Roger Craig - US grants

myofibrils; muscle proteins;

This award will support collaborative research between Professor Roger Craig of the University of Massachusetts Medical School and Professor Raul Padron of the Instituto Venezolano de Investigaciones Cientificas (IVIC) in Caracas, Venezuela. The objective of this research is to study the structural mechanism of contractile cells in muscle and nonmuscle tissue. These cells are able to contract by the sliding of contractile filaments past one another without any change in filament length. The sliding force is generated by the cyclic interaction of myosin crossbridges with actin filaments and is powered by the splitting of ATP during each crossbridge cycle. Using ultrarapid freezing techniques, the investigators are able to arrest the crossbridges at transient points in the cycle and directly examine, via an electron microscope, intermediate crossbridge states. The physiological techniques and apparatus for freezing the muscles, as well as the facilities and expertise for image processing of the micrographs will be done at IVIC while the U.S. side will carry out most of the freezing and the subsequent electron microscopy of the samples. This research should provide insights into the molecular basis of cellular movement and a better understanding defects in structure that occur in diseased states.

The long term goal of this project is to provide new insights into the molecular structure of the contractile filaments of muscle and the structural changes that underlie contraction and its regulation. Specific aims are: (1) to describe the structural changes that occur in myosin crossbridges during their cycle of attachment to actin when a muscle contracts; (2) to define the regulatory structural changes that occur in the contractile filaments when they are activated; (3) to elucidate further the crossbridge arrangement and backbone structure of the myosin filaments of smooth and striated muscles. These questions will be approached using high resolution electron microscopy, combined with image analysis, to elucidate the structures of contractile molecules, filaments and whole muscles in relaxed and activated states. Intact smooth and striated muscle tissue, in the relaxed or contracting state, will be studied by rapid freezing and cryosectioning methods; isolated filaments by negative staining or "frozen hydrated" methodology combined with minimal electron dose methods; and purified muscle proteins by rotary shadowing. The results of this project should broaden our understanding of the structure of healthy muscle and of the molecular processes underlying contraction and its regulation. Such knowledge is essential to an understanding of defects in structure that occur in diseased states.

Funds are requested for the purchase of a high resolution cryo-electron microscope. Research proposals of the users require the ability to observe particulate and sectioned material in the frozen-hydrated state. The microscope will also be available for low dose and conventional microscopy. Users with peer-reviewed NIH funding have been investigating structures at the molecular level using negative staining and shadowing techniques. These approaches have now been largely superseded by observation of unstained specimens in the frozen-hydrated state, which preserves them in an essentially native form. This approach is necessary in order to answer questions without staining, fixation or drying artifacts and because is capable of providing significantly improved resolution in many cases. It is also a powerful technique for observing time resolved structural changes that can be captured by combining rapid freezing with, for example, the release of metabolites from caged precursors. Projects requiring the use of this technique include studies of the molecular structure of muscle filaments, hemoglobin crystals, DNA-protein interactions and coated vesicles. The cryo-electron microscope will be the first at the University of Massachusetts Medical Center. The acquisition of this instrument is essential to provide for the current needs of users and for the initiation of new directions in projects that are now impossible to realize. This technology will provide a natural and powerful complement to the structural techniques that are already in use on this campus, including X-ray crystallography, 3D light microscopic studies of cellular structure, high resolution NMR and conventional and freeze-substitution and freeze fracture electron microscopic studies of subcellular structure.

Smooth muscle is essential to the normal operation of the body, playing crucial roles in vascular, respiratory, digestive, and other functions. Malfunction of smooth muscle is implicated in major diseases such as hypertension, asthma and arteriosclerosis. Smooth muscle contraction is ultimately a result of the sliding of actin filaments past myosin filaments. The long term objective of this project is to elucidate the structural basis of smooth muscle contraction and its regulation at the molecular level. Exciting new insights into smooth muscle function have recently become possible by solution of the crystal structures of several contractile and regulatory proteins and by recent advances in electron microscopy and image processing. We will take advantage of these advances to carry out the following Specific Aims: (1) To define the three-dimensional molecular architecture and composition of native smooth muscle myosin filaments; (2) To determine the molecular basis of the side-polar structure of smooth muscle myosin filaments and the structural significance of SM1 and SM2 myosin isoforms; (3) To determine the three- dimensional molecular structure of the actin filaments in smooth muscle; and (4) To elucidate the three-dimensional organization of the actin and myosin filaments in smooth muscle cells, and the structural basis of their interaction. Electron microscopy will be used to determine the molecular structure of the actin and myosin filaments, and the structural changes that accompany contraction. Muscle filaments will be preserved in close-to their native states using state-of-the-art techniques, and their three- dimensional structures will be computed by helical or tomographic reconstruction. A near-atomic understanding of filament structure and its relation to function will be gained by fitting the atomic structures of actin and the myosin head to the filament structures determined by electron microscopy. Novel structural insights into molecular assembly, organization and function will be obtained using complementary molecular biological and immunological approaches. Both vascular and gastrointestinal smooth muscle model systems will be studied. The results of this project should provide fundamental insights into the molecular mechanism of smooth muscle contraction and regulation, and into structural changes that may occur in diseased states. Preliminary data are presented showing the feasibility of our goals.

DESCRIPTION (provided by applicant): Muscles must be able to relax as well as contract. Contraction and relaxation are regulated by molecular switches on the thick (myosin-containing) and thin (actin-containing) filaments, which together make up the contractile machinery. Defects in regulation can lead to muscle disease. Our long term goal is to understand the structural basis of muscle regulation; our focus in this application is the relaxed state. During the current grant period we achieved two key breakthroughs in defining the molecular structure and regulatory mechanism of myosin filaments, resolving basic questions dating back more than 40 years. We also gained new insights into troponin-tropomyosin regulation of actin filaments. Our insights raise key new mechanistic questions, which we propose to investigate in this renewal. To understand how muscles relax, the structures of the actin and myosin filaments in the relaxed state must be determined. To achieve this, state-of-the-art electron microscopic and 3D image reconstruction techniques will be applied to native filaments and their isolated component molecules. Using these approaches: (1) The molecular interactions underlying the relaxed state of striated and smooth muscle myosin filaments will be defined in detail. (2) The specific amino acid residues and protein domains essential to these relaxed-state interactions will be determined by analyzing the effects of targeted mutations on the structure of expressed single myosin molecules. (3) The structural basis of thin filament relaxation will be defined by analyzing the native, relaxed-state organization of the regulatory components troponin and tropomyosin, and the thin filament template protein, nebulin. In each study, reconstructions will be fitted to atomic resolution crystal structures of thick and thin filament subunits, defining molecular contacts at near-atomic resolution. Our studies of myosin molecules and filaments will provide new insights into general mechanisms of thick filament regulation, and especially into the nature of the relaxed state in vertebrate striated muscle. Further advances in our studies of troponin-tropomyosin regulated thin filaments will provide new information on native thin filament structure, the 3D organization of troponin and tropomyosin, and the structural contribution of nebulin to the relaxed state. Preliminary data demonstrate the feasibility of our aims. The new insights arising from these proposals will provide a deeper understanding of the structural basis of muscle relaxation. Defining molecular mechanisms in healthy muscle is essential to understanding structural defects that underlie muscle disorders.

DESCRIPTION (provided by applicant): This application is a request for funds to provide CCD digital imaging capability for 11 major and 5 minor users of TEMs in the Core Electron Microscopy Facility of the University of Massachusetts Medical School (UMMS). Ultrastructural projects range from the tissue to the molecular level, and include low dose, tomographic, cryo-EM, serial section, immunolabeling, and morphological studies of a wide range of biomedically important systems. The major benefit of direct digital image acquisition is the instantaneous feedback that it provides, in the form of immediate images and instant knowledge of imaging parameters and specimen characteristics. These benefits make a critical difference to specialist applications, such as cryo-EM and tomography, where they can save hundreds of hours of work. In some cases studies become possible for the first time using CCD technology. The benefits of CCD imaging are also key to enhancing productivity in more routine applications. Digital acquisition not only adds convenience and saves time (by eliminating the film processing, printing, and scanning necessary for analysis and publication of images), but also results in major cost savings. In the past year, UMMS researchers spent approximately $40,000 in supplies and personnel costs for developing, printing and scanning the approximately 10,000 micrographs that were taken, all on film. Digital imaging would have eliminated almost all of these costs. Finally, digital imaging is important in teaching, facilitating instruction of novice microscopists and providing stimulating images for visiting medical, graduate and high school students. To provide for the needs of both specialist and routine TEM users alike, we are requesting funds to acquire digital imaging capability for two microscopes. These microscopes perform complementary tasks: one is reserved specifically for specialist applications, including low dose microscopy, cryo-EM and tomography, while the other is used for routine microscopy. This division of labor avoids any compromise to the functioning of the specialist microscope, where optimal optical, vacuum and mechanical performance are crucial. Equipping both microscopes with slow scan CCD cameras will extend the advantages of this technology to all researchers who require it. A third TEM will continue to operate with film for researchers who prefer this medium.

[unreadable] DESCRIPTION (provided by applicant): We request funds for the purchase of an FEI Quanta 200 field emission scanning electron microscope (FESEM) together with a Gatan Model 681 ion beam sputter coater. The microscope and sputter coater will provide for the scanning electron microscopy needs of nine major and three minor users at the University of Massachusetts Medical School. The microscope will be housed in the Core Electron Microscopy Facility of the Medical School, and will replace the only other instrument available - an obsolete 30-year old SEM that is out of service much of the time, lacks key capabilities, and is proving impossible to repair due to its age and the unavailability of spare parts. The ion beam sputter coater will replace a conventional 15-year old sputter coater and is required to provide the fine grain coatings needed for high resolution studies. The projects that will use SEM include studies of the functioning of cell surface receptors involved in immune recognition, the development and function of cilia, the structure of normal and diseased bone, structural changes in mutants of signaling and other proteins in mice and Drosophila, and cell surface structures involved in bacterial infection. These projects require FESEM for two main purposes - the imaging of cell surface structure at high resolution, and the detection and localization of specific cell surface antigens by immuno-FESEM. These images will provide essential insights into the mechanisms of cell development, function, infection, and disease. In addition to the users on this grant, there are approximately twenty other investigators at the medical school who would use SEM if we had a functioning instrument. A state-of-the-art SEM, with field emission gun, has become essential to meet this need. It will transform the ability of investigators to carry out their SEM-related studies, it will greatly improve the quality and speed of data collection, and it will make possible observations that are impossible on our current instrument. All of the projects that will use this microscope aim to provide fundamental insights into cellular structure and function. Many have a direct bearing on the understanding of human disease. Results provided by this instrument will play a key role in advancing our knowledge in these areas. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We request funds for the purchase of an FEI Tecnai G2 Spirit BioTwin transmission electron microscope (TEM) with embedded CCD camera. This will serve the biomedical research needs of eleven NIH-funded investigators at the University of Massachusetts Medical School (UMMS) and Boston University School of Medicine. The microscope will be housed in the Core EM Facility of UMMS, and maintained and monitored by the Facility Manager, ensuring efficient use by investigators. It will replace a 23-year old, second-hand Philips CM12, which is based on outdated technology, lacks key capabilities required by users, and for which maintenance is likely to be discontinued or become difficult in the near future. The new instrument would immediately solve these problems. The projects that will use the Tecnai include fundamental studies of autophagic and apoptotic cell death, mechanisms of synapse formation, the regulation of contraction in smooth and striated muscle, the role of MAP kinase signaling pathways in neurodegeneration, the function of centrosomes in normal and diseased cells, the regulation of G-protein-coupled receptors, the structure and function of spliceosomes, and the structure and function of cilia and flagella in normal and diseased states. These projects require TEM as an essential tool for investigating cellular and molecular architecture at high resolution and as a means of detecting and localizing specific cell molecules by immuno-gold labeling. The images obtained will provide essential insights into mechanisms of cell development, function, infection and disease. In addition to the users in this application, there are numerous other investigators at UMMS who use TEM on a more occasional basis and who would also benefit from modernization of our TEM technology. The instrument proposed represents the state-of-the-art for a conventional TEM, and has many features that would greatly enhance TEM productivity at UMMS. These include a modern, user-friendly computer interface, motorized stage controls with 180o tilt capability, the ability to store and recall grid locations and alignment and imaging parameters (especially useful in several studies requiring serial sectioning), and full integration of an embedded CCD camera, making it possible to record images rapidly and with full documentation. All of the projects that will use the microscope aim to provide fundamental insights into cellular structure and function. Most have a direct bearing on the understanding of human disease. Access to this state-of-the-art instrument will play a key role in advancing our knowledge in these biomedically important areas.

? DESCRIPTION (provided by applicant): Myosin-binding protein C (MyBP-C) is a thick (myosin) filament component of vertebrate striated muscle that plays a key role in modulating contraction. Three distinct isoforms are encoded by different genes, resulting in the expression of fast and slow skeletal muscle MyBP-C isoforms and a third (cardiac) isoform. Since its discovery in skeletal muscle 40 years ago, most studies of MyBP-C have focused on the cardiac isoform, because mutations in this isoform are a prime cause of inherited cardiomyopathies. However, the recent discovery that mutations in slow skeletal MyBP-C cause skeletal muscle myopathies, one of which is neonatally lethal, makes it clear that defining the molecular structure and function of the skeletal MyBP-C isoforms is critically important. Therefore, in this dual-PI proposal, PIs Craig (UMMS) and Warshaw (UVM), in collaboration with Drs. Irving (Illinois) and Sadayappan (Loyola), will combine their labs' expertise in high resolution imaging and single molecule biophysics coupled with X-ray diffraction, molecular biology and mass spectrometry to elucidate the molecular structure and function of skeletal MyBP-C. In Aim 1, in situ and in vitro model systems will help determine if MyBP-C activates and/or mechanically modulates the calcium- dependent sliding of native thin (actin) filaments over native thick filaments from fast and slow rat skeletal fibers and whether contractile modulation occurs only where MyBP-C exists in the thick filament. In Aim 2, through a novel super-resolution light microscopic technique, we will determine whether the MyBP-C N terminus functions by binding to actin and/or myosin. In complementary experiments, fiber X-ray analysis and EM 3D reconstruction of native thin and thick filaments will determine if MyBP-C displaces tropomyosin to activate the thin filament and/or directly influences myosin head interactions to modulate head function. In Aim 3, the structural and functional consequences of MyBP-C N-terminal domain isoform differences between fast and slow MyBP-C will be characterized with special emphasis on 2 slow MyBP-C splice variants thought to affect actin and myosin binding. Through structural mutagenesis, N-terminal fragments will be expressed with domain deletions and slow MyBP-C splice inserts in an effort to define the domains and inserts that confer MyBP-C's modulation of actomyosin function. Although skeletal MyBP-C's clinical impact is apparent, its functional role is far from certain and thus this dual-PI proposal, tightly integrating MyBP-C structure and function, offers an opportunity to rapidly advance our understanding of both fast and slow skeletal MyBP-C isoforms in their normal state.

The interacting-heads motif (IHM) is a configuration of myosin heads in relaxed thick filaments of muscle, in which ATP turnover and actin binding are inhibited by the interaction of each head with the other. In a dramatic development in the field, this motif has become recognized as a fundamental feature of normal muscle relaxation and contraction, through its regulation of thick filament activity. In addition to its role in thick filaments, the IHM also underlies the structure of single molecules of myosin II in almost all types of animal cell. In this monomeric form, the myosin tail folds up, forming a compact molecule, in which ATPase activity is again inhibited by similar head-head interactions. The IHM appears to play two key roles in the body. In thick filaments, it contributes to energy conservation in the relaxed state of muscle. As a monomer, it functions as a storage form of myosin whose compact form facilitates transport to its site of filament assembly. These critical new findings are the motivation for this application: our goal is to elucidate the structure of this fundamental regulatory motif in skeletal muscle thick filaments and single myosin molecules, thus illuminating how it functions. We will do this using state-of-the-art cryo-EM and 3D reconstruction techniques, studying selected model systems and integrating the information gained from each. In Aim 1 we will determine the 3D structure of the IHM in native thick filaments using novel cryo-EM technology that is currently revolutionizing structural biology. Using tarantula skeletal muscle filaments, the most stable species known, we will determine at better than 10 Å resolution the interactions between the two heads, and between the heads and the tail, that create the IHM. With help from the insights gained, we will determine the structure of frog skeletal thick filaments, the most stable vertebrate filament. And we will build on this information to determine the structure of the IHM in (less stable) mammalian thick filaments. In Aim 2, we will determine the 3D structure of the IHM in isolated myosin molecules, using three complementary systems: smooth muscle myosin as the most stable single molecule, which will provide the highest resolution; tarantula myosin as a direct link to the filament structure in Aim 1, aiding its interpretation; and mammalian myosin, which will reveal the structure in vertebrate skeletal muscle. We will also examine molecules in which putative head interaction sites have been mutated, to test their importance in formation of the IHM. In Aim 3, we will use single molecule EM to test the hypothesis that disease mutations in the head region of skeletal myosin impact the stability of the IHM. The IHM is now recognized as a fundamental motif of normal muscle function. Our proposal will elucidate its interactions, providing new insights into the structural basis of contraction and relaxation, and of the potential impact of disease mutations on these functions.