Mechanochemical Transduction Research Articles

Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells

The molecular mechanisms by which bone cells transduce mechanical stimuli into intracellular biochemical responses have yet to be established. There is evidence that mechanical stimulation acts synergistically with parathyroid hormone PTH(1-34) in mediating bone growth. Using picosecond time-resolved fluorescence microscopy and G protein-coupled receptor conformation-sensitive fluorescence resonance energy transfer (FRET), we investigated conformational transitions in parathyroid hormone type 1 receptor (PTH1R). 1) A genetically engineered PTH1R sensor containing an intramolecular FRET pair was constructed that enabled detection of conformational activity of PTH1R in single cells. 2) The nature of ligand-dependent conformational change of PTH1R depends on the type of ligand: stimulation with the PTH(1-34) leads to conformational transitions characterized by decrease in FRET efficiency while NH(2)-terminal truncated ligand PTH(3-34) stimulates conformational transitions characterized by higher FRET efficiencies. 3) Stimulation of murine preosteoblastic cells (MC3T3-E1) with fluid shear stress (FSS) leads to significant changes in conformational equilibrium of the PTH1R in MC3T3-E1 cells, suggesting that mechanical perturbation of the plasma membrane leads to ligand-independent response of the PTH1R. Conformational transitions induced by mechanical stress were characterized by an increase in FRET efficiency, similar to those induced by the NH(2)-terminal truncated ligand PTH(3-34). The response to the FSS stimulation was inhibited in the presence of PTH(1-34) in the flow medium. These results indicate that the FSS can modulate the action of the PTH(1-34) ligand. 4) Plasma membrane fluidization using benzyl alcohol or cholesterol extraction also leads to conformational transitions characterized by increased FRET levels. We therefore suggest that PTH1R is involved in mediating primary mechanochemical signal transduction in MC3T3-E1 cells.

NSC 622124 Inhibits Human Eg5 and Other Kinesins via Interaction with the Conserved Microtubule-Binding Site

Kinesin-5 proteins are essential for formation of a bipolar mitotic spindle in most and, perhaps all, eukaryotic cells. Several Kinesin-5 proteins, notably the human version, HsEg5, are targets of a constantly expanding group of small-molecule inhibitors, which hold promise both as tools for probing mechanochemical transduction and as anticancer agents. Although most such compounds are selective for HsEg5 and closely related Kinesin-5 proteins, some, such as NSC 622124, exhibit activity against at least one kinesin from outside the Kinesin-5 family. Here we show NSC 622124, despite identification in a screen that yielded inhibitors now known to target the HsEg5 monastrol-binding site, does not compete with [(14)C]monastrol for binding to HsEg5 and is able to inhibit the basal and microtubule-stimulated ATPase activity of the monastrol-insensitive Kinesin-5, KLP61F. NSC 622124 competes with microtubules, but not ATP, for interaction with HsEg5 and disrupts the microtubule binding of HsEg5, KLP61F, and Kinesin-1. Proteolytic degradation of an HsEg5.NSC622124 complex revealed that segments of the alpha3 and alpha5 helices map to the inhibitor-binding site. Overall, our results demonstrate that NSC 622124 targets the conserved microtubule-binding site of kinesin proteins. Further, unlike compounds previously reported to target the kinesin microtubule-binding site, NSC 622124 does not produce any enhancement of basal ATPase activity and thus acts solely as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators (i.e., microtubules). Our work emphasizes the concept that microtubule-dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.

New Mechanism of Actin Activation of Myosin

Actin activation of myosin ATPase activity is a general property of actomyosin systems, however, its role in the mechanochemical transduction is still not unveiled. Recently, we showed that in the absence of actin the rate limiting step of the ATPase cycle is not the phosphate release step but the preceding conformational change and actin activates directly this step to constitute the power stroke. Here we report that the rate of the power stroke step is initiated directly by the interaction of actin and the prolin rich (PR) loop which is located in the proximal part of the relay helix. Deletion of the PR loop does not change any of the kinetic properties of Dictyostelium myosin motor domain in the absence of actin. Actin binding of the PR loop deleted mutant decreased only three fold in rigor and in the presence of nucleotides. Also, ATP induced actin dissociation was only slightly affected by the deletion. Nevertheless, we found that PR loop deletion caused dramatic effect on the actin activation: basal ATPase activity is not increased by actin. Surprisingly, the motility is not reduced if this interaction between the PR loop and the N-terminus of actin is abolished. These results indicate a new communication pathway in myosin. Based on the seesaw model of the relay helix movement, actin pulls the PR loop which directly moves the relay region and accelerates the rate of the power stroke. The kinetic scheme suggests that actin activation is not required directly for the motor functions and motility. The increased ATPase biases the kinetic paths towards the actin bound forms even in weak actin bound states, thus this effect reduces the possibility of futile cycles of the actomyosin ATPase.

Importance In The Powerstroke Of Interaction Between The Relay Helix And Helix HQ Of Myosin

Ideas on mechano-chemical transduction by myosin have matured greatly through crystallography and are ripe for testing in vivo. One approach for doing so is termed reversion analysis, in which pairs of compensating mutations are identified. Suppression of one missense mutation by another reveals an interaction at the amino acid level that may be direct or indirect, long-lived or fleeting, but nonetheless physiologically relevant. Reversion analysis thus can test current ideas and, importantly, uncover interactions underlying dynamic or strain-dependent myosin states likely not represented in crystals. We used random mutagenesis to induce suppressors of Caenorhabditis elegans myosin/UNC-54 mutation E524K (=E500 of chicken myosin V), located on helix HQ at the predicted actin-binding region.Worms with E524K alone display disorganized A-bands and have a paralysis that worsens with increasing temperature. Thermodynamically, the heat-sensitivity suggests loss of a salt-bridge. The comparable residue in other myosins forms in the rigor-like and post-rigor crystallographic structures, but not in the pre-powerstroke one, a salt-bridge with a lysine on the relay helix (K460 of myosin V; =K483 of UNC-54). Thus, in the paralyzed worms, electrostatic repulsion between E524K and K483 potentially destabilizes interactions between helix HQ and the relay helix, thereby hindering the powerstroke.Twenty independent lines of suppressed worms were recovered from a screen of 106 mutagenized haploid genomes, and the suppressors mapped to seven residues: near the P-loop, V187I; in the actin-binding domain, E524K to E/T, L547F, A548V, and M579I/L/V; on the SH1-helix, C712Y; and in the converter domain, D724N. Consistent with the crystallographic structures of myosin, the suppressors can be interpreted as diminishing unfavorable interaction between E524K and K483, thus permitting the relay helix to reorganize properly as myosin progresses through the crossbridge cycle.

Monitoring of mechanically induced transitions in biology using Raman tweezers

Biological systems function within a delicate balance between mechanical forces and chemical reactions. Some well-known examples are the stretching and twisting of DNA during its protein binding actions and protein conformational changes in response to biochemical processes. Indeed, most biological systems could act as mechanochemical transducers since many aspects of chemical binding and enzymatic processes involve changes in structure dimensions.In this work, optical tweezers are used to controllably apply forces to biological systems and the resulting chemical and structural changes are monitored using Raman spectroscopy. The forces are applied in vivo and simulate stretching and compression effects that are normally experienced by the matter.The first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is induced through stretching of a red blood cell (1). The applied force mimics that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. The transition is due to hemoglobin-membrane and hemoglobin neighbor-neighbor interactions that are enhanced upon stretching. The latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods.This technique is also applied to study DNA stretching. Results will be presented that indicate conformational changes in the DNA structure that are evidenced through changes in its Raman spectrum, upon stretching. The typically low Raman scattering cross section of DNA is countered with the incooperation of silver colloids that enhance the scattered fields. The utilization of surface-enhanced Raman scattering (SERS) allows fast acquisition of spectra during the DNA intermediate stretched states which aides in elucidating its conformational change pathways.1. S. Rao, et. al., Biophys. J., in press.

2008 George E. Brown Memorial Lecture—Flow-Induced Vasodilation in the Human Heart: Unique Endothelial Mechanisms and Clinical Insights

The most physiologically important stimulus for endothelium-dependent dilation is shear stress. Flow across the endothelial surface triggers a mechanochemical signal transduction pathway that results in release of nitric oxide (NO), prostacyclin, and/or endothelial-derived hyperpolarizing factors (EDHFs) such as hydrogen peroxide (H 2 O 2 ) and epoxyeicosatrienoic acid (EET). In most normal arteries, NO is the prominent mediator; however, in disease states, NO may be quenched by an elevation in superoxide. In these situations, compensatory dilator mechanisms may emerge, using EDHFs. One mechanism of this compensation may involve an important dilator pathway interaction whereby NO inhibits CYP450 monooxygenase. This is particularly prominent in the microcirculation, where disease-induced loss of NO often results in enhanced endothelial CYP450 activity and generation of EET as an alternate dilator. Recent data indicate that H 2 O 2 can also inhibit CYP450. In the human coronary microcirculation from subjects with coronary artery disease, NO and prostacyclin play little role in the dilation to most pharmacological agonists or to shear stress. Instead, EETs and H 2 O 2 are critical mediators of flow-induced dilation. Interestingly, the dilation requires ROS generation from the endothelial mitochondrial electron transport chain. Dual vessel bioassay studies suggest that H 2 O 2 is the transferable compound responsible for hyperpolarizing and relaxing the underlying vascular smooth muscle cells. In contrast, dilation to bradykinin also involves both H 2 O 2 and EET, but the H 2 O 2 originates from endothelial nicotinamide adenine dinucleotide phosphateoxidase oxidase, and EETs serve as an important transferable dilator agent. Endothelial release of NO, EET, and H 2 O 2 has implications beyond vasodilation. NO and EETs have potent antiatherosclerotic properties. Hydrogen peroxide, on the other hand, although a potent dilator, has proatherogenic properties. Therefore, it is hypothesized that the profile of endothelial factors released by shear stress and other stimuli may help determine the balance between proatherosclerotic and anti-atherosclerotic factors in vascular homeostasis.

Microscopic mechanics of biomolecules in living cells

The exporting of theoretical concepts and modelling methods from physics and mechanics to the world of biomolecules and cell biology is increasing at a fast pace. The role of mechanical forces and stresses in biology and genetics is just starting to be appreciated, with implications going from cell adhesion, migration, division, to DNA transcription and replication, to the mechanochemical transduction and operation of molecular motors, and more. Substantial advances in experimental techniques over the past 10 years allowed to get unprecedented insight into the elasticity and mechanical response of many different proteins, cytoskeletal filaments, nucleic acids, both in vitro and, more recently, directly inside the cell. In a parallel effort, also theoretical models and computational methods are evolving into a rather specialized toolbox. However, several key issues need to be addressed when applying to life sciences the theories and methods typically originating from the fields of condensed matter and solid mechanics. The presence of a solvent and its dielectric properties, the many subtle effects of entropy, the non-equilibrium thermodynamics conditions, the dominating role of weak forces such as Van der Waals dispersion, hydrophobic interactions, and hydrogen bonding, impose a special caution and a thorough consideration, up to possibly rethinking some basic physics concepts. Discussing and trying to elucidate at least some of the above issues is the main aim of the present, partial and non-exhaustive, contribution.

PTH Receptor Responds To Shear Stress Perturbation of Plasma Membrane

The precise molecular mechanisms by which bone cells transduce mechanical stimulus into intracellular biochemical response have not been established yet. Here we show that mechanical perturbation of the plasma membrane leads to ligand‐independent conformational transitions in G protein coupled receptor (GPCR) such as parathyroid hormone type 1 receptor (PTH1r). By using picosecond time‐resolved fluorescence microscopy and GPCR conformation‐sensitive fluorescence energy transfer (FRET) we found that stimulation of MC3T3 cells with fluid shear stress or membrane fluidizing agent leads to a significant changes in conformational equilibrium of PTH1r in MC3T3 cells. The PTH1r conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single cell under fluid shear stress in real time using genetically engineered PTH1r containing intramolecular FRET pair. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of PTH1r. Therefore we suggest that PTH1r is involved in mediating primary mechanochemical signal transduction in MC3T3 cells. Furthermore we show that conformational transitions induced by mechanical stress are different from those induced by its ligand PTH(1‐34).This research was supported by the NIH grant HL086943.

Building a radial spoke: Flagellar radial spoke protein 3 (RSP3) is a dimer

Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial spokes are mechanochemical transducers that transmit signals from the central pair apparatus to the outer doublet microtubules for local control of dynein activity. Recently, progress has been made in identifying individual components of the radial spoke, yet little is known about how the radial spoke is assembled or how it performs in signal transduction. Here we focus on radial spoke protein 3 (RSP3), a highly conserved AKAP located at the base of the radial spoke stalk and required for radial spoke assembly on the doublet microtubules. Biochemical approaches were taken to further explore the functional role of RSP3 within the radial spoke structure and for control of motility. Chemical crosslinking, native gel electrophoresis, and epitope-tagged RSP3 proteins established that RSP3 forms a dimer. Analysis of truncated RSP3 proteins indicates the dimerization domain coincides with the previously characterized axoneme binding domain in the N-terminus. We propose a model in which each radial spoke structure is built on an RSP3 dimer, and indicating that each radial spoke can potentially localize multiple PKAs or AKAP-binding proteins in position to control dynein activity and flagellar motility.

BaHigh-force magnetic tweezers with force feedback for biological applications

Magnetic micromanipulation using magnetic tweezers is a versatile biophysical technique and has been used for single-molecule unfolding, rheology measurements, and studies of force-regulated processes in living cells. This article describes an inexpensive magnetic tweezer setup for the application of precisely controlled forces up to 100 nN onto 5 microm magnetic beads. High precision of the force is achieved by a parametric force calibration method together with a real-time control of the magnetic tweezer position and current. High forces are achieved by bead-magnet distances of only a few micrometers. Applying such high forces can be used to characterize the local viscoelasticity of soft materials in the nonlinear regime, or to study force-regulated processes and mechanochemical signal transduction in living cells. The setup can be easily adapted to any inverted microscope.

Mechanical stimuli of skeletal muscle: implications on mTOR/p70s6k and protein synthesis

The skeletal muscle is a tissue with adaptive properties which are essential to the survival of many species. When mechanically stimulated it is liable to undergo remodeling, namely, changes in its mass/volume resulting mainly from myofibrillar protein accumulation. The mTOR pathway (mammalian target of rapamycin) via its effector p70s6k (ribosomal protein kinase S6) has been reported to be of importance to the control of skeletal muscle mass, particularly under mechanical stimulation. However, not all mechanical stimuli are capable of activating this pathway, and among those who are, there are differences in the activation magnitude. Likewise, not all skeletal muscle fibers respond to the same extent to mechanical stimulation. Such evidences suggest specific mechanical stimuli through appropriate cellular signaling to be responsible for the final physiological response, namely, the accumulation of myofibrillar protein. Lately, after the mTOR signaling pathway has been acknowledged as of importance for remodeling, the interest for the mechanical/chemical mediators capable of activating it has increased. Apart from the already known MGF (mechano growth factor), some other mediators such as phosphatidic acid (PA) have been identified. This review article comprises and discusses relevant information on the mechano-chemical transduction of the pathway mTOR, with special emphasis on the muscle protein synthesis.

The shear stress of it all: the cell membrane and mechanochemical transduction

As the inner lining of the vessel wall, vascular endothelial cells are poised to act as a signal transduction interface between haemodynamic forces and the underlying vascular smooth-muscle cells. Detailed analyses of fluid mechanics in atherosclerosis-susceptible regions of the vasculature reveal a strong correlation between endothelial cell dysfunction and areas of low mean shear stress and oscillatory flow with flow recirculation. Conversely, steady shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. The molecular basis of shear-induced mechanochemical signal transduction and the endothelium's ability to discriminate between flow profiles remains largely unclear. Given that fluid shear stress does not involve a traditional receptor/ligand interaction, identification of the molecule(s) responsible for sensing fluid flow and mechanical force discrimination has been difficult. This review will provide an overview of the haemodynamic forces experienced by the vascular endothelium and its role in localizing atherosclerotic lesions within specific regions of the vasculature. Also reviewed are several recent lines of evidence suggesting that both changes in membrane microviscosity linked to heterotrimeric G proteins, and the transmission of tension across the cell membrane to the cell-cell junction where known shear-sensitive proteins are localized, may serve as the primary force-sensing elements of the cell.

Microarray analysis of Myf5-/-:MyoD-/- hypoplastic mouse lungs reveals a profile of genes involved in pneumocyte differentiation.

Fetal breathing-like movements (FBMs) are important in normal lung growth and pneumocyte differentiation. In amyogenic mouse embryos (designated as Myf5-/-:MyoD-/-, entirely lacking skeletal musculature and FBMs), type II pneumocytes fail to differentiate into type I pneumocytes, the cells responsible for gas exchange, and the fetuses die from asphyxia at birth. Using oligonucleotide microarrays, we compared gene expression in the lungs of Myf5-/-:MyoD-/- embryos to that in normal lungs at term. Nine genes were found to be up-regulated and 54 down-regulated at least 2-fold in the lungs of double-mutant embryos. Since many down-regulated genes are involved in lymphocyte function, immunohistochemistry was employed to study T- and B-cell maturity in the thymus and spleen. Our findings of normal lymphocyte maturity implied that the down-regulation was specific to the double-mutant lung phenotype and not to its immune system. Immunostaining also revealed altered distribution of transcription and growth factors (SATB1, c-Myb, CTGF) from down-regulated genes whose knockouts are now known to undergo embryonic or neonatal death secondary to respiratory failure. Together, it appears that microarray analysis has identified a profile of genes potentially involved in pneumocyte differentiation and therefore in the mechanisms that may be implicated in the mechanochemical signal transduction pathways underlying FBMs-dependent pulmonary hypoplasia.

Ionotropic Stress and Integrin

Cardiac remodeling is an adaptive response to chronically increased wall stress. Adaptive remodeling of the myocardium, caused either by increased workload or ionotropic stress, requires mediators for the communication of cardiac cells with their surrounding extracellular matrix.1 Integrins are likely candidates that transduce increased mechanical force into the intracellular biochemical signals. In this issue of Hypertension , Krishnamurthy et al2 report a potential role of myocardial β1-integrin in the adaptive and maladaptive remodeling that occurs in isoproterenol-induced left ventricular hypertrophy. To place the findings of this study in context with emerging studies regarding myocardial remodeling and the progression to left ventricular failure, a brief review of the biology of β1-integrin with respect to myocardial hypertrophy and remodeling is presented here. Integrins are heterodimeric cell-surface receptors composed of α and β subunits that function as adhesive and signaling molecules, as well as mechanotransducers.3 In noncardiac cells, it has been demonstrated that integrins respond to abnormal strain in a manner similar to that which would be found during pressure or volume overload in the heart.4 Integrins are expressed in various types of cells, including leukocytes, endothelial cells, vascular smooth muscle …

Mechanically induced chemical oscillations and motion in responsive gels.

Mechanochemical transduction plays a vital role in biological processes. There have, however, been few studies on exploiting mechanical stimuli to trigger chemical signals in non-biological systems. Using computational modeling, we investigate how an applied mechanical pressure can be harnessed to initiate traveling chemical waves in polymer gels undergoing the Belousov-Zhabotinsky (BZ) reaction. We uncover a rich dynamic behavior, isolating systems where the applied pressure induces chemical oscillations in an initially non-oscillatory system. We also pinpoint a scenario where the compression induces both oscillations and the autonomous rotation of the entire sample. Determining factors that control mechanochemical transduction in BZ gels is necessary for establishing guidelines to create self-adjusting or adaptive materials that not only "sense" a localized impact, but also transmit a global chemical signal in response to the local mechanical perturbation. Such materials can potentially be used to fabricate touch-sensitive sensors and membranes, as well as self-reinforcing materials.

Cellular Biophysics: Bacterial Endospore, Membranes and Random Fluctuation

Purposeful motion of biological processes can be driven by Brownian motion of macromolecular complexes with one-sided binding biasing movement in one direction: a Brownian ratchet, now proposed to explain membrane motion during a phagocytosis-like process in bacteria.

Fetal ocular movements and retinal cell differentiation: analysis employing DNA microarrays.

As developmental biologists we study the role of fetal movements in providing continuity between prenatal and postnatal life. There are two major categories of fetal motility. The first category consists of movements that have an obvious effect on the survival or development of the fetus (e.g., changes of position, sucking and swallowing). The second category consists of fetal movements that anticipate postnatal functions. For example, fetal ocular movements (FOMs) predict postnatal eye function (e.g., motion vision) of the newborn and therefore represent an important indicator of fetal health. However, while the clinical significance of fetal motility is obvious, its biological significance is elusive. We propose to use retina of genetically modified mouse embryos to study the biological role of FOMs in the genesis of cell diversity and organ functional maturation. Our results have already demonstrated the importance of fetal eye motility in the differentiation of cholinergic amacrine cells (CACs) in the retina (Kablar, 2003). Apparently, these cells are sensitive to motion and also responsible for motion vision. In the current report, we suggest employing the unique opportunity provided by the mouse Myf5-/-:MyoD-/- knock-outs that lack skeletal musculature and FOMs, microarray analysis and the follow-up experiments to identify a group of candidate genes that are essential for the molecular regulation of CAC differentiation and in turn for the functional maturation of the visual system towards its ability to perform motion vision. Finally, the molecules identified via this approach may be important in the mechanochemical signal transduction pathways employed during the process of conversion of a mechanical stimulus into an instruction understandable by the developing retinal neurons and glia cells.

Mapping ATP-dependent Activation at a σ54 Promoter

The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in sigma(54) and core RNA polymerase. The organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using sigma(54) single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early- and late-melted promoters. The results show that sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation. sigma(54) and sigma(54)-RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues. Nucleotide-dependent alterations in fluorescence resonance energy transfer efficiencies identify different functional states of the activator-sigma(54)-RNAP-promoter complex that exist throughout the mechano-chemical transduction pathway of transcriptional activation, i.e. from closed to open promoter complexes. The results suggest that open complex formation only occurs efficiently on replacement of a repressive fork junction with down-stream melted DNA.

G protein-coupled receptors sense fluid shear stress in endothelial cells

Hemodynamic shear stress stimulates a number of intracellular events that both regulate vessel structure and influence development of vascular pathologies. The precise molecular mechanisms by which endothelial cells transduce this mechanical stimulus into intracellular biochemical response have not been established. Here, we show that mechanical perturbation of the plasma membrane leads to ligand-independent conformational transitions in a G protein-coupled receptor (GPCR). By using time-resolved fluorescence microscopy and GPCR conformation-sensitive FRET we found that stimulation of endothelial cells with fluid shear stress, hypotonic stress, or membrane fluidizing agent leads to a significant increase in activity of bradykinin B2 GPCR in endothelial cells. The GPCR conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single endothelial cell under fluid shear stress in real time. We show that this response can be blocked by a B(2)-selective antagonist. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of GPCRs. Therefore, we suggest that GPCRs are involved in mediating primary mechanochemical signal transduction in endothelial cells. We anticipate our experiments to be a starting point for more sophisticated studies of the effects of changes in lipid bilayer environment on GPCR conformational dynamics. Furthermore, because GPCRs are a major target of drug development, a detailed characterization of mechanochemical signaling via the GPCR pathway will be relevant for the development of new antiatherosclerosis drugs.

Adhesion contact kinetics of HepG2 cells during Hepatitis B virus replication: Involvement of SH3-binding motif in HBX

It has been shown that Hepatitis B virus (HBV) replication directly alters the expression of key cytoskeleton-associated proteins which play key roles in mechanochemical signal transduction. Nevertheless, little is known on the correlation between HBV replication and the subsequent adhesion mechanism of HBV-replicating cells. In this study, it is demonstrated that the lag time of adhesion contact evolution of HepG2 cells with HBV replication is significantly increased by two times compared to that of normal HepG2 cell on collagen coated substrate. During the initial 20 min of cell seeding, only diffuse forms of vinculin was detected in HBV replicating cells while vinculin-associated focal complexes were found in normal and control cells. Similar delay in cell adhesion in HBV-replicating cells was observed in cells transfected with HBX, the smallest HBV protein, suggesting its involvement in this cellular process. In addition, a proline rich region found in many SH3 binding proteins was identified in HBX. HBX was found to interact with the focal adhesion protein, vinexin-β, through the SH3 binding. Furthermore, HepG2 cells with HBV replication showed evidence of cell rounding up, possibly resulting from cytoskeletal reorganizations associated with interaction between HBX and vinexin-β. Taken together, our results suggest that HBX is involved in the cytoskeletal reorganization in response to HBV replication.