The mechanical properties of the tissue vary widely around the body, from the soft brain to the rigid bone. Tissue cells are able to sense mechanical signals from their environment, which influence many aspects of cell behaviour such as migration, proliferation and differentiation. Focal adhesions (FAs) are large protein complexes that form the bridge between the extracellular matrix (ECM)-binding integrins and the contractile actin cytoskeleton. Here, they sense the rigidity of the local environment and translate this information into a cellular response, a process known as mechanotransduction. However, the FA proteins required for mechanotransduction, and the molecular mechanisms involved in this fundamental process, remain to be elucidated. Talin, vinculin, FAK and paxillin are four core FA-associated proteins that are thought to be involved in mechanotransduction. These proteins associate and dissociate from the complex in a constant state of flux. Using a live-cell imaging approach, I found that the rate of dynamic exchange of an FA protein correlates to its function. The FA appears to have a modular organisation; the slowest proteins have a structural role, such as talin and vinculin, responsible for directly linking integrin to actin and sensing the ECM stiffness. The signalling proteins are turned over more rapidly, including FAK and paxillin, and are responsible for directing the cellular response to force-generated signals from the ECM.The second results chapter focused on the force-dependent interactions between talin, vinculin and actin. The talin domains R2R3 were identified as the key mechanosensitive vinculin-binding sites, which are exposed upon the application of force across the talin rod. Vinculin binding to R2R3 led to actin associating with the central actin-binding site in the talin rod (ABS2), which is required for the transmission of actomyosin tension onto the underlying substrate as cellular traction force. Finally, the protein turnover data were incorporated into two mathematical models, describing talin and vinculin turnover, which were able to simulate the dynamic exchange of various talin and vinculin mutants in response to changing ECM stiffness. Using these models, the talin ABS2-actin and vinculin tail-actin interactions were found to be extremely important for sensing the stiffness of the ECM. These findings significantly increase our knowledge of the molecular mechanisms underpinning cellular mechanotransduction. Increased understanding of how mechanical signals are sensed and interpreted by the cell could lead to a number of novel therapies for a wide range of associated diseases, such as atherosclerosis, muscular dystrophy and cancer.

Dans les organismes multicellulaires, les cellules génèrent et subissent des forces mécaniques qui se propagent aux cellules voisines. Ces forces peuvent déterminer la forme des tissus et organes, et aussi être converties en signaux biochimiques. Dans un épithélium, les cellules forment un tissu en adhérant directement les unes aux autres grâce à des complexes d’adhérence, tels que les Jonctions Adhérentes. Ces Jonctions Adhérentes sont composées de protéines transmembranaires les E-cadhérines, dont la partie cytoplasmique est sous tension générée par le cytosquelette d’actomyosine par un lien assurée par la β-caténine. La β-caténine est aussi un cofacteur de transcription majeur qui régule l’activité de gènes impliqués dans la transition épithélio-mésenchymateuse une fois dans le noyau. L’accumulation nucléaire et l’activité transcriptionnelle de la β-caténine peuvent avoir lieu à la suite de stimulations mécaniques dans des situations physiologiques et pathologiques, et ont été proposées comme la conséquence d’une libération de la β-caténine des Jonctions Adhérentes suite à sa phosphorylation. Néanmoins, les preuves directes de ce phénomène et ses mécanismes manquent, et le rôle qu’y tient la tension des E-cadhérines n’est pas connu.Dans cette thèse, nous avons établi la relation entre la tension des E-cadhérines et la localisation nucléaire et l’activité de la β-caténine, prouvé l’existence d’une translocation de la membrane au noyau de la β-caténine, et caractérisé les mécanismes moléculaires sous-jacents dans des cellules en migration induite par un facteur de croissance ou par blessure sur un épithélium, deux conditions qui récapitulent au moins partiellement une transition épithélio-mésenchymateuse.Nous avons montré que l’accumulation nucléaire de la β-caténine est due à un départ substantiel de celle-ci de la membrane, spécifiquement dans les cellules en migration. Cette translocation a lieu en aval d’une voie de signalisation impliquant les kinases Src et FAK, et qui conduit à une relaxation de tension des E-cadhérines. Le mécanisme sous-jacent implique une réorganisation du cytosquelette d’actine, caractérisé par un enrichissement des fibres des stress ventrales, soutenant les protrusions, en phospho-myosine, au détriment du cortex d’actine des Jonctions Adhérentes. En revanche, les phosphorylations dans le complexe cadhérine/caténine ne sont pas requises. Ces résultats démontrent que les E-cadhérines ont un rôle de senseur de la mécanique intracellulaire, et que les adhésions focales sont impliquées dans l’activation de la voie de signalisation β-caténine / In multicellular organisms, cells generate and experience mechanical forces that propagate between and within cells. These forces may shape cells, tissues and organs, and also convert into biochemical signals. In a simple epithelium, cells form tissue sheets by directly adhering to one another through adhesion complexes, such as the Adherens Junctions. Adherens Junctions comprise transmembrane proteins E-cadherins, which are under actomyosin-generated tension via a link that contains β-catenin. β-catenin is also a major transcription cofactor that regulates gene activity associated with Epithelial-to-Mesenchyme Transition when translocated in the nucleus. β-catenin nuclear localization and transcriptional activity are mechanically inducible in a variety of healthy and disease models and were proposed to follow phosphorylation-induced -catenin release from E-cadherin. However, direct evidence for this translocation and these mechanisms are lacking, and whether E-cadherin tension is involved is unknown.In this thesis, we assess the relationship between E-cadherin tension and β-catenin nuclear localization and activity, determine the relevance of β-catenin shuttling between membrane and nucleus, and characterize the underlying molecular mechanisms in cells migrating in an at least partial EMT-like fashion upon hepatocyte growth factor (HGF) or wound stimulation. We showed that β-catenin nuclear activity follows a substantial release from the membrane that is specific to migrating cells. This translocation occurs downstream of the Src-FAK pathway, which targets E-cadherin tension relaxation. The underlying mechanisms sufficiently involve actomyosin remodeling, characterized by an enrichment of ventral stress fibers that capture phosphomyosin at the expense of the cortex at Adherens Junctions. In contrast, phosphorylations of the cadherin/catenin complex are not substantially required. These data demonstrate that E-cadherin acts as a sensor of intracellular mechanics in a crosstalk with cell-substrate adhesions that targets β-catenin signaling

La motilité cellulaire directionnelle au cours du développement de l'organisme et des tissus, l'homéostasie et la maladie nécessite une rupture de symétrie. Ce processus repose sur la capacité des cellules individuelles à établir une polarité avant-arrière, et peut se produire en l'absence de signaux externes. L'initiation de la migration a été attribuée à la polarisation spontanée des composants du cytosquelette, tandis que l'évolution spatio-temporelle des forces du cytosquelette résultant de l'interaction mécanique cellule-substrat continue n'a pas encore été résolue. Ici, nous établissons un test de migration microfabriqué unidimensionnel qui imite un environnement fibrillaire complexe in vivo tout en étant compatible avec les mesures de force à haute résolution, la microscopie quantitative et l'optogénétique. La quantification des paramètres morphométriques et mécaniques révèle un comportement de stick-slip générique initié par un détachement stochastique des contacts adhésifs d'un côté de la cellule dépendant de la contractilité, qui est suffisant pour conduire la motilité cellulaire directionnelle en absence de polarité du cytosquelette préétablie ou de gradients morphogènes. Un modèle théorique valide le rôle crucial de la dynamique d'adhésion au cours de la rupture de symétrie spontanée, en proposant que le phénomène examiné puisse émerger indépendamment d'un système auto-polarisant complexe. / Directional cell motility during organism and tissue development, homeostasis and disease requires symmetry breaking. This process relies on the ability of single cells to establish a front-rear polarity, and can occur in absence of external cues. The initiation of migration has been attributed to the spontaneous polarization of cytoskeleton components, while the spatio- temporal evolution of cytoskeletal forces arising from continuous mechanical cell-substrate interaction has yet to be resolved. Here, we establish a one- dimensional microfabricated migration assay that mimics complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to drive directional cell motility in absence of pre-established cytoskeleton polarity or morphogen gradients. A theoretical model validates the crucial role of adhesion dynamics during spontaneous symmetry breaking, proposing that the examined phenomenon can emerge independently of a complex self-polarizing system.

Osteocytes have been proposed as the mechanosensory cells during the process of bone adaption. In this thesis, a microfluidics chamber system (MCS) device was designed, fabricated and tested as a means to maximally simulate the in vivo osteocytic ultrastructure and reproduce the in vivo shear stress experienced by osteocyte, providing an ideal platform for in vitro study on osteocyte mechanotransduction. By employing a micropipette aspiration technique, single osteocyte adhesion and osteocytic process formation were achieved on PDMS with MCS structure. In this study, the involvement of sphingosine-1-phosphate (S1P) signaling pathway in osteocytes responding to oscillatory fluid flow (OFF) was also examined. Firstly, MLO-Y4 osteocytes like cells were demonstrated to express integrated and functional S1P cascade. By modulating S1P cascade components and testing a series of cellular outcomes, it was indicated that exogenous S1P, endogenous S1P and S1P receptor S1P2 were involved in the regulation of loading induced osteocytic responses.

Osteocytes have been proposed as the mechanosensory cells during the process of bone adaption. In this thesis, a microfluidics chamber system (MCS) device was designed, fabricated and tested as a means to maximally simulate the in vivo osteocytic ultrastructure and reproduce the in vivo shear stress experienced by osteocyte, providing an ideal platform for in vitro study on osteocyte mechanotransduction. By employing a micropipette aspiration technique, single osteocyte adhesion and osteocytic process formation were achieved on PDMS with MCS structure. In this study, the involvement of sphingosine-1-phosphate (S1P) signaling pathway in osteocytes responding to oscillatory fluid flow (OFF) was also examined. Firstly, MLO-Y4 osteocytes like cells were demonstrated to express integrated and functional S1P cascade. By modulating S1P cascade components and testing a series of cellular outcomes, it was indicated that exogenous S1P, endogenous S1P and S1P receptor S1P2 were involved in the regulation of loading induced osteocytic responses.

<p>The research presented here represents a quest to understand and address limitations in the field of skeletal muscle tissue engineering, with hopes to better understand the factors involved in producing viable engineered skeletal muscle tissue. The driving force behind this research was to address two of the many factors important in muscle cell proliferation and differentiation, toward developing mature and functional bioartificial skeletal muscles (BAMs). Our work focused on understanding the individual effects of mechanical stimulation and microRNAs (miRNAs), as well as the synergistic relationship between the two factors. We hypothesized that (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via temporally regulated miRNAs and that (2) modulating these miRNAs can enhance skeletal muscle function in a 3D tissue-engineered system.</p><p>We first established a BAM system using C2C12 mouse myoblasts in a collagen gel, showing that these cells were able to produce mature sarcomeres when cultured under steady, passive tension for up to 36 days. Staining muscle-specific proteins and electron microscopy showed distinct striations and myofiber organization as early as 6 days, post-differentiation. At 33 days, cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming.</p><p>We then focused on the effects of mechanical stimulation on C2C12 myoblasts in a more simple, 2D system. In particular, we assessed miRNA and muscle-specific gene expression over time and in response to two cyclic stretch regimens using miRNA microarray technology and quantitative real time RT-PCR. Both miRNAs and certain genes, such as SRF and Mef2c, had differential responses to the two regimens. Over-expression and inhibition studies of one muscle-specific miRNA, miR-1, abrogated the stretch response and suggest that a balancing mechanism is in place to avoid large fluctuations in miRNA levels. </p><p>Finally, since miRNA modulation quenched the stretch-mediated response in myoblasts, we chose to examine 3D BAM function when miRNA levels were altered to promote differentiation. Using the same collagen gel model established previously, a muscle-specific miRNA, miR-133, known to promote proliferation, was transiently inhibited (anti-miR-133) to encourage differentiation. Forces in the anti-miR-133 BAMs were, on average, 20% higher over the negative control. Further, myofiber diameters were significantly greater and striations were more organized in the anti-miR-133 BAMs, suggesting that transient, exogenous delivery of miRNAs may be a viable approach to create a more fully differentiated muscle.</p> / Dissertation

Bladder obstruction (neurogenic or anatomic) induces strain injury in detrusor smooth muscle cells. Signaling via strain injury in other systems has been highly studied, while in bladder obstruction, it has been quite limited to a small number of pathways. In our study we have examined the effects of strain injury using a combination of in vivo, ex vivo and in vitro models, with the aim of understanding disease pathogenesis in the bladder. Using a combination of literature searches, phospho-protein screens and pathway analysis, we uncovered three pathways activated by mechanical strain, ERK, STAT3 and mTOR, with potential for changing not only the way we understand but also the way we treat obstructive myopathies of the bladder. We found that not only were these pathways activated in response to strain and distension injury of BSMC, but they were also responsible for proliferation and sometimes de-differentiation. Included herein are three chapters, published in 2006 and 2010, on the role of ERK, STAT3 and mTOR pathways in bladder smooth muscle cell proliferation and differentiation, 8 Appendices containing the first pages of other papers and reviews published during the course of my studies.

Background: Stretching may be viewed as an external/internal force influencing the range of motion of the connective tissue (muscles, tendons and the myotendon unit (MTU)) The magnitude and rate of stretching may potentially induce mechanical responses of the musculoskeletal system, such as increased range of motion (ROM). The degree of the intensity of stretch (low, medium, or high) may be used to optimize recovery from muscle damage via ameliorating inflammation; this is however, a plausible hypothesis that needs to be appropriately investigated. Aims: The present project aimed to investigate: 1) whether intense stretching (IS) causes an acute inflammatory response (study 1), 2) the effects of stretching intensity (low, medium, or high) in the onset of inflammation (study 2), and 3) investigate whether stretching intensity is responsible for aiding in the recovery of the muscle, post muscle damage (study 3). Methods: Studies one and two were randomized crossover trials consisting of 12 and 11 recreational male athletes, respectively. The former investigated whether high intensity stretching can cause an acute inflammatory response, with study two examining the effects of different stretching intensities (30%, 60% and 90%) based on a participant’s perceived maximum range of motion (mROM). Blood for both studies was collected at pre-, post, and 24h post intervention, and analyzed for high sensitivity C-reactive protein (hsCRP) (study 1 and 2), and for interleukin (IL)-1β, IL-6, and tumour necrosis factor (TNF)-α (study 1). In study three, a randomized controlled trial investigated whether stretching intensity (low or high), can influence the recovery from muscle damage. Thirty participants were randomized into three groups, a) low intensity stretching (LiS) (30-40% ROM), b) high intensity stretching (HiS) (70-80% ROM) and c) Control group. All participants performed both eccentric (EPT) and isometric peak torque (IPT) tests prior to a muscle damage protocol (MDP) (baseline). Participants were then assessed for EPT and IPT for three consecutive days post MDP. Soreness levels were recorded immediately post muscle damage and at 24, 48, and 72h, with blood samples collected at pre, 24, 48, and 72h post muscle damage and analyzed for Creatine Kinase (CK) and hsCRP. Results: Study one revealed a significant increase in hsCRP (P = 0.006) when comparing IS to Control condition, also confirmed by the effect size analyses. In study two, low (30% of mROM), and medium (60% of mROM) intensity stretching did not elicit an inflammatory response while a pronounced inflammatory response was observed when comparing 30 to 90 and 60 to 90% mROM. In study three, LiS showed a significant increase in EPT compared to both HiS and Control, and these findings were confirmed by magnitude based inferences analyses (i.e. LiS was associated with a positive effect for both IPT and soreness levels compared to Control and HiS). Blood biomarkers were associated with inconsistent effects compared to Control and HiS for all three-time periods. Conclusions: This thesis provides preliminary results suggesting that increased stretching intensity may be responsible for causing an acute inflammatory response. In addition, it was observed that LiS might be associated with faster recovery from muscle damage with respect to muscle function (EPT and IPT) and soreness levels. More research is needed to investigate these findings further.

The Intervertebral disc (IVD), comprised of two distinct regions, namely the fibrous annulus fibrosus (AF) and the gelatinous nucleus pulposus (NP), is a fibrocartilage pad located between adjoining vertebrae of the spine. The function of the IVD is to provide stability to the spine, while maintaining movement. IVD degeneration, also known as degenerative disc disease (DDD), is the process whereby the IVD tissue degrades, resulting in loss of function to the disc. Low back pain (LBP) is associated with the degeneration of the IVD, making it important to investigate the pathogenesis of DDD, as this could lead to novel therapies for the prevention and/or treatment of LBP. Mechanical stimuli (MS) are known to be important for IVD cell matrix homeostasis, with cells of the AF and NP responding to physiological forces with a trend towards increased matrix anabolism, while non-physiological forces lead to matrix catabolism. Furthermore, recent evidence suggests that IVD cells derived from degenerate tissue may have lost their ability to respond to physiological MS in the 'normal' anabolic manner, potentially leading to the progression of DDD. It is therefore important to investigate the response of IVD cells derived from both non-degenerate and degenerate tissue to MS, to ascertain whether there is a difference with degeneration. If the response is found to be altered with degeneration, then elucidation of the potentially altered mechanotransduction pathway utilised by degenerate cells could lead to the discovery of novel therapeutic targets for the treatment of DDD. To date, the majority of IVD MS studies have concentrated on the response of NP cells to hydrostatic pressure, with only a limited number of AF studies available. Thus, the first aim of this PhD was to investigate the response of human AF cells derived from non-degenerate and degenerate IVDs to the physiologically relevant mechanical stimulus of cyclic tensile strain (CTS), to ascertain whether the response (regulation of matrix protein and matrix degrading enzyme gene expression) was frequency-dependent or altered with IVD degeneration. Using an in vitro mechanical loading system (Flexcell® Tension Plus™ system, Flexcell International) capable of delivering a CTS of 10% strain, 0.33Hz or 1.0Hz for 20 minutes, the response of AF cells derived from non-degenerate IVDs was found to be frequency-dependent (reduced catabolism at 1.0Hz, with decreased MMP -3 and ADAM-TS -4 gene expression; and catabolic at 0.33Hz, with decreased types I and II collagen and increased MMP -9 gene expression). Furthermore, the response of AF cells to 1.0Hz CTS was shown to be altered with IVD degeneration, depicted by a switch from reduced catabolism (decreased MMP -3 and ADAM-TS -4) in non-degenerate AF cells, to reduced anabolism (decreased aggrecan and type I collagen gene expression) in degenerate AF cells. Subsequently, the second aim of the PhD was to attempt to elucidate the mechanotransduction pathways operating in human AF cells derived from non-degenerate and degenerate IVDs, to ascertain whether the mechanotransduction pathway was altered with IVD degeneration. An identical mechanical stimulation regime was used (1.0Hz CTS) in parallel with functional inhibitors against the cytokines interleukin (IL) -1 and -4, and the cell surface receptors, RGD-recognising integrins. Additionally, the involvement of the cytokine associated transcription factors, nuclear factor kappa beta (NFκB) and signal transducer and activator of transcription (STAT) -6, as well as the integrin-associated kinase, focal adhesion kinase (FAK) was investigated in 1.0Hz CTS-treated non-degenerate AF cells. The response to 1.0Hz CTS (reduced catabolism) of AF cells derived from non-degenerate IVDs occurred in an IL -1, IL -4 and RGD-recognising integrin-dependent manner, with FAK being phosphorylated. Of significant interest, the altered response of AF cells derived from degenerate IVDs to 1.0Hz CTS (reduced anabolism) occurred independently of either cytokine and independently of RGD-recognising integrins, suggesting an altered mechanotransduction pathway in operation and warranting further investigation.

No description available.