Tensile Loads on Tethered Actin Filaments Induce Accumulation of Cell Adhesion-Associated Proteins in Vitro.

Focal adhesions (FAs) and adherens junctions (AJs), which serve as a mechanical interface of cell-matrix and cell-cell interactions, respectively, experience tensile force either originating from the deformation of the surrounding tissues or generated by the actomyosin machinery in the cell. These mechanical inputs cause enlargement of FAs and AJs, while the detailed mechanism for the force-dependent development of FAs and AJs remain unclear. Both FAs and AJs provide sites for tethering of actin filaments and actin polymerization. Here, we develop a cell-free system, in which actin filaments are tethered to glass surfaces, and show that application of tensile force to the tethered filaments in the cell extract induces accumulation of several FA and AJ proteins, associated with further accumulation of actin filaments via de novo actin polymerization. Decline in the tensile force results in a decrease in the amount of the accumulated proteins. These results suggest that the tensile force acting on the tethered actin filaments plays a crucial role in the accumulation of FA and AJ proteins.

Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics.

Single-molecule imaging and kinetic analysis of cooperative cofilin-actin filament interactions.

The actin filament-severing protein actin depolymerizing factor (ADF)/cofilin is ubiquitously distributed among eukaryotes and modulates actin dynamics. The cooperative binding of cofilin to actin filaments is crucial for the concentration-dependent unconventional modulation of actin dynamics by cofilin. In this study, the kinetic parameters associated with the cooperative binding of cofilin to actin filaments were directly evaluated using a single-molecule imaging technique. The on-rate of cofilin binding to the actin filament was estimated to be 0.06 µM(-1)⋅s(-1) when the cofilin concentration was in the range of 30 nM to 1 µM. A dwell time histogram of cofilin bindings decays exponentially to give an off-rate of 0.6 s(-1). During long-term cofilin binding events (>0.4 s), additional cofilin bindings were observed in the vicinity of the initial binding site. The on-rate for these events was 2.3-fold higher than that for noncontiguous bindings. Super-high-resolution image analysis of the cofilin binding location showed that the on-rate enhancement occurred within 65 nm of the original binding event. By contrast, the cofilin off-rate was not affected by the presence of prebound cofilin. Neither decreasing the temperature nor increasing the viscosity of the test solution altered the on-rates, off-rates, or the cooperative parameter (ω) of the binding. These results indicate that cofilin binding enhances additional cofilin binding in the vicinity of the initial binding site (ca. 24 subunits), but it does not affect the off-rate, which could be the molecular mechanism of the cooperative binding of cofilin to actin filaments.

Non-channel mechanosensors working at focal adhesion-stress fiber complex.

Mechanosensitive ion channels (MSCs) have long been the only established molecular class of cell mechanosensors; however, in the last decade, a variety of non-channel type mechanosensor molecules have been identified. Many of them are focal adhesion-associated proteins that include integrin, talin, and actin. Mechanosensors must be non-soluble molecules firmly interacting with relatively rigid cellular structures such as membranes (in terms of lateral stiffness), cytoskeletons, and adhesion structures. The partner of MSCs is the membrane in which MSC proteins efficiently transduce changes in the membrane tension into conformational changes that lead to channel opening. By contrast, the integrin, talin, and actin filament form a linear complex of which both ends are typically anchored to the extracellular matrices via integrins. Upon cell deformation by forces, this structure turns out to be a portion that efficiently transduces the generated stress into conformational changes of composite molecules, leading to the activation of integrin (catch bond with extracellular matrices) and talin (unfolding to induce vinculin bindings). Importantly, this structure also serves as an "active" mechanosensor to detect substrate rigidity by pulling the substrate with contraction of actin stress fibers (SFs), which may induce talin unfolding and an activation of MSCs in the vicinity of integrins. A recent study demonstrates that the actin filament acts as a mechanosensor with unique characteristics; the filament behaves as a negative tension sensor in which increased torsional fluctuations by tension decrease accelerate ADF/cofilin binding, leading to filament disruption. Here, we review the latest progress in the study of those non-channel mechanosensors and discuss their activation mechanisms and physiological roles.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions.

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

Analyses of a gravistimulation-specific Ca2+ signature in Arabidopsis using parabolic flights.

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca(2+)]c). However, the [Ca(2+)]c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca(2+) response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10(-4)g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca(2+)]c increase, which corresponds closely to the second sustained [Ca(2+)]c increase observed in ground experiments. The [Ca(2+)]c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g-2g) into Ca(2+) signals on a subsecond time scale.

Force- and Ca²âº-dependent internalization of integrins in cultured endothelial cells.

The effects of mechanical force applied to the integrin clusters at focal contacts were examined in cultured human umbilical vein endothelial cells. When a fibronectin-coated glass bead was attached to the apical cell surface, focal contacts formed beneath the bead that became linked to focal contacts at the basal cell membrane by actin stress fibers in 5 minutes. Integrin dynamics at the basal focal contacts were monitored in live cells in response to a localized mechanical stimulus generated by displacing the glass bead. Traction force transmitted to the basal focal contacts through the stress fibers was monitored by measuring the deformation of the polyacrylamide gel substratum. The force declined in a few seconds, probably owing to decreases in the elastic modulus of the stress fibers. This transient mechanical stimulus caused the dephosphorylation of paxillin and disassembly of integrin clusters at the basal cell membrane in 20 minutes. The disassembly was mediated mainly by clathrin-dependent endocytosis of integrins. The integrin internalization was inhibited in Ca(2+)- and K(+)-free solution, and by phenylarsine oxide, a phosphatase inhibitor. These results suggest that a transient mechanical stimulus applied to focal contacts induces Ca(2+)-dependent dephosphorylation of some proteins, including paxillin, and facilitates clathrin-dependent endocytosis of integrins.

Surface-dependent quenching of Qdot emission can be a new tool for high resolution measurements.

Single quantum dots (Qdots) are often used in the field of single-molecule imaging. Qdots are sensitive to changes in the physical interactions between the Qdots and the surrounding materials. However, the spectral changes in a single Qdot emission have not been studied in detail. Low-temperature plasma treatment of glass surfaces reduced the intensity of the 655 nm emission peak of Qdot655 on glass surfaces, but did not significantly change the intensity of the 580 nm emission. Silanization of the glass surface increases the thickness of the silane layer, and the 655 nm emission peak increased. When single Qdots on the untreated glass were imaged, plasma treatment decreased the intensity of red emission and increased yellow emission. When Qdots were brought close to the glass surface in the range of 28-0 nm, the red emission intensity decreased and the yellow emission intensity increased slightly. When single actin filaments were labeled with Qdots, fluctuations of the yellow and red emission of the Qdot were detected, which reflected the very small distance changes. Our results indicate that the local interaction of Qdots with the glass surface improves the spatial and temporal resolution of optical measurements of biomolecules labeled with Qdots.

Mechanical Stress Decreases the Amplitude of Twisting and Bending Fluctuations of Actin Filaments.

A variety of biological roles of mechanical forces have been proposed in cell biology, such as cell signaling pathways for survival, development, growth, and differentiation. Mechanical forces alter the mechanical conditions within cells and their environment, which strongly influences the reorganization of the actin cytoskeleton. Single-molecule imaging studies of actin filaments have led to the hypothesis that the actin filament acts as a mechanosensor; e.g., increases in actin filament tension alter their conformation and affinity for regulatory proteins. However, our understanding of the molecular mechanisms underlying how tension modulates the mechanical behavior of a single actin filament is still incomplete. In this study, a direct measurement of the twisting and bending of a fluorescently labeled single actin filament under different tension levels by force application (0.8-3.4 pN) was performed using single-molecule fluorescence polarization (SMFP) microscopy. The results showed that the amplitude of twisting and bending fluctuations of a single actin filament decreased with increasing tension. Electron micrograph analysis of tensed filaments also revealed that the fluctuations in the crossover length of actin filaments decreased with increasing filament tension. Possible molecular mechanisms underlying these results involving the binding of actin-binding proteins, such as cofilin, to the filament are discussed.

Amyloid-ß slows cilia movement along the ventricle, impairs fluid flow, and exacerbates its neurotoxicity in explant culture.

Alzheimer's disease (AD) is characterized by extensive and selective death of neurons and deterioration of synapses and circuits in the brain. The Aß1-42 concentration is higher in an AD brain than in cognitively normal elderly individuals, and Aß1-42 exhibits neurotoxicity. Brain-derived Aß is transported into the cerebrospinal fluid (CSF), and CSF flow is driven in part by the beating of cilia and CSF secretion into ventricles. Ventricles are lined with ependyma whose apical surface is covered with motile cilia. Herein, we constructed an experimental system to measure the movement of ependymal cilia and examined the effects of Aß1-42 to the beating of cilia and neurons. The circadian rhythm of the beating frequency of ependymal cilia was detected using brain wall explant-cultures containing ependymal cilia and neurons; the beating frequency was high at midday and low at midnight. Aß1-42 decreased the peak frequency of ciliary beating at midday and slightly increased it at midnight. Aß1-42 exhibited neurotoxicity to neurons on the non-ciliated side of the explant culture, while the neurotoxicity was less evident in neurons on the ciliated side. The neurotoxic effect of Aß1-42 was diminished when 1 mPa of shear stress was generated using a flow chamber system that mimicked the flow by cilia. These results indicate that Aß1-42 affects the circadian rhythm of ciliary beating, decreases the medium flow by the cilia-beating, and enhances the neurotoxic action of Aß1-42 in the brain explant culture.

Entanglement of Arabidopsis Seedlings to a Mesh Substrate under Microgravity Conditions in KIBO on the ISS.

The International Space Station (ISS) provides a precious opportunity to study plant growth and development under microgravity (micro-G) conditions. In this study, four lines of Arabidopsis seeds (wild type, wild-type MCA1-GFP, mca1-knockout, and MCA1-overexpressed) were cultured on a nylon lace mesh placed on Gelrite-solidified MS-medium in the Japanese experiment module KIBO on the ISS, and the entanglement of roots with the mesh was examined under micro-G and 1-G conditions. We found that root entanglement with the mesh was enhanced, and root coiling was induced under the micro-G condition. This behavior was less pronounced in mca1-knockout seedlings, although MCA1-GFP distribution at the root tip of the seedlings was nearly the same in micro-G-grown seedlings and the ground control seedlings. Possible involvement of MCA1 in the root entanglement is discussed.

The gravistimulation-induced very slow Ca2+ increase in Arabidopsis seedlings requires MCA1, a Ca2+-permeable mechanosensitive channel.

Gravity is a critical environmental factor affecting the morphology and function of plants on Earth. Gravistimulation triggered by changes in the gravity vector induces an increase in the cytoplasmic free calcium ion concentration ([Ca2+]c) as an early process of gravity sensing; however, its role and molecular mechanism are still unclear. When seedlings of Arabidopsis thaliana expressing apoaequorin were rotated from the upright position to the upside-down position, a biphasic [Ca2+]c-increase composed of a fast-transient [Ca2+]c-increase followed by a slow [Ca2+]c-increase was observed. We find here a novel type [Ca2+]c-increase, designated a very slow [Ca2+]c-increase that is observed when the seedlings were rotated back to the upright position from the upside-down position. The very slow [Ca2+]c-increase was strongly attenuated in knockout seedlings defective in MCA1, a mechanosensitive Ca2+-permeable channel (MSCC), and was partially restored in MCA1-complemented seedlings. The mechanosensitive ion channel blocker, gadolinium, blocked the very slow [Ca2+]c-increase. This is the first report suggesting the possible involvement of MCA1 in an early event related to gravity sensing in Arabidopsis seedlings.

Structural changes in the cytoplasmic domain of the mechanosensitive channel MscS during opening.

The bacterial mechanosensitive channel MscS forms a homoheptamer of subunits composed of a transmembrane (TM) domain and a large cytoplasmic (CP) domain. Recent studies suggest that a lateral expansion of the TM domain, structural change in the CP domain, and TM-CP interactions are essential to open the channel. However, it has not been examined whether the CP domain undergoes structural changes during channel opening. The aim of this study was to estimate structural changes in the CP domain during channel opening using fluorescence resonance energy transfer (FRET) spectroscopy. To monitor changes in the horizontal diameter of the CP domain, four point mutants (A132C, F178C, L246C, and R259C), all of which had channel activity, were created and labeled with Alexa488 and Alexa568 for FRET analysis. The FRET efficiency of these mutants decreased when lysophosphatidylcholine was applied to open the channel, suggesting that the CP domain swells up when the channel opens. The degree of the decease in FRET efficiency after lysophosphatidylcholine treatment was smaller in the D62N/F178C mutant, which was deficient in the TM-CP interactions, than in the F178C mutant. These findings provide the first, to our knowledge, experimental evidence that the CP domain swells up during channel opening, and the swelling is mediated by the TM-CP interactions.

Real-Time Single-Molecule Kinetic Analyses of AIP1-Enhanced Actin Filament Severing in the Presence of Cofilin.

Rearrangement of actin filaments by polymerization, depolymerization, and severing is important for cell locomotion, membrane trafficking, and many other cellular functions. Cofilin and actin-interacting protein 1 (AIP1; also known as WDR1) are evolutionally conserved proteins that cooperatively sever actin filaments. However, little is known about the biophysical basis of the actin filament severing by these proteins. Here, we performed single-molecule kinetic analyses of fluorescently labeled AIP1 during the severing process of cofilin-decorated actin filaments. Results demonstrated that binding of a single AIP molecule was sufficient to enhance filament severing. After AIP1 binding to a filament, severing occurred with a delay of 0.7 s. Kinetics of binding and dissociation of a single AIP1 molecule to/from actin filaments followed a second-order and a first-order kinetics scheme, respectively. AIP1 binding and severing were detected preferentially at the boundary between the cofilin-decorated and bare regions on actin filaments. Based on the kinetic parameters explored in this study, we propose a possible mechanism behind the enhanced severing by AIP1.

Dynamics of actin filaments during tension-dependent formation of actin bundles.

The actin cytoskeleton stress fiber is an actomyosin-based contractile structure seen as a bundle of actin filaments. Although tension development in a cell is believed to regulate stress fiber formation, little is known for the underlying biophysical mechanisms. To address this question, we examined the effects of tension on the behaviors of individual actin filaments during stress fiber (actin bundle) formation using cytosol-free semi-intact fibroblast cells that were pre-treated with the Rho kinase inhibitor Y-27632 to disassemble stress fibers into a meshwork of actin filaments. These filaments were sparsely labeled with quantum dots for live tracking of their motions. When ATP and Ca(2+) were applied to the semi-intact cells to generate actomyosin-based forces, actin meshwork in the protruded lamellae was dragged toward the cell body, while the periphery of the meshwork remained in the original region, indicating that centripetally directed tension developed in the meshwork. Then the individual actin filaments in the meshwork moved towards the cell body accompanied with sudden changes in the direction of their movements, finally forming actin bundles along the direction of tension. Dragging the meshwork by externally applied mechanical forces also exerted essentially the same effects. These results suggest the existence of tension-dependent remodeling of cross-links within the meshwork during the rearrangement of actin filaments, thus demonstrating that tension is a key player to regulate the dynamics of individual actin filaments that leads to actin bundle formation.

Mechano-sensitive channels regulate the stomatal aperture in Vicia faba.

In the bright fields, stomata of the plants are fully opened to raise the transpiration rate and CO(2) uptake required for photosynthesis. Stomatal opening is driven by the activation of plasma membrane H(+)-ATPase and K(+)(in) channels, and the Ca(2+)-dependent inactivation and blockage of both components were supposed to be inevitable function to regulate the stomatal aperture. Although, it is still obscure how these activities are regulated at the open state. Application of an amphipathic membrane creator, trinitrophenol (TNP), instantly generates the convex curvature in the plasma membrane, which occurs in the phases of stomatal opening and closure. TNP surely activates mechanosensitive Ca(2+)-permeable channels and attenuates the promotion of stomatal opening, but does not inhibit and promote stomatal closure. These results suggest that activation of mechanosensitive Ca(2+)-permeable channels regulates the opening phase of stomata in plants.

Functional characterization of calcium channels localized on the growth cones of cultured rat dorsal root ganglion cells.

Ca2+ channels on growth cones of cultured rat dorsal root ganglion (DRG) neurons were functionally characterized with an optical method using Fura-2. An increase in intracellular calcium concentration ([Ca2+](i)) of the growth cone was induced in response to electrical stimulation to the DRG cell body. The ([Ca2+](i))-increase was partly inhibited by either of omega - conotoxin GVIA (omega - CgTx, 3 microM) or omega - agatoxin IVA (omega - aga IVA, 300 nM) and completely blocked by both present at the same time, but was not affected by nicardipine (30 microM). The omega - CgTx - as well as omega - aga IVA - sensitive Ca2+ channels were immunologically localized on the growth cones using field emission scanning electron microscopy. It is concluded that the omega - CgTx - as well as omea - aga IVA - sensitive Ca2+ channels are involved in the ([Ca2+](i))-increase in the growth cones of the cultured DRG neurons, leading to glutamate release before synapse formation.

Calcium mobilizations in response to changes in the gravity vector in Arabidopsis seedlings: possible cellular mechanisms.

Gravity influences the growth direction of higher plants. Changes in the gravity vector (gravistimulation) immediately promote the increase in the cytoplasmic free calcium ion concentration ([Ca(2+)]c) in Arabidopsis (Arabidopsis thaliana) seedlings. When the seedlings are gravistimulated by reorientation at 180°, a transient two peaked (biphasic) [Ca(2+)]c-increase arises in their hypocotyl and petioles. Parabolic flights (PFs) can generate a variety of gravity-stimuli, and enables us to measure gravity-induced [Ca(2+)]c-increases without specimen rotation, which demonstrate that Arabidopsis seedlings possess a rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes into Ca(2+) signals on a sub-second timescale. Hypergravity by centrifugation (20 g or 300 g) also induces similar transient [Ca(2+)]c-increases. In this review, we propose models for possible cellular processes of the garavi-stimulus-induced [Ca(2+)]c-increase, and evaluate those by examining whether the model fits well with the kinetic parameters derived from the [Ca(2+)]c-increases obtained by applying gravistimulus with different amplitudes and time sequences.

Mechano-sensing by actin filaments and focal adhesion proteins.

Mechanosensitive ion channels have long been the only established molecular class of cell mechanosensors with known molecular entities. However, recent advances in the state-of-the-art techniques, including single-molecule manipulation and imaging, have enabled an investigation of non-channel type cell mechanosensors and the underlying biophysical mechanisms of their activation. To date, two focal adhesion proteins, talin and p130Cas, have been postulated to act as putative mechanosensors, acting through mechano-induced unfolding of their particular soft domain(s) susceptible to phosphorylation. More recently, the actin filament has been demonstrated to act as a mechanosensor in the presence of the soluble actin-severing protein, cofilin. The cofilin severing activity negatively depends on the tension in the actin filament through tension-dependent binding/unbinding of cofilin to/from the actin filament. As a result, relaxed actin filaments are severed, while tensed ones are either not severed or severed after a long delay. Here we review the latest progress in the mechanosensing by non-channel type proteins and discuss the possible physiological roles of the mechanosensing performed by actin filaments in the course of cell migration.

Expression of Arabidopsis MCA1 enhanced mechanosensitive channel activity in the Xenopus laevis oocyte plasma membrane.

Higher plants sense and respond to osmotic and mechanical stresses such as turgor, touch, flexure and gravity. Mechanosensitive (MS) channels, directly activated by tension in the cell membrane and cytoskeleton, are supposed to be involved in the cell volume regulation under hypotonic conditions and the sensing of these mechanical stresses based on electrophysiological and pharmacological studies. However, limited progress has been achieved in the molecular identification of plant MS channels. Here, we show that MCA1 (mid1-complementing activity 1; a putative mechanosensitive Ca ( 2+) -permeable channel in Arabidopsis thaliana) increased MS channel activity in the plasma membrane of Xenopus laevis oocytes. The functional and kinetic properties of MCA1 were examined by using a Xenopus laevis oocytes expression system, which showed that MCA1-dependent MS cation currents were activated by hypo-osmotic shock or by membrane stretch produced by pipette suction. Single-channel analyses suggest that MCA1 encodes a possible MS channel with a conductance of 34 pS.