Ravikrishnan Elangovan

Skeletal muscle resists stretch by rapid binding of the second motor domain of myosin to actin

A shortening muscle is a machine that converts metabolic energy into mechanical work, but, when a muscle is stretched, it acts as a brake, generating a high resistive force at low metabolic cost. The braking action of muscle can be activated with remarkable speed, as when the leg extensor muscles rapidly decelerate the body at the end of a jump. Here we used time-resolved x-ray and mechanical measurements on isolated muscle cells to elucidate the molecular basis of muscle braking and its rapid control. We show that a stretch of only 5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere is sufficient to double the number of myosin motors attached to actin within a few milliseconds. Each myosin molecule has two motor domains, only one of which is attached to actin during shortening or activation at constant length. A stretch strains the attached motor domain, and we propose that combined steric and mechanical coupling between the two domains promotes attachment of the second motor domain. This mechanism allows skeletal muscle to resist external stretch without increasing the force per motor and provides an answer to the longstanding question of the functional role of the dimeric structure of muscle myosin.

The mechanism of the resistance to stretch of isometrically contracting single muscle fibres.

Rapid attachment to actin of the detached motor domain of myosin dimers with one motor domain already attached has been hypothesized to explain the stretch‐induced changes in X‐ray interference and stiffness of active muscle. Here, using half‐sarcomere mechanics in single frog muscle fibres (2.15 μm sarcomere length and 4°C), we show that: (1) an increase in stiffness of the half‐sarcomere under stretch is specific to isometric contraction and does not occur in rigor, indicating that the mechanism of stiffness increase is an increase in the number of attached motors; (2) 2 ms after 100 μs stretches (amplitude 2–8 nm per half‐sarcomere) imposed during an isometric tetanus, the stiffness of the array of myosin motors in each half‐sarcomere (em) increases above the isometric value (em0); (3) em has a sigmoidal dependence on the distortion of the motor domains (Δz) attached in isometric contraction, with a maximum ∼2 em0 for a distortion of ∼6 nm; em is influenced by detachment of motors at Δz > 6 nm; (4) at the end of the 100 μs stretch the relation between em/em0 and Δz lies slightly but not significantly above that at 2 ms. These results support the idea that stretch‐induced sliding of the actin filament distorts the actin‐attached motor domain of the myosin dimers away from the centre of the sarcomere, providing the steric conditions for rapid attachment of the second motor domain. The rate of new motor attachment must be as high as 7.5 × 104 s−1 and explains the rapid and efficient increase of the resistance of active muscle to stretch.

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

•  Force and shortening in muscle are due to the ATP‐powered motor protein myosin II, polymerized in two bipolar arrays of motors that pull the two overlapping actin filaments toward the centre of the sarcomere. •  The parameters of the myosin motor in situ have been best characterized for the skeletal muscle of the frog, from which single intact cells can be isolated allowing fast sarcomere level mechanics to be applied. •  Up to now no reliable methods have been developed for the study of frog myosin with single molecule techniques. •  In this work a new protocol for extraction and conservation of frog muscle myosin allows us to estimate the sliding velocity of actin on myosin (VF) and its modulation by pH, myosin density, temperature and substrate concentration. •  By integrating in vitro and in situ parameters of frog muscle myosin we can relate kinetic and mechanical steps of the acto‐myosin ATPase.

New perspectives on photosynthetic phosphorylation in the light of a torsional mechanism of energy transduction and ATP synthesis

New perspectives on photophosphorylation have been offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis. New experimental data on the involvement of malate anions in ATP synthesis in an acid-base malate bath procedure has been reported on spinach chloroplast thylakoids as the model system. The data cannot be reconciled with the chemiosmotic theory but has been shown to be naturally explained by the torsional mechanism. The path of malic acid in the acid and base stages of the experiment has been traced, offering further strong support to the new paradigm. Classical observations in the field have been re-interpreted in the light of these findings. A new concept of ion translocation, energy transduction and coupling at the overall physiological level in photophosphorylation has been presented and a large number of novel experimentally testable predictions have been made and shown to arise as logical consequences of the new perspectives.

Highly-sensitive detection of Salmonella typhi in clinical blood samples by magnetic nanoparticle-based enrichment and in-situ measurement of isothermal amplification of nucleic acids

Enteric fever continues to be a major cause of mortality and morbidity globally, particularly in poor resource settings. Lack of rapid diagnostic assays is a major driving factor for the empirical treatment of enteric fever. In this work, a rapid and sensitive method ‘Miod’ ‘has been developed. Miod includes a magnetic nanoparticle-based enrichment of target bacterial cells, followed by cell lysis and loop-mediated isothermal amplification (LAMP) of nucleic acids for signal augmentation along with concurrent measurement of signal via an in–situ optical detection system. To identify positive/negative enteric fever infections in clinical blood samples, the samples were processed using Miod at time = 0 hours and time = 4 hours post-incubation in blood culture media. Primers specific for the STY2879 gene were used to amplify the nucleic acids isolated from S. typhi cells. A limit of detection of 5 CFU/mL was achieved. No cross-reactivity of the primers were observed against 106 CFU/mL of common pathogenic bacterial species found in blood such as E. coli, P. aeruginosa, S. aureus, A. baumanni, E. faecalis, S. Paratyphi A and K. pneumonia. Miod was tested on 28 human clinical blood samples. The detection of both pre-and post-four-hours incubation confirmed the presence of viable S. typhi cells and allowed clinical correlation of infection. The positive and negative samples were successfully detected in less than 6 hours with 100% sensitivity and specificity.

Compact 3D printed module for fluorescence and label-free imaging using evanescent excitation

Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid-solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (<0.1 mm2). Objective-based TIRFs are also expensive as they require dichroic mirrors and efficient notch filters to prevent specular reflection within the objective lenses. We have developed a compact 3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.

Maximum limit to the number of myosin II motors participating in processive sliding of actin.

In this work, we analysed processive sliding and breakage of actin filaments at various heavy meromyosin (HMM) densities and ATP concentrations in IVMA. We observed that with addition of ATP solution, the actin filaments fragmented stochastically; we then determined mean length and velocity of surviving actin filaments post breakage. Average filament length decreased with increase in HMM density at constant ATP, and increased with increase in ATP concentration at constant HMM density. Using density of HMM molecules and length of actin, we estimated the number of HMM molecules per actin filament (N) that participate in processive sliding of actin. N is solely a function of ATP concentration: 88 ± 24 and 54 ± 22 HMM molecules (mean ± S.D.) at 2 mM and 0.1 mM ATP respectively. Processive sliding of actin filament was observed only when N lay within a minimum lower limit (Nmin) and a maximum upper limit (Nmax) to the number of HMM molecules. When N < Nmin the actin filament diffused away from the surface and processivity was lost and when N > Nmax the filament underwent breakage eventually and could not sustain processive sliding. We postulate this maximum upper limit arises due to increased number of strongly bound myosin heads.

Interference X-ray Diffraction from Single Muscle Cells Reveals the Molecular Basis of Muscle Braking

Muscle is a machine that converts metabolic energy into mechanical work by cyclic ATP-driven interactions of the molecular motor, myosin II, with the actin filament. Muscle can also act as a brake, generating a high resistive force with reduced ATP consumption, when the load is increased above the isometric force. To investigate the molecular basis of the braking action of muscle, we used time-resolved X-ray diffraction from intact cells isolated from skeletal muscle of the frog. The results indicate that a stretch of 2–6nm per half-sarcomere imposed on the actively contracting cell induces a rapid attachment to actin of the second motor domain of the myosin molecules that have the first motor domain already attached before the stretch. This mechanism allows skeletal muscle to almost instantaneously resist an external stretch, while minimising the stress on an individual motor.