrupt myosin function and lead to skeletal muscle disease or cardiomyopathy. We will employ a multidisciplinary approach that takes advantage of the powerful genetic tools available for Drosophila melanogaster along with a broad range of expertise that allows us to study myosin in an integrative manner, from its crystal structure and kinetic function through its effects upon muscle ultrastructure, fiber mechanical properties and locomotion. For Aim 1, we will build the first models of human distal arthrogryposis syndromes, which cause skeletal muscle contractures of varying severity. Using transgenic Drosophila, we will assess effects upon myosin structure, ATPase activity, in vitro motility, indirect flight and jump muscle ultrastructure, fiber mechanics and organism locomotion. We will test the hypothesis that distal arthrogryposis mutations cause abnormal interactions with nucleotides yielding enhanced ADP binding, reduced sliding velocity, slowed relaxation dynamics and hypercontraction and that the severity of the defects correlates with the severity of the human syndromes. For Aim 2, we will examine the mechanistic basis of myosin dysfunction caused by two hypertrophic cardiomyopathy mutations. We will construct organisms expressing point mutations that change the charge of the disease-causing residues and then test predicted suppressor mutations for functional rescue in vitro and in vivo. This will reveal interactions altered by the initial mutations and provide direct insight into protein-protein interactions that are critical for myosin function. We will test the hypothesis that mutations in residues that cause human hypertrophic cardiomyopathy alter contacts between the strands of the central b-sheet of myosin or interactions between the N-terminal domain of myosin and the lever arm, resulting in increases in ATPase activity, actin sliding velocity, fiber power levels and myofibrillar disarray. For Aim 3, we will examine the ability of the Drosophila heart to hypertrophy as a result of expressing myosin mutations known to cause human hypertrophic cardiomyopathy. This will be the first attempt to determine how the simple Drosophila heart tube, which is known to exhibit dilated, constricted or hypertrophic phenotypes, reacts to human contractile protein mutations that cause hypertrophy. We will monitor effects upon RNA transcription, calcium handling, ultrastructure and cardiac physiology and determine whether manipulating signaling pathways by gene knockdown or pharmacological intervention ameliorates observed defects. We will test the hypothesis that myosin mutations that cause human hypertrophic cardiomyopathy result in significant wall thickening, abnormal myofibrillar arrays, arrhythmias, disrupted calcium signaling, and transcript profiles that mimic the human condition, and that these pathologies can be suppressed by pharmacological treatment or genetic intervention. Overall our project will take advantage of an innovative experimental system to test significant questions regarding basic myosin function and disease.

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Title: Mechanistic basis and potential therapies for myosin storage myopathy

Funding Source: National Institutes of Health Research Grant

Abstract:

We propose to build and analyze the first reported animal models of myosin storage myopathy (MSM), a degenerative disease of human skeletal and cardiac muscles that arises from point mutations in the C-terminal rod region of slow/beta-cardiac myosin heavy chain. Our transgenic Drosophila melanogaster models will be used to dissect the molecular and developmental bases of MSM and to test possible therapeutic modalities. The Drosophila system will allow us to examine both homozygotes and heterozygotes (mutant/+) for each MSM allele in a standardized genetic background. This will define the importance of interactions between wild-type and mutant myosin molecules to disease pathology and will obviate genetic heterogeneity that leads to phenotypic variability in the human disease. We will test the hypotheses that: 1) MSM mutant myosin expressed in Drosophila leads to specific cell biological and physiological abnormalities similar to those seen in human MSM patients, 2) MSM myosin molecules a) are defective in filament assembly, b) show abnormal filament degradation and/or c) are prone to aggregation, 3) preventing MSM myosin aggregate formation or enhancing MSM myosin turnover can improve mutant muscle structure and performance. To test these hypotheses, we will pursue the following specific aims: 1) Examine the structural and functional effects of four different MSM mutations on skeletal and cardiac muscles during aging. We will explore the progressive nature of MSM via microscopy and physiological assays and correlate our results with extant human data. 2) Isolate mutant myosin molecules and assess their filament-forming ability, filament stability to proteolysis and aggregation propensity. This will help define the molecular basis of the disease. 3) Attempt to ameliorate disease phenotypes in organisms a) by over-expressing the molecular chaperones alphaB-crystallin, Hsp70, Hsp90 or UNC-45 (all known to aid in myosin folding and/or protection from stress); b) by using small molecule inducers of the heat shock response to more broadly elicit expression of molecular chaperones; c) by using transgenic or pharmacological approaches to induce autophagy as a mechanism to clear myosin aggregates. This multifaceted approach will provide novel insights into the developmental and biophysical bases of MSM and yield potential therapeutic approaches that may be useful for treating MSM and other inclusion body diseases.

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Title: Disease mechanism and therapy development for inclusion body myopathy type 3

Funding Source: Muscular Dystrophy Association Research Grant

Abstract:

The E706K mutation in the SH1 helix of a human muscle myosin motor causes dominant hereditary inclusion body myopathy 3 (IBM-3). Myofibrillar degeneration and rimmed vacuoles are disease hallmarks. Analysis of human biopsies suggests the mutant motor has reduced functionality. Presumably muscles attempt to degrade this myosin, leading to myofibril degeneration and protein aggregation in inclusion bodies. By inserting the human mutation into Drosophila muscle myosin we developed a model that we used to determine the biochemical defects arising from this mutation and the structural and functional abnormalities in heterozygous and homozygous organisms. Drosophila is an advantageous model since we can control for presence of a wild-type allele, other myosin isoforms and modifier genes. Further, the short life cycle simplifies study of age-specific defects. Also, Drosophila is amenable to drug treatments, as well as tissue-specific gene therapy approaches to mitigating myopathic phenotypes.

We found that IBM-3 myosin displays reduced ATPase activity and in vitro actin filament sliding velocity. It is highly susceptible to unfolding and aggregation. IBM-3 Drosophila homozygotes display progressive myofibrillar abnormalities, with increased myofibrillar disarray and membranous structures reminiscent of rimmed vacuoles. Further, Ref(2)P antibody staining shows high numbers of protein aggregates. Flight ability is absent and muscle fibers produce no power. Heterozygotes show better myofibrillar morphology, but display poor flight ability and oscillatory power generation. Overall the Drosophila model recapitulates many of the hallmarks of the human disease.

Here we propose to test the hypotheses that 1) IBM-3 myosin is ubiquitinated and accumulates in vacuoles and/or autophagosomes, 2) expression of IBM-3 myosin leads to abnormal accumulation of other proteins within aggregates, and 3) manipulation of particular proteins or pathways in IBM-3 muscle can enhance removal of mis-folded myosin and/or improve muscle structure and function.

Our specific aims are to use Drosophila indirect flight muscles to: 1) Examine the relationship among myosin modification, aggregation, localization and the degradation pathways in the IBM-3 disease model (by analysis of ubiquitination and localization of extra-myofibrillar myosin, the prevalence and composition of aggregates and vacuolar bodies in aging mutants, and proteasome and autophagosome involvement in the disease state). 2) Test therapeutic modalities by up- or down-regulating key genes/pathways by pharmacological or genetic approaches in an attempt to improve clearance of unfolded/aggregated protein and/or muscle performance.

Our studies should be relevant to understanding and treating other inclusion body diseases such as nemaline myopathy and inclusion body myositis.

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Title: SDSU Initiative for Maximizing Student Development (SDSU IMSD).

Funding Source: National Institutes of Health Training Grant

Abstract:

The overall aim of the SDSU Minority Biomedical Research Support Program (MBRS) IMSD program is to increase the number of students from underrepresented groups who attain Ph.D. degrees in the biomedical, behavioral and physical sciences. We will facilitate their exposure to science, academia and careers as researchers. The program will provide 20 undergraduates with faculty mentoring, customized advising, extracurricular experiences and opportunities for communicating with engaged peers each year during the course of the project period. We have assembled a dedicated group of active research mentors who will provide our students with hands-on research training. Our management and assessment plan will measure and ensure program and student success. We will provide a highly personalized experience to the students so that they can become the successful science leaders of tomorrow, and thereby, improve the diversity of academic scientists. Furthermore, we provide documentation of the effects our program has had upon students. Our training program will result in students with strong, highly competitive applications for doctoral programs such that at least 60% of our graduating seniors will directly enter into Ph.D. programs in the biomedical, behavioral and physical sciences. To accomplish this long-term goal, we plan to (1) implement a Pre-IMSD Biomedical Exploration Program (BEP) for freshman/sophomore students to introduce biomedical research and the pathway to preparing early for graduate school in the biomedical/behavioral sciences, (2) provide year-round mentored research experiences that will allow 20 IMSD undergraduate students to acquire excellent research and laboratory skills, and (3) develop the critical thinking and oral and writte communication skills of all program participants, by providing opportunities to present their research before professional audiences and to contribute to manuscripts for publication. We also propose three aims related to program evaluation: (1) to systematically measure program outcomes with mixed methods of quantitative and qualitative data collection and the establishment of a comprehensive electronic tracking database, (2) to use the data to continuously improve the program by maintaining elements that are most effective and modifying those that are least effective, and (3) to document the process of the SDSU IMSD program so that it becomes a model of accountability and to assure future success.

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Title: Structure of the UNC-45 Chaperone and its Interaction with Skeletal Muscle Myosin

Funding Source: National Institutes of Health Research Grant

Abstract:

We propose to study the structure and mechanism of action of the UNC-45 molecular chaperone, a myosin- affiliated "UCS domain" protein. Molecular chaperones play key roles in muscle development and function by aiding protein folding and inhibiting protein denaturation and aggregation. We have demonstrated that UNC-45 is critical for skeletal muscle myosin accumulation and myofibril assembly and that it interacts with myosin to protect it from heat-induced aggregation. However, the structure of UNC-45, its mode of interaction with myosin and its roles in skeletal muscle disease are largely unexplored. Our goal is to define the molecular structure of Drosophila UNC-45 and to study its physical interaction with myosin. We will test the hypotheses that 1) ATP binding, which enhances UNC-45 chaperone function, causes structural elements of the protein to undergo conformational change and 2) the UCS domain of UNC-45 interacts with myosin. To this end, we will examine UNC-45 in the apo- and nucleotide-bound states at atomic-level resolution by crystallization, x-ray diffraction and computational analysis. This will be the first high-resolution structure of a UCS domain protein, a class of myosin-associated proteins found in fungi through mammals. We will also image UNC-45 complexed with myosin S-1 by negative staining, electron microscopy and single particle image analysis followed by docking the crystal structures into class averaged projections. This research program will take advantage of our expertise in contractile protein analysis and the capabilities of highly qualified collaborators. Our studies will elucidate the structure of UNC-45, provide an understanding of its interaction with ATP and define ATP- induced conformational changes. Further, our efforts will yield insight into UNC-45's physical interaction with the myosin substrate, which is critical to myofibril assembly and resistance to stress.

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Title: Disease mechanism and therapy development for inclusion body myopathy type 3

Funding Source: Muscular Dystrophy Association Research Grant

Abstract:

A point mutation in the highly conserved SH1 helix of a human skeletal muscle myosin motor domain causes dominant hereditary inclusion body myopathy type 3. Myofibrillar degeneration and rimmed vacuoles associated with filamentous material are disease hallmarks. Preliminary analysis of human biopsies suggests the mutant motor has reduced functionality. Presumably the cell attempts to degrade this myosin, leading to myofibril degeneration and protein aggregation in inclusion bodies. By inserting the human mutation into Drosophila muscle myosin we will develop a model for accurately determining the biochemical and biophysical defects arising from this mutation and the resulting structural and functional abnormalities in the heterozygous and homozygous states. Unlike the situation for human studies, the Drosophila system can control for presence of a wild-type allele, other myosin isoforms and modifier genes. Further, as chaperone induction alleviates some inclusion body neuropathies, it is appropriate to test if specific chaperones or small molecule chaperone inducers reduce myofibril disruption, inclusion body formation and muscle dysfunction.

We will test the hypotheses that 1) specific functional defects in myosin cause inclusion body myopathy type 3, 2) defective myosin leads to specific cell biological and physiological abnormalities, and 3) manipulation of molecular chaperone levels or other gene products can prevent myosin degradation/aggregation and thereby improve muscle structure and function. We are expert in production of transgenic Drosophila lines expressing mutant myosin and in studying the biochemical, biophysical, structural and physiological effects of such mutations. We also have expertise in analyzing the in vivo and in vitro functions of chaperones.

Our specific aims are to:

1) Produce the disease model:
Clone a muscle myosin gene with the Glu706Lys mutation that causes human inclusion body myopathy type 3. We will express this gene in transgenic Drosophila lines lacking endogenous myosin in indirect flight muscles and jump muscles.

2) Determine the biochemical and biophysical defects in the mutant myosin:
Isolate the mutant myosin from indirect flight muscles to define its ATPase properties, actin affinity, actin filament in vitro motility, force-generating capability, detachment rate and step size.

3) Determine the structural and functional defects in muscle induced by the mutation:
Study the structure (light and electron microscopy) and function (flight and jump ability, fiber mechanics) of homozygous and heterozygous mutant muscles. We will also discern if the levels and locations of various chaperones are perturbed by expression of the mutant myosin.

4) Identify therapeutic modalities:
Determine if the myopathy can be ameliorated by over-expression of _B-crystallin, Hsp70, Hsp90 or UNC-45, all known to aid in myosin folding and/or protection from stress. If excess UNC-45 is naturally produced in mutant muscle, we will test the effect of reducing it, as very high levels of UNC-45 target myosin for degradation in some systems. Further, we will use small molecule inducers of the heat shock response to determine if more generally inducing chaperone function ameliorates in vivo defects associated with myosin-based inclusion body myopathies. Finally, we will use random chemical mutagenesis to select for flies with improved muscle function and determine which mutated gene(s) suppress the myopathic phenotype.

Our studies will directly determine the biochemical and biophysical defects in myosin that result in inclusion body myopathy type 3 and define the structural and muscle mechanical perturbations that the myosin mutation causes. Further, we will obtain insight into whether modulating the levels of specific chaperones or other gene products can overcome defects associated with inclusion body disease. This may yield possible therapeutic strategies for treating protein mis-folding and aggregation myopathies.

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Title: Myosin Hinge Region and Contraction

Funding Source: National Institutes of Health Research Grant

Abstract:

We propose a multifaceted and integrative approach to determine the function of the "hinge" region of the myosin heavy chain protein in muscle contraction. While much effort has focused on the role of the myosin head in dictating muscle contractile properties, our novel results indicate that the hinge of the rod also is critical for muscle function. We employ Drosophila melanogaster, which is amenable to genetic, transgenic and muscle mechanical approaches. Its single myosin gene encodes alternative myosin hinge regions, one expressed in slow twitch embryonic muscles and one expressed in fast twitch and oscillatory adult muscles. We produced a transgenic line that expresses myosin with the embryonic hinge in muscles that normally express the adult hinge (indirect flight and jump muscles). Transgenic muscles assemble normal-looking myofibrils, but muscle function is severely compromised. We propose to determine if hinge function is critical at the level of the intact organism, the isolated fiber, the single myofibril, the thick filament and/or the myosin molecule, by testing the following hypotheses: 1) the myosin hinge is not important in myofibril assembly; 2) the hinge influences the mechanical properties of muscle fibers and myofibrils [in collaboration with Dr. David Maughan (U. Vermont) and Dr. Gerald Pollack (U. Washington), experts in biophysical measurements of muscle fibers and myofibrils, respectively]; 3) the hinge contributes significantly to physical properties of myosin molecules and thick filaments, specifically to differences in the shortening of isolated myosin molecules or elasticity of intact thick filaments (in collaboration with Dr. Pollack); 4) the propensity to form a coiled-coil is critical to defining the differences between alternative hinge domains; 5) hinge-specific protein interactions impart functional differences between alternative hinge regions [we will test for interaction with the thick filament protein flightin (in collaboration with Dr. Jim Vigoreaux, U. Vermont) and perform genetic suppression studies in flies]. Our integrative approach should elucidate the role of the myosin hinge in muscle function and the mechanism by which it acts. Our work is relevant to human disease since mutations in myosin can cause hypertrophic cardiomyopathy in heart muscle and central core disease in skeletal muscle. Further, since the indirect flight muscle has oscillatory and stretch-activated properties similar to human cardiac muscle, understanding myosin's involvement in generating these properties may lead to insights into human heart function.

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Title: Analysis and Amelioration of Defective Protein Folding in Skeletal Muscle

Funding Source: Muscular Dystrophy Association Research Grants

Abstract:

Defects in protein folding are the basis of several neuromuscular diseases. These defects can result from mutant proteins that fold improperly or mutations in molecular chaperones that normally facilitate protein folding in vivo. We are studying UNC-45, a molecular chaperone/co-chaperone of muscle cells that aids in folding of the muscle myosin heavy chain motor and possibly other muscle or non-muscle proteins. The overall goals of our research are to provide insights into the molecular mechanisms of chaperone-based genetic disease and to help develop therapeutic strategies for such disease.

We propose to provide a detailed structure/function analysis of UNC-45 in vivo and in vitro as well as to determine mechanisms whereby defects in protein folding can be ameliorated via genetic and transgenic suppression. We are using a system that is amenable to genetic manipulation, Drosophila melanogaster. Our aims are to:

1) define the role of UNC-45 within muscle cells through analysis of the phenotypes engendered by mutations in the functional domains of this protein.

2) elucidate the biochemical basis of mutant defects by isolating wild-type and mutant UNC-45 proteins and studying their chaperone activity in an in vitro system.

3) define which proteins interact with UNC-45 during muscle differentiation by genetically selecting for and characterizing mutations that suppress the recessive lethality of UNC-45 mutations.

4) test the interchangeability of chaperones, by determining whether over-expression of chaperones known to be critical for protein folding in skeletal muscle cells allows UNC-45 mutant rescue.

Our research will lead to insights as to how mutations that result in production of abnormally folded contractile proteins cause phenotypic defects and how these may be ameliorated. Since several neuromuscular diseases arise from aberrant protein folding and accumulation of misfolded protein aggregates, the proposed study will be an important contribution to understanding the disease process and potential therapeutic modalities.

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Title: Genetics and Molecular Biology of Myosin

Funding Source: National Institutes of Health Research Grants

Abstract:

We will use an integrative and multidisciplinary approach to investigate how the S1 head domain of the myosin heavy chain (MHC) protein drives muscle function. Myosin is the molecular motor of muscle and the major component of myofibrillar thick filaments. Its ATP-dependent interaction with actin-containing thin filaments powers muscle contraction. Our studies use the model organism Drosophila melanogaster, which has a single muscle Mhc gene but produces multiple forms of the protein (isoforms) by alternative RNA splicing. Using MHC null mutants in conjunction with germline transformation, we express engineered versions of the protein and employ them to test basic and novel hypotheses that predict structural, biochemical, fiber mechanical, physiological and locomotory properties imparted by specific myosin domains and amino acid residues. An innovative aspect of our system is that functions will be tested in vitro, in skeletal and cardiac muscle and in intact organisms. Therefore, we can determine directly and to what degree a specific biochemical property defines a physiological or locomotory characteristic. To this end, we will utilize a battery of in vitro and in vivo assays: ATPase, actin and nucleotide affinity, in vitro motility, x-ray crystallography, molecular modeling, electron microscopy, isolated fiber mechanics, video-based cardiac imaging and organismal locomotion. Our first aim is to elucidate the role of a critical communication element of the myosin motor called the relay domain. We will determine the importance of specific transient interactions of key amino acid residues of the relay that we hypothesize to interact with the converter domain or with the SH1-SH2 helix region during the mechanochemical cycle. For this, we will combine the transgenic approach with classical genetics to introduce and suppress mutations. Our second aim will test predicted isoform-specific interactions during the mechanochemical cycle. To this end, we will exploit the Drosophila system to express flight and embryonic muscle myosin isoforms that will be crystallized and compared in multiple nucleotide binding states. Our third aim will test our hypotheses about the effects of a mutation in myosin that is known to cause restrictive cardiomyopathy. We will create a Drosophila model of this human disease by mutating the invariant proline at the myosin head-rod junction. We will define the biochemical, biophysical, mechanical and locomotory defects engendered by the myosin mutation. We will also examine whether the mutation affects the flexibility of the myosin head and determine how it influences Drosophila heart (dorsal vessel) structure and function. Overall, our novel integrative analyses will permit testing of models for the transduction of chemical energy into movement and will yield insight into how myosin functions in muscle. Further, we will directly address the role of myosin in human muscle disease, by defining the molecular basis of a restrictive cardiomyopathy.

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Title: Myosin Isoform Structure and Function

Funding Source: National Institutes of Health Minority Biomedical Research Support Grant

Abstract:

DESCRIPTION: The San Diego State University MBRS/IMSD program submits this competitive renewal after nine years of successful minority biomedical student research development. The overall goal is to rectify the underrepresentation of minorities in biomedical research. We will continue our successful model of the mentor-protege concept to achieve this major objective. As will be evident in this application, our institution is evolving from a major teaching/ research institution with an emphasis on undergraduate training, to one of the top ranking research institutions in California. Because of this fact, we will continue developing undergraduate talent but will expand the graduate component of the program (including the Ph.D.). Therefore, the major value added component of this application is the increased emphasis on graduate training of master's and Ph.D. level MBRS research participants. This increase on graduate training will assist the NIH/NIGMS/MORE programs to achieve their goal of helping to resolve the underrepresentation of biomedical researchers in a more timely manner. In summary, we have added several new aims and components, and a strong evaluation component to assure that the objectives of the MORE programs and our specific SDSU program are met.

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Title: Functional Analysis of Miniparamyosin in vivo

Funding Source: National Science Foundation Research Grant

Abstract:

The goal of this project is to determine the function of miniparamyosin, a thick filament protein that is likely to be important in myofibril assembly and muscle contraction. This protein was discovered a few years ago in Dr. Bernstein's laboratory. The N-terminal quarter of this protein is unique, while the remainder is identical to the C- terminal half of the thick filament core protein paramyosin. From the work of others, miniparamyosin is now known to be a component of many invertebrate muscles, and its presence in a number of phyla suggests that it serves an important role in myofibril assembly and/or function. In situ hybridization studies showed that miniparamyosin mRNA is expressed in only a subset of adult Drosophila muscle cell types. Antibody studies from other laboratories have shown that miniparamyosin is a component of the thick filament and that it has a different localization pattern in indirect flight muscles and tubular muscles of the adult. The overall aim of this project is to directly test the role of miniparamyosin in muscle structure and function and its interplay with other elements of the thick filament in vivo. The project will employ transgenic, ultrastructural, and physiological approaches designed to directly test the function of this protein in vivo in Drosophila melanogaster. The specific objectives for the grant period are as follows: 1, to isolate and/or construct Drosophila strains which are mutant for miniparamyosin and analyze their genetic, ultrastructural and physiological defects; 2, produce transgenic flies that express greatly increased levels of miniparamyosin in their indirect flight muscles. By analyzing the ultrastructure of such muscles, it will be determined whether thick filament length, diameter or electron density are affected as a result of changing the ratio of this protein to that of myosin heavy chain and paramyosin. Antibodies against miniparamyosin will be used to define whether over-expression results in protein relocalization within the thick filament. It will also be determined how over-expression of miniparamyosin in flight muscles affects the biophysical properties of these muscles; 3, determine whether the amount of paramyosin influences the accumulation and localization of miniparamyosin within thick filaments. This will be accomplished by over-expression of paramyosin in the indirect flight muscles in lines producing normal and elevated levels of miniparamyosin. This goal is based on observations that the amount of paramyosin correlates with the level and pattern of miniparamyosin accumulation. The subcellular machinery of muscle cells that allows them to carry out their contractile function consists of two sets of complex filaments, termed thick and thin filaments, which are arranged in parallel arrays within a larger structure termed the myofibril. In fully "relaxed" muscles, these parallel arrays only partially overlap each other Contraction occurs when the thick and thin filaments slide over each other in a coordinated manner to increase the extent of overlap, thereby shortening the myofibrils (and thus shortening the cell), a process which requires hydrolysis of ATP. The "motor" which transduces the energy of hydrolysis of ATP into movement is myosin, the major component of the thick filaments. In order to fully understand how muscle cells function, it is necessary to understand the fine molecular structure of the myofibril. Recently, a new protein, termed miniparamyosin, has been discovered as a component of thick filaments in a model invertebrate, the fruit fly Drosophila melanogaster. This project will explore the role that this newly-discovered thick filament protein plays in the assembly and function of myofibrils, and will undoubtedly lead to new insights into those processes.

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