RESUMO
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 â¼0.3 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.
Assuntos
Miofibrilas , Sarcômeros , Animais , Diástole , Camundongos , Contração Muscular , Miócitos CardíacosRESUMO
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 Nav1.5 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 Nav1.5 by promoting ephaptic conduction in coordination with electronic conduction, and, thereby, efficiently transmitting excitation-contraction coupling between cardiomyocytes.
RESUMO
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 µL ADV at viral titers of 2 to 4 × 1011 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 µm 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 µm, 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.
Assuntos
Miócitos Cardíacos/fisiologia , Nanotecnologia , Sarcômeros/fisiologia , Actinina/metabolismo , Adenoviridae , Animais , Vetores Genéticos , Ventrículos do Coração , CamundongosRESUMO
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 (â¼2.7 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.
RESUMO
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 (Kdâ¯=â¯98 pM). Herein, we engineered the BR binding and fluorescence of UnaG to be Ca2+-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, Ca2+ ion. The affinity for BR differed by three orders of magnitude between the Ca2+-free state (Kdâ¯=â¯9.70â¯nM) and Ca2+-saturated state (Kdâ¯=â¯9.65⯵M). The chimeric protein can release nano- to micro-molar levels of BR with Ca2+ control, and is thus named BReleaCa (BR + releaser + Ca2+). Such a protein hybridization technique will be generally applicable to change the ligand binding properties of a variety of ligand-inducible functional proteins.
Assuntos
Sinalização do Cálcio/fisiologia , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Fluorescência/métodos , Cálcio/metabolismo , Calmodulina/química , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Ligantes , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Engenharia de Proteínas/métodosRESUMO
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 holoUnaG1 and holoUnaG2. The holoUnaG1 initially forms after binding BR and then changes to the brighter holoUnaG2 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 (holoUnaG1/holoUnaG2) 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 holoUnaG2.
Assuntos
Bilirrubina/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Relação Dose-Resposta a Droga , Ligantes , Cloreto de Sódio/farmacologia , Espectrometria de Fluorescência , Água/químicaRESUMO
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.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/análogos & derivados , Proteínas de Transporte/metabolismo , Profilinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fenômenos Biomecânicos , Cinética , Modelos Biológicos , Modelos Moleculares , Músculo Esquelético/metabolismo , Polimerização , Multimerização Proteica , CoelhosRESUMO
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.
Assuntos
Espectrometria de Massas em Tandem , Talidomida/química , Talidomida/metabolismo , Cromatografia Líquida , Hidrólise , Hidroxilação , EstereoisomerismoRESUMO
The cardiac pump function is a result of a rise in intracellular Ca2+ 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 â¼100 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 â¼100 nm on a beat-to-beat basis. Recent advances in optical technologies have enabled measurements of intracellular Ca2+ 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.
Assuntos
Imagem Molecular/métodos , Nanotecnologia/métodos , Sarcômeros/metabolismo , Animais , Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismoRESUMO
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 (â¼50%) of the magnitude of displacement because of the different timing of individual SL changes, regardless of the absence or presence of positive inotropy (via ß-adrenergic stimulation or enhanced actomyosin interaction). Finally, we find that ß-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.
Assuntos
Cálcio/metabolismo , Proteínas Luminescentes/fisiologia , Miócitos Cardíacos/citologia , Sarcômeros/metabolismo , Actinina/metabolismo , Animais , Animais Recém-Nascidos , Nanotecnologia , RatosRESUMO
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.
Assuntos
Frequência Cardíaca/fisiologia , Ventrículos do Coração/fisiopatologia , Sarcômeros/fisiologia , Animais , Diástole/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio , Miócitos Cardíacos/fisiologia , Sístole/fisiologiaRESUMO
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.
Assuntos
Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Sobrevivência Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião de Mamíferos/citologia , Embrião não Mamífero/citologia , Proteínas de Fluorescência Verde , Camundongos , Fótons , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Miocárdio/ultraestrutura , Pontos Quânticos , Sarcômeros/química , Animais , Camundongos , Imagem Molecular/métodos , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Sarcômeros/metabolismoRESUMO
As the dynamic properties of cardiac sarcomeres are markedly changed in response to a length change of even â¼0.1 µm, it is imperative to quantitatively measure sarcomere length (SL). Here we show a novel system using quantum dots (QDs) that enables a real-time measurement of the length of a single sarcomere in cardiomyocytes. First, QDs were conjugated with anti-α-actinin antibody and applied to the sarcomeric Z disks in isolated skinned cardiomyocytes of the rat. At partial activation, spontaneous sarcomeric oscillations (SPOC) occurred, and QDs provided a quantitative measurement of the length of a single sarcomere over the broad range (i.e., from â¼1.7 to â¼2.3 µm). It was found that the SPOC amplitude was inversely related to SL, but the period showed no correlation with SL. We then treated intact cardiomyocytes with the mixture of the antibody-QDs and FuGENE HD, and visualized the movement of the Z lines/T tubules. At a low frequency of 1 Hz, the cycle of the motion of a single sarcomere consisted of fast shortening followed by slow relengthening. However, an increase in stimulation frequency to 3-5 Hz caused a phase shift of shortening and relengthening due to acceleration of relengthening, and the waveform became similar to that observed during SPOC. Finally, the anti-α-actinin antibody-QDs were transfected from the surface of the beating heart in vivo. The striated patterns with â¼1.96-µm intervals were observed after perfusion under fluorescence microscopy, and an electron microscopic observation confirmed the presence of QDs in and around the T tubules and Z disks, but primarily in the T tubules, within the first layer of cardiomyocytes of the left ventricular wall. Therefore, QDs are a useful tool to quantitatively analyze the movement of single sarcomeres in cardiomyocytes, under various experimental settings.
Assuntos
Miócitos Cardíacos/fisiologia , Pontos Quânticos , Sarcômeros/fisiologia , Animais , Masculino , Contração Miocárdica/fisiologia , Miócitos Cardíacos/ultraestrutura , Ratos , Ratos Wistar , Sarcômeros/ultraestruturaRESUMO
Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.
Assuntos
Movimento Celular , Polaridade Celular , Epêndima/crescimento & desenvolvimento , Epêndima/metabolismo , Miosina Tipo II/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Cílios/metabolismo , Epêndima/citologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Varredura , Miosina Tipo II/genética , Biossíntese de ProteínasRESUMO
Using small-angle X-ray scattering (SAXS), we have studied the initial stage (nucleation and oligomerization) of actin polymerization induced by raising temperature in a stepwise manner from 1°C to 30°C at low ionic strength (4.0 mg ml-1 actin in G-buffer). The SAXS experiments were started from the mono-disperse G-actin state, which was confirmed by comparing the scattering pattern in q- and real space with X-ray crystallographic data. We observed that the forward scattering intensity I(q â 0), used as an indicator for the extent of poly-merization, began to increase at â¼14°C for Mg-actin and â¼20°C for Ca-actin, and this critical temperature did not depend on the nucleotide species, i.e., ATP or ADP. At the temperatures higher than â¼20°C for Mg-actin and â¼25°C for Ca-actin, the coherent reflection peak, which is attributed to the helical structure of F-actin, appeared. The pair-distance distribution functions, p(r), corresponding to the frequency of vector lengths (r) within the molecule, were obtained by the indirect Fourier transformation (IFT) of the scattering curves, I(q). Next, the size distributions of oligomers at each temperature were analyzed by fitting the experimentally obtained p(r) with the theoretical p(r) for the helical and linear oligomers (2-13mers) calculated based on the X-ray crystallographic data. We found that p(r) at the initial stage of polymerization was well accounted for by the superposition of monomer, linear/helical dimers, and helical trimer, being independent of the type of divalent cations and nucleotides. These results suggest that the polymerization of actin in G-buffer induced by an increase in temperature proceeds via the elongation of the helical trimer, which supports, in a structurally resolved manner, a widely believed hypothesis that the polymerization nucleus is a helical trimer.
RESUMO
Actin is a major component of the cytoskeleton that transmits mechanical stress in both muscle and nonmuscle cells. As the first step toward developing a "bio-nano strain gauge" that would be able to report the mechanical stress imposed on an actin filament, we quantitatively examined the fluorescence intensity of dyes attached to single actin filaments under various tensile forces (5-20 pN). Tensile force was applied via two optically trapped plastic beads covalently coated with chemically modified heavy meromyosin molecules that were attached to both end regions of an actin filament. As a result, we found that the fluorescence intensity of an actin filament, where 20% of monomers were labeled with tetramethylrhodamine (TMR)-5-maleimide at Cys(374) and the filamentous structure was stabilized with nonfluorescent phalloidin, decreased by approximately 6% per 10 pN of the applied force, whereas the fluorescence intensity of an actin filament labeled with either BODIPY TMR cadaverin-iodoacetamide at Cys(374) or rhodamine-phalloidin showed only an approximately 2% decrease per 10 pN of the applied force. On the other hand, spectroscopic measurements of actin solutions showed that the fluorescence intensity of TMR-actin increased 1.65-fold upon polymerization (G-F transformation), whereas that of BODIPY-actin increased only 1.06-fold. These results indicate that the external force distorts the filament structure, such that the microenvironment around Cys(374) approaches that in G-actin. We thus conclude that the fluorescent dye incorporated into an appropriate site of actin can report the mechanical distortion of the binding site, which is a necessary condition for the bio-nano strain gauge.
Assuntos
Citoesqueleto de Actina/metabolismo , Estresse Mecânico , Actinas/química , Actinas/metabolismo , Animais , Sítios de Ligação , Fenômenos Biomecânicos , Corantes Fluorescentes/metabolismo , Masculino , Microscopia , Coelhos , Espectrometria de FluorescênciaRESUMO
The interaction between actin and myosin V has been probed by measuring the unbinding force of individual actomyosin complexes using optical tweezers. Surprisingly, we found that in both the nucleotide-free and ADP-bound states single- and double-headed binding occurs with approximately the same probability. Estimation of the spring constant of individual actomyosin complexes confirmed that in each of the nucleotide states two distinct populations exist. These results confirm that optical nanometry can be used to reliably study the mechanism of how cytoskeleton molecular motors interact with their associated polymer lattices under solution conditions more closely resembling the intracellular environment.
RESUMO
Actin is a ubiquitous protein that is a major component of the cytoskeleton, playing an important role in muscle contraction and cell motility. At steady state, actin monomers and filaments (F-actin) coexist, and actin subunits continuously attach and detach at the filament ends. However, the size distribution of actin oligomers in F-actin solution has never been clarified. In this study, we investigated the size distribution of actin oligomers using photon-counting histograms. For this purpose, actin was labeled with a fluorescent dye, and the emitted photons were detected by confocal optics (the detection volume was of femtoliter (fL) order). Photon-counting histograms were analyzed to obtain the number distribution of actin oligomers in the detection area from their brightness, assuming that the brightness of an oligomer was proportional to the number of protomers. We found that the major populations at physiological ionic strength were 1-5mers. For data analysis, we successfully applied the theory of linear and helical aggregations of macromolecules. The model postulates three states of actin, i.e., monomers, linear polymers, and helical polymers. Here we obtained three parameters: the equilibrium constants for polymerization of linear polymers, K(l)=(5.2 +/- 1.1) x 10(6) M(-1), and helical polymers, K(h)=(1.6 +/- 0.5) x 10(7) M(-1); and the ratio of helical to linear trimers, gamma = (3.6 +/- 2.3) x 10(-2). The excess free energy of transforming a linear trimer to a helical trimer, which is assumed to be a nucleus for helical polymers, was calculated to be 2.0 kcal/mol. These analyses demonstrate that the oligomeric phase at steady state is predominantly composed of linear 1-5mers, and the transition from linear to helical polymers occurs on the level of 5-7mers.