Yale E. Goldman - US grants

The overall aims of this research are to understand the molecular mechanism by which muscle proteins convert chemical energy into mechanical work, and to obtain a precise correlation between the physiological, biochemical and structural events of muscular contraction. Novel methods will be applied to striated muscle fibers to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. Rapid changes in the chemical concentrations of pertinent biochemical species, such as ATP, will be made by laser pulse photolysis of photolabile "caged" precursors. A newly developed method to secure the fiber ends will be used to improve the uniformity and reproducibility of the contractions. The orientation of fluorescent molecules covalently bound to the myosin heads will be monitored at high time resolution to determine the rates and identity of specific structural changes of the protein molecules. This method combined with laser photolysis, new chromophoric probes, and additional labelling sites made available by recombinant technology will provide orientation and mobility information from several regions of the myosin head. The experiments will be carried out on single muscle fibers of rabbit psoas and frog semitendinosus muscles. Results from this project should significantly advance knowledge of the contractile process and thus bring a greater understanding of both normal and pathological states of striated muscle and other types of cell motility.

The proposed conference is the 17th in a series of Gordon Research Conferences that originated in 1966 with a conference on cardiac muscle. The major focus of the 1987 conference will be: The molecular basis of the contractile process. The purpose of this conference is to summarize the major areas of progress during the past 3-5 years, and to point the way to those areas which should prove productive for research in the immediate future. The work to be presented will deal with mammalian skeletal, cardiac and smooth muscle as well as with selected non-muscle contractile systems. In the past three years significant progress has been made in a broad range of areas in muscle research. Particular emphasis will be given to new methods involving genetics and molecular biology to discover the relationship between the amino acid sequence of the contractile proteins, their three-dimensional structures and their enzymatic and mechanical activity. A high resolution crystal structure of G-actin may be completed and promises to aid in defining the structure of F-actin filaments. The myosin subfragment-1 head has also been crystalized and progress toward solving the structure of these crystals will be presented. Chemical cross-linking studies, limited proteolysis and new photoaffinity labels have given new insight into the possible domain structure of myosin and to the location of the actin and nucleotide binding sites. New applications of flourescent and spin-labeled probes as well as time-resolved low angle x-ray diffraction are determining cross-bridge orientation in defined mechanical states. The possible regulation of smooth muscle contraction by systems other than light chain phosphorylation will be addressed as will the function of auxiliary and cytoskeletal proteins. A major theme will be the correlation of biochemical information from purified systems with similar studies in skinned muscle fibers. New photolabile precursors of nucleotide analogs allow kinetic experiments in fibers to focus on specific chemical reaction steps. The conference will include a limited number of formal presentations with emphasis being placed on discussion and informal interchanges. Ample use will be made of poster presentations and discussion of this material will be incorporated into the main program. This conference has become a major triennial meeting which attracts the leading investigators in muscle research from around the world. In allowing scientists of diverse backgrounds to focus on the molecular mechanism of muscle contraction for a week, this conference should help to open new areas of research as well as provide a meaningful reexamination of previously accepted ideas.

The Program Project (Pennsylvania Muscle Institute) functions as a multi-institutional, interdisciplinary program for collaborative studies on normal and diseased vascular smooth, cardiac and skeletal muscle and platelet function. Major areas investigated are: the structure and the biochemical and mechanical kinetics of myosin crossbridges, excitation-contraction coupling, stimulation-secretion coupling and shape change in blood platelets, the interaction of actin with actin-binding proteins and the developmental differentiation of sarcomeres and other organized components of contractile cells. The development of the following novel methods and instrumention, for this research, is a specific goal of the Program: biological electron probe and electron energy loss analysis, the synthesis of inert photolabile precursors caged compounds) of biologically active molecules for kinetic studies through activation with laser flash photolysis; scanning confocal and fluorescent light microscopy, microinjection of antibodies, mRNA and antisense messengers for developmental studies, and the development and use of antibodies directed to contractile, structural and regulatory proteins as probes of cell differentiation and contractile function. The longterm scientific aims and technologies developed in the Program Project are directed to understanding the pathophysiology of diseases of the heart and vascular smooth muscle.

The Program Project (Pennsylvania Muscle Institute) functions as a multi- institutional, interdisciplinary program for collaborative studies on normal and diseased cardiac and skeletal muscle and blood platelets. The major theme of the Program is the assembly and mechano-chemistry of contractile proteins. It integrates investigations of contractile proteins from genetic regulation of cell differentiation, development, isoform diversity and growth, assembly of the components in vitro, myofibrillogenesis in live, differentiating cells and the molecular motions and reaction kinetics in fully constituted myofibrils. The objective is to understand how contractile proteins form highly specific, functional macromolecular assemblies in vitro and in vivo and, how these complex assemblies perform their task of chemical-to-mechanical energy transduction at the molecular level. The projects and investigators within the Program are interdependent and closely linked through complimentary research goals and shared technologies, resources and training programs. Novel instrumentation to be developed for the research will include microscopically localized photolysis of photolabile ("caged") precursors of fluorescent labels for dynamic studies of incorporation and exchange of contractile proteins, time-resolved and scanning confocal fluorescence polarization microscopy and ultra-rapid freezing electron microscopy synchronized to transient molecular events initiated by laser pulse photolysis of "caged" substrate or signal molecules. Novel techniques will include new eukaryotic co-expression systems for protein structure-function studies, transfection and micro- injection of cDNA plasmids coding for truncated, mutated or chimeric proteins into primary cultured myocytes, and mass spectrometry of nanomolar stable phosphate isotopes. The long-term scientific aims and technologies developed in the Program Project are directed to understanding the normal growth, assembly and function as well as pathophysiology of muscles relevant to the cardiovascular and pulmonary systems.

The overall aims of this research are to understand the molecular mechanism by which muscle proteins convert chemical energy into mechanical work, and to obtain a precise correlation between the physiological and biochemical events of muscular contraction. Novel methods will be applied to striated muscle fibers to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. Rapid changes in the chemical concentrations of pertinent biochemical species, such as ATP, will be made by laser pulse photolysis of photolabile "caged" precursors. A newly developed method to secure the fiber ends will be used to improve the uniformity and reproducibility of the contractions.Rapid mechanical transients initiated by quick length changes will be obtained under conditions with altered concentrations of the biochemical substrate and products. Kinetics modelling, involving a novel method to determine theoretical values directly from the experiments, will be used to help interpret the results. The isotonic filament sliding distance per substrate molecule utilized by the contractile proteins will be determined to relate the number of enzymatic cycles to the number of mechanical interfilament interactions. Rapidly repeated steps of shortening after photo release of known and limited concentrations of substrate will provide a direct indication of the mechano-chemical coupling ratio. The experiments will be carried out on single muscle fibers of rabbit psoas and frog semitendinosus muscles that have had the surface membrane removed to allow alterations to the biochemical environment of the proteins. Results from this project should significantly advance knowledge of the contractile process and thus bring a greater understanding of both normal and pathological states of striated muscle and other types of cell motility.

method development; fluorescence microscopy; macromolecule; molecular dynamics; single cell analysis; time resolved data; fluorescent dye /probe; cell motility; bioengineering /biomedical engineering; myosins; protein structure; orientation; optics; bioenergetics; biophysics; video recording system; radiation detector;

The Program Project (Pennsylvania Muscle Institute) functions as in interdisciplinary program for collaborative studies on the molecular mechanism of muscle contraction and myosin-based cell motility. The major themes of the Program are the structural biology, mechano-chemistry, protein design and sequence specificity of the contractile proteins. It integrates investigations from genetic expression, isoform diversity, enzymatic activity, molecular motions and reaction kinetics in isolated contractile protein molecules fully constituted myofibrils. The objective is to understand how these proteins, individually and organized in highly specific, macromolecular assemblies, perform their task of chemical, structural and mechanical energy transduction at the molecular level. The projects and investigators within the Program are interdependent and closely linked through complementary research goals and shared technologies, resources and training programs. Novel instrumentation to be developed for the research will include laser optical traps, determination of force, translocation distance and inter-domain motions of both single molecules and ordered arrays of contractile proteins, time-resolved fluorescence polarization microscopy, and ultra-rapid freezing electron microscopy synchronized to transient molecular events. These transients will be initiated by laser pulse photolysis of "caged" substrate or signaling molecules and by rapid mechanical perturbations. Novel techniques will include new eukaryotic co-expression systems for protein structure-function studies, transgenic expression of truncated, mutated or chimeric proteins in genetically amenable organisms, bifunctional fluorescence labeling for quantitative protein orientational dynamics and mechanical analysis of single myofibrils. Myosin from specialized tissues, such as smooth and oscillatory muscles, will be studied intensively. The long-term scientific aims and technologies developed in the Program Project are directed to understanding the normal assembly and function as well as pathophysiology of contractile proteins relevant to the cardiovascular and pulmonary systems.

The goals of the work are to develop methods and apply them to understand how ribosomes translate the genetic code into amino acid sequences. Many aspects of ribosomal functions are mysterious, such as how fidelity of translation is achieved and how translocation along a messenger RNA maintains the exact 3-base reading frame. GTP-binding proteins, termed elongation factors (EFs), utilize cellular energy to achieve this performance in an unknown way. The current research will elucidate these and other aspects of ribosomal function by novel real- time single-molecule structural biophysics. Protein translation is a ubiquitous function in cell biology and it serves as a prototypical cellular machine transferring information and transducing energy. The functions of EFs also bear directly on other G-proteins, such the proto- oncogene Ras, the visual transducer, transducin, beta-adrenergic signalling, and ATPase motor proteins, such as myosin and kinesin. These proteins all share a common fold and many functional similarities. Thus this fundamental research has very broad impact in biomedicine.

The research will test crucial functional aspects of mRNA-directed ribosomal protein biosynthesis at the level of individual ribosomes. Two new methodological developments, single molecule fluorescence polarization and force-feedback infrared laser optical trap will be applied, providing information currently unavailable about the mechanism of the ribosomal elongation cycle. Fluorescent probes will be inserted into elongation factors (EFs) and transfer RNA (tRNA) with known, predetermined orientations. The mechanism of proof-reading the genetic code and of translocation along the mRNA will be determined by investigating the kinetics, and angular relationships between, structural changes in EFs and tRNA. Whether EFs are motors or switches will be elucidated. The timing of tRNA motions through the ribosome will be compared to those of structural changes in the EFs. Using the optical trap, the relationship between mechanical tension in mRNA and velocity of peptide elongation (the force-velocity curve) of single ribosomes will be determined. The influence of altering substrate and product concentrations on the force velocity curve will be used in distinguishing hypothetical mechanisms of translocation. Protein synthesis is ubiquitous among living organism and the similarities between all ribosomes indicate that the elongation cycle is one of the most fundamental biological processes. Thus the collaboration proposed in this application between laboratories having expertise in motor protein and ribosome function, in vitro single molecule mechanics, and novel polarization spectroscopy will advance the understanding of ribosomal and G-protein function, and should impact very broadly in biophysics and biomedicine.

DESCRIPTION (provided by applicant): The overall aims of this research are to understand the molecular mechanism by which actomyosin motility systems convert chemical energy into mechanical work, and to obtain a precise correlation between the mechanical, biochemical and structural events of actomyosin motility at the molecular level. Novel methods will be applied to striated muscle fibers and to isolated motor proteins to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. The orientation of fluorescent molecules covalently bound to subunits within the myosin heads will be monitored at high time resolution by novel multi-molecule and single-molecule fluorescence polarization techniques to determine the rates and identity of specific protein structural changes. Newly developed methods of orienting spectroscopic probe molecules relative to the protein, using bifunctional attachment to engineered protein residues will be used to determine the motions of the myosin head with high time and angular resolution. An infrared optical trap will be combined with single-molecule fluorescence polarization by total internal reflection microscopy to directly evaluate the influence of mechanical stress and strain on protein orientation changes that relate to chemomechanical transduction. The experiments will be carried out on single fibers of rabbit psoas muscle and on myosins isolated from muscle, neural tissue and heterologous expression systems. Results from this project should significantly advance knowledge of cell motility processes and thus bring a greater understanding of both normal and pathological states of striated muscle, neuronal development and other types of cell motility.

The University of Pennsylvania's Nano Science and Engineering Center on Molecular Function at the Nano/Bio Interface will exploit Penn's strengths in design of molecular functionality, quantifying behavior of individual molecules, and interactions at organic/inorganic interfaces to perform research that establishes the foundation for understanding molecular function in the context of interfacing with physical systems. The NSEC unites 18 investigators from three schools (the School of Engineering and Applied Science, the School of Medicine, and the School of Arts and Sciences). Two multidisciplinary research teams are focused on aspects of the fundamental issues outlined above. Additionally, two cross cutting initiatives develop ideas integral to the research themes and make explicit links between them. The two fundamental themes are: optoelectronic function in synthetic biomolecules and mechanical motion of molecules from physiological systems. The two cross-cutting initiatives are on Molecular Nano Property Probes and Ethics in Nanotechnology.

The impact of these efforts will be felt in biophysics, bioengineering, chemistry, electrical and mechanical engineering and materials science. Discoveries from this effort will provide a sound basis for the development of new technologies for nanoscale device manufacture, drug delivery and integrated chemical sensors, enabling several near term practical applications as well provide the basis for future practical implementation. Furthermore, these issues are also at the core of understanding many complex biological/physiological processes.

The broad impact of the NSEC will occur on several levels. From a technical perspective it will articulate the critical issues that define the field at the interface of nanotechnology and biology at the molecular level. As such it will focus the attention of many disciplines to an area that is at the core of future of the field. The NSEC will impact public education, social discourse, workforce development and diversity, both locally and nationally. The implementation of educational activities in an urban environment will target a highly diverse audience at the early stage when exposure to exciting science can influence interests and future career choices, while developing models that can be implemented across the country. This NSEC will take a leadership position in the social discussion of ethics in nanoscience and technology.

DESCRIPTION (provided by applicant): Targeted transport of cellular constituents to specific locations is an essential function for normal activity and growth in every eukaryotic cell type. The three motor families, myosins, dyneins and kinesins, cooperate in directing cargoes along microtubules and the actin cytoskeleton. Although multiple motors are required to confer bidirectional motion and to switch between these cytoskeletal tracks, the coordination and competition between them is not understood. Many of the molecular motors are regulated by Ca 2+, phosphorylation, or recruitment to their sites of action by protein scaffolds. The domains that target the motor proteins, the scaffolds that assemble the specific linkages between motors and cargoes, and the chemistry of association between macromolecular motors and lipids are major open questions. Novel biophysical, molecular, and cell biological techniques developed for earlier studies open exciting opportunities for understanding targeted intracellular transport. In this program project, the actin-based motors, myosin I, myosin V and myosin VI, and the microtubule-based motor, cytoplasmic dynein, and its accessory protein complex, dynactin, will be studied intensively by a battery of state-of-the-art approaches. Single-molecule fluorescence polarization, nanometer-resolved fluorophore localization, infrared optical traps, rapid biochemical reaction kinetics, nanosecond time-resolved fluorescence anisotropy, dynamic light scattering, genetic manipulations, and detailed electron and atomic force microscopy will be applied in collaborative studies to understand the individual mechanisms of molecular motors and their mutual interactions. An understanding of the assembly process of every cell, including the muscle sarcomere and cytoskeleton, will require an in-depth study of the function of a number of different motor proteins. As cell proliferation, assembly, protein expression, motility, energy metabolism, defense, nourishment, and secretion all involve such localized motor-driven complexes, the impact of understanding the mechanisms and control of targeted intracellular transport spreads across all of cell biology. Cancer invasion and metastasis, neuronal and muscle development, pathogen attack by host defenses and many other systems make extensive use of the same molecular motors. Thus, detailed understanding of the function and interaction of these proteins have specific and broad-reaching implications in human disease and treatment. We will discover how motor proteins, dynein and unconventional myosins work individually and in brother/sisterhood.

This award is for the development of a new instrument capable of single-molecule fluorescence measurements of angular (polarization), translational, and conformational motions collected during Atomic Force Microscopy (AFM) imaging and manipulation of individual biomolecules and their complexes. AFM is the only technique that can image wet, native samples with nanometer resolution, and it has also emerged as a key technology for manipulation-type studies of single molecules and complexes, including protein and RNA folding-unfolding under tension, and studies of enzyme activity under compressive confinement. Fluorescence microscopy has also progressed over the last decade to single molecule localization considerably beyond the classical diffraction limit, and structural dynamics by intramolecular distance, spatial orientation and mobility. The goal of this award is to design and develop a new instrument that will report the internal structural changes of single macromolecules and surface layers by single molecule fluorescence microscopy, simultaneous with application and detection of relevant mechanical forces and distances by AFM. The long term objective for this new microscope is to increase fundamental understanding of assembly, folding and function of macromolecules and surface layers.

Beyond research discoveries made possible with the new microscope, the broader impacts of the award include training, outreach and communication. Students and post-doctoral fellows will be integrated into the mechanical, optical, electronic and software development. The cross-disciplinary experience of conceiving a new technology, bringing it to practical fruition, and applying it directly to a current scientific problem cannot be obtained in any other way. The new microscope will be in the Penn Nano-Bio Interface Center, which provides community outreach that educates under-represented groups and high school students and teachers.

DESCRIPTION (provided by applicant): Our goal is exploit the power of single molecule observation to elucidate the mechanism by which specific sequences within mRNA modulate the rate of translation by E. coli ribosomes, through use of an approach coupling Total Internal Reflection Fluorescence Microscopy (TIRFM) with Fluorescence Resonance Energy Transfer (FRET). In our approach, fluorescent groups are introduced in the ribosome that, by FRET interaction with fluorescently-labeled tRNAs, allow initial aminoacyl-tRNA binding to the ribosomal A-site, tRNA translocation to the P-site, and release of discharged tRNA from the E-site to be monitored on single ribosomes in real time. This approach will provide a detailed, continuous kinetic profile of mRNA translation during continuous elongation, providing unique insights into the regulation of translation rates in protein synthesis that are important for cell function. We will determine kinetic profiles for expression of i) short model mRNAs containing known pausing elements regulating translation; ii) complete mRNAs coding for full proteins; and iii) designed mutations of such mRNAs that permit rigorous evaluation of the effects of pausing elements, singly or in groups, in the context of full protein synthesis. Such determinations will allow new understanding of the roles such pauses play in biologically important processes and providing suggestions for optimizing cell-free protein synthesis systems. Our specific aims are to: 1. Determine translation profiles for model mRNAs. We will determine translation kinetic profiles for model mRNAs incorporating known pausing elements (rare codons, downstream mRNA 2o structure, upstream nascent peptides) either one at a time or in tandem. The information obtained will quantify effects of such elements on translation and elucidate the mechanism of the intrinsic ribosomal helicase. 2. Determine translation rates for full-length mRNAs. We will determine translation kinetic profiles for full length mRNAs and specifically designed mutants of such mRNAs in order to determine how surrounding context influences the effect of a given pausing element or group of pausing elements on translation rate, beginning with the mRNAs coding for E. coli dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CATIII). 3. Optimize the reagents employed in the TIRFM-FRET approach. The principal improvements over currently available reagents will be directed toward i. reducing background from fluorescently-labeled tRNA by derivatizing EF-Tu with a fluorescence quencher; ii. synthesizing a larger variety of fluorescent tRNAs; and iii. labeling ribosomes with quantum dots for increased stability toward photobleaching. 4. Optimize the apparatus and methods needed for the TIRFM-FRET approach. Several improvements to the apparatus, software and procedures will be accomplished to enable collection of long kinetic sequences from the onset of elongation, with high fidelity and minimum perturbation by photobleaching. The image processing software used to quantify single molecule FRET pairs and their efficiencies will be improved, optimized and statistically validated. PUBLIC HEALTH RELEVANCE: Three major consequences will flow from our work. First, we will be able to examine the effects of known translational pausing elements in the context of complete protein chain expression, and to determine if new elements and synergistic effects can be identified. Second, we will be able to systematically explore the role translational pausing plays in the integration of protein synthesis and cellular function, focusing on such issues as the tradeoff between speed and accuracy in translation at functionally crucial residues, and the possible coupling of translational pausing and co-translational protein folding. Third, we will be able to provide important information with respect to optimizing cell-free protein translation systems.

Core A will provide the overall and day-to-day administration of the Program Project "Molecular Motors in Cell Biology". This includes oversight of the scientific and budgetary aspects of the Program, the provision of administrative services to the Director and Principal Investigators for work directly related to the Program Project, and coordination of the Seminar, Visiting Scientist Programs and the Internal and External Advisory Board Site meetings. Core A will develop and maintain a web site for data sharing within the program and for public asses to data and high resolution images. Core A will administer and facilitate sharing of scientific resources developed within the program.

The structural changes in unconventional myosins are beginning to be elucidated using a combination of single-molecule mechanical and spectroscopic approaches along with ensemble biochemistry and structural biology. The mechanisms of their regulation and interaction with other molecular motors are still largely unknown. We hypothesize that structural changes in calmodulin molecules bound to IQ motifs in the neck of myosin V and I alter the mechanics of the lever arm when Ca2+ ions bind. Techniques we previously devised for investigation of the basic motility properties will be applied to the mechanism of this modulation. Combined optical trap and single molecule fluorescence microscopy will determine the stiffness changes, rotational motions and rotational mobility of the calmodulin subunits and the relationship of these parameters to modulating motility. Cytoplasmic dynein is a molecular motor that uses the free energy of ATP hydrolysis to drive movement of cargo along microtubules. For a wide range of cellular functions, an activator complex, dynactin, which binds both to dynein and to microtubules is necessary. Dynactin has a role in cargo-binding, but also may also have a more active role in the mechano-chemistry of force generation. Dynein, dynactin, unconventional myosins, and kinesins interact by binding to each other, to individual vesicle cargoes and through their mutual interactions in the cytoplasm. By incorporating several molecular motor types onto manipulatable cargoes in vitro, we will seek to understand these interactions. We will develop assays in vitro that implement aspects of cellular complexity, such as intersections between actin filaments and microtubules near to a surface and away from any surfaces. This work begins a 'bottom up'route to understanding the complexities of cellular motility. We will study the molecular mechanisms of these systems using newly developed technologies, combined optical trap and polarized TIRF microscopy, and 3-D single molecule tracking at nanometer accuracy in vitro and in live cells. These studies complement and link strongly to all of the other sections and cores by providing mechanisms that apply to the cell biological and structural studies with simpler in vitro assemblies of purified cytoskeletal and motor components.

Members of the superfamilies of molecular motors, kinesins, dyneins and myosins, are the machines that drive many forms of crucial intracellular transport. The three motor families coordinate their actions with dynamic (and highly regulated) cytoskeletal filaments to control cell growth, define cell shape, deliver and polarize intracellular cargoes, traffic endosomal membranes, and participate in signaling cascades. Many critical cellular processes involve the regulated switching of cargo organelles from one type of cytoskeletal filament to another, but the requisite coordination and competition among multiple motors are not understood. This integrated program project will study the interactions, structure, regulation, and biophysical mechanisms of the molecular motors in growing and functioning cells. The cytoskeletal tracks for intracellular motility, actin and microtubules, the actin-based motors, myosin I, myosin V, and the microtubule-based motors, cytoplasmic dynein and kinesin, will be studied intensively using a battery of state-of-the-art approaches that open exciting research opportunities. Single-molecule fluorescence polarization, nanometer-resolved fluorophore localization, infrared optical traps, rapid biochemical reaction kinetics, genetic manipulations, and novel forms of electron microscopy, correlated with hyper-resolution light microscopy in the same regions, will be applied in collaborative studies to understand the mechanisms of individual molecular motors and their mutual interactions. These approaches yield high temporal and spatial resolution that enables us to dissect mechanisms in assays of increasing molecular complexity that model aspects of the intracellular environment. Particular biological systems, selected for facility of study as well as relevance to broader mechanisms of intracellular motility are endocytosis and vesicle trafficking in neurons and insulin-stimulated fusion of glucose transporter vesicles with the surface membrane in adipocytes. There are close synergies and practical links between all of the sections and cores in this program. We anticipate that the proposed work will take us significantly further toward our goal of understanding motility in the normal and pathological function of cells.

Project Summary Active transport of vesicular cargoes is vital to the targeted delivery of organelles, proteins, and signaling mol- ecules in the complex and crowded cellular environment. Accordingly, defects are linked to developmental, neurodegenerative, pigmentation, immunological, and other diseases. Knowing the detailed mechano- chemistry and structural dynamics of isolated motor proteins is essential for integrating the divergent mechani- cal and kinetic properties of cytoskeletal motor families. We have developed a number of powerful new bio- physical tools that reveal the modulation of mechanical and ATPase reaction kinetics under applied mechani- cal force, elucidate the essential rotational conformational transitions of specific domains within the motor pro- teins and produce reliable force, stepping dynamics, and local viscoelastic parameters of the cellular environ- ment. We will apply these unique tools to investigate the divergent biochemical and mechanical properties of myosin-I (Myo1) and dynein isoforms that have that have not yet been approached at the mechanistic detail now possible. Aim 1: Using simultaneous optical trapping and TIRF microscopy, determine stress- and strain-dependence of the binding and dissociation of ATP and ADP that fuel motion and control motor stepping; Aim 1A: in myosins 1b and 1c; Aim 1B: in budding yeast and mammalian cytoplasmic dynein. Aim 2: To determine the rotational motions of motor heads and lever arms that generate force and cargo translocation using single molecule polarized TIRF (polTIRF) microscopy; 2A: in myosins 1b and 1c; 2B: in yeast cytoplasmic dynein. We discovered that Myo1b and Myo1c have markedly different strain-dependent attachment lifetime, even though their unloaded kinetics and protein sequences are quite similar. We will apply our formidable single-molecule mechanical and fluorescence technologies to understand how these closely related motors have optimized their kinetic and structural variations for their different cellular roles. Mammalian and Saccharomyces cerevisiae cytoplasmic dynein have overlapping roles in their respec- tive cells, but their properties are very different. We are in the unique position to compare them using advanced biophysical methods. We will determine their strain-dependent ATPase dynamics and rotational motions. We anticipate that these studies will provide insight into the motors' mechanisms, and will also reveal biochemical and mechanical adaptations to their distinct functions. Aim 3: We will use our unique new technology for measuring and calibrating optical trap signals within live cells to 3A: determine the functional speciali- zations that vary the complement of motors among early and late endosomes, and small and large la- tex bead compartments (LBCs) endocytosed or phagocytosed into live cells. 3B: measure forces and motor dynamics during remodeling of endoplasmic reticulum (ER) by interaction with endosomal vesi- cles. All three aims represent close, essential collaborations with Drs. Ostap, Holzbaur and Shuman. Many cargos have opposing motors bound simultaneously; these motors may operate in teams functioning either cooperatively or competitively. We will focus on the role of oppositely directed motors at critical junctures in cellular organelle trafficking: early and late endosomes and the remodeling of endoplasmic reticulum we have observed when transport vesicles interact with the ER. Together, these studies will provide insights into the roles of the molecular motor families in regulating organelle motility, morphology and remodeling. These stud- ies will lead to a much improved understanding of myosin I and dynein isoforms that operate very differently from the better understood kinesin and myosin motors, leading to a more rigorous understanding of their func- tions in cell biology and disease.

The Science and Technology Center for Engineering Mechano-Biology (CEMB) brings together leading researchers from a diverse group of disciplines and institutions to investigate, understand, and innovate at the intersection of biology, mechanics, and engineering. The mission of this Center is to discover the governing principles of molecular and cellular communication, provide the intellectual foundations and materials for engineering new and powerful cell-based devices, and to train students in the multilingual foundations of engineering mechano-biology preparing them to be innovative leaders able to explore and exploit these interconnections to impact society. This award supports an innovative network of researchers and educators to investigate the fundamental relationships between cells, their environment, and the forces that act upon them. The team will train a new generation of scientists and engineers in the emerging discipline of Mechano-biology, and will partner with industry to translate new scientific discoveries into products and solutions for the health and prosperity of the nation.

Engineering Mechano-biology, with its focus on the interactions between structure, mechanics, and function, will have a major impact on our ability to construct and repair tissues, organs, and implants; to adapt plants to changing environments; to treat inflammation and fibrosis; to understand the effects of exercise, activity, and trauma; and to engineer optimized synthetic and biomimetic materials. This interdisciplinary field will enable fundamental discoveries in biological function and spur the development of cutting edge technologies for interrogation and guidance of plant and animal structures on multiple scales. Projects will span the length and time scales over which forces operate: from single molecules to supramolecular complexes, cells, tissues and whole organisms, and from milliseconds to hours, weeks or months. Major research efforts will focus on three Integrated Research Thrusts (IRTs) requiring new interfaces across disciplines and organized following a cell's hierarchical perspective from mechano-responsive molecules to signaling pathways to the extracellular niche. Thrust 1: Mechano-biology of Biomolecules and Nanostructures will characterize and engineer proteins and molecules, enabling detection and manipulation of the pN and nm mechano-responsiveness of proteins, scaffolds, and cells to probe or generate increasingly complex engineered mechano-biological reagents, materials, and systems. Thrust 2: Mechano-biology of Cells and Signaling will elucidate how cells dynamically react to mechanical forces through feedback between the cytoskeleton, the nucleus and the surrounding matrix, uncover the ways mechano-signaling contributes to cell-cell communication, and discover how cells distinguish and integrate mechano-signals across length and time scales. Thrust 3: Mechano-biology of Tissues, Materials and Microenvironments will identify matrix-based mechanical cues in plants and animals and investigate the fabrication of bulk gels, fibrous networks, and engineered micro-devices. This will lead to the generation of materials with mechano-responsive properties as well as novel means for studying the structural and biophysical cues in mechano-transduction.

? DESCRIPTION (provided by applicant): Protein synthesis and active transport of vesicular cargoes are vital to development of all tissues and to the targeted delivery of organelles, proteins, and signaling molecules in eukaryotes. Accordingly, defects in protein expression and transport are linked to developmental, neurodegenerative, pigmentation, immunological, and other diseases. Knowing the detailed mechano-chemistry and structural dynamics of the ribosome and motor proteins is essential for understanding and interpreting their roles in the cell. We developed a number of powerful new biophysical tools that reveal the modulation of structural dynamics and reaction kinetics of the protein synthesis elongation cycle and molecular motors under applied mechanical force, discriminate models of energy transduction and elucidate the essential rotational motions of conformational transitions of specific domains within the elongation factors and motor proteins. We will apply these unique tools to investigate the rhythm of protein synthesis in bacteria and eukaryotes and mechanisms that functionally modulate it and the divergent biochemical and mechanical properties of myosins-I, V, VI and X, and dynein isoforms. Understanding functional properties that have that have not yet been approached at the mechanistic detail is now possible. This application combines and extends three NIGMS grants, and so has a number of aims. Our Aims in protein synthesis are to Determine the mechanisms of modulation of protein synthesis rate of 1) downstream mRNA 2o structure and 2) upstream Shine-Delgarno (SD) sequences. Aim 3) is to Develop a eukaryotic single molecule FRET platform for detailed study of peptide elongation in higher organisms. Aim 4) is to Define how read-through of premature stop codons takes place and how small molecule enhancers of read-through operate, which are promising therapies in many diseases, e.g. Duchenne muscular dystrophy and cyctic fibrosis. For molecular motors, for each of the target myosins I, V, VI and X, to 5) test the thermal search hypothesis, determine their flexibility and solve the mechanisms of their high directionality; 6) Determine the mechanical force-dependence of association of motors with their cytoskeletal tracks and the energetics of their mechanical stroke using an ultra-high-speed optical trap; 7) Determine the dynamics of ATP association and ADP and Pi dissociation from the motors under force using combined optical trapping and single molecule TIRF microscopy; 8) Determine the rotational motions of motor heads and lever arms that generate force and cargo translocation using single molecule polarized TIRF microscopy; 9) Determine motor force and force regulation on intracellular cargos, and 10) Determine how many kinesin motors are actively engaged on intracellular cargos and their spatial distribution around the cargo surface. Overall, these studies will lead to a much more general view of the mechanisms and characteristics of the ribosome and molecular motors in vitro and in live cells leading to a more rigorous understanding of their functions in cell biology and disease.

Project Summary An invaluable opportunity has emerged in advanced optical microscopy for obtaining accurate and precise measurements of distance, occupancy and dynamics in biologically important macromolecules. Förster resonance energy transfer (FRET) has been available for some time to measure distances in macromolecular assemblies that are labeled with fluorescent donor-acceptor probe pairs separated by ~2 ? 8 nm. However, artifacts and uncertainties arise in classical ensemble (cuvette) FRET experiments due to non-stoichiometric labeling, contaminants, anomalous photo-physical behavior, unknown rotational mobilities, and averaging over static and dynamic inhomogeneities. Moreover, inhomogeneities may represent normal functional variations or sample degradation. These problems are overcome in the requested instrument by simultaneously recording spectral properties of the probes as individual macromolecules diffuse through a microscopic volume. A single molecule multi-parameter fluorescence detection (smMFD) system with confocal detection (a) dramatically reduces quantitative uncertainties, (b) greatly increases accuracy, (c) is much faster, (d) greatly simplifies sample quantity and preparation requirements, and (e) ensures identical sample conditions because all measurements are made over a short time interval on the same sample. Sample immobilization is not required, which both simplifies sample preparation and eliminates artifacts. Two correlated detectors are required for the donor and two for the acceptor to eliminate detector dead time and after-pulsing. When a polarizing beam splitter projects the emission onto each pair of detectors, rotational anisotropy is also obtained in the same experiment. Therefore, each detection event informs an 8-fold parameter space consisting of anisotropy, lifetime, intensity (stoichiometry), detection time (related to diffusion coefficient), excitation spectrum, emission spectrum, quantum yield, and distance between the two fluorophores. An analysis of these signals enables the identification of individual species present in the population of molecules, and determination of the interconversion rates between these species. The multi-parameter fluorescence detection approach enables us to discriminate among heterogeneous species, correct for labeling stoichiometry, and quantify dynamic motions as the molecule explores its conformational space. Energy transfer efficiency is converted to the distance between the labeling sites by computational analysis of a dynamic structural model with tethered probes. The instrument requested in this proposal will make this powerful technology available to all interested investigators.

Project Summary Hypertrophic cardiomyopathy (HCM) is a prevalent genetic cardiac disease affecting 1 in every 300-500 people. The disease is characterized by left ventricular hypertrophy, cardiomyocyte disarray, and interstitial fibrosis resulting in impaired diastolic function often with preserved or enhances systolic function. Dilated cardiomyopathy (DCM) has a similar occurrence and is characterized by thinning of one or both ventricular walls producing insufficient systolic function and diminished ejection fraction, hallmarks of a failing heart. Genetic mutations in sarcomere proteins have been identified to be associated with HCM and DCM, with mutations in ?- cardiac myosin (MYH7) strongly implicated as drivers of both conditions. A widely cited model relating myosin function to disease proposes that HCM arises from myosin mutations that enhance activity yielding hypercontractile myocytes, whereas DCM arises from loss-of-function mutations that diminish activity yielding hypo-contractility. Contractile abnormalities are proposed to be due to MYH7 gene mutations that affect ATPase activity, velocity, force production, the number or availability of motor domains, and thin filament activation. These molecular changes ultimately affect power output in a manner that impacts tissue architecture, electrophysiological signaling, and cardiac performance. Emerging research has provided examples that do not fit clearly into the prevailing model. For example, HCM mutations with decreased activity have been described in molecular assays and at the level of isolated myofibrils. To delineate the mechanisms by which myosin mutations lead to HCM and DCM, it is important to determine how changes in protein sequence lead to changes in activity at both the molecular and ensemble levels. We selected specific HCM and DCM mutations that are predicted to affect the mechanochemistry of the myosin motor. Our goal is to determine the effect of these mutations on myosin activity, and to test whether the HCM gain-of-function and DCM loss-of-function paradigm holds. We will utilize biochemical and biophysical approaches to assess the effect of mutations on the activity of single motors and regulated filament assemblies. Aim 1 will determine the biochemical and mechanical effects of key HCM and DCM mutations in human ?-cardiac myosin. We will measure the ensemble kinetics and motility using biochemical and gliding assays. Changes in unitary forces, step-size, and force dependent kinetic steps will be measured using single molecule optical-trapping. Aim 2 will determine the effect of HCM/DCM mutations on heterogenous myosin assembles and thin filament activation. We will examine the effect the regulation of single molecules within a regulated system, and we will construct a myosin nanomachine using DNA origami that mimics cardiac muscle. We will use regulated thin filaments and myofilaments containing defined ratios of WT and mutant myosin. We expect that our approach will result in an unprecedented understanding of the underlying etiology of HCM and DCM.