Switch between large hand-over-hand and small inchworm-like steps in myosin VI.

Many biological motor molecules move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.

The Synergic Role of Actomyosin Architecture and Biased Detachment in Muscle Energetics: Insights in Cross Bridge Mechanism Beyond the Lever-Arm Swing.

Muscle energetics reflects the ability of myosin motors to convert chemical energy into mechanical energy. How this process takes place remains one of the most elusive questions in the field. Here, we combined experimental measurements of in vitro sliding velocity based on DNA-origami built filaments carrying myosins with different lever arm length and Monte Carlo simulations based on a model which accounts for three basic components: (i) the geometrical hindrance, (ii) the mechano-sensing mechanism, and (iii) the biased kinetics for stretched or compressed motors. The model simulations showed that the geometrical hindrance due to acto-myosin spatial mismatching and the preferential detachment of compressed motors are synergic in generating the rapid increase in the ATP-ase rate from isometric to moderate velocities of contraction, thus acting as an energy-conservation strategy in muscle contraction. The velocity measurements on a DNA-origami filament that preserves the motors' distribution showed that geometrical hindrance and biased detachment generate a non-zero sliding velocity even without rotation of the myosin lever-arm, which is widely recognized as the basic event in muscle contraction. Because biased detachment is a mechanism for the rectification of thermal fluctuations, in the Brownian-ratchet framework, we predict that it requires a non-negligible amount of energy to preserve the second law of thermodynamics. Taken together, our theoretical and experimental results elucidate less considered components in the chemo-mechanical energy transduction in muscle.

Theoretical modeling reveals that regulatory T cells increase T-cell interaction with antigen-presenting cells for stable immune tolerance.

The immune system in tolerance maintains cell diversity without responding to self-antigens. Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) inhibit T-cell activation through various molecular mechanisms. However, several key questions are still not resolved, including how Tregs control the immune response on the basis of their self-skewed T-cell receptor repertoire and how Tregs avoid impeding relevant immunity against pathogens. Here, we show that Tregs promote the proliferation of conventional T cells in the presence of excessive co-stimulation when murine T cells are stimulated in vitro with allogeneic antigen-presenting cells (APCs). Antigen-specific Tregs increase the number of cells interacting with dendritic cells (DCs) by increasing the number of viable DCs and the expression of adhesion molecules on DCs. Theoretical simulations and mathematical models representing the dynamics of T-APC interaction and T-cell numbers in a lymph node indicate that Tregs reduce the dissociation probability of T cells from APCs and increase the new association. These functions contribute to tolerance by enhancing the interaction of low-affinity T cells with APCs. Supporting the theoretical analyses, we found that reducing the T-cell numbers in mice increases the ratio of specific T cells among CD4+ T cells after immunization and effectively induces autoimmune diabetes in non obese diabetes mice. Thus, as a critical function, antigen-specific Tregs stabilize the immune state, irrespective of it being tolerant or responsive, by augmenting T-APC interaction. We propose a novel regulation model in which stable tolerance with large heterogeneous populations proceeds to a specific immune response through a transient state with few populations.

Live single cell mass spectrometry reveals cancer-specific metabolic profiles of circulating tumor cells.

Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics-based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type.

Single-Cell Screening of Tamoxifen Abundance and Effect Using Mass Spectrometry and Raman-Spectroscopy.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.

Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging.

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s-1). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1 s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations. SIGNIFICANCE STATEMENT: The transcription factor, cAMP response element-binding protein (CREB) is known to regulate gene expression in neuronal activity-dependent processes. However, its spatiotemporal interactions with the genome remain unknown. Single-molecule imaging in cortical neurons revealed that fluorescent-tagged CREB spots frequently reside at fixed nuclear locations in the time range of several seconds. Neuronal activity had little effect on the CREB residence time, but increased the rapid and frequent reappearance of long-residence CREB spots at the same nuclear locations. Thus, activity-dependent transcription is attributable to frequent binding of CREB to specific genome loci.

Including Thermal Fluctuations in Actomyosin Stable States Increases the Predicted Force per Motor and Macroscopic Efficiency in Muscle Modelling.

Muscle contractions are generated by cyclical interactions of myosin heads with actin filaments to form the actomyosin complex. To simulate actomyosin complex stable states, mathematical models usually define an energy landscape with a corresponding number of wells. The jumps between these wells are defined through rate constants. Almost all previous models assign these wells an infinite sharpness by imposing a relatively simple expression for the detailed balance, i.e., the ratio of the rate constants depends exponentially on the sole myosin elastic energy. Physically, this assumption corresponds to neglecting thermal fluctuations in the actomyosin complex stable states. By comparing three mathematical models, we examine the extent to which this hypothesis affects muscle model predictions at the single cross-bridge, single fiber, and organ levels in a ceteris paribus analysis. We show that including fluctuations in stable states allows the lever arm of the myosin to easily and dynamically explore all possible minima in the energy landscape, generating several backward and forward jumps between states during the lifetime of the actomyosin complex, whereas the infinitely sharp minima case is characterized by fewer jumps between states. Moreover, the analysis predicts that thermal fluctuations enable a more efficient contraction mechanism, in which a higher force is sustained by fewer attached cross-bridges.

Single molecule detection, thermal fluctuation and life.

Single molecule detection has contributed to our understanding of the unique mechanisms of life. Unlike artificial man-made machines, biological molecular machines integrate thermal noises rather than avoid them. For example, single molecule detection has demonstrated that myosin motors undergo biased Brownian motion for stepwise movement and that single protein molecules spontaneously change their conformation, for switching to interactions with other proteins, in response to thermal fluctuation. Thus, molecular machines have flexibility and efficiency not seen in artificial machines.

Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis.

Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light-induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light's intensity and position.

Direct visualization of secondary structures of F-actin by electron cryomicroscopy.

F-actin is a helical assembly of actin, which is a component of muscle fibres essential for contraction and has a crucial role in numerous cellular processes, such as the formation of lamellipodia and filopodia, as the most abundant component and regulator of cytoskeletons by dynamic assembly and disassembly (from G-actin to F-actin and vice versa). Actin is a ubiquitous protein and is involved in important biological functions, but the definitive high-resolution structure of F-actin remains unknown. Although a recent atomic model well reproduced X-ray fibre diffraction intensity data from a highly oriented liquid-crystalline sol specimen, its refinement without experimental phase information has certain limitations. Direct visualization of the structure by electron cryomicroscopy, however, has been difficult because it is relatively thin and flexible. Here we report the F-actin structure at 6.6 Å resolution, made obtainable by recent advances in electron cryomicroscopy. The density map clearly resolves all the secondary structures of G-actin, such as α-helices, ß-structures and loops, and makes unambiguous modelling and refinement possible. Complex domain motions that open the nucleotide-binding pocket on F-actin formation, specific D-loop and terminal conformations, and relatively tight axial but markedly loose interprotofilament interactions hydrophilic in nature are revealed in the F-actin model, and all seem to be important for dynamic functions of actin.

Local heat activation of single myosins based on optical trapping of gold nanoparticles.

Myosin is a mechano-enzyme that hydrolyzes ATP in order to move unidirectionally along actin filaments. Here we show by single molecule imaging that myosin V motion can be activated by local heat. We constructed a dark-field microscopy that included optical tweezers to monitor 80 nm gold nanoparticles (GNP) bound to single myosin V molecules with nanometer and submillisecond accuracy. We observed 34 nm processive steps along actin filaments like those seen when using 200 nm polystyrene beads (PB) but dwell times (ATPase activity) that were 4.5 times faster. Further, by using DNA nanotechnology (DNA origami) and myosin V as a nanometric thermometer, the temperature gradient surrounding optically trapped GNP could be estimated with nanometer accuracy. We propose our single molecule measurement system should advance quantitative analysis of the thermal control of biological and artificial systems like nanoscale thermal ratchet motors.

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V.

Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.

mNUDC is required for plus-end-directed transport of cytoplasmic dynein and dynactins by kinesin-1.

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein-LIS1-microtubule complex in a kinesin-1-dependent manner. However, the underlying mechanism by which a cytoplasmic dynein-LIS1-microtubule complex binds kinesin-1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin-1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin-1. mNUDC is also required for anterograde transport of a dynactin-containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin-1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin-1 and supports their transport by kinesin-1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin-1.

Four-dimensional spatial nanometry of single particles in living cells using polarized quantum rods.

Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule's function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees.

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility.

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

Self-organization of the phosphatidylinositol lipids signaling system for random cell migration.

Phosphatidylinositol (PtdIns) lipids have been identified as key signaling mediators for random cell migration as well as chemoattractant-induced directional migration. However, how the PtdIns lipids are organized spatiotemporally to regulate cellular motility and polarity remains to be clarified. Here, we found that self-organized waves of PtdIns 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] are generated spontaneously on the membrane of Dictyostelium cells in the absence of a chemoattractant. Characteristic oscillatory dynamics within the PtdIns lipids signaling system were determined experimentally by observing the phenotypic variability of PtdIns lipid waves in single cells, which exhibited characteristics of a relaxation oscillator. The enzymes phosphatase and tensin homolog (PTEN) and phosphoinositide-3-kinase (PI3K), which are regulators for PtdIns lipid concentrations along the membrane, were essential for wave generation whereas functional actin cytoskeleton was not. Defects in these enzymes inhibited wave generation as well as actin-based polarization and cell migration. On the basis of these experimental results, we developed a reaction-diffusion model that reproduced the characteristic relaxation oscillation dynamics of the PtdIns lipid system, illustrating that a self-organization mechanism provides the basis for the PtdIns lipids signaling system to generate spontaneous spatiotemporal signals for random cell migration and that molecular noise derived from stochastic fluctuations within the signaling components gives rise to the variability of these spontaneous signals.

Myosin-V as a mechanical sensor: an elastic network study.

According to recent experiments, the molecular-motor myosin behaves like a strain sensor, exhibiting different functional responses when loads in opposite directions are applied to its tail. Within an elastic-network model, we explore the sensitivity of the protein to the forces acting on the tail and find, in agreement with experiments, that such forces invoke conformational changes that should affect filament binding and ADP release. Furthermore, conformational responses of myosin to the application of forces to individual residues in its principal functional regions are systematically investigated and a detailed sensitivity map of myosin-V is thus obtained. The results suggest that the strain-sensor behavior is involved in the intrinsic operation of this molecular motor.

Activation kinetics of RAF protein in the ternary complex of RAF, RAS-GTP, and kinase on the plasma membrane of living cells: single-molecule imaging analysis.

RAS is an important cell signaling molecule, regulating the activities of various effector proteins, including the kinase c-RAF (RAF). Despite the critical function of RAS signaling, the activation kinetics have not been analyzed experimentally in living cells for any of the RAS effectors. Here, we analyzed the kinetics of RAF activation on the plasma membrane in living HeLa cells after stimulation with EGF to activate RAS. RAF is recruited by the active form of RAS (RAS-GTP) from the cytoplasm to the plasma membrane through two RAS-binding sites (the RAS-binding domain and the cysteine-rich domain (CRD)) and is activated by its phosphorylation by still undetermined kinases on the plasma membrane. Using single-molecule imaging, we measured the dissociation time courses of GFP-tagged molecules of wild type RAF and fragments or mutants of RAF containing one or two of the three functional domains (the RAS-binding domain, the CRD, and the catalytic domain) to determine their interaction with membrane components. Each molecule showed a unique dissociation time course, indicating that both its interaction with RAS-GTP and its phosphorylation by the kinases are rate-limiting steps in RAF activation. Based on our experimental results, we propose a kinetic model for the activation of RAF. The model suggests the importance of the interaction between RAS-GTP and CRD for the effective activation of RAF, which is triggered by rapid RAS-GTP-induced conformational changes in RAF and the subsequent presentation of RAF to the kinase. The model also suggests necessary properties of the kinases that activate RAF.

A protein switch with tunable steepness reconstructed in Escherichia coli cells with eukaryotic signaling proteins.

An important goal of synthetic biology is to construct reaction circuits with artificial responses by assembling modulated biological elements into living cells. While many such attempts have been based upon the cellular transcriptional apparatus, the use of the post-translational machinery remains relatively rare. Here we report the reconstruction in Escherichia coli of a protein-based artificial module based upon elements of a eukaryotic cell signaling pathway. The module shows a switch-like ultrasensitive response, using the opposing functions of a protein kinase and a phosphatase. The switch is acutely responsive to the kinase:phosphatase ratio, and can be modulated as a function of the expression level of the substrate. We can theoretically predict the response of this module and can control its steepness based on these predictions. Future work will demonstrate the potential of this controllable protein-based switch to be incorporated into artificial circuits.

Simultaneous observation of the lever arm and head explains myosin VI dual function.

Myosin VI is an adenosine triphosphate (ATP)-driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand-over-hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.