Krishna Midde

Comparison of orientation and rotational motion of skeletal muscle cross-bridges containing phosphorylated and dephosphorylated myosin regulatory light chain.

Background: Myosin cross-bridges containing phosphorylated and dephosphorylated regulatory light chain may be different. Results: Relaxed cross-bridges, but not active ones, are better ordered in muscle containing dephosphorylated RLC than phosphorylated RLC; they both rotate equally slowly. During contraction phosphorylated cross-bridges rotate faster. Conclusion: Both types of cross-bridges are functionally different. Significance: Phosphorylation of skeletal myosin RLC plays a role in regulation of skeletal muscle contraction. Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca2+ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca2+ saturation. A simple theory was developed to account for this fact.

Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti:sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM).

Evidence for Pre- and Post-Power Stroke of Cross-Bridges of Contracting Skeletal Myofibrils

We examined the orientational fluctuations of a small number of myosin molecules (approximately three) in working skeletal muscle myofibrils. Myosin light chain 1 (LC1) was labeled with a fluorescent dye and exchanged with the native LC1 of skeletal muscle myofibrils cross-linked with 1-ethyl-3-[3(dimethylamino) propyl] carbodiimide to prevent shortening. We observed a small volume within the A-band (∼10(-15) L) by confocal microscopy, and measured cyclic fluctuations in the orientation of the myosin neck (containing LC1) by recording the parallel and perpendicular components of fluorescent light emitted by the fluorescently labeled myosin LC1. Histograms of orientational fluctuations from fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms from contracting muscles were best fit by at least two Gaussians. These results provide direct evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power-stroke states.

Myosin Cross-Bridges Do Not Form Precise Rigor Bonds in Hypertrophic Heart Muscle Carrying Troponin T Mutations

Distribution of orientations of myosin was examined in ex-vivo myofibrils from hearts of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C. Humans are heterozygous for sarcomeric FHC mutations and so hypertrophic myocardium contains a mixture of the wild-type (WT) and mutated (MUT) TnT. If mutations are expressed at a low level there may not be a significant change in the global properties of heart muscle. In contrast, measurements from a few molecules avoid averaging inherent in the global measurements. It is thus important to examine the properties of only a few molecules of muscle. To this end, the lever arm of one out of every 60,000 myosin molecules was labeled with a fluorescent dye and a small volume within the A-band (~1 fL) was observed by confocal microscopy. This volume contained on average 5 fluorescent myosin molecules. The lever arm assumes different orientations reflecting different stages of acto-myosin enzymatic cycle. We measured the distribution of these orientations by recording polarization of fluorescent light emitted by myosin-bound fluorophore during rigor and contraction. The distribution of orientations of rigor WT and MUT myofibrils was significantly different. There was a large difference in the width and of skewness and kurtosis of rigor distributions. These findings suggest that the hypertrophic phenotype associated with the TnT mutations can be characterized by a significant increase in disorder of rigor cross-bridges.

Mesoscopic analysis of motion and conformation of cross-bridges

The orientation of a cross-bridge is widely used as a parameter in determining the state of muscle. The conventional measurements of orientation, such as that made by wide-field fluorescence microscopy, electron paramagnetic resonance (EPR) or X-ray diffraction or scattering, report the average orientation of 1012–109 myosin cross-bridges. Under conditions where all the cross-bridges are immobile and assume the same orientation, for example in normal skeletal muscle in rigor, it is possible to determine the average orientation from such global measurements. But in actively contracting muscle, where a parameter indicating orientation fluctuates in time, the measurements of the average value provide no information about cross-bridge kinetics. To avoid problems associated with averaging information from trillions of cross-bridges, it is necessary to decrease the number of observed cross-bridges to a mesoscopic value (i.e. the value affected by fluctuations around the average). In such mesoscopic regimes, the averaging of the signal is minimal and dynamic behavior can be examined in great detail. Examples of mesoscopic analysis on skeletal and cardiac muscle are provided.

Membrane Topology of Human Presenilin-1 in SK-N-SH Cells Determined by Fluorescence Correlation Spectroscopy and Fluorescent Energy Transfer

Presenilin-1 (PS1) protein acts as passive ER Ca2+ leak channels that facilitate passive Ca2+ leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer’s disease (FAD). FADPS1 mutations abrogate the function of ER Ca2+ leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487–500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca2+) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP–PS1, and PS1–CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca2+ leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1–CFP was either expressed alone or together with YFP–PS1 into SK-N-SH cell line and the interaction between YFP–PS1 and PS1–CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP–PS1 and PS1–CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1–CFP and YFP–PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.

Effect of a Myosin Regulatory Light Chain mutation K104E on Actin-Myosin Interactions

Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC.

A Novel Method of Determining the Functional Effects of a Minor Genetic Modification of a Protein

Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being “kicked” by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either “Standard” (Std) (decaying monotonically in time) or “Non-standard” (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.

Myosin Cross-Bridges do not Form Precise Rigor Bonds in Hypertrophic Heart Muscle Carrying Troponin T Mutations

Distribution of orientations of myosin was examined in ex-vivo myofibrils from hearts of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C. Humans are heterozygous for sarcomeric FHC mutations and so hypertrophic myocardium contains a mixture of the wild-type (WT) and mutated (MUT) TnT. If mutations are expressed at a low level there may not be a significant change in the global properties of heart muscle. In contrast, measurements from a few molecules avoid averaging inherent in the global measurements. It is thus important to examine the properties of only a few molecules of muscle. To this end, the lever arm of one out of every 60,000 myosin molecules was labeled with a fluorescent dye and a small volume within the A-band (∼ 1 fL) was observed by confocal microscopy. This volume contained on average 5 fluorescent myosin molecules. The lever arm assumes different orientations reflecting different stages of acto-myosin enzymatic cycle. We measured the distribution of these orientations by recording polarization of fluorescent light emitted by myosin-bound fluorophore during rigor and contraction. The distribution of orientations of rigor WT and MUT myofibrils was significantly different. There was a large difference in the width and of skewness and kurtosis of rigor distributions. These findings suggest that the hypertrophic phenotype associated with the TnT mutations can be characterized by a significant increase in disorder of rigor cross-bridges.