Mechanosensory Response Research Articles

Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function.

A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, we discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, our results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the "histone code," in regulating dendrite morphology.

Assaying mechanosensation.

C. elegans detect and respond to diverse mechanical stimuli using neuronal circuitry that has been defined by decades of work by C. elegans researchers. In this WormMethods chapter, we review and comment on the techniques currently used to assess mechanosensory response. This methods review is intended both as an introduction for those new to the field and a convenient compendium for the expert. A brief discussion of commonly used mechanosensory assays is provided, along with a discussion of the neural circuits involved, consideration of critical protocol details, and references to the primary literature.

Myosin II Functions as a Direct Mechanosensor for Intercellular Invasion during Cell-Cell Fusion

How cells sense and react to external mechanical stimuli is a fundamental question in cellular biophysics. Here, we describe a previously unrecognized mechanosensory response to a localized cellular protrusive force during cell-cell fusion. Our previous studies of myoblast fusion in Drosophila embryos and cell fusion in a reconstituted culture system revealed that cell-cell fusion is an asymmetric process, in which one fusion partner (the attacking cell) extends invasive finger-like protrusions into the other (the receiving cell) to promote plasma membrane juxtaposition and fusion. Here, we demonstrate that the Rho-Rok-Myosin II (MyoII) pathway is specifically activated in the receiving cell in response to the invasive force from the attacking cell. Disrupting the function of this pathway renders less cortical resistance in the receiving cell and defects in cell-cell fusion, despite deeper invasion of the attacking cell. Increasing the cortical tension in the receiving cell by overexpressing an actin crosslinker significantly rescued such fusion defect. We show that MyoII accumulates to the cell cortex earlier than its upstream biochemical regulators Rok and Rho in response to applied force, and that MyoII is required for the steady-state accumulation of Rok and Rho. Furthermore, the motor domain of MyoII is indispensible for its cortical accumulation triggered by intercellular invasion. These results strongly suggest that MyoII functions as a direct mechanosensor that feeds back to its upstream regulators, and that the mechanosensory function of MyoII is mediated by its binding to actin filaments under mechanical stress. This newly discovered mechanosensory system, consisting of a well-defined source of protrusive force and the corresponding activation of the Rho-Rok-MyoII pathway in the neighboring cell, highlights a central role of MyoII in the mechanosensory response that ultimately leads to cortical tension generation in animal cells.

The Microtubule-Associated Protein END BINDING1b, Auxin, and Root Responses to Mechanical Cues

The ability of roots to penetrate through the soil and maneuver around rocks and other impenetrable objects requires a system for modulating output from mechanosensory response networks. The microtubule-associated protein END BINDING1b (EB1b) has a role in this process; it represses root responses to mechanical cues. In this study, a possible relationship between EB1b and auxin during root responses to mechanical cues was investigated. We found that eb1b-1-mutant roots are more sensitive than wild-type roots to chemicals that disrupt auxin transport, whereas the roots of mutants with defects in auxin transport are resistant to these treatments. Using seedlings that express the auxin-sensitive DR5rev::GFP construct, we also found that wild-type and eb1b-1 roots treated with the auxin transport inhibitor naphthylphthalamic acid exhibited dose-dependent reductions in basipetal auxin transport that were indistinguishable from each other. The responses of eb1b-1 roots to mechanical cues were also enhanced over wild type in the presence of p -chlorophenoxyisobutyric acid, a chemical thought to inhibit auxin signaling. Finally, roots of eb1b-1 and wild-type plants exhibited slight increases in loop formation in response to increasing levels of exogenously applied indole-3-acetic acid or 1-naphthalene acetic acid. Taken together, these results suggest that the repression of loop formation by EB1b and auxin transport/signaling occurs by different mechanisms.

The Molecular Basis for Cellular Contractility Driven Mechanosensing

Internal and external mechanical forces govern many cellular processes in normal and diseased states such as muscle contraction, tissue invasion and metastasis of tumor cells. Thus a cell's ability to sense and respond to mechanical stimuli is critical for cell behavior and survival. In Dictyostelium, the mechanoenzyme myosin II and the actin crosslinker cortexillin I work cooperatively to control cellular contractility in response to deformation. The scaffolding proteins IQGAP1 and IQGAP2, which can bind to cortexillin I, further regulate this mechanosensory system through feedback loops. The IQGAPs are not required for mechanosensing, but affect the stress-dependent myosin accumulation in the cortex. IQGAP1 inhibits mechanosensing, while IQGAP2 is required for suppressing this inhibition. A molecular understanding of how this mechanosensory system works to modulate contractility remains elusive. By measuring protein dynamics using fluorescence recovery after photobleaching (FRAP), we observed stress-dependent reduction in the mobility of many of the proteins in the mechanosensory system. Further, these dynamics are dependent on the integrity of the mechanosensory system, as mutant cells with disrupted mechanosensing typically show slower protein dynamics and increased cortical association of the proteins than in wild type cells. In addition, we are also characterizing the biochemical interactions important for mechanosensory response. These experiments will allow us to develop a molecular model to explain how mechanical forces are transmitted within the cell. The knowledge of the molecular underpinnings of this myosin II-based feedback control system will be applicable to other contractile systems such as those involved in development and morphogenesis of multicellular organisms.

Hyperpolarization-activated cyclic nucleotide-gated cation channel subtypes differentially modulate the excitability of murine small intestinal afferents

To assess the role of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels in regulating the excitability of vagal and spinal gut afferents. The mechanosensory response of mesenteric afferent activity was measured in an ex vivo murine jejunum preparation. HCN channel activity was recorded through voltage and current clamp in acutely dissociated dorsal root ganglia (DRG) and nodose ganglia (NG) neurons retrogradely labeled from the small intestine through injection of a fluorescent marker (DiI). The isoforms of HCN channels expressed in DRG and NG neurons were examined by immunohistochemistry. Ramp distension of the small intestine evoked biphasic increases in the afferent nerve activity, reflecting the activation of low- and high-threshold fibers. HCN blocker CsCl (5 mmol/L) preferentially inhibited the responses of low-threshold fibers to distension and showed no significant effects on the high-threshold responses. The effect of CsCl was mimicked by the more selective HCN blocker ZD7288 (10 μmol/L). In 71.4% of DiI labeled DRG neurons (n = 20) and 90.9% of DiI labeled NG neurons (n = 10), an inward current (I(h) current) was evoked by hyperpolarization pulses which was fully eliminated by extracellular CsCl. In neurons expressing I(h) current, a typical "sag" was observed upon injection of hyperpolarizing current pulses in current-clamp recordings. CsCl abolished the sag entirely. In some DiI labeled DRG neurons, the I(h) current was potentiated by 8-Br-cAMP, which had no effect on the I(h) current of DiI labeled NG neurons. Immunohistochemistry revealed differential expression of HCN isoforms in vagal and spinal afferents, and HCN(2) and HCN(3) seemed to be the dominant isoform in DRG and NG, respectively. HCNs differentially regulate the excitability of vagal and spinal afferent of murine small intestine.

Xaml1/Runx1 is required for the specification of Rohon-Beard sensory neurons in Xenopus

Lower vertebrates develop a unique set of primary sensory neurons located in the dorsal spinal cord. These cells, known as Rohon-Beard (RB) sensory neurons, innervate the skin and mediate the response to touch during larval stages. Here we report the expression and function of the transcription factor Xaml1/Runx1 during RB sensory neurons formation. In Xenopus embryos Runx1 is specifically expressed in RB progenitors at the end of gastrulation. Runx1 expression is positively regulated by Fgf and canonical Wnt signaling and negatively regulated by Notch signaling, the same set of factors that control the development of other neural plate border cell types, i.e. the neural crest and cranial placodes. Embryos lacking Runx1 function fail to differentiate RB sensory neurons and lose the mechanosensory response to touch. At early stages Runx1 knockdown results in a RB progenitor-specific loss of expression of Pak3, a p21-activated kinase that promotes cell cycle withdrawal, and of N-tub, a neuronal-specific tubulin. Interestingly, the pro-neural gene Ngnr1, an upstream regulator of Pak3 and N-tub, is either unaffected or expanded in these embryos, suggesting the existence of two distinct regulatory pathways controlling sensory neuron formation in Xenopus. Consistent with this possibility Ngnr1 is not sufficient to activate Runx1 expression in the ectoderm. We propose that Runx1 function is critically required for the generation of RB sensory neurons, an activity reminiscent of that of Runx1 in the development of the mammalian dorsal root ganglion nociceptive sensory neurons.

PKC-1 acts with the ERK MAPK signaling pathway to regulate Caenorhabditis elegans mechanosensory response

In most animals, multiple genes encode protein kinase C (PKC) proteins. Pharmacological studies have revealed numerous roles for this protein family, yet the in vivo roles of specific PKC proteins and the functional targets of PKC activation are poorly understood. We find that in Caenorhabditis elegans, two PKC genes, pkc-1 and tpa-1, are required for mechanosensory response; the role of the nPKCε/η ortholog, pkc-1, was examined in detail. pkc-1 function is required for response to nose touch in adult C. elegans and pkc-1 likely acts in the interneurons that regulate locomotion which are direct synaptic targets of mechanosensory neurons. Previous studies have suggested numerous possible targets of pkc-1; our analysis indicates that pkc-1 may act via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway. We find that ERK/MAPK pathway function is required for mechanosensory response in C. elegans and that at least one component of this pathway, lin-45 Raf, acts in interneurons of the mechanosensory circuit. Genetic analysis indicates that lin-45 and pkc-1 act together to regulate nose touch response. Thus, these results functionally link two conserved signaling pathways in adult C. elegans neurons and define distinct roles for PKC genes in vivo.

Squeezing and Detachment of Living Cells

The interaction of living cells with their environment is linked to their adhesive and elastic properties. Even if the mechanics of simple lipid membranes is fairly well understood, the analysis of single cell experiments remains challenging in part because of the mechanosensory response of cells to their environment. To study the mechanical properties of living cells we have developed a tool that borrows from micropipette aspiration techniques, atomic force microscopy, and the classical Johnson-Kendall-Roberts test. We show results from a study of the adhesion properties of living cells, as well as the elastic response and relaxation. We present models that are applied throughout the different stages of an experiment, which indicate that the contribution of the different components of the cell are active at various stages of compression, retraction, and detachment. Finally, we present a model that attempts to elucidate the surprising logarithmic relaxation observed when the cell is subjected to a given deformation.

Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner

Cell surface receptors integrate chemical and mechanical cues to regulate a wide range of biological processes. Integrin complexes are the mechanotransducers between the extracellular matrix and the actomyosin cytoskeleton. By analogy, cadherin complexes may function as mechanosensors at cell-cell junctions, but this capacity of cadherins has not been directly demonstrated. Furthermore, the molecular composition of the link between E-cadherin and actin, which is needed to sustain such a function, is unresolved. In this study, we describe nanomechanical measurements demonstrating that E-cadherin complexes are functional mechanosensors that transmit force between F-actin and E-cadherin. Imaging experiments reveal that intercellular forces coincide with vinculin accumulation at actin-anchored cadherin adhesions, and nanomechanical measurements show that vinculin potentiates the E-cadherin mechanosensory response. These investigations directly demonstrate the mechanosensory capacity of the E-cadherin complex and identify a novel function for vinculin at cell-cell junctions. These findings have implications for barrier function, morphogenesis, cell migration, and invasion and may extend to all soft tissues in which classical cadherins regulate cell-cell adhesion.

Myosin II is an Active Stress Sensor at the Core of a Cell Division Control System

Mechanosensing is important in many cellular processes such as cell motility and cell division. Cells experience mechanical stress from the surrounding environment and also its internal cytoskeleton structure. Previous studies from our lab showed that cellular mechanosensing is crucial for regulating cytokinesis shape change. Using micropipette aspiration (MPA) to generate stress on the cell cortex, we discovered that mechanical stress stimulates the accumulation of myosin-II (a contractile force generating protein) and cortexillin-I (an actin bundling protein) at the deformation site. These proteins then contract the cortex, correcting the shape of the cell. Both myosin-II and cortexillin-I are required for this mechanosensory system during cytokinesis. Recently, we demonstrated that this mechanosensory response is tunable by varying the lever arm length of myosin-II heavy chain, showing that myosin-II is an active force sensor in this mechanosensory system. We now focus on how mechanical inputs mediated through myosin-II lead to changes in biochemical signaling pathways, specifically the cortexillin-I regulatory and spindle signaling pathways. Rac1A (a small GTPase), IQGAP1, and IQGAPA (GTPase effectors) can form complexes with cortexillin-I. In the absence of both IQGAP1 and IQGAPA, cortexillin-I does not localize normally to the cleavage furrow during cell division. However, IQGAPA, but not IQGAP1, is required for myosin-II mechanosensing. Kif12, a mitotic-kinesin-like protein in Dictyostelium cells, is part of the chromosomal passenger complex, including INCENP and Aurora kinase. Kif12 is also recruited to the cell cortex inside the micropipette in a myosin-II-dependent and/or IQGAPA-dependent manner during cell division. Thus, mitotic spindle signaling proteins are responsive to mechanical stress sensed by myosin-II. Overall, myosin-II is a key mechanical stress sensor and these mechanical inputs are fed back to the mitotic spindle signaling system.

Role of TRPV1 in high-threshold rat colonic splanchnic afferents is revealed by inflammation

The vanilloid-1 receptor TRPV1 is known to play a role in extrinsic gastrointestinal afferent function. We investigated the role of TRPV1 in mechanosensitivity in afferents from normal and inflamed tissue. Colonic mechanosensitivity was determined in an in vitro rat colon preparation by recording from attached splanchnic nerves. Recordings were made from serosal/mesenteric afferents responding only at high thresholds to graded mechanical stimulation with von Frey probes. Colonic inflammation was induced by adding 5% dextran sulphate sodium (DSS) to the drinking water for 5 days, and was confirmed by histopathology. The selective TRPV1 antagonist, SB-750364 (10 −8 to 10 −6 M), was tested on mechanosensory stimulus response functions of afferents from normal and inflamed preparations ( N = 7 each). Mechanosensory responses had thresholds of 1–2 g, and maximal responses were observed at 12 g. The stimulus response function was not affected by DSS-induced colitis. SB-750364 had no effect on stimulus response functions in normal preparations, but reduced (up to 60%) in a concentration-dependent manner those in inflammation (2-way ANOVA, p < 0.05). Moreover, in inflamed tissue, spontaneous afferent activity showed a dose-dependent trend toward reduction with SB-750364. We conclude that mechanosensitivity of high-threshold serosal colonic splanchnic afferents to graded stimuli is unaffected during DSS colitis. However, there is a positive influence of TRPV1 in mechanosensitivity in inflammation, suggesting up-regulation of excitatory TRPV1-mediated mechanisms.

In vivo ablation of mechanosensory neurons in C. elegans by an optogenetics tool KillerRed

Event Abstract Back to Event In vivo ablation of mechanosensory neurons in C. elegans by an optogenetics tool KillerRed Vahagn Karapetyan1* and Hiroshi Suzuki2 1 University of Toronto, Undergraduate Program in Neuroscience, Canada 2 University of Toronto, Department of Physiology, Faculty of Medicine, Canada Optogenetics is a newly emerging field to optically manipulate cell physiology by using genetically encoded molecular tools which are represented by a light-gated cation channel, Channelrhodopsin (ref-1). In this session, we discuss a novel procedure to induce spatially and temporally specific oxidative stress using an optogenetics tool KillerRed which generates Reactive oxygen species (ROS) on light-activation (Fig.1). ROS plays an important role in various diseases including neurodegenerative diseases like Parkinson’s and Alzheimer’s (ref-2). However, administration of ROS-generating reagents, such as Paraquat, affects various tissues and cell compartments over a fairly long time, and a new approach to allow us spatial and temporal control of ROS generation has been desired. KillerRed is a red fluorescent protein that is molecularly engineered to produces large quantities of ROS upon excitation light-activation (Fig.1). Its ROS production and cell-toxic effect was confirmed in mammalian cell-culture (ref-3). The genetically encoded molecular tool can be expressed in specific cells allowing us spatially and temporally controlled ROS production by light illumination. To demonstrate the efficacy of KillerRed in living animals, we attempted to ablate mechanosensory neurons in the transparent round worm C. elegans, which mediate escaping behaviour from “touch” stimulation on body (Fig 2). We generated transgenic worms expressing KillerRed in mitochondria of the mechanosensory neurons, and studied the KillerRed mediated in vivo ablation of the sensory neurons in living animals by (i) scoring the behaviour response and (ii) direct observation of cell morphology of the neurons stained by fluorescent marker. The worms illuminated by the KillerRed excitation light developed and behaved normally except for the marked loss of the mechanosensory behaviour response and the neurons themselves. We are further attempting to use the novel approach to study of the controlled production of ROS in the dopaminergic neurons in C.elegans. fig5 fig4

Domain Mapping of the Polycystin-2 C-terminal Tail Using de Novo Molecular Modeling and Biophysical Analysis

In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca2+) by isothermal titration calorimetry and undergo Ca2+-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca2+ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca2+-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease.

Selective Role for TRPV4 Ion Channels in Visceral Sensory Pathways

Although there are many candidates as molecular mechanotransducers, so far there has been no evidence for molecular specialization of visceral afferents. Here, we show that colonic afferents express a specific molecular transducer that underlies their specialized mechanosensory function: the transient receptor potential channel, vanilloid 4 (TRPV4). We found TRPV4 mRNA is highly enriched in colonic sensory neurons compared with other visceral and somatic sensory neurons. TRPV4 protein was found in colonic nerve fibers from patients with inflammatory bowel disease, and it colocalized in a subset of fibers with the sensory neuropeptide CGRP in mice. We characterized the responses of 8 subtypes of vagal, splanchnic, and pelvic mechanoreceptors. Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and dramatically reduced by targeted deletion of TRPV4 or by a TRP antagonist. Other subtypes of vagal and pelvic afferents, by contrast, were unaffected by these interventions. The behavioral responses to noxious colonic distention were also substantially reduced in mice lacking TRPV4. These data indicate that TRPV4 contributes to mechanically evoked visceral pain, with relevance to human disease. In view of its distribution in favor of specific populations of visceral afferents, we propose that TRPV4 may present a selective novel target for the reduction of visceral pain, which is an important opportunity in the absence of current treatments.

Cellular suicide pact

Mass suicides are not the reserve of cults, it appears. On page 567, Link and colleagues report that cells do it too. Figure 1 When communal cell death fails in mutant chimeric flies, epithelial tissue remains in patches (circled) across the wing. Cellular suicide—known as apoptosis—is necessary during development for sculpting the shape of organs and limbs. Most of the time, individual cell suicides are dotted throughout the tissue, and cell corpses are then cleared away by phagocytosis. Link et al., however, have now captured by live imaging a mass cellular suicide in flies. Epithelial wing cells died in a wave that swept across the developing wing in a matter of minutes. The dead cells were not phagocytosed but were instead swept into the wing veins, which drain into the body. The authors hypothesize that a released signaling factor or mechanosensory response propagates the swift en masse suicides. A mutant screen for defects in this collective cell death has not yet identified any such instigators but has revealed novel cell death genes, including homeodomain-interacting protein kinase. This gene's product functions in collective cell death and in more conventional examples of programmed cell death. The authors speculate that communal cell death might be a more widespread phenomenom. Massive cellular suicides might occur, for example, in mammals during development or after rapid hormonal changes, such as mammary gland involution after nursing and the shedding of the uterine lining at menstruation.

Delta expression in post‐mitotic neurons identifies distinct subsets of adult‐specific lineages in Drosophila

The Drosophila ventral nerve cord is comprised of numerous neuronal lineages, each derived from a stereotypically positioned neuroblast (NB). At the embryonic stage the unique identities of each NB, and several of their neuronal progeny, are well characterized by spatial and temporal expression patterns of molecular markers. These patterns of expression are not preserved at the larval stage and thus the identity of adult-specific lineages remains obscure. Recent clonal analysis using MARCM has identified 24 adult-specific lineages arising from thoracic NBs at the larval stage. In this study, we have explored a role for the Delta protein in development of the post-embryonic Drosophila ventral nerve cord. We find that Delta expression identifies 7 of the 24 adult-specific lineages of the thoracic ganglia by being highly enriched in clusters of newly born post-mitotic neurons and their neurite bundles. The Delta lineages constitute the majority of bundles projecting to the ventral neuropil, consistent with a role in processing leg sensory information. Targeted knockdown of Delta in neurons using RNAi results in significantly decreased leg chemosensory response and a relatively unaffected leg mechanosensory response. Delta RNAi knockdown in Delta lineages also gives a more diffuse bundle terminal morphology while the overall path-finding of neurite bundles is unaffected. We also identify a male-specific Delta lineage in the terminal abdominal ganglia, implicating a role for Delta in development of sexually dimorphic neural networks. Examples of Delta-expressing neurites contacting Notch-expressing glia are also seen, but are not common to all Delta lineages. Altogether, these data reveal a fundamental pattern of Delta expression that is indicative of an underlying developmental program that confers identity to adult lineage neurons.

Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress

Interactions of cell adhesions, Rho GTPases and actin in the endothelial cells’ response to external forces are complex and not fully understood, but a qualitative understanding of the mechanosensory response begins to emerge. Here, we formulate a mathematical model of the coupled dynamics of cell adhesions, small GTPases Rac and Rho and actin stress fibers guiding a directional reorganization of the actin cytoskeleton. The model is based on the assumptions that the interconnected cytoskeleton transfers the shear force to the adhesion sites, which in turn transduce the force into a chemical signal that activates integrins at the basal surface of the cell. Subsequently, activated and ligated integrins signal and transiently de-activate Rho, causing the disassembly of actin stress fibers and inhibiting the maturation of focal complexes into focal contacts. Focal complexes and ligated integrins activate Rac, which in turn enhances focal complex assembly. When Rho activity recovers, stress fibers re-assemble and promote the maturation of focal complexes into focal contacts. Merging stress fibers self-align, while the elevated level of Rac activity at the downstream edge of the cell is translated into an alignment of the cells and the newly forming stress fibers in the flow direction. Numerical solutions of the model equations predict transient changes in Rac and Rho that compare well with published experimental results. We report quantitative data on early alignment of the stress fibers and its dependence on cell shape that agrees with the model.

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.

Signal transduction in the Caenorhabditis elegans nervous system.

Caenorhabditis elegans interacts with its environment by sensing chemicals, touch, and temperature; genetic analysis of each of these responses has led to the identification of candidate signaling molecules within sensory neurons. A molecular model for touch sensation has emerged from studies of the mechanosensory response; the receptors and signal transduction mechanisms in olfactory neurons are being elucidated; and an unusual neuroendocrine role for a TGF-beta-related peptide in chemosensory neurons has been discovered. Presynaptic and postsynaptic components of neuronal synapses have been identified in behavioral and pharmacological mutant screens. Mutations have been found in multiple classes of nicotinic acetylcholine receptor genes, excitatory and inhibitory glutamate receptor genes, and candidate gap junction genes, allowing their function to be studied in vivo. Different G-protein signaling pathways have characteristic effects on behavior, neuronal degeneration, and embryonic development.