<p>Physical forces play a key role in the morphogenesis of embryos. As cells and tissues change shape, grow, and migrate, they exert and respond to forces via mechanosensitive proteins and protein complexes. How the response to force is regulated is not completely understood. </p><p>Dorsal closure in Drosophila is a model system for studying cell sheet forces during morphogenesis. We demonstrate a role for mechanically gated ion channels (MGCs) in dorsal closure. Microinjection of GsMTx4 or GdCl₃, inhibitors of MGCs, blocks closure in a dose-dependent manner. UV-mediated uncaging of intracellular Ca<super>2+</super> causes cell contraction whereas the reduction of extra- and intracellular Ca<super>2+</super> slows closure. Pharmacologically blocking MGCs leads to defects in force generation via failure of actomyosin structures during closure, and impairs the ability of tissues to regulate forces in response to laser microsurgery.</p><p>We identify three genes which encode candidate MGC subunits that play a role in dorsal closure, <italic>ripped pocket</italic>, <italic>dtrpA1</italic>, and <italic>nompC</italic>. We find that knockdown of these channels either singly or in combination leads to defects in force generation and cell shapes during closure. </p><p>Our results reveal a key role for MGCs in closure, and suggest a mechanism for the coordination of force producing cell behaviors across the embryo.</p> / Dissertation

Cartilage tissue provides compressive resistance in diarthrodial joints, and has been shown to be regulated by mechanical signals, in particular with regard to production of extracellular matrix proteins. However, less is understood about how chondrocytes in regions not solely purposed to provide compressive resistance may also be affected by mechanical forces. The growth plate is a small layer of cartilage that functions to facilitate longitudinal growth of the long bones from in utero through post-adolescent development. The growth plate maintains distinct regions of chondrocytes at carefully regulated stages of endochondral ossification that are in part characterized by their morphology and differential responsiveness to vitamin D metabolites. Understanding if mechanical cues could be harnessed to accelerate or delay the process of endochondral ossification might be beneficial for optimizing tissue engineering of cartilage or osteochondral interfaces. This study focused on three aims to provide a basis for future work in this area: 1) Develop a cell line culture model useful for studying growth plate chondrocytes, 2) Determine the response of primary growth plate chondrocytes and the cell line model to fluid shear stress, and 3) determine if expression of integrin beta 1 is important for the observed responses to shear stress. The findings of this study suggest that inorganic phosphate can promote differentiation in coordination with the 24,25(OH)2D3 metabolite of vitamin D, and that fluid shear stress generally inhibits differentiation and proliferation of growth plate chondrocytes in part through an integrin beta 1 mediated pathway.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 180-204). Also available online.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

La capacité des cellules à sentir les propriétés physiques de leur environnement est un facteur déterminant de l'homéostasie tissulaire. Ainsi, la rigidité de la matrice extracellulaire (forces exogènes) et les tensions du cytosquelette (forces endogènes) coopèrent de manière fonctionnelle modulant les transformations phénotypiques. Les cellules perçoivent et transmettent des forces en développant des structures d'adhérences appelées adhésions focales. Ces adhésions sont composées de protéines transmembranaires, les intégrines, qui font le lien entre le cytosquelette et la matrice extracellulaire.La partie centrale de mon projet de thèse aborde la question du couplage des intégrines b1 et b3 dans la mécanotransduction. Les données actuelles plaident fortement en faveur d'une relation bidirectionnelle entre l'adhésion intégrine-dépendante et les forces mécaniques générées dans ce processus. Les approches génétiques classiques ont souligné le rôle majeur des intégrines b1 et b3 dans mécanosensibilité cellulaire, sans préciser leur contribution relative. Par exemple, la manière dont la modulation de l'expression de l'intégrine b3 affecte la génération des forces de traction cellulaires et la distribution des adhésions intégrines-dépendantes reste à être explorées. Dans ce travail de thèse nous avons montré que les intégrines b1 ont un rôle essentiel dans la génération de forces cellulaires, que les intégrines b1 sont régulées négativement par les intégrines b3 en affectant la distribution spatiale des intégrines b1 à travers leur capacité à lier à la fois la taline et la kindline. Et enfin, nous avons montré que les intégrines b3 régulent temporellement l'activité contractile de la cellule.J'ai également participé à deux autres études dans le cadre de collaborations avec le Pr. Holmgren et le Dr. Debili, au cours desquelles j'ai utilisé la microscopie à traction de forces comme un outil diagnostique afin d'observer l'effet des forces contractiles dans la formation de la lumen aortique et de la formation des plaquettes sanguines. J'ai ainsi pu confirmer que la protéine amotL2, reliant les fibres contractiles aux VE-cadhérines, est impliquée dans la force intercellulaire nécessaire à la formation de la lumen aortique. Et lors d'une deuxième collaboration, j'ai pu montrer que la contractilité des mégacaryocytes, via leur système actomyosine, est nécessaire pour la formation des proplaquettes. / Cell ability to sense mechanical properties of their microenvironment is crucial for tissue homeostasis which means their capacity to maintain mechanical integrity as they are submitted to external forces.Integrins have been highlighted as mechanotransducers able to form micro-scale structures called focal adhesion sites which mechanically link cells to the extracellular matrix by recruiting various adaptors. Both b1 and b3 integrins have been identified as the principal actors of tensional homeostasis. However as the resulting mechanotransduction processes are intrinsically dynamic, the respective and cooperative roles b1 and b3 integrins need to be addressed over time and space.In the present work, coupling time-resolved traction force microscopy and genetics approaches, we investigated the respective role of b1 and b3 integrins in active force generation at the single cell level. Our findings show that b1 integrins has an essential role in generation of cellular traction forces, b1 integrin-generated force is negatively regulated by b3 integrins which impacts the redistribution of b1 integrin containing adhesion through its ability to bind to talin and kindlin, b3 integrin supports min-scale temporal regulation of cellular contractile activity generated by b1 integrin. Finally, cell mechanical equilibrium relies on the ability of cells to maintain a fixed contractile moment.I also participated in two others studies in the framework of collaborations in which I used the traction force microscopy as a diagnostic tool to observe the effect of contractile forces in the formation of the aortic lumen and the formation of proplatelets. I was able to confirm that the protein amotL2 connecting the contractile fibers to VE-cadherin, is involved in intercellular forces necessary for the formation of the aortic lumen. And in a second collaboration, where I found by using traction force microscopy that the contractility of megakaryocytes via its actomyosin system, is necessary for the formation proplatelets.

La mécanotransduction est une propriété essentielle au développement des tissus, leur homéostasie et leur physiopathologie. La voie de signalisation YAP (Yes-Associated Protein) est apparue comme un régulateur particulièrement important de la mécano-réponse. Un défaut de mécanosensibilité défectueuse, associant une signalisation aberrante de la voie YAP, a récemment été rapportée dans des myoblastes humains de patients souffrant de dystrophie musculaire congénitale liée à des mutations du gène de la lamine (L-CMD) (Bertrand et al., 2014). La L-CMD est une forme grave de dystrophie musculaire à début précoce. Mon projet de doctorat visait à disséquer les défauts de la mécanosensibilité de cellules précurseurs du muscle présentant la mutation ΔK32. Mes résultats ont montré que les myoblastes mutants ΔK32 présentaient des un défaut de contact cellule-cellul, attestant d’anomalies de transmission des forces mécaniques entre cellules. Contrairement à ce que l’on observe dans les cellules contrôles à confluence, la voie YAP reste activée dans les myoblastes mutants ΔK32. Cela s‘est traduit par une activité transcriptionnelle accrue de YAP et une localisation nucléaire persistante de YAP dans les myoblastes ΔK32. La suractivité de YAP dans les myoblastes mutants ΔK32 n'était pas liée à une altération de la voie Hippo, voie de signalisation canonique qui régule YAP. La signalisation YAP défectueuse a été associée à une désorganisation de différents sous-ensembles du cytosquelette d'actine, incluant l'actine supranucléaire, l'actine basale et les fibres d'actine de la jonction cellule-cellule. La formation et la maturation de jonctions cellule-cellule était défectueuse dans les myoblates ΔK32, et les expressions protéiques des deux principales cadhérines, M et N-cadhérins, étaient significativement réduites à confluence. De plus, les myoblastes mutants ΔK32 présentaient une perte accrue de contact cellule-cellule pendant la migration, responsable d’une perte de la migration collective dans les cellules mutantes. Enfin, nous avons rapporté une augmentation de l'activité transcriptionnelle de la signalisation Smad 1/5/8 dans les myoblastes mutants ΔK32. En conclusion, ces résultats de thèse suggèrent que les défauts de mécanosensibilité dans les myoblastes mutants ΔK32 affectent la capacité des myoblastes à former des contacts cellule-cellule et à migrer collectivement. Ces défauts de mécanosensibilité peuvent contribuer à la physiopathologie de la L-CMD. / Mechanotransduction is critical for tissue development, homeostasis and diseases. YAP (Yes-Associated Protein) signaling has emerged as a particularly important regulator of the mechano-response. A defective mechanosensing response, including aberrant YAP signaling, has been recently reported in human myoblasts from patients suffering from LMNA related congenital dystrophy (L-CMD) (Bertrand et al., 2014). L-CMD is a severe early-onset form of muscular dystrophies caused by mutations in A-type lamins. My PhD project aims to further dissect mechanosensing defects of immortalized muscle precursor cells which carry the L-CMD causing ∆K32 mutation. My results showed that ∆K32 mutant myoblasts had a defective translation of mechanical forces at cell-cell contact sides. ∆K32 mutant myoblasts failed to inactivate YAP in high cell-cell contact conditions, as attested by an increased transcriptional activity of YAP and a persistent nuclear localization. YAP overactivity in ∆K32 mutant myoblasts was not related to an impaired activation of the Hippo signaling pathway. Defective YAP signaling was associated with a disorganization of different subsets of the actin cytoskeleton, including the supranuclear actin, the basal actin and the actin fibers at cell-cell junction. The formation of mature cell-cell contacts in ∆K32 myoblats was defective, and the protein expressions of both M- and N-cadherins were significantly reduced in high cell-cell contact conditions. Moreover, ∆K32 mutant myoblasts showed an increased loss of cell-cell contact during migration, which caused a shift from a sheet-like to a single cell migration pattern. Finally, we reported an increased transcriptional activity of mechanosensitive Smad 1/5/8 signaling in ∆K32 mutant myoblasts. Taken together, these results suggest that mechanosensing defects in ∆K32 mutant myoblasts affect the ability of myoblast to form cell-cell contacts and to migrate collectively. These mechanosensing defects may contribute to the pathophysiology of L-CMD.

No description available.

Biomaterials have evolved over the years from the passive role of mere biocompatibility to an increasingly active role of presenting instructive cues to elicit precise responses at the molecular and cellular levels. Various characteristics common to synthetic biomaterials in vitro and extracellular matrices in vivo, such as immobilized functional or peptide groups, mechanical stiffness, bulk physical properties and topographical features, are key players that regulate cell response. The dynamics in the cell microenvironment and at the cell adhesive interface trigger a web of cell-material and cell-cell information exchanges that have a profound impact in directing the ultimate cell fate decision. Therefore, comprehension of cell substrate interactions is crucial to propel forward the evolution of new instructive biomaterials. Combinatorial biomaterials that encompass a wide range of properties can help to recapitulate the complexity of a cell microenvironment. The objective of this research was to fabricate combinatorial biomaterials with properties that span wide ranges in both surface chemistries and mechanical moduli. These materials were based on polydimethyl siloxane (PDMS), an elastomeric silicone biomaterial with physiologically relevant stiffness. After developing these mechano-chemical gradient biomaterials, we conducted high throughput screening of cell responses on them to elucidate cell substrate interactions and material directed behaviors. Our central hypothesis was that materials encompassing monotonic gradients in mechanical elastic modulus and orthogonal surface chemistry gradients could be engineered using the soft biomaterial, polydimethyl siloxane (PDMS) and that these gradient biomaterials would evoke a varied cell response. Furthermore, we expected high throughput screening of cell-material interactions using these materials would elucidate patterns and thresholds of synergy or antagonism in the overall cell response to the increased complexity presented by combinatorial materials. First, reproducible gradients in surface chemistry were generated on PDMS through surface modification techniques. Cell response to PDMS surface chemistry gradients was then screened in a rapid high throughput manner. Additionally, characteristics of the adhesive interface were probed to understand its role in cell response. Finally, a 2D combinatorial gradient with a gradient in mechanical elastic modulus and an orthogonal gradient in surface chemistry was fabricated with PDMS. High throughput screening of the synergistic influence of the varied mechanical and biochemical extracellular signals presented by the combinatorial biomaterial on cell response was conducted in a systematic manner. This research demonstrates the fabrication of combinatorial biomaterials with a wide range of mechanochemical properties for rapid screening of cell response; a technique that will facilitate the development of biomaterial design criteria for numerous biomedical engineering applications including in vitro cell culture platforms and tissue engineering.

Traumatic Brain Injury (TBI) is linked to a diverse range of diffuse pathological damage for which there is a severe lack of therapeutic options. A major limitation to drug development is the inability to identify causal mechanisms that link head trauma to the multitude of secondary injury cascades that underlie neuropathology. To elucidate these relationships, it is important to consider how physical forces are transmitted through the brain across multiple spatial scales ranging from the whole head to the sub-cellular level. In doing so, the mechanical behavior of the brain is typically characterized solely by its material properties and biological structure. Alternatively, forces transmitted through distinct cellular and extracellular structures have been shown to influence physiological processes in multiple cell types through the transduction of mechanical forces into cellular chemical responses. As an essential component of various biological processes, these mechanotransduction events are regulated by mechanical cues directed through extracellular matrix (ECM) and cell adhesion molecules (CAM) to mechanosensitive intra-cellular structures such as focal adhesions (FAs). Using a series of in vitro models, we have implicated FAs in the cellular mechanism of traumatic axonal injury by showing that forces directed through these structures potentiate injury levels and, moreover, that inhibition of FA-mediated signaling pathways may be neuroprotective. In addition, we show that localizing trauma forces through specific brain ECM results in differential injury rates, further implicating mechanosensitive cell-ECM linkages in the mechanism of TBI. Therefore, we show that FAs play a major role in axonal injury at low strain magnitudes indicating that cellular mechanotransduction may be an important mechanism underlying the initiation of cell and sub-cellular injuries ultimately responsible for the diffuse pathological damage and clinical symptoms observed in diffuse axonal injury. Furthermore, since these mechanisms may present the earliest events in the complex sequelae associated with TBI, they also represent potential therapeutic opportunities. / Engineering and Applied Sciences

Mechanical cues have been recognized to be critically important in the regulation of cells. A myriad of cellular processes including differentiation, proliferation, and gene expression are all affected by physical forces from the extra- and intra-cellular microenvironments. Despite recent advances in nano-technologies, many questions still surround how cells sense and respond to forces. Through a series of studies, we demonstrate how both the structure and inherent mechanical properties of the cell affect their response to mechanical cues. We first develop a methodology to mechanically manipulate cells while simultaneously characterizing their deformations. Using combined atomic force and confocal microscopy techniques and through systematic examination we demonstrate the role of the cytoskeleton and nucleus in the deformability and shape change of epithelial cells. Mechanical properties have been used in recent years to identify diseased states, including cancer. With this in mind, we used HeLa cells as a model and characterized significant deformability of their plasma membrane and underlying cortex. Importantly, we demonstrate and characterize their ability to recover from large shape changes, which we also observed in other epithelial cells. Shape recovery is shown to be rapid and reliant upon the actin cytoskeleton and intracellular fluid flow. Although the nucleus does not contribute significantly to the deformation and recovery of HeLa cells, the importance of nuclear mechanics cannot be forgone. In vitro studies have shown that mechanical forces transmitted through the cell’s cytoskeleton critically affect nuclear mechanics and gene transcription processes. Many others have used simple models and isolated nuclei in an attempt to characterize nuclear properties. Thus, in a subsequent study, we examine the nucleus within intact cells. Nuclear shape change, in response to force, is shown to be complex and cannot be well-characterized by isotropic mechanical properties. Characterization of the mechanics of the cell, as demonstrated through our findings, is crucial in the field of biological physics. The aforementioned studies, written as scientific articles, are presented in the body of this thesis (Chapters 2-5). A review article that focuses on mechanotransduction and relevant examples using AFM as a tool for its examination acts as an introductory chapter.