Mechanosensing Proteins Research Articles

Molecular Counting in Traction Force Microscopy

The mechanical respons of the micro environment of a cell is of great influence for its differentiation and mobility. However, the mechanism that a cell uses to sense these mechanical properties remains largely unkown. Mechanosensing could happen by direct coupling to the nucleus via the actin cytoskeleton or by activating molecular signaling cascades through protein complexes.A recent combination of traction force microscopy with super resolution imaging allowed for novel research into the nanoscale architecture of force-bearing focal adhesions. Here we apply this technique to investigate the relation between local force exertion by the cell and the number of signal transduction proteins inside the integrin adhesome.Integrin adhesomes are mechanosensory protein complexes that couple the intracelular force bearing actomyosin structures to the extracelular environment. These complexes contain signal transduction proteins like paxillin, vinculin, talin and FAK, that change conformation when force is exerted on the complex.By immunostaining these proteins with Alexa647 their position can be localized to 20nm in fixed cells. However, due to the photophysical properties of Alexa647, and the binding stoichiometry in antibody staining the number of localizations does not scale linearly with the number of signal transduction proteins. To overcome this problem we present a novel analysis method to relate the number of localizations to the minimal number of signal transduction proteins.

INCREASED PASSIVE AND CONTRACTILE STRESS WITHIN THE MYOFILAMENTS CONTRIBUTE TO THE LOSS OF MECHANOSENSING IN THE HEART DURING THE PROGRESSION TO HEART FAILURE

Mechanosensing in the heart is an essential process allowing it to adapt to changes in the passive forces generated by changes in blood, and the forces produced by crossbridge cycling. We hypothesized that different mechanosensors may respond differently to these forces. We examined a guinea pig model of angiotensin II infusion (400ng/kg/min) spanning from early hypertrophy to heart failure. Using Western blotting, we show that desmin was upregulated during heart failure by 55% (n=7, P=0.03), while a second mechanosensor protein, Muscle LIM Protein (MLP) was found to have increased cytoplasmic to nuclear ratio by 88% (n=4, P=0.009) during compensatory hypertrophy, while this ratio decreased significantly during heart failure. We uncoupled the two sources of mechanical stress using cultured neonatal rat myocytes. To mimic passive stretch, we exposed myocytes to 48 hours of cyclic mechanical stretch at 1Hz, and to mimic the crossbridge cycling force, we electrically paced myocytes at 6.8 V (1Hz) for 48 hours. Results show that levels of desmin increased 23% (n=5, P=0.02) only in response to passive stretch. In contrast, the ratio of cytoplasmic to nuclear MLP decreased 50% (n=4, p=0.02) only in response to pacing. We inhibited crossbridge cycling by treating myocytes with blebbistatin (10 µM). This resulted in a ∼3 fold increase in the cytoplasmic to nuclear MLP ratio (n=8, p=0.02), while the desmin level remained unchanged. These data suggest that the failing heart may experience a prolonged increase in passive stretch and increased mechanical stress within the sarcomeres leading to a progressive loss of mechanosensing.

ZASP interacts with the mechanosensing protein Ankrd2 and p53 in the signalling network of striated muscle.

ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein.

Mechanical Stress-Driven Changes in the Dynamics of Cytoskeletal Proteins

Physical forces drive cell shape across diverse processes such as cytokinesis, cell migration and tissue invasion. The actin cytoskeleton provides the framework for regulating cell shape in response to mechanical stress. In the social amoeba Dictyostelium discoideum, a myosin II-based mechanosensory system controls cellular contractility and cell shape. The IQGAP proteins bind to the actin crosslinker, cortexillin I, and regulate myosin accumulation under stress. To determine how these proteins interact to regulate contractility, we studied the protein dynamics using fluorescence recovery after photobleaching (FRAP). We observe mechanical stress-dependent reduction in the mobility and network release of these proteins from the cytoskeleton. These altered dynamics could reflect a new mechanism for the accumulation of the mechanosensory proteins under stress by local rearrangement of the actin cytoskeleton microstructure. Additionally, IQGAP2 regulates the dynamics of cortexillin I under stress, indicating a functional relevance for the biochemical interaction between these proteins. These studies will help determine how cell shape is regulated various mechanical contexts, which will be valuable for understanding processes such as metastasis and tissue remodeling.

Disorders of red cell volume regulation

Regulation of erythrocyte volume homeostasis is critical for survival of the erythrocyte. Inherited or acquired disorders that perturb this homeostasis jeopardize the erythrocyte, leading to its premature destruction. This report reviews recent insights into pathways that influence cellular water and solute homeostasis and cell volume. The molecular and genetic bases of primary disorders of erythrocyte hydration are beginning to be revealed. Recent studies have implicated roles for a new protein PIEZO1, a long sought after mammalian mechanosensory protein; GLUT1, the glucose transporter; SLC4A1, the anion transporter; RhAG, the Rh-associated glycoprotein; and ABCB6, an ATP-binding cassette family member. Secondary disorders associated with perturbed cellular volume and volume regulation include sickle cell disease, thalassemia, and hereditary spherocytosis, in which dehydration contributes to disease pathology and clinical complications. Advances in understanding the mechanisms regulating erythrocyte solute and water content, particularly associated with mechanotransduction pathways, have revealed novel mechanisms controlling erythrocyte hydration. Understanding these processes may provide innovative strategies to maintain normal erythrocyte volume in disorders associated with primary or secondary cellular dehydration. Understanding the mechanisms controlling erythrocyte volume regulation will serve as a paradigm for other cells and may reveal new therapeutic targets for disease prevention and treatment.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of α-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure.

Probing the Interaction between the Auto-Inhibitory Filamin a Immunoglobulin Repeats Pair 20 and 21 under Tension

The Immunoglobulin Repeats 16-23 of Filamin A (FLNa) interact with numerous binding partners. Recent studies suggest that inter-domain interactions of these repeats under tension is crucial for the mechanosensing function of FLNa: the accessibility by FLNa binding partners is inhibited when the domain pairs are in closed conformation or permitted when they are in opened conformation. For example, it was proposed that in a closed stacked conformation, the A-strand of IgFLNa 20 interacts with the CD interface of IgFLNa 21, blocking its accessibility by other molecules such as b7 integrin tail and GP1Ba peptide. Prompted by these results, we examined the inter-domain interaction between repeats 20 and 21 (IgFLNa 20-21) using a magnetic tweezers setup. We show that the stacking between the two repeats can be disrupted at ∼15 pN at a loading rate range 0.1 - 2.1 pN/second and it can reform at decreased force values. Our results support the previously proposed force-dependent regulation of the accessibility of cryptic binding sites in the domain pair 20 and 21 of FLNa, which may have broad biological implications in the mechanisms of other mechanosensing proteins.

A Molecular Trajectory of α-Actinin Activation

The mechanisms by which living cells respond to mechanical stimuli are not yet fully understood. It has been suggested that mechanosensing proteins play an important role in mechanotransduction because their binding affinities are directly affected by the external stress. α-Actinin is an actin cross-linker and may act as a mechanosensor in adhesion sites. Its interaction with vinculin is suggested to be mechanically regulated. In this study, the free energy of activation is explored using the umbrella sampling method. An activation trajectory is generated in which α-actinin’s vinculin-binding site swings out of the rod domain, leading to approximately an 8 kcal/mol free energy release. The activation trajectory reveals several local and global conformational changes along the activation pathway accompanied by the breakage of a number of key interactions stabilizing the inhibited structure. These results may shed light on the role of α-actinin in cellular mechanotransduction and focal adhesion formation.

Tandem zyxin LIM sequences do not enhance force sensitive accumulation

The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano-signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing protein candidate. Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force-bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity.

Abstract 306: Atheroprone Hemodynamics Regulate Smooth Muscle Cell Phenotype Through Endothelial PECAM and Fibronectin

Introduction: Atherosclerosis is an inflammatory disease that develops preferentially in regions of disturbed hemodynamic shear stress. The extracellular matrix protein fibronectin (FN) is deposited in the sub-endothelial layer of pre-atherosclerotic and advanced lesions. Atheroprone shear stress promotes FN deposition and inflammatory signaling pathways in endothelial cells (ECs). Platelet endothelial cell adhesion molecule (PECAM), a mechanosensory protein, is necessary for the production, secretion, and assembly of FN matrix by ECs. Similar to ECs, vascular smooth muscle cells (SMCs) also display a pro-inflammatory phenotype in regions of atherogenesis, and this phenotype is key to the progression of atherosclerosis. Hypothesis: We hypothesize that endothelial PECAM and FN signaling promotes a pro-inflammatory smooth muscle cell phenotype in response to atheroprone shear stress patterns. Methods: An in vitro cone-and-plate viscometer model was used to apply human-derived atheroprone or atheroprotective shear stress patterns to human umbilical vein ECs co-cultured with underlying human umbilical vein SMCs. Results: Atheroprone flow increased SMC NFkB activity compared to atheroprotective flow (1.94±0.35 fold, p&lt;0.05). This increase of SMC NFkB activity under atheroprone flow was attenuated by siRNA knockdown of either endothelial PECAM (0.56±0.08 fold, p&lt;0.01) or FN (0.50±0.10 fold, p&lt;0.01). Endothelial PECAM knockdown also reduced genes marking the SMC inflammatory phenotype, including smooth muscle alpha actin, vascular cell adhesion molecule 1, and cyclooxygenase-2. Conclusion: These results suggest that SMCs switch to a pro-inflammatory phenotype when overlying ECs are exposed to atheroprone shear stress, and that PECAM and FN signaling in the ECs regulate this phenotype.

Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling

To remodel endothelial cell-cell adhesion, inflammatory cytokine- and angiogenic growth factor-induced signals impinge on the vascular endothelial cadherin (VE-cadherin) complex, the central component of endothelial adherens junctions. This study demonstrates that junction remodeling takes place at a molecularly and phenotypically distinct subset of VE-cadherin adhesions, defined here as focal adherens junctions (FAJs). FAJs are attached to radial F-actin bundles and marked by the mechanosensory protein Vinculin. We show that endothelial hormones vascular endothelial growth factor, tumor necrosis factor α, and most prominently thrombin induced the transformation of stable junctions into FAJs. The actin cytoskeleton generated pulling forces specifically on FAJs, and inhibition of Rho-Rock-actomyosin contractility prevented the formation of FAJs and junction remodeling. FAJs formed normally in cells expressing a Vinculin binding-deficient mutant of α-catenin, showing that Vinculin recruitment is not required for adherens junction formation. Comparing Vinculin-devoid FAJs to wild-type FAJs revealed that Vinculin protects VE-cadherin junctions from opening during their force-dependent remodeling. These findings implicate Vinculin-dependent cadherin mechanosensing in endothelial processes such as leukocyte extravasation and angiogenesis.

Myosin IIA Modulates T Cell Receptor Transport and CasL Phosphorylation during Early Immunological Synapse Formation

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88–103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL.

Multi-tasking role of the mechanosensing protein Ankrd2 in the signaling network of striated muscle.

BackgroundAnkrd2 (also known as Arpp) together with Ankrd1/CARP and DARP are members of the MARP mechanosensing proteins that form a complex with titin (N2A)/calpain 3 protease/myopalladin. In muscle, Ankrd2 is located in the I-band of the sarcomere and moves to the nucleus of adjacent myofibers on muscle injury. In myoblasts it is predominantly in the nucleus and on differentiation shifts from the nucleus to the cytoplasm. In agreement with its role as a sensor it interacts both with sarcomeric proteins and transcription factors.Methodology/Principal FindingsExpression profiling of endogenous Ankrd2 silenced in human myotubes was undertaken to elucidate its role as an intermediary in cell signaling pathways. Silencing Ankrd2 expression altered the expression of genes involved in both intercellular communication (cytokine-cytokine receptor interaction, endocytosis, focal adhesion, tight junction, gap junction and regulation of the actin cytoskeleton) and intracellular communication (calcium, insulin, MAPK, p53, TGF-β and Wnt signaling). The significance of Ankrd2 in cell signaling was strengthened by the fact that we were able to show for the first time that Nkx2.5 and p53 are upstream effectors of the Ankrd2 gene and that Ankrd1/CARP, another MARP member, can modulate the transcriptional ability of MyoD on the Ankrd2 promoter. Another novel finding was the interaction between Ankrd2 and proteins with PDZ and SH3 domains, further supporting its role in signaling. It is noteworthy that we demonstrated that transcription factors PAX6, LHX2, NFIL3 and MECP2, were able to bind both the Ankrd2 protein and its promoter indicating the presence of a regulatory feedback loop mechanism.Conclusions/SignificanceIn conclusion we demonstrate that Ankrd2 is a potent regulator in muscle cells affecting a multitude of pathways and processes.

Zyxin-mediated Actin Assembly Is Required for Efficient Wound Closure

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.

The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells

We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.

Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot formechanosensitive proteins

Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions andcytoskeletal structures upon substrate deformation. For example, tissue formation,directed cell orientation or cell differentiation are regulated by such mechanosensingprocesses. Focal adhesions and the actin cytoskeleton are believed to be involved inthese processes, but where mechanosensing molecules are located and how elasticsubstrate, focal adhesions and the cytoskeleton couple with each other upon substratedeformation still remains obscure. To approach these questions we have developed asensitive method to apply defined spatially decaying deformation fields to cellscultivated on ultrasoft elastic substrates and to accurately quantify the resultingdisplacements of the actin cytoskeleton, focal adhesions, as well as the substrate.Displacement fields were recorded in live cell microscopy by tracking either signalsfrom fluorescent proteins or marker particles in the substrate. As model cell typewe used myofibroblasts. These cells are characterized by highly stable adhesionand force generating structures but are still able to detect mechanical signalswith high sensitivity. We found a rigid connection between substrate and focaladhesions. Furthermore, stress fibers were found to be barely extendable almost overtheir whole lengths. Plastic deformation took place only at the very ends of actinfilaments close to focal adhesions. As a result, this area became elongated withoutextension of existing actin filaments by polymerization. Both ends of the stress fiberswere mechanically coupled with detectable plastic deformations on either site.Interestingly, traction force dependent substrate deformation fields remained mostlyunaffected even when stress fiber elongations were released. These data arguefor a location of mechanosensing proteins at the ends of actin stress fibers anddescribe, except for these domains, the whole system to be relatively rigid fortensile strain with a mechanical coupling between the front and rear end of a cell.

Cell type-specific expression of acid-sensing ion channels in hippocampal interneurons

Pacinian corpuscles are innervated by large myelinated Aα-β axons from the large- and intermediate-sized sensory neurons of dorsal root ganglia. These neurons express different members of the degenerin/epithelial Na+ channel (DEG/ENa+C) superfamily of proteins with putative mechanosensory properties, whose expression is regulated by the TrkB–BDNF system. Thus, we hypothesized that BDNF and/or NT-4 signalling through activation of TrkB may regulate the expression of molecules supposed to be necessary for the mechanosensory function of Pacinian corpuscles. To test this hypothesis we analyzed the expression and distribution of ENa+C subunits and acid-sensing ion channel 2 (ASIC2) in Pacinian corpuscles from 25 days old mice deficient in TrkB, BDNF and NT-4. Pacinian corpuscles in these animals are normal in number, structure, and expression of several immunohistochemical markers. Using immunohistochemistry we observed that the β-ENa+C and γ-ENa+C subunits, but not the α-ENa+C subunit, were expressed in wild-type animals, and they were always found in the central axon. ASIC2 immunoreactivity was found in both the central axon and the inner core cells. The absence of TrkB or BDNF abolished expression of β-ENa+C and ASIC2, whereas expression of γ-ENa+C did not change. Expression of β-ENa+C and γ-ENa+C subunits in NT-4 deficient mice was found in the axons but also in the inner core cells whereas levels of expression of ASIC2 were increased in these animals. This study suggests that expression in Pacianian corpuscles of some potential mechanosensory proteins is regulated by BDNF, NT-4 and TrkB.

The expression of ENa +C and ASIC2 proteins in Pacinian corpuscles is differently regulated by TrkB and its ligands BDNF and NT-4

Pacinian corpuscles are innervated by large myelinated Aα-β axons from the large- and intermediate-sized sensory neurons of dorsal root ganglia. These neurons express different members of the degenerin/epithelial Na + channel (DEG/ENa +C) superfamily of proteins with putative mechanosensory properties, whose expression is regulated by the TrkB–BDNF system. Thus, we hypothesized that BDNF and/or NT-4 signalling through activation of TrkB may regulate the expression of molecules supposed to be necessary for the mechanosensory function of Pacinian corpuscles. To test this hypothesis we analyzed the expression and distribution of ENa +C subunits and acid-sensing ion channel 2 (ASIC2) in Pacinian corpuscles from 25 days old mice deficient in TrkB, BDNF and NT-4. Pacinian corpuscles in these animals are normal in number, structure, and expression of several immunohistochemical markers. Using immunohistochemistry we observed that the β-ENa +C and γ-ENa +C subunits, but not the α-ENa +C subunit, were expressed in wild-type animals, and they were always found in the central axon. ASIC2 immunoreactivity was found in both the central axon and the inner core cells. The absence of TrkB or BDNF abolished expression of β-ENa +C and ASIC2, whereas expression of γ-ENa +C did not change. Expression of β-ENa +C and γ-ENa +C subunits in NT-4 deficient mice was found in the axons but also in the inner core cells whereas levels of expression of ASIC2 were increased in these animals. This study suggests that expression in Pacianian corpuscles of some potential mechanosensory proteins is regulated by BDNF, NT-4 and TrkB.

A single conserved proline residue determines the membrane topology of stomatin

Stomatin is an integral membrane protein which is widely expressed in many cell types. It is accepted that stomatin has a unique hairpin-loop topology: it is anchored to the membrane with an N-terminal hydrophobic domain and the N- and C-termini are cytoplasmically localized. Stomatin is a prototype for a family of related proteins, containing among others MEC-2 (mechanosensory protein 2) from Caenorhabditis elegans, SLP (stomatin-like protein)-3 and podocin, all of which interact with ion channels to regulate their activity. Members of the stomatin family partly localize in DRMs (detergent-resistant membrane domains) enriched in cholesterol and sphingolipids. It has been proposed that a highly conserved proline residue in the middle of the hydrophobic domain directly binds cholesterol and that cholesterol binding is necessary for the regulation of ion channels. In the present study we show that a small part of the stomatin pool exists as a single-pass transmembrane protein rather than a hairpin-loop protein. The highly conserved proline residue is crucial for adopting the hairpin-loop topology: substitution of this proline residue by serine transfers the whole stomatin pool to the single-pass transmembrane form, which no longer localizes to DRMs. These results suggest that formation of the hairpin loop is inefficient and that the conserved proline residue is indispensable for formation of the hairpin loop. The single-pass transmembrane form exists also for SLP-3 and it should be considered that it mediates part of the physiological functions of stomatin and related proteins.

Extracellular Matrix Rigidity Promotes Invadopodia Activity

Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) [1]. Molecular components of invadopodia include branched actin-assembly proteins, membrane trafficking proteins, signaling proteins, and transmembrane proteinases [1]. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2-4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM-rigidity signals depends on the cellular contractile apparatus [5-7], given that inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia, and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM-rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.