Optical Diffraction Studies of Muscle Fibers

A new technique to monitor light diffraction patterns electrically is applied to frog semitendinosus muscle fibers at various levels of stretch. The intensity of the diffraction lines, sarcomere length change, and the length-dispersion (line width) were calculated by fast analogue circuits and displayed in real time. A heliumneon laser (wavelength 6328 Å) was used as a light source. It was found that the intensity of the first-order diffraction line drops significantly (30-50%) at an optimal sarcomere length of 2.8 μm on isometric tetanic stimulation. Such stimulation produced contraction of half-sarcomeres by about 22 nm presumably by stretching inactive elements such as tendons. The dispersion of the sarcomere lengths is extremely small, and it is proportional to the sarcomere length (less than 4%). The dispersion increases on stimulation. These changes on isometric tetanic stimulation were dependent on sarcomere length. No vibration or oscillation in the averaged length of the sarcomeres was found during isometric tetanus within a resolution of 3 nm; however, our observation of increased length dispersion of the sarcomeres together with detection of the averaged shortening of the sarcomere lengths suggests the presence of asynchronous cyclic motions between thick and thin filaments. An alternative explanation is simply an increase of the length dispersion of sarcomeres without cyclic motions.     

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Single molecule force spectroscopy methods, such as optical and magnetic tweezers and atomic force microscopy, have opened up the possibility to study biological processes regulated by force, dynamics of structural conformations of proteins and nucleic acids, and load-dependent kinetics of molecular interactions. Among the various tools available today, optical tweezers have recently seen great progress in terms of spatial resolution, which now allows the measurement of atomic-scale conformational changes, and temporal resolution, which has reached the limit of the microsecond-scale relaxation times of biological molecules bound to a force probe. Here, we review different strategies and experimental configurations recently developed to apply and measure force using optical tweezers. We present the latest progress that has pushed optical tweezers’ spatial and temporal resolution down to today’s values, discussing the experimental variables and constraints that are influencing measurement resolution and how these can be optimized depending on the biological molecule under study.     

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Single molecule force spectroscopy methods, such as optical and magnetic tweezers and atomic force microscopy, have opened up the possibility to study biological processes regulated by force, dynamics of structural conformations of proteins and nucleic acids, and load-dependent kinetics of molecular interactions. Among the various tools available today, optical tweezers have recently seen great progress in terms of spatial resolution, which now allows the measurement of atomic-scale conformational changes, and temporal resolution, which has reached the limit of the microsecond-scale relaxation times of biological molecules bound to a force probe. Here, we review different strategies and experimental configurations recently developed to apply and measure force using optical tweezers. We present the latest progress that has pushed optical tweezers’ spatial and temporal resolution down to today’s values, discussing the experimental variables and constraints that are influencing measurement resolution and how these can be optimized depending on the biological molecule under study.     

Association Kinetics from Single Molecule Force Spectroscopy Measurements

Single molecule force spectroscopy is often used to study the dissociation of single molecules by applying mechanical force to the intermolecular bond. These measurements provide the kinetic parameters of dissociation. We present what to our knowledge is a new atomic force microscopy-based approach to obtain the activation energy of the association reaction and approximate grafting density of reactive receptors using the dependence of the probability to form molecular bonds on probe velocity when one of the interacting molecules is tethered by a flexible polymeric linker to the atomic force microscopy probe. Possible errors in the activation energy measured with this approach are considered and resulting corrections are included in the data analysis. This new approach uses the same experimental setup as traditional force spectroscopy measurements that quantify dissociation kinetics. We apply the developed methodology to measure the activation energy of biotin-streptavidin association (including a contribution from the steric factor) and obtain a value of 8 ± 1 kT. This value is consistent with the association rate measured previously in solution. Comparison with the solution-derived activation energy indicates that kinetics of biotin-streptavidin binding is mainly controlled by the reaction step.     

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.     

Effects of External Calcium Deprivation on Single Muscle Fibers

Deprivation of external calcium causes sudden potentiation of the twitch response of single muscle fibers. The potentiation was 64 ± 8%. Potentiation is simultaneous with membrane depolarization occurring after Ca⁺⁺ removal. This depolarization amounted to 9 ± 2 mv. Ca⁺⁺ removal also alters the action potential. 3 min after calcium withdrawal, action potential amplitude fell by 36 ± 3 mv; maximum rates of rise and fall of the spike decreased by 55 ± 5 and 63 ± 5% respectively. Changes in shape of the A. P. differ from those seen with other potentiators of the twitch response, such as Zn⁺⁺. After short exposure to calcium-free media, potassium-induced contractures show potentiation of peak tension. The S-shaped curve relating potassium contracture tension to log [K]_o shifts to the left after such treatment. Calcium deprivation also increased the rate of relaxation of the contractures. This effect depends on the duration of calcium deprivation, and is probably related to the effect of calcium lack on the membrane. The change in relaxation occurred immediately after calcium deprivation, and was reversed by sudden readmission of calcium. Relaxation of twitch and tetanus responses also were affected by Ca lack, but not as rapidly as potassium contractures. The results suggest that external calcium is not directly involved in the process responsible for tension development, supporting the view that this process is mediated by translocation of intracellular calcium. The relaxation process, however, appears to be rapidly affected by deprivation of external calcium.     

Single Cell Measurements in Research on Calcium-Mobilising Purinoceptors

Abstract

The Ca-sensitive photoprotein aequorin has been used to record repetitive free Ca transients in single rat liver cells responding to either ATP or ADP. The time-course of individual transients is longer when ATP is the agonist. If both agonists operate through the same receptor, then curtailment of the receptors' activity occurs more rapidly when ADP is the agonist, and we infer that an agonist-occupied receptor linked to a GTP-liganded G protein is the moiety responsible for activating a phosphatidylinositol bisphosphate-specific phospholipase C. The alternative explanation is that ATP and ADP act through different receptors.     

Voltage Clamp Experiments on Single Muscle Fibers of Rana pipiens

A voltage clamp for single muscle fibers has been developed. Stability of the system was achieved when an artificial node was created by enclosing a single muscle fiber in a petroleum jelly seal which served as an analogue of the myelin sheath. Typical voltage clamp records were obtained with large inward transient currents followed by a delayed rectification of the outward currents. These currents looked qualitatively similar when the transverse tubular system was destroyed. Errors in current measurement, especially those due to anomalous rectification, are discussed.     

生物单分子光学探测方法的进展

活细胞中单分子的实时显视是单分子生物学的关键技术,本文针对单分子显视的光学方法做了评述。分别描述了共焦荧光显微术、荧光全内反射显微术以及荧光共振能量转移探测的技术细节,分析了这些技术对于单分子探测所具备的优势和不足。并对单分子方法的未来发展给出预测。指出包括原于力在内的各种探测手段的联合使用和创新荧光染料技术是进一步提高分辨率的突破口。而随着高灵敏和低噪音探测器的发展,各种新方法的出现也有可能突破目前荧光染料尺度给予的分辨极限。     

In-Situ Optical and Acoustical Measurements of the Buoyant Cyanobacterium P. Rubescens: Spatial and Temporal Distribution Patterns

Optical (fluorescence) and acoustic in-situ techniques were tested in their ability to measure the spatial and temporal distribution of plankton in freshwater ecosystems with special emphasis on the harmful and buoyant cyanobacterium P. rubescens. Fluorescence was measured with the multi-spectral FluoroProbe (Moldaenke FluoroProbe, MFP) and a Seapoint Chlorophyll Fluorometer (SCF). In-situ measurements of the acoustic backscatter strength (ABS) were conducted with three different acoustic devices covering multiple acoustic frequencies (614 kHz ADCP, 2 MHz ADP, and 6 MHz ADV). The MFP provides a fast and reliable technique to measure fluorescence at different wavelengths in situ, which allows discriminating between P. rubescens and other phytoplankton species. All three acoustic devices are sensitive to P. rubescens even if other scatterers, e.g., zooplankton or suspended sediment, are present in the water column, because P. rubescens containing gas vesicles has a strong density difference and hence acoustic contrast to the ambient water and other scatterers. After calibration, the combination of optical and acoustical measurements not only allows qualitative and quantitative observation of P. rubescens, but also distinction between P. rubescens, other phytoplankton, and zooplankton. As the measuring devices can sample in situ at high rates they enable assessment of plankton distributions at high temporal (minutes) and spatial (decimeters) resolution or covering large temporal (seasonal) and spatial (basin scale) scales.     

Measurements of Electrical Potential Differences on Single Nephrons of the Perfused Necturus Kidney

Stable electrical potential differences can be measured by means of conventional glass microelectrodes across the cell membrane of renal tubule cells and across the epithelial wall of single tubules in the doubly perfused kidney of Necturus. These measurements have been carried out with amphibian Ringer's solution, and with solutions of altered ionic composition. The proximal tubule cell has been found to be electrically asymmetrical inasmuch as a smaller potential difference is maintained across the luminal cell membrane than across the peritubular cell boundary. The tubule lumen is always electrically negative with respect to the peritubular extracellular medium. Observations on the effectiveness of potassium ions in depolarizing single tubule cells indicate that the transmembrane potential is essentially an inverse function of the logarithm of the external potassium concentration. The behavior of the peritubular transmembrane potential resembles more closely an ideal potassium electrode than that of the luminal transmembrane potential. From these results, and the effects of various ionic substitutions on the electrical profile of the renal tubular epithelium, a thesis concerning the origin of the observed potential differences is presented. A sodium extrusion mechanism is considered to be located at the peritubular cell boundary, and reasons are given for the hypothesis that the electrical asymmetry across the proximal renal tubule cell could arise as a consequence of differences in the relative sodium and potassium permeability at the luminal and peritubular cell boundaries.     

Muscle Fiber Types in Crabs: Studies on Single Identified Muscle Fibers

SYNOPSIS. Based on electrophysiological and histochemical data,four types of muscle fibers (types I, II, III and IV) can beidentified in the closer of the crab Eriphia. Although characteristicsused for typing vary among the fibers of a particular type,the combination of several parameters permits an assignment.Of particular significance for typing is the myosin ATPase activityand its stability after preincubation at different pH levels.The fiber types defined for the closer muscle can also be foundin the other leg muscles of riphia. Single, electrophysiologically identified fibers of each typewere quantitatively analyzed for several key enzymes of oxidativeand glycolytic energy metabolism (GAPDH, LDH, CS, IDH, HAD).Despite the variations found, different metabolic types canbe defined. The typing derived from biochemical studies correlateswell with that obtained electrophysiologically and histochemically.The variability of the biochemical properties, however, seemsto be considerably larger. The type I fibers can be regarded as slow oxidative, the typeII and III fibers as fast oxidative glycolytic, and the typeIV fibers as fast glycolytic.     

Action Potential-Evoked Calcium Release Is Impaired in Single Skeletal Muscle Fibers from Heart Failure Patients

Background

Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium (Ca²⁺) release, mandating investigations in skeletal muscle from HF patients. The purpose of this study was to test the hypothesis that Ca²⁺ release is significantly impaired in the skeletal muscle of HF patients in whom exercise capacity is severely diminished compared to age-matched healthy volunteers.

Methods and Findings

Using state-of-the-art electrophysiological and optical techniques in single muscle fibers from biopsies of the locomotive vastus lateralis muscle, we measured the action potential (AP)-evoked Ca²⁺ release in 4 HF patients and 4 age-matched healthy controls. The mean peak Ca²⁺ release flux in fibers obtained from HF patients (10±1.2 µM/ms) was markedly (2.6-fold) and significantly (p<0.05) smaller than in fibers from healthy volunteers (28±3.3 µM/ms). This impairment in AP-evoked Ca²⁺ release was ubiquitous and was not explained by differences in the excitability mechanisms since single APs were indistinguishable between HF patients and healthy volunteers.

Conclusions

These findings prove the feasibility of performing electrophysiological experiments in single fibers from human skeletal muscle, and offer a new approach for investigations of myopathies due to HF and other diseases. Importantly, we have demonstrated that one step in the ECC process, AP-evoked Ca²⁺ release, is impaired in single muscle fibers in HF patients.     

Polarization of Tryptophan Fluorescence from Single Striated Muscle Fibers : A molecular probe of contractile state

Instrumentation has been developed to detect rapidly the polarization of tryptophan fluorescence from single muscle fibers in rigor, relaxation, and contraction. The polarization parameter (P) obtained by exiciting the muscle tryptophans with light polarized perpendicular to the long axis of the muscle fiber had a magnitude P (relaxation) > P (contraction) > P (rigor) for the three types of muscle fibers examined (glycerinated rabbit psoas, glycerinated dorsal longitudinal flight muscle of Lethocerus americanus, and live semitendinosus of Rana pipiens). P from single psoas fibers in rigor was found to increase as the sarcomere length increased but in relaxed fibers P was independent of sarcomere length. After rigor, pyrophosphate produced little or no change in P, but following an adenosine triphosphate (ATP)-containing solution, pyrophosphate produced a value of P that fell between the contraction and relaxation values. Sinusoidal or square wave oscillations of the muscle of amplitude 0.5–2.0% of the sarcomere length and frequency 1, 2, or 5 Hz were applied in rigor when the myosin cross-bridges are considered to be firmly attached to the thin filaments. No significant changes in P were observed in either rigor or relaxation. The preceding results together with our present knowledge of tryptophan distribution in the contractile proteins has led us to the conclusion that the parameter P is a probe of the contractile state of myosin which is probably sensitive to the orientation of the myosin S1 subfragment.     

Effects of Temperature and Humidity on Laser Diffraction Measurements to Jet Nebulizer and Comparison with NGI

Laser diffraction (LD) and next generation impactor (NGI) are commonly used for the evaluation of inhaled drug formulations. In this study, the effect of temperature and humidity on the assessment of the nebulizer particle size distribution (PSD) by LD was investigated, and the consistency between NGI and LD measurements was evaluated. There was an increase in particle size with higher temperature or lower humidity. The particle population with a diameter less than 1 μm was significant at a temperature of 5°C or at relative humidity >90%; however, the same particle population became undetectable when temperature increased to 39°C or at relative humidity of 30–45%. The results of the NGI and LD measurements of aerosol generated from three types of jet nebulizers were compared. A poor correlation between the NGI and LD measurements was observed for PARI LC (2.2 μm) (R ²?=?0.893) and PARI LC (2.9 μm) (R ²?=?0.878), while a relatively good correlation (R ²?=?0.977) was observed for the largest particle size nebulizer (PARI TIA (8.6 μm)). We conclude that the ambient environment and the nebulizer have significant impacts on the performance and consistency between these instruments. These factors should be controlled in the evaluation of inhaled aerosol drug formulations when these instruments are used individually or in combination.     

The Role of Cell Calcium in the Contraction of Single Cannulated Muscle Fibers

Intracellular injections of the calcium-binding agent, EGTA,into single cannulated fibers of Balanus and Maia were ableto suppress, almost completely, the contractions induced byvarious contractile agents. The amount oE EGTA required in Maia fibers for the suppressionof the contractile response produced by caffeine and high-Ksaline, as has already been reported, was similar to the meanfiber calcium level. In Balanus fibers, however, although theamount of EGTA needed for the suppression of the caffeine-salineresponse was similar to the estimated level of fiber calcium,the amount required in the raised-K-saline experiments was considerablygreater. It has been suggested that membrane depolarizationunder these conditions allows calcium to enter the fiber fromthe external saline, the amount entering being related, at leastin part, to a large effective sarcolemmal surface area and thepresence of binding agent internally. The results of intracellularand plate-electrode stimulation of Belanus fibers also suggestedthat the fiber under certain conditions could utilize externalcalcium ions, while the results of plate-electrode stimulationof Maia fibers could be explained most easily in terms of mobilizationof mainly intracellular calcium for the process of contraction. Efflux of Sr⁸⁹ ions from Balanus fibers under various conditionssuggested that this ion is bound and mobilized internally ina manner similar to calcium. The results are seen not to contradict a chanelled-current theoryfor e-c coupling of the type proposed for crayfish fibers.