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Hypertrophic cardiomyopathies (HCMs) are the leading cause of acute cardiac failure in young individuals. Over 300 mutations throughout β-cardiac myosin, including in the motor domain, are associated with HCM. A β-cardiac myosin motor mutation (R712L) leads to a severe form of HCM. Actin-gliding motility of R712L-myosin is inhibited, despite near normal ATPase kinetics. By optical trapping, the working stroke of R712L-myosin was decreased 4-fold, but actin-attachment durations were normal. A prevalent hypothesis that HCM mutants are hypercontractile is thus not universal. R712 is adjacent to the binding site of the heart failure drug omecamtiv mecarbil (OM). OM suppresses the working stroke of normal β-cardiac myosin, but remarkably, OM rescues the R712L-myosin working stroke. Using a flow chamber to interrogate a single molecule during buffer exchange, we found OM rescue to be reversible. Thus, the R712L mutation uncouples lever arm rotation from ATPase activity and this inhibition is rescued by OM.

Hypertrophic cardiomyopathies (HCMs) are the leading cause of acute cardiac failure in young individuals. Over 300 mutations throughout β-cardiac myosin, including in the motor domain, are associated with HCM. A β-cardiac myosin motor mutation (R712L) leads to a severe form of HCM. Actin-gliding motility of R712L-myosin is inhibited, despite near-normal ATPase kinetics. By optical trapping, the working stroke of R712L-myosin was decreased 4-fold, but actin-attachment durations were normal. A prevalent hypothesis that HCM mutants are hypercontractile is thus not universal. R712 is adjacent to the binding site of the heart failure drug omecamtiv mecarbil (OM). OM suppresses the working stroke of normal β-cardiac myosin, but remarkably, OM rescues the R712L-myosin working stroke. Using a flow chamber to interrogate a single molecule during buffer exchange, we found OM rescue to be reversible. Thus, the R712L mutation uncouples lever arm rotation from ATPase activity and this inhibition is rescued by OM.

Key steps of cardiac mechanochemistry, including the force-generating working stroke and the release of phosphate (Pi), occur rapidly after myosin-actin attachment. An ultra-high-speed optical trap enabled direct observation of the timing and amplitude of the working stroke, which can occur within i which, along with other findings, indicates the stroke precedes phosphate release. After Pi release, Pi can rebind enabling reversal of the working stroke. Detecting these rapid events under physiological loads provides definitive indication of the dynamics by which actomyosin converts biochemical energy into mechanical work.

Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin’s working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

Myosin folding and assembly in striated muscle are mediated by the general chaperones Hsc70 and Hsp90 and involve a myosin-specific co-chaperone related to the Caenorhabditis elegans gene unc-45. Two unc-45 genes are found in vertebrates, a general cell isoform, unc45a, and a striated muscle-specific isoform, unc45b. We have investigated the role of both isoforms of mouse Unc45 in myosin folding using an in vitro synthesis and folding assay. A smooth muscle myosin motor domain (MD) fused to green fluorescent protein (GFP) (MD::GFP) was used as substrate, and folding was measured by native gel electrophoresis and functional assays. In the absence of Unc45, the MD::GFP chimera folds poorly. Addition of either Unc45a or Unc45b dramatically enhances the folding in a reaction that is dependent on Hsp90 ATPase activity. Unc45a is more effective than Unc45b with a higher apparent affinity and greater extent of folding. The Unc45-Hsp90 chaperone complex acts late in the folding pathway and promotes motor domain maturation after release from the ribosome. Unc45a behaves kinetically as an activator of the folding reaction by stimulating the rate of the Hsp90-dependent folding by >20-fold with an apparent Kact of 33 nm. This analysis of vertebrate Unc45 isoforms clearly demonstrates a direct role for Unc45 in Hsp90-mediated myosin motor domain folding and highlights major differences between the isoforms in substrate specificity and mechanism.

Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin., Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin in clinical trials for treatment of systolic heart failure. Here the authors determine the crystal structure of an OM-bound human β-cardiac myosin motor domain to provide molecular level insight into the mechanism of drug action.

We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector of cardiac muscle myosin, on the kinetic and "in vitro" motility properties of the porcine ventricular heavy meromyosin (PV-HMM). Omecamtiv Mecarbil increases the equilibrium constant of the hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as determined by quench flow, but the maximal rates of both the hydrolysis step and tryptophan fluorescence increase are unchanged by the drug. OM also increases the amplitude of the fast phase of phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production in muscle by 4-fold. These results suggest a mechanism in which hydrolysis of M-ATP to M-ADP-Pi occurs both before and after the recovery stroke, but rapid acceleration of phosphate dissociation by actin occurs only on post-recovery stroke A-M-ADP-Pi. One of the more dramatic effects of OM on PV-HMM is a 14-fold decrease in the unloaded shortening velocity measured by the in vitro motility assay. The increase in flux through phosphate dissociation and the unchanged rate of ADP dissociation (AM-ADP → AM + ADP) by the drug produce a higher duty ratio motor in which a larger fraction of myosin heads are strongly bound to actin filaments. The increased internal load produced by a larger fraction of strongly attached crossbridges explains the reduced rate of in vitro motility velocity in the presence of OM and predicts that the drug will produce slower and stronger contraction of cardiac muscle.

We have investigated the functional impact on cardiac myofibril organization and myosin motor activity of point mutations associated with familial hypertrophic cardiomyopathies (FHC). Embryonic chicken cardiomyocytes were transfected with vectors encoding green fluorescent protein (GFP) fused to a striated muscle myosin heavy chain (GFP-myosin). Within 24 hours of transfection, the GFP-myosin is found co-assembled with the endogenous myosin in striated myofibrils. The wild-type GFP-myosin had no effect on the organization of the contractile cytoskeleton of the cardiomyocytes. However, expression of myosin with the R403Q FHC mutation resulted in a small but significant decrease in myofibril organization, and the R453C and G584R mutations caused a more dramatic increase in myofibril disarray. The embryonic cardiomyocytes beat spontaneously in culture and this was not affected by expression of the wild-type or mutant GFP-myosin. For the biochemical analysis of myosin motor activity, replication defective adenovirus was used to express the wild-type and mutant GFP-myosin in C2C12 myotubes. The R403Q mutation enhanced actin filament velocity but had no effect on the myosin duty ratio. The R453C and G584R mutations impaired actin filament movement and both increased the duty ratio. The effects of these mutations on myosin motor activity correlate with changes in myofibril organization of live cardiomyocytes. Thus, mutations associated with hypertrophic cardiomyopathies that alter myosin motor activity can also impair myofibril organization.

Most studies of regulation of vertebrate striated muscle contraction have focussed on activation of the thin filament via calcium binding to the troponin-tropomyosin complex. There is accumulating evidence, however, that thick filament activation is an additional regulatory mechanism. Under relaxing conditions, the two heads of a myosin can interact to form an inactive asymmetric off-state, called the interacting-heads motif (IHM). The off-state inhibits cross-bridge formation and cycling by blocking actin binding in one head and ATPase activity in the other. Docking of the IHM onto the thick filament backbone results in the super-relaxed state (SRX) observed in muscle fibres, where the myosin ATPase is strongly inhibited. What regulates formation and disruption of the IHM is largely uncharacterised. Myosin binding protein C, also known as C-protein, is a key sarcomere component, bound at sites spaced by 43 nm in the cross-bridge regions of thick filaments. The arrangement, mode of action, and interactions of C-protein with myosin are poorly understood. In the heart, phosphorylation of C-protein increases the rate of contraction and its ablation disrupts the SRX. To investigate the interaction of human cardiac C-protein with β-cardiac heavy meromyosin (HMM), we have imaged the complex by electron microscopy and single particle averaging. Cardiac HMM in the IHM motif has the characteristic blocked and inhibited head conformations. The fraction of HMM in the IHM motif is increased when dephosphorylated C-protein is bound, suggesting it stabilises the IHM. Characterization of the complex should provide insight into the regulatory and structural roles of the cardiac C-protein/myosin interaction in heart.

Cytoplasmic dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly [1]. To study the dynamic behavior of cytoplasmic dynein in the filamentous fungus Aspergillus nidulans , we replaced the gene for the cytoplasmic dynein heavy chain, nudA, with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP–nudA . The GFP–NUDA fusion protein is fully functional in vivo : strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy showed GFP–NUDA to be in comet-like structures that moved in the hyphae toward the growing tip. Retrograde movement of some GFP–NUDA comets after they arrived at the tip was also observed. These dynamics of GFP–NUDA were not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP–NUDA tip-ward movement is similar to the rate of cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP–NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP–NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex.

The transition of laminin from a monomeric to a polymerized state is thought to be a crucial step in the development of basement membranes and in the case of skeletal muscle, mutations in laminin can result in severe muscular dystrophies with basement membrane defects. We have evaluated laminin polymer and receptor interactions to determine the requirements for laminin assembly on a cell surface and investigated what cellular responses might be mediated by this transition. We found that on muscle cell surfaces, laminins preferentially polymerize while bound to receptors that included dystroglycan and α7β1 integrin. These receptor interactions are mediated through laminin COOH-terminal domains that are spatially and functionally distinct from NH2-terminal polymer binding sites. This receptor-facilitated self-assembly drives rearrangement of laminin into a cell-associated polygonal network, a process that also requires actin reorganization and tyrosine phosphorylation. As a result, dystroglycan and integrin redistribute into a reciprocal network as do cortical cytoskeleton components vinculin and dystrophin. Cytoskeletal and receptor reorganization is dependent on laminin polymerization and fails in response to receptor occupancy alone (nonpolymerizing laminin). Preferential polymerization of laminin on cell surfaces, and the resulting induction of cortical architecture, is a cooperative process requiring laminin– receptor ligation, receptor-facilitated self-assembly, actin reorganization, and signaling events.

Tissue architecture, stability, and mechanical attributes are all determined by the structure and organization of collagen fibrils. Therefore, the characterization of fibril growth steps and determination of how this growth is regulated is essential to the elucidation of how tissues are assembled. We have proposed that fibril segments are intermediates in the formation of mature fibrils. The purpose of this study was to determine the length and structure of fibrils within a relatively mature tendon. The in situ determination of length performed here was only the second direct determination of fibril length in a vertebrate connective tissue and the first for a relatively mature tissue. The data demonstrate that the fibrils were discontinuous at 18 days of tendon development. However, both ends were not present in any of the analyzed fibrils within the 18-day tendon. Because the data set was 50-60 microm, this indicates a mean fibril length greater than 60 microm. These data are in contrast to data from the 14-day tendon, in which 80% of the fibrils had both ends in a 26-microm data set and the mean segment length was shown to be 10-30 microm. There were equal numbers of alpha and beta ends in the 18-day tendon. The structure of the ends was comparable to that in the less mature tendon. The data also indicate that fibril asymmetry and structure were maintained. The increase in fibril length is interpreted as being the result of a post-depositional, regulated assembly of segments via a lateral association/fusion to form mature fibrils. This hypothesis predicts an increase in diameter at this stage of development. The diameter increases have been documented, but this is the first demonstration of increases in length and maintenance of segment structure during this important stage of tendon development.

Omecamtiv mecarbil (OM), a small-molecule increases cardiac contractility by directly activating cardiac myosin by accelerating phosphate (Pi) release (Malik et al., 2011). Here we analyzed the affect of OM on the mechanochemical cycle and motility of human β-cardiac heavymeromyosin expressed in a adenoviral/C2C12 muscle cell based system, and purified porcine ventricular myosin (PV-HMM). Double-mixing stopped-flow fluorescence and phosphate binding protein (MDCC-PBP) were used to determine the maximum rate (kmax) of phosphate release. Rates of 30s−1 and 35s−1 were obtained for human and porcine cardiac HMM respectively. Addition of 20 µM OM resulted in a 2-fold increase in the maximal rate of actin activated Pi release to kmax = 70 s−1 (AM-ADP-Pi _> AM-ADP + Pi) for both human and porcine cardiac HMM. The unloaded actin filament velocity measured in the in vitro motility assay was 0.76 ± 0.37 µm/sec for human β-cardiac HMM and 0.97 ± 0.23 µm/sec for PV-HMM. OM has two effects on the motor activity: 1) The actin filament velocity was significantly slower (∼ 16 -fold at saturation); 2) the filament motion became more persistent with long periods of uninterrupted movement. This suggests that OM recruits more crossbridges to participate in the movement of the actin filaments resulting in higher forces per filament, which overcomes the pinning defects that invariably trap actin filaments in normal motility assays. The increase in attached crossbridges/filament also slows the filament velocity. The motility data are consistent with the kinetic analysis indicating that OM accelerates Pi release without dramatically enhancing the rate of the ATPase hydrolysis. This produces a higher duty ratio in which the motor spends more of the ATPase cycle in tight binding states. Supported by AHA-BGIA to EF and AHA-GI to DAW.

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the “open” (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca 2+ is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.

We designed a system to examine the structural properties of human β-cardiac myosin using fluorescence spectroscopy. Expression of human β-cardiac myosin subfragment 1 (M2B-S1) was achieved through the generation of recombinant adenovirus with subsequent infection in the C2C12 embryonic muscle cell line. A tetracysteine site, which enables site-specific labeling of the fluorescein biarscenical hairpin-binding dye (FlAsH), was introduced in the upper 50 kDa (U50) region (residues 316-321) corresponding to the previously utilized site in myosin V. Key conformational changes in the myosin motor, including those associated with nucleotide and actin binding, can be examined by fluorescence resonance energy transfer (FRET) using M2B-S1 FlAsH and fluorescent nucleotides or IAEDANs-labeled actin. We demonstrate the enzymatic properties of M2B-S1 FlAsH are similar to unlabeled M2B-S1, and both are capable of moving actin filaments in the in vitro motility assay. We utilized FRET between M2B-S1 FlAsH and Cy3ATP to examine structural changes in the U50 domain. The kinetics of structural changes were examined during the ATP-binding and actin-activated product release steps. We also designed a system to study structural changes in the force-generating lever arm using FlAsH labeling at the N-terminus of a human β-cardiac myosin construct containing a C-terminal GFP (M2B-GFP). The spectroscopic approach can be used as a tool to study the effects of hypertrophic cardiomyopathy (HCM) mutations on myosin structure or to study the impact of drugs that alter myosin motor activity.

Image analysis of electron micrographs of thin-sectioned myosin subfragment-1 (S1) crystals has been used to determine the structure of the myosin head at approximately 25-A resolution. Previous work established that the unit cell of type I crystals of myosin S1 contains eight molecules arranged with orthorhombic space group symmetry P212121 and provided preliminary information on the size and shape of the myosin head (Winkelmann, D. A., H. Mekeel, and I. Rayment. 1985. J. Mol. Biol. 181:487-501). We have applied a systematic method of data collection by electron microscopy to reconstruct the three-dimensional (3D) structure of the S1 crystal lattice. Electron micrographs of thin sections were recorded at angles of up to 50 degrees by tilting the sections about the two orthogonal unit cell axes in sections cut perpendicular to the three major crystallographic axes. The data from six separate tilt series were merged to form a complete data set for 3D reconstruction. This approach has yielded an electron density map of the unit cell of the S1 crystals of sufficient detail. to delineate the molecular envelope of the myosin head. Myosin S1 has a tadpole-shaped molecular envelope that is very similar in appearance to the pear-shaped myosin heads observed by electron microscopy of rotary-shadowed and negatively stained myosin. The molecule is divided into essentially three morphological domains: a large domain on one end of the molecule corresponding to approximately 60% of the total molecular volume, a smaller central domain of approximately 30% of the volume that is separated from the larger domain by a cleft on one side of the molecule, and the smallest domain corresponding to a thin tail-like region containing approximately 10% of the volume. This molecular organization supports models of force generation by myosin which invoke conformational mobility at interdomain junctions within the head.

De novo folding and assembly of striated muscle myosin was analyzed by expressing a GFP-tagged embryonic myosin heavy chain (GFP-myosin) in post-mitotic C2C12 myocytes using replication defective adenoviruses. In the early stages of muscle differentiation, the GFP-myosin accumulates in bright globular foci and short filamentous structures that are later replaced by brightly fluorescent myofibrils. Time-lapse microscopy shows that the intermediates are dynamic and are present in elongating and fusing myocytes and in multinucleated myotubes. Immunostaining reveals the co-localization of the molecular chaperones Hsc70 and Hsp90 with the GFP-myosin in the intermediates, but not in the mature myofibrils. Uninfected cells have similar intermediates suggesting a common pathway for myosin maturation. Two conformation-sensitive antibodies that bind the unfolded motor domain and the coiled-coil conformation of the rod demonstrate that in the intermediates, the myosin rod is folded but the motor domain is not folded. Electron microscopy reveals that the intermediates contain loose filament bundles surrounded by a protein rich matrix. Geldanamycin, a specific inhibitor of Hsp90, reversibly blocks myofibril assembly and triggers accumulation of myosin folding intermediates. We conclude that multimeric complexes of nascent myosin filaments associated with Hsc70 and Hsp90 are intermediates in the folding and assembly pathway of muscle myosin.

The folding pathway of the heavy meromyosin subfragment (HMM) of a skeletal muscle myosin has been investigated by in vitro synthesis of the myosin heavy and light chains in a coupled transcription and translation assay. Analysis of the nascent translation products for folding intermediates has identified a major intermediate that contains all three myosin subunits in a complex with the eukaryotic cytosolic chaperonin. Partially folded HMM is released from this complex in an ATP-dependent manner. However, biochemical and functional assays reveal incomplete folding of the myosin motor domain. Dimerization of myosin heavy chains and association of heavy and light chains are accomplished early in the folding pathway. To test for other factors necessary for the complete folding of myosin, a cytoplasmic extract was prepared from myotubes produced by a mouse myogenic cell line. This extract dramatically enhanced the folding of HMM, suggesting a role for muscle-specific factors in the folding pathway. We conclude that the molecular assembly of myosin is mediated by the eukaryotic cytosolic chaperonin with folding of the motor domain as the slow step in the pathway.

Most observers agree that, barring catastrophe, the global population will number more than 8 billion by 2025. There is, however, less agreement about the consequences for food security. Some, for example, Brown and Kane of the World Watch Institute (Brown and Kane, 1994), argue that there will be widespread shortages of foodstuffs, accompanied by higher global prices. Others, like Alexandratos of the Food and Agriculture Organization of the United Nations (FAO) (Alexandratos, 1995), claim that production will match rising demands and that prices will continue to decline in real terms. All agree, however, that the world’s poor, especially those in the poorest countries, will be without adequate food. This chapter links food security with poverty and argues that increased productivity in agriculture is the most effective way for the poor to achieve food security. What is food security? The International Food Policy Research Institute, IFPRI (IFPRI, 1995) defines food security as “economic and physical access at all times to the food required for a healthy and productive life.” Food security has two dimensions: the availability of food and access to food. Availability depends on production, and, while the debate about global availability continues, the view in this chapter, like that of the FAO, is that adequate food will be available globally at real prices roughly comparable to those prevailing in the 1990s. (See Dyson, 1996, p. 167, for perhaps the latest systematic study holding this view.) To achieve food adequacy, the major assumptions here are that research will continue to turn out the elements of improved technologies and that Eastern Europe, especially Russia and Ukraine, will move closer to its potential as a food supplier. Lack of access to food because of poverty, however, is a major challenge. Some people in the developed world and in higher-income developing countries do not have access to food, but policy changes could resolve that problem; certainly, such societies have the resources. This is not the case for the poor in the world’s poorest countries; for them, the combination of individual and societal poverty severely limits access to adequate foodstuffs.

A network-based prototype has been developed to automate the process of generating and maintaining distributed databases of medical images and clinical reports, and for conducting interactive consultation among disparate clinical and research sites irrespective of the architecture of interacting computers. Pathologists routinely interpret gross and microscopic specimens to tender diagnoses and to engage in a broad spectrum of research. This assessment process leads to clinical decisions often limited by time constraints and by the availability of local expertise. Consultation with peers at other institutions is typically achieved by direct transfer of slides rather than images. A network of heterogeneous computer platforms, graphical user interfaces, and operating systems was established to test the performance of the software. Clinical diagnoses rendered by pathologists using the prototypical system and software were consistent with those reported two years earlier using conventional light microscopy in more than 97% of the cases studied.

In a commonly used motility assay, cytoskeletal filaments are observed as they glide over a surface coated with motor proteins. Defects in the motion frequently interrupt the flow of filaments. Examination of one such defect, in which a filament adopts a spiral form and rotates about a fixed point, provides a simple measure of the force exerted by the motor proteins. We demonstrate the universality of this approach by estimating the elementary forces of both myosin and kinesin.

We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4–5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement.

Tropomyosins (Tm) are α-helical coiled-coil proteins that associate end-to-end along the length of actin filaments in muscle and non-muscle eukaryotic cells. Conserved functions include binding and stabilization of actin filaments and cooperative regulation of actomyosin. From a phylogenetic analysis we identified the most conserved Tm residues and tested the hypothesis that residues important for actin-related functions should be conserved (Barua et al., 2011). We introduced Ala mutations at conserved b, c, f surface coiled-coil positions of rat striated αTm. The mutations were grouped according to their positions in the first or second half of periods 2-6 in a 7-period model of Tm's sequence. The E. coli -expressed Tms had an N-terminal Ala-Ser to increase actin affinity. We previously reported that most mutations in the first half of periods 2-6 reduce actin affinity greater than 4-fold. Since mutations in the second halves had a less than 2-fold effect, we postulated the residues may be important for another conserved function, such as myosin regulation. Here we report results of in vitro motility assays to study the effects of mutations in the second halves of periods 2-6 on the cooperative regulation of actomyosin. At surface myosin concentrations that allow maximal velocity of naked actin filaments, the velocity of actin-Tm (A-Tm) filaments was ∼40-50% lower. Addition of N-ethylmaleimide-modified myosin S1 (NEM-S1) increased the velocity of A-Tm filaments over that of actin alone, in a concentration-dependent manner, illustrating activation. The velocity of A-Tm(mut) filaments was further inhibited by ∼50-80% relative to A-Tm, depending on the mutant. Addition of NEM-S1 increased the velocity. The results indicate that conserved residues in the second half of the periodic repeats are required for normal cooperative regulation of actomyosin by Tm. Supported by NIH.

Conformational transitions in defined regions of the motor domain of skeletal muscle myosin involved in ATP hydrolysis, actin binding and motility were probed with monoclonal antibodies. Competition binding assays demonstrate that three different monoclonal antibodies react with spatially related sites on the myosin heavy chain. One recognizes a sequential epitope between residues 65 and 80 and has no effect on actin filament movement in an in vitro motility assay despite tight binding to myosin and acto-S1. The other two monoclonal antibodies react with sequential epitopes between residues 29 and 60 and both inhibit actin filament movement. A fourth monoclonal antibody reacts with the N-terminus of the heavy chain (residues 1–12) at a spatially distinct site on the myosin head and also inhibits actin filament movement. These four monoclonal antibodies have been mapped by immunoelectron microscopy to the large, actin binding region of the myosin head; however, the antibody binding sites remain accessible on rigor complexes of acto-S1. Thus, this group of monoclonal antibodies identify sequential epitopes in a mobile segment of the myosin heavy chain. In addition, two conformation-sensitive monoclonal antibodies are described that are affected by ATP and actin binding to myosin S1, and display distinct and marked inhibitory effects on actin filament movement. In contrast, an anti-light chain monoclonal antibody that binds near the myosin head-rod junction has little effect on the number and velocity of moving actin filaments. These results identify mobile regions on the myosin head that are perturbed by antibody binding and that may be linked to force production and motion.

Directed movement is a characteristic of many living organisms and occurs as a result of the transformation of chemical energy into mechanical energy. Myosin is one of three families of molecular motors that are responsible for cellular motility. The three-dimensional structure of the head portion of myosin, or subfragment-1, which contains both the actin and nucleotide binding sites, is described. This structure of a molecular motor was determined by single crystal x-ray diffraction. The data provide a structural framework for understanding the molecular basis of motility.

The interaction of two monoclonal antibodies (mAbs) with actin has been characterized to map the epitopes defined by these mAbs and to determine the accessibility of these sites in the actin filament (F-actin). Both mAbs react specifically with actin in radioimmunoassays and Western blot assays, and by immunoprecipitation. The location of the epitopes within the primary structure of actin has been determined using limited proteolysis of actin and Western blots, or using immunoprecipitation of truncated actin fragments synthesized in a cell free translation assay. Both mAbs bind to the C-terminal fragment of actin (residues 68-375) produced by chymotrypsin cleavage. One epitope is further localized to a 9.9 kD peptide corresponding to residues 5-93. Therefore, the epitope defined by this mAb (2G11.4) lies between residues Lys68 and Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal of residues 356-365 from the C-terminus of actin completely abolished the binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of these epitopes in native F-actin was determined with solution binding assays and characterized by immunoelectron microscopy. Monoclonal antibody 4E3.adl binds strongly to filaments, resulting in bundling or decoration of F-actin depending on the valency of the mAb, and indicating that the epitope is readily accessible in F-actin. In contrast, mAb 2G11.4 disrupts F-actin structure, resulting in the formation of an amorphous immunoprecipitate. These results place constraints on models of actin filament structure.

Myosin folding and assembly in striated muscle is mediated by the general chaperones Hsc70 and Hsp90 and a myosin specific co-chaperone, UNC45. Two UNC45 genes are found in vertebrates, including a striated muscle specific form, Unc45b. We have investigated the role of Unc45b in myosin folding. Epitope tagged murine Unc45b (Unc45b(Flag)) was expressed in muscle and non-muscle cells and bacteria, isolated and characterized. The protein is a soluble monomer in solution with a compact folded rod-shaped structure of approximately 19 nm length by electron microscopy. When over-expressed in striated muscle cells, Unc45b(Flag) fractionates as a cytosolic protein and isolates as a stable complex with Hsp90. Purified Unc45b(Flag) re-binds Hsp90 and forms a stable complex in solution. The endogenous Unc45b in muscle cell lysates is also found associated with Hsp90. The Unc45b(Flag)/Hsp90 complex binds the partially folded myosin motor domain when incubated with myosin subfragments synthesized in a reticulocyte lysate. This binding is independent of the myosin rod or light chains. Unc45b(Flag) does not bind native myosin subfragments consistent with a chaperone function. More importantly, Unc45b(Flag) enhances myosin motor domain folding during de novo motor domain synthesis indicating that it has a direct role in myosin maturation. Thus, mammalian Unc45b is a cytosolic protein that forms a stable complex with Hsp90, selectively binds the unfolded conformation of the myosin motor domain, and promotes motor domain folding.

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.

We have developed a novel technique which allows one to direct the two dimensional motion of actin filaments on a myosin coated sheet using a weak electric field parallel to the plane of motion. The filament velocity can be increased or decreased, and even reversed, as a function of orientation and strength of the field. PMMA (poly(methylmethacrylate)) gratings, which act as rails for actin, allow one for the first time to explore three quadrants of the force velocity diagram. We discuss effective friction, duty ratio and stall force at different myosin densities. A discontinuity in the velocity force relationship suggests the existence of dynamical phase transition.

Monoclonal antibodies were used to identify and localize by immunoelectron microscopy epitopes on myosin isozymes. An antibody that reacts with an amino-terminal fragment of the myosin heavy chain maps on the myosin head 140 A distal to the head-rod junction. It identifies an epitope that is shared on adult and embryonic myosin, and detects two transitions in myosin expression during avian pectoralis myogenesis. Another antibody maps to the carboxyl terminus of the myosin rod. It is specific for an adult fast myosin epitope that is not detected in early developing pectoralis muscle. In contrast, an epitope that is present throughout development is identified by an antibody that reacts with a myosin light chain. This light chain epitope is localized at the head-rod junction. These results demonstrate structural changes in widely separated regions of the myosin molecule accompanying the sequential expression of developmental myosin isozymes.

Crystals of myosin subfragment-1 have been examined by X-ray diffraction and electron microscopy to determine how the molecules pack in the unit cell and to gain preliminary information on the size and shape of the myosin head. Subfragment-1 crystallizes in space group P212121. Analysis of the X-ray diffraction photographs shows that there are eight molecules in the unit cell with two in the asymmetric unit related by a non-crystallographic or local 2-fold axis. It also indicates that in projection down the a axis, two molecules of myosin subfragment-1 lie almost directly on top of one another except for a translation of about 9 A along c. Small crystals were fixed and embedded in the presence of tannic acid, and thin sections were cut perpendicular to each of the three crystallographic axes. Image analysis of micrographs recorded from these sections confirm the packing arrangement deduced from X-ray diffraction, and give the approximate size and shape of the molecule in the crystal lattice. They show that the molecule is at least 160 A long with a maximum thickness of about 60 A, and that it has marked curvature in the unit cell.

The location of protein S4 in the small ribosomal subunit has been identified by immunoelectron microscopy. Although intact small subunits are not reactive with antibodies directed against protein S4, subribosomal particles reconstituted without proteins S5 and S12 are reactive. By using these "incomplete" subparticles, we have mapped the position of S4. It is located at a single site on the exterior (cytoplasmic) side of the subunit, at the partition that separates the one-third, or head, from two-thirds, or base, of the subunit. In this location, protein S4 is "beneath" proteins S5 and S12. All three proteins are members of a complex on, or near, the external surface of the small ribosomal subunit that plays an important role in regulation of translational fidelity.

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.

Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.

The three-dimensional locations of Escherichia coli ribosomal proteins S3, 86, S8 and S10 on the surface of the small subunit were determined by immune electron microscopy. All four proteins are located on the “external surface” of the small subunit; i.e. on the side of the subunit in contact with the cytosol in the 70 S ribosome. Proteins S3, S6, S8 and S10 map at single sites, although the S3 site is extended approximately 40A along the long axis of the subunit. S8 is located near the base of the cleft separating the platform from the upper one-third or head; protein S10 is located in the head, near the site previously mapped for S14; S3 extends from the level of the constriction to near the top of the head in the vicinity of S10; and S6 is located on the platform of the small subunit near the site previously mapped for S11. The locations of these proteins correlate well with other information on their spatial relationships obtained from assembly interactions, neutron diffraction, crosslinking and protein associations.

Antibodies to Escherichia coli ribosomal protein S4 react with S4 in subribosomal particles, eg, the complex of 16S RNA with S4, S7, S8, S15, S16, S17, and S19 and the RI* reconstitution intermediate, but they do not react with intact 30S subunits. Antibodies were isolated by three different methods from antisera obtained during the immunization of eight rabbits. Some of these antibody preparations, which contained contaminant antibodies directed against other ribosomal proteins, reacted with subunits, but this reaction was not affected by removal of the anti-S4 antibody population. Other antibody preparations did not react with subunits. It is concluded that the antigenic determinants of S4 are accessible in some protein deficient subribosomal particles but not in intact 30S subunits.