Clara Franzini-Armstrong - US grants

The proposed conference is under the sponsorship of the Gordon Research Conference and is scheduled for July 11-15 1988 at Tilton, New Hampshire. The subject of the conference is excitation-contraction coupling; the last conference on this subject was held in 1985. The timing of the current conference is particular appropriate in view of the recent convergence of biochemical and physiological approaches in the identification of dihydropiridine and ryanodine receptors and their role in e-c coupling. Themes to be developed are: channels in surface and T tubule membranes; relationship between nifedipine receptors, charge movement and Ca release; macromolecules and Ca release channels in triads and SR membranes; time course of Ca release and reuptake, Ca and pH signals; role of second messengers in e-c coupling; time course of calcium and regulatory proteins interaction, molecular structure, regulation and mechanisms of the Ca pump; comparative aspects of e-c coupling, including non- muscle cells. Two special aspects of this conference are 1) a round table discussion of e-c coupling parameters designed to bridge the gap between the mostly structural and physiological data of the last 30 years and the molecular data of the last 5 years; 2) an oral discussion of posters presented by a selected group of young investigators. The conference will be divided into sessions with scheduled speakers, poster sessions and free discussion time. The Gordon Conference's format is conducive to free and informal exchange of ideas and unpublished data and this is particularly valuable at present time in the e-c coupling field because recent progress has opened new avenues of investigation. These meetings have a highly successful history in bringing together active investigators from all over the world in a format which is designed to optimize discussion. No other conference bringing together a wide range of expertise in this area of research is planned in the near future.

DESCRIPTION (adapted from the applicant's description): The overall goal of this proposal is to elucidate the basis for structural organization of the calcium release units (CRUs) of cardiac and skeletal muscle which underlie excitation-contraction coupling. CRUs are comprised of junctional sarcoplasmic reticulum (jSR) which are composed of a luminal calcium-binding protein, calsequestrin (CSQ), and several membrane proteins which include the calcium-release channel (ryanodine receptor; RyRs), and supporting proteins junctin and triadin. The adjacent areas of sarcolemma include L-type calcium channels (dihydropyridine receptors; DHPRs). To determine the basis for the assembly, organization, and maintenance of structural integrity of these units, structure will be assessed by electron microscopy, using thin sections and freeze fracture methods, and protein composition will be assessed using immunofluorescence coupled to confocal microscopy and by Western blotting. Three strategies will be used to assess the effect of altered protein composition on the structure of CRUs. 1) Transgenic mice which overexpress one or more of the CRU proteins will be studied. 2) Muscle specific CRU proteins will be expressed in non-muscle cells in order to determine the proteins necessary for formation of CRUs. 3) The composition of CRUs in skeletal muscle of Amphioxus will be examined using molecular biology and ultrastructural approaches.

A relationship between specific states of the actomyosin cross-bridge cycle and their conformation is not yet established. Using photolysis of caged compounds and rapid freezing we will examine the structure of cross-bridges in various states by electron microscopy and image analysis. In the process of excitation-contraction coupling, depolarization of the surface membrane is translated into a signal for release of calcium from the sarcoplasmic reticulum (SR). The SR calcium release channel has been identified: it is the foot protein, whose cytoplasmic domain spans the gap between the SR surface membrane and/or its invaginations, the transverse (T) tubules. We seek identification of a component of the surface membrane/T tubules (the junctional tetrads) with the L type calcium channel (or dihydropyridine receptor) which is thought to be the voltage sensor of E-C coupling. Using electron microscopy of muscle fibers from the E-C coupling defective mutation muscular dysgenesis, of dysgenic muscle fibers rescued by transfection with the cDNA for the alpha 1 subunit of DHPRs, and of transfected chinese hamster ovary cells, we will establish if a correlation exists between junctional tetrads and DHPRs. Development of the membrane systems in skeletal muscle fibers in vivo proceeds through several well coordinated steps. We will explore whether a relationship exists between specific events in membrane development and the appearance and rearrangement of the intermediate filament system.

This sections uses ultrastructural approaches to test for motions of the myosin head and thus provides the basis for modeling the force bearing transition in the myosin cross bridge activity cycle. Implementation of rapid freezing techniques, in combination with caged compounds has provided the exceptional opportunity for time resolved structural analysis of myosin motors in the context of the intact myofibril. We propose to use rapid freezing in combination with rapid stretch and release protocols to synchronize cross bridges in frog fibers and to correlate their structural states and disposition with tension development. Image analysis will be used to dissect the contribution of various regions of the myosin head to the shape changes during tension development. We will also take advantage of the unexcelled paracrystalline order of insect flight muscle (IFM) in a collaborative exploration of myosin cross bridges, that will eventually lead to 3-D EM tomography and 3-D classification of cross-bridges by correspondence analysis (with Dr. M.K Reedy). Cross bridges will be either trapped in relaxed (MgATP-relaxed), early "pre-force" states (glycol- AMPPNP cold or with Ca2+; ADP-AlF/4 with Ca2+), strong binding states (rigor) or following a rapid stretch of fully activated fibers. A second component of the project explores structure and function of myosin heavy chain isoforms and of the regulatory light chain in the processes of myofibril assembly and contraction in Drosophila. Collaborations within the program project allow a fruitful combination of genetic and transgenic approaches with functional and structural assays. A novel myosin, myosin rod protein or MRP, in which the catalytic and actin binding head region is substituted by an N terminal extension homologous to that of a light chain, offers a unique opportunity for tested whether this extension allows a direct, tethering interaction with actin. The structural effects of genetic and transgenic manipulations that vary the amount of MRP normally expressed in special flight muscle will be assessed by a battery of techniques, from thin sectioning of intact muscles to rotary shadowing and negative staining of isolated filaments, complemented by diffraction analysis. Ultrastructure of myofibrils and thick filaments from the indirect flight muscle (IFM) of transgenic flies will serve as basis for assessing effects of misexpression of various proteins on the stretch activation responses and on the specific myofibril architecture of these muscles. The IFMs will be induced to express myosin heavy chains with alternative exon 11S belonging to other muscles without stretch activation properties, over a null background.

[unreadable] DESCRIPTION (provided by applicant): This project probes the arrangement of proteins and the biogenesis of membranes composing the macromolecular complexes responsible for regulated calcium release in skeletal and cardiac muscle. The basic approach is to establish molecular-structural and structural- functional correlations, under the premise that structure can be best understood by observing its changes following specific molecular perturbations, and that function cannot be fully modeled without knowledge of the underlying structure. Regarding the arrangement of proteins, the key hypotheses to be tested are: 1. calsequestrin's disposition depends on a combination of links to the SR membrane and intermolecular bonding in the presence of bivalent cations; 2. junctophilin 1 and 2 are essential for the docking of SR to surface membranes. In the absence of junctophilin the permanent association between surface and internal membrane systems may not be possible; 3. skeletal type excitation-contraction coupling, based on a molecular link between proteins of two different membrane systems, evolved at the transition between low chordates and vertebrates and it required the evolution of a new type of ryanodine receptor; 4. caveolin-3 is necessary for the development of T tubules. The experimental strategies involve perturbation of the molecular composition and developmental events by selected addition, removal and substitution of key components and perturbation of the functional state by changes in ionic composition. The structural approaches to be used center on techniques of transmission and freeze-fracture electron microscopy, correlated with immunofluorescence and confocal light microscopy. [unreadable] [unreadable]

PROJECT SUMMARY (See instructions): Core D will perform the qualitative and quantitative histological and ultrastructural checks that are necessary to support all other Projects. The Core will not only supply various techniques of electron, phase contrast and confocal microscopy, but it will also operate at a high standard of quality and offer an expert critical evaluation of the results of experimental and molecular alterations. The uniform and consistent use of high quality images will allow the detection of even subtle alterations in protein-protein interactions and in the response of individual cell organelles that either are at the basis of altered functions or are the long term results of such alterations. It is clear that the overall ultrastructural response of the muscle fiber to mutations affecting excitation-contraction coupling are quite specific and offer considerable insight into causative effects. In the past period we have evidenced a strong fiber type- and gender-dependence of the pathology, that correspindes quite well with similar variations in function. Two general approaches are proposed. One is to define any alterations in the relationships between the major protein components of calcium release units (CRUs, triads in skeletal muscle) within the context of the mutation and the other is to follow the development of pathology (most specifically mitochondrial, myofibrillar and CRUs' alterations) through development and aging and in relation to the known functional effects of the mutation on CRUs' channels.This will be achieved by combining light microscope techniques (phase contrast of fibers whole mounts and confocal imaging of fluorescently immunolabeled fibers) with thin sectioning and freeze-fracture for electron microscopy supplemented by quantitative morphometry techniques. The core aims at defining the primary impact of each mutation on the macromelcular assembly of calcium release units within a short term and the secondary impact on SR, mitochondria and contractile material on the long term