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The brains of several representative species of echolocating bats were found to contain regions concerned with the emission of species-specific orientation sounds. When the dorsal part of the reticular formation in the midbrain or the lateral part of the central gray matter is stimulated with a short train of electric pulses, the bat moves its mouth and pinnae and emits sounds identical to orientation sounds, with a latency of 25 to 60 msec. The acoustic parameters of the electrically elicited sounds and the temporal patterns of emissions vary with the location of the stimulating electrodes. Bats of the genus Myotis (little brown bat) emitted short frequency-modulated (FM) sounds both in nature and when electrically stimulated. Pteronotus parnellii (mustache bat) responded with sounds containing long constant-frequency (CF) and short FM components that were typical of the species. Eptesicus fuscus (big brown bat), Pteronotus suapurensis (naked-backed bat), and Noctilio leporinus (fish-catching bat) also made characteristic orientation sounds when electrically stimulated. It appears that the midbrain contains neural circuits for the emission of species-specific orientation sounds and that a part of the vocalization system is located in the regions juxtaposed to the inferior colliculus, an important neural center for echolocation. [ABSTRACT FROM AUTHOR]

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart's pump functions. It has generally been considered that in cardiac muscle as well as in skeletal muscle, sarcomeres equally contribute to myofibrillar dynamics in myocytes at varying loads by producing similar levels of active and passive force. In the present study, we expressed α-actinin-AcGFP in Z-disks to analyze dynamic behaviors of sequentially connected individual sarcomeres along a myofibril in a left ventricular (LV) myocyte of the in vivo beating mouse heart. To quantify the magnitude of the contribution of individual sarcomeres to myofibrillar dynamics, we introduced the novel parameter "contribution index" (CI) to measure the synchrony in movements between a sarcomere and a myofibril (from -1 [complete asynchrony] to 1 [complete synchrony]). First, CI varied markedly between sarcomeres, with an average value of ∼0.3 during normal systole. Second, when the movements between adjacent sarcomeres were asynchronous (CI < 0), a sarcomere and the ones next to the adjacent sarcomeres and farther away moved in synchrony (CI > 0) along a myofibril. Third, when difference in LV pressure in diastole and systole (ΔLVP) was lowered to <10 mm Hg, diastolic sarcomere length increased. Under depressed conditions, the movements between adjacent sarcomeres were in marked asynchrony (CI, -0.3 to -0.4), and, as a result, average CI was linearly decreased in association with a decrease in ΔLVP. These findings suggest that in the left ventricle of the in vivo beating mouse heart, (1) sarcomeres heterogeneously contribute to myofibrillar dynamics due to an imbalance of active and passive force between neighboring sarcomeres, (2) the force imbalance is pronounced under depressed conditions coupled with a marked increase in passive force and the ensuing tug-of-war between sarcomeres, and (3) sarcomere synchrony via the distal intersarcomere interaction regulates the heart's pump function in coordination with myofibrillar contractility., (© 2021 Kobirumaki-Shimozawa et al.)

Various insects utilise hydrophobic biological surfaces to live on the surface of water, while other organisms possess hydrophilic properties that enable them to live within a water column. Dixidae larvae reside, without being submerged, just below the water surface. However, little is known about how these larvae live in such an ecological niche. Herein, we use larvae of Dixa longistyla (Diptera: Dixidae) as experimental specimens and reveal their characteristics. A complex crown-like structure on the abdomen consists of hydrophobic and hydrophilic elements. The combination of these contrasting features enables the larvae to maintain their position as well as to move unidirectionally. Their hydrophobic region leverages water surface tension to function as an adhesive disc. By using the resistance of water, the hydrophilic region serves as a rudder during locomotion.

Myocardial contraction is initiated by action potential propagation through the conduction system of the heart. It has been thought that connexin 43 in the gap junctions (GJ) within the intercalated disc (ID) provides direct electric connectivity between cardiomyocytes (electronic conduction). However, recent studies challenge this view by providing evidence that the mechanosensitive cardiac sodium channels Na v 1.5 localized in perinexii at the GJ edge play an important role in spreading action potentials between neighboring cells (ephaptic conduction). In the present study, we performed real-time confocal imaging of the CellMask-stained ID in the living mouse heart in vivo. We found that the ID structure was not rigid. Instead, we observed marked flexing of the ID during propagation of contraction from cell to cell. The variation in ID length was between ~30 and ~42 μm (i.e., magnitude of change, ~30%). In contrast, tracking of α -actinin-AcGFP revealed a comparatively small change in the lateral dimension of the transitional junction near the ID (i.e., magnitude of change, ~20%). The present findings suggest that, when the heart is at work, mechanostress across the perinexii may activate Na v 1.5 by promoting ephaptic conduction in coordination with electronic conduction, and, thereby, efficiently transmitting excitation-contraction coupling between cardiomyocytes.

Many insects possess a hyper-elongated intromittent organ with a diameter of only a few micrometers. Using morphological and theoretical approaches, we investigated the biomechanics of sperm transfer through such organs by calculating (1) how far and how fast sperm could fill in the penis by capillary action, (2) how much capillary pressure is generated in the penis, and (3) how much pressure is needed to pump sperm out of the penis. The results enabled us to propose the following hypotheses: (1) penile filling basically occurs by capillary action, and (2) sperm transport to females occurs by contracting the sperm pump muscles or by active propulsion of spermatozoa. Potential experimental approaches to test these hypotheses are discussed.

Formins are actin-binding proteins that construct nanoscale machinery with the growing barbed end of actin filaments and serve as key regulators of actin polymerization and depolymerization. To maintain the regulation of actin dynamics, formins have been proposed to processively move at every association or dissociation of a single actin molecule toward newly formed barbed ends. However, the current models for the motile mechanisms were established without direct observation of the elementary processes of this movement. Here, using optical tweezers, we demonstrate that formin mDia1 moves stepwise, observed at a nanometer spatial resolution. The movement was composed of forward and backward steps with unitary step sizes of 2.8 and -2.4 nm, respectively, which nearly equaled the actin subunit length (∼2.7 nm), consistent with the generally accepted models. However, in addition to steps equivalent to the length of a single actin subunit, those equivalent to the length of two or three subunits were frequently observed. Our findings suggest that the coupling between mDia1 stepping and actin polymerization is not tight but loose, which may be achieved by the multiple binding states of mDia1, providing insights into the synergistic functions of biomolecules for the efficient construction and regulation of nanofilaments.

The present study was conducted to systematically investigate the optimal viral titer as well as the volume of the adenovirus vector (ADV) that expresses α -actinin-AcGFP in the Z-disks of myocytes in the left ventricle (LV) of mice. An injection of 10  μ L ADV at viral titers of 2 to 4 × 10 11 viral particles per mL (VP/mL) into the LV epicardial surface consistently expressed α -actinin-AcGFP in myocytes in vivo , with the fraction of AcGFP-expressing myocytes at ~10%. Our analysis revealed that SL was ~1.90-2.15  μ m upon heart arrest via deep anesthesia. Likewise, we developed a novel fluorescence labeling method of the T-tubular system by treating the LV surface with CellMask Orange (CellMask). We found that the T-tubular distance was ~2.10-2.25  μ m, similar to SL, in the healthy heart in vivo . Therefore, the present high-precision visualization method for the Z-disks or the T-tubules is beneficial to unveiling the mechanisms of myocyte contraction in health and disease in vivo .

UnaG is a green-emitting fluorescent protein that utilizes unconjugated bilirubin (BR) as its fluorophore. While BR has captured the attention of physiologists as an important antioxidant that scavenges reactive oxygen species in biological membranes, its excessive accumulation causes several clinical symptoms. Although the optimal regulation of BR concentration would result in clinical therapies for aging as well as reduce risks of clinical symptoms, UnaG hardly releases BR owing to its extremely high affinity for BR (K d  = 98 pM). Herein, we engineered the BR binding and fluorescence of UnaG to be Ca 2+ -sensitive via a genetic insertion of calmodulin (CaM). The resultant UnaG/CaM hybrid protein is a dual-ligand modulable fluorescent protein; binding of the fluorogenic ligand BR is negatively regulated by the other ligand, Ca 2+ ion. The affinity for BR differed by three orders of magnitude between the Ca 2+ -free state (K d  = 9.70 nM) and Ca 2+ -saturated state (K d  = 9.65 μM). The chimeric protein can release nano- to micro-molar levels of BR with Ca 2+ control, and is thus named BReleaCa (BR + releaser + Ca 2+ ). Such a protein hybridization technique will be generally applicable to change the ligand binding properties of a variety of ligand-inducible functional proteins., (Copyright © 2018 Elsevier Inc. All rights reserved.)

UnaG is a recently discovered ligand-induced fluorescent protein that utilizes bound bilirubin (BR) as its fluorophore. The fluorescence of the UnaG-BR complex (holoUnaG) compares in quantum efficiency to that of enhanced green fluorescent protein, but it is superior in that the fluorophore formation is instantaneous and not dependent on oxygen; hence, much attention has been paid to UnaG as a new fluorescent probe. However, many important molecular properties of fluorescent probes remain unknown, such as the association/dissociation rates of BR, which determine the stability thereof, and the dispersibility of UnaG in aqueous solutions, which influence the functions of labeled proteins. In this study, we found, in the process of investigating the association rate, that the holoUnaG takes two distinct fluorescence states, which we named holoUnaG 1 and holoUnaG 2 . The holoUnaG 1 initially forms after binding BR and then changes to the brighter holoUnaG 2 by a reversible intra-molecular reaction, thereby finally reaching an equilibrium between the two states. Spectroscopic analysis indicated that the intra-molecular reaction was associated not with a chemical change of BR but with a change in the environmental conditions surrounding BR. We also revealed that the molecular brightness ratio and equilibrium population ratio of the two states (holoUnaG 1 /holoUnaG 2 ) were 1:3.9 and 6:4, respectively, using photon number counting analysis. From these results, we have suggested a novel schema, to our knowledge, for the formation of the UnaG and BR complex system and have determined the various rate constants associated therein. Additionally, using analytical ultracentrifugation, we established that UnaG in the apo-state (apoUnaG) and the holoUnaG are monomeric in aqueous solution. These findings provide not only key information for the practical use of UnaG as a fluorescent probe, but also the possibility for development of a brighter UnaG mutant by genetic engineering to constitutive holoUnaG 2 ., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Formins are force-sensing proteins that regulate actin polymerization dynamics. Here, we applied stretching tension to individual actin filaments under the regulation of formin mDia1 to investigate the mechanical responses in actin polymerization dynamics. We found that the elongation of an actin filament was accelerated to a greater degree by stretching tension for ADP-G-actin than that for ATP-G-actin. An apparent decrease in the critical concentration of G-actin was observed, especially in ADP-G-actin. These results on two types of G-actin were reproduced by a simple kinetic model, assuming the rapid equilibrium between pre- and posttranslocated states of the formin homology domain two dimer. In addition, profilin concentration dramatically altered the force-dependent acceleration of actin filament elongation, which ranged from twofold to an all-or-none response. Even under conditions in which actin depolymerization occurred, applications of a several-piconewton stretching tension triggered rapid actin filament elongation. This extremely high force-sensing mechanism of mDia1 and profilin could be explained by the force-dependent coordination of the biphasic effect of profilin; i.e., an acceleration effect masked by a depolymerization effect became dominant under stretching tension, negating the latter to rapidly enhance the elongation rate. Our findings demonstrate that the biphasic effect of profilin is controlled by mechanical force, thus expanding the function of mDia1 as a mechanosensitive regulator of actin polymerization., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Enantiomeric thalidomide undergoes various kinds of biotransformations including chiral inversion, hydrolysis, and enzymatic oxidation, which results in several metabolites, thereby adding to the complexity in the understanding of the nature of thalidomide. To decipher this complexity, we analyzed the multidimensional metabolic reaction networks of thalidomide and related molecules in vitro. Characteristic patterns in the amount of various metabolites of thalidomide and related molecules generated during a combination of chiral inversion, hydrolysis, and hydroxylation were observed using liquid chromatography-tandem mass spectrometry and chiroptical spectroscopy. We found that monosubstituted thalidomide derivatives exhibited different time-dependent metabolic patterns compared with thalidomide. We also revealed that monohydrolyzed and monohydroxylated metabolites of thalidomide were likely to generate mainly by a C-5 oxidation of thalidomide and subsequent ring opening of the hydroxylated metabolite. Since chirality was conserved in most of these metabolites during metabolism, they had the same chirality as that of nonmetabolized thalidomide. Our findings will contribute toward understanding the significant pharmacological effects of the multiple metabolites of thalidomide and its derivatives., (© 2017 Wiley Periodicals, Inc.)

The cardiac pump function is a result of a rise in intracellular Ca 2+ and the ensuing sarcomeric contractions [i.e., excitation-contraction (EC) coupling] in myocytes in various locations of the heart. In order to elucidate the heart's mechanical properties under various settings, cardiac imaging is widely performed in today's clinical as well as experimental cardiology by using echocardiogram, magnetic resonance imaging and computed tomography. However, because these common techniques detect local myocardial movements at a spatial resolution of ∼100 μm, our knowledge on the sub-cellular mechanisms of the physiology and pathophysiology of the heart in vivo is limited. This is because (1) EC coupling occurs in the μm partition in a myocyte and (2) cardiac sarcomeres generate active force upon a length change of ∼100 nm on a beat-to-beat basis. Recent advances in optical technologies have enabled measurements of intracellular Ca 2+ dynamics and sarcomere length displacements at high spatial and temporal resolution in the beating heart of living rodents. Future studies with these technologies are warranted to open a new era in cardiac research., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In cardiac muscle, contraction is triggered by sarcolemmal depolarization, resulting in an intracellular Ca(2+) transient, binding of Ca(2+) to troponin, and subsequent cross-bridge formation (excitation-contraction [EC] coupling). Here, we develop a novel experimental system for simultaneous nano-imaging of intracellular Ca(2+) dynamics and single sarcomere length (SL) in rat neonatal cardiomyocytes. We achieve this by expressing a fluorescence resonance energy transfer (FRET)-based Ca(2+) sensor yellow Cameleon-Nano (YC-Nano) fused to α-actinin in order to localize to the Z disks. We find that, among four different YC-Nanos, α-actinin-YC-Nano140 is best suited for high-precision analysis of EC coupling and α-actinin-YC-Nano140 enables quantitative analyses of intracellular calcium transients and sarcomere dynamics at low and high temperatures, during spontaneous beating and with electrical stimulation. We use this tool to show that calcium transients are synchronized along the length of a myofibril. However, the averaging of SL along myofibrils causes a marked underestimate (∼50%) of the magnitude of displacement because of the different timing of individual SL changes, regardless of the absence or presence of positive inotropy (via β-adrenergic stimulation or enhanced actomyosin interaction). Finally, we find that β-adrenergic stimulation with 50 nM isoproterenol accelerated Ca(2+) dynamics, in association with an approximately twofold increase in sarcomere lengthening velocity. We conclude that our experimental system has a broad range of potential applications for the unveiling molecular mechanisms of EC coupling in cardiomyocytes at the single sarcomere level., (© 2016 Tsukamoto et al.)

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart's pump functions in mammals. Although it plays a critical role in the circulatory system, myocardial sarcomere length (SL) change has not been directly measured in vivo under physiological conditions because of technical difficulties. In this study, we developed a high speed (100-frames per second), high resolution (20-nm) imaging system for myocardial sarcomeres in living mice. Using this system, we conducted three-dimensional analysis of sarcomere dynamics in left ventricular myocytes during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. We found that (a) the working range of SL was on the shorter end of the resting distribution, and (b) the left ventricular-developed pressure was positively correlated with the SL change between diastole and systole. The present findings provide the first direct evidence for the tight coupling of sarcomere dynamics and ventricular pump functions in the physiology of the heart., (© 2016 Kobirumaki-Shimozawa et al.)

Some small animals only use water transport mechanisms passively driven by surface energies. However, little is known about passive water transport mechanisms because it is difficult to measure the wettability of microstructures in small areas and determine the chemistry of biological surfaces. Herein, we developed to directly analyse the structural effects of wettability of chemically modified biological surfaces by using a nanoliter volume water droplet and a hi-speed video system. The wharf roach Ligia exotica transports water only by using open capillaries in its legs containing hair- and paddle-like microstructures. The structural effects of legs chemically modified with a self-assembled monolayer were analysed, so that the wharf roach has a smart water transport system passively driven by differences of wettability between the microstructures. We anticipate that this passive water transport mechanism may inspire novel biomimetic fluid manipulations with or without a gravitational field.

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

We here review the use of quantum dots (QDs) for the imaging of sarcomeric movements in cardiac muscle. QDs are fluorescence substances (CdSe) that absorb photons and reemit photons at a different wavelength (depending on the size of the particle); they are efficient in generating long-lasting, narrow symmetric emission profiles, and hence useful in various types of imaging studies. Recently, we developed a novel system in which the length of a particular, single sarcomere in cardiomyocytes can be measured at ~30 nm precision. Moreover, our system enables accurate measurement of sarcomere length in the isolated heart. We propose that QDs are the ideal tool for the study of sarcomere dynamics during excitation-contraction coupling in healthy and diseased cardiac muscle.