The assembly, form, and function of the heart is regulated by complex mechanical signals originating from intrinsic and extrinsic sources, such as the cytoskeleton and the extracellular matrix. During development, mechanical forces influence the self-assembly of highly organized ventricular myocardium. However, mechanical overload induces maladaptive remodeling of tissue structure and eventual failure. Thus, mechanical forces potentiate physiological or pathological remodeling, depending on factors such as frequency and magnitude. We hypothesized that mechanical stimuli in the form of microenvironmental stiffness, cytoskeletal architecture, or cyclic stretch regulate cell-cell junction formation and cytoskeletal remodeling during development and disease. To test this, we engineered cardiac tissues in vitro and quantified structural and functional remodeling over multiple spatial scales in response to diverse mechanical perturbations mimicking development and disease. We first asked if the mechanical microenvironment impacts tissue assembly. To investigate this, we cultured two-cell cardiac µtissues on flexible substrates with tunable stiffness and monitored cell-cell junction formation over time. As myocytes transitioned from isolated cells to interconnected µtissues, focal adhesions disassembled near cell-cell interfaces and mechanical forces were transmitted almost completely through cell-cell junctions. However, µtissues cultured on stiff substrates mimicking fibrotic microenvironments retained focal adhesions near the cell-cell interface, potentially to reinforce the cell-cell junction in response to excessive forces generated by myofibrils in stiff microenvironments. Intercellular electrical conductance between myocytes was measured as a function of connexin 43 immunosignal and the length-to-width ratio of cell pairs. We observed that conductance was correlated to connexin 43 immunosignal and cell pair length-to-width ratio, indicating that tissue architecture can affect electrical coupling. The impact of mechanical overload was also determined by applying chronic cyclic stretch to engineered cardiac tissues. Stretch activated gene expression patterns characteristic of pathological remodeling, including up-regulation of focal adhesion genes, and impacted sarcomere alignment and myocyte shape. Furthermore, chronic cyclic stretch altered intracellular calcium cycling in a manner similar to heart failure and decreased contractile stress generation, suggestive of maladaptive remodeling. In summary, we show that the assembly, form, and function of cardiac tissue is sensitive to a wide range of mechanical cues that emerge during physiological and pathological growth. / Engineering and Applied Sciences

Ο αυξητικός παράγοντας πλειοτροπίνη (Pleiotrophin, PTN) έχει μοριακή μάζα 18 kDa και ανήκει σε μια διακριτή οικογένεια αυξητικών παραγόντων που δεσμεύονται στην ηπαρίνη και σχετίζονται με αγγειογένεση και καρκινική ανάπτυξη. Στην παρούσα εργασία, μελετήσαμε τη συμμετοχή των ενδοκυτταρικών κινασών FAK και ILK στην επαγόμενη από PTN κυτταρική μετανάστευση σε ανθρώπινα ενδοθηλιακά κύτταρα από φλέβα ομφάλιου λώρου (HUVEC). Εξωγενής χορήγηση ΡΤΝ επάγει τη φωσφορυλίωση της κινάσης FAK στις τυροσίνες 397 και 925, ενώ μειώνει τη φωσφορυλίωση της κινάσης FAK στη τυροσίνη 576. Η κινάση ILK φαίνεται να εμπλέκεται στη διεγερτική δράση της PTN στη μετανάστευση των κυττάρων HUVEC, αφού μείωση της έκφρασης της κινάσης ILK με παρεμβαλλόμενο RNA στα κύτταρα HUVEC, οδήγησε σε αναστολή της επαγόμενης από ΡΤΝ κυτταρικής μετανάστευσης. Επιπλέον, διέγερση των κυττάρων HUVEC με PTN είχε ως αποτέλεσμα την επαγωγή της ενεργοποίησης της κινάσης ILK. Με σκοπό να διερευνηθεί η θέση της κινάσης ILK στο μονοπάτι μεταγωγής σήματος που ενεργοποιείται από τη PTN στα κύτταρα HUVEC, μελετήσαμε την πιθανή αλληλεπίδραση της ILK με μόρια που είναι γνωστό ότι συμμετέχουν σε αυτό το μονοπάτι. Παρατηρήθηκε ότι η κινάση ILK αλληλεπιδρά σε μεγάλο βαθμό με την κινάση FAK, ενώ μικρού βαθμού αλληλεπίδραση φαίνεται και με τις ιντεγκρίνη β3 και κινάση c-Src. Ενδιαφέρον παρουσιάζει το γεγονός ότι η διέγερση με PTN των κυττάρων HUVEC αυξάνει την αλληλεπίδραση μεταξύ των κινασών FAK και ILK. Τέλος, με δεδομένο ότι η β-κατενίνης εμπλέκεται στη κυτταρική μετανάστευση, ερευνήσαμε κατά πόσο η PTN και η κινάση ILK εμπλέκονται στο σηματοδοτικό μονοπάτι της β-κατενίνης στα κύτταρα HUVEC. Η ΡΤΝ αυξάνει με δοσο-εξαρτώμενο και χρονο-εξαρτώμενο τρόπο τη φωσφορυλίωση της β-κατενίνης σε κύτταρα HUVEC, φαινόμενο που αναστέλλεται μετά τη μείωση της έκφρασης της κινάσης ILK με siRNA. Συμπερασματικά, στην παρούσα εργασία καταδεικνύεται ότι η ΡΤΝ έχει διαφορική δράση στη φωσφορυλίωση τυροσινών της κινάσης FAK διαφορετικών θέσεων και ότι η κινάση ILK συμμετέχει στη διεγερτική δράση της PTN στη μετανάστευση των κυττάρων. / Pleiotrophin (PTN) is an 18 kDa secreted growth factor that displays high affinity for heparin. A growing body of evidence indicates that PTN is involved in cell proliferation, migration and differentiation. In the present work, we studied the possible role of two intracellular kinases, focal adhesion kinase (FAK) and integrin-linked kinase (ILK), in the PTN-induced migration of human umbilical vein endothelial cells (HUVEC). Exogenous administration of PTN significantly increased the phosphorylation of FAK kinase in tyrosines 397 and 925 and decreased phosphorylation in tyrosine 576. ILK seems to be involved in PTN-induced migration of HUVEC, since suppression of the ILK kinase using small interfering RNA (siRNA) abolished the stimulatory effect of PTN in migration of HUVEC. In addition, stimulation of HUVEC with PTN increased the ILK kinase activity. In order to determine which other signaling mediators are involved in the PTN signaling pathway, we studied the interaction of ILK with other proteins that have been implicated in the PTN-induced signal transduction. ILK strongly interacted with FAK kinase and to a lesser extent with c-src kinase and integrin ανβ3. Interestingly, PTN increased the degree of interaction between ILK and FAK kinases. Finally, it has been well described that β-catenin is involved in cell migration and that PTN increases β-catenin phosphorylation. We therefore investigated whether PTN affects β-catenin phosphorylation in HUVEC through activation of ILK kinase. PTN significantly increased phosphorylation of β-catenin in a concentration and time dependent manner, which seemed to be abolished after suppression of the ILK kinase using siRNA. Collectively, these results suggest a role of FAK and ILK kinases in the PTN-related signaling cascade which leads to cell migration both human endothelial cells.

Focal adhesion kinase (FAK), a non-receptor kinase, is a key regulator of integrin and focal adhesion signaling required for cancer cell survival, cell migration, and cell invasion. Amplification/Overexpression of FAK occurs in a wide variety of human cancers, supporting a role in carcinogenesis. Moreover, preclinical studies using cancer models where FAK is genetically inhibited indicate that this kinase is a potential therapeutic target to interfere with cancer progression. However, very little progress has been made in the identification of chemical inhibitors for potential therapeutic applications, in contrast to other kinases. Herein, I report optimization of the high-throughput in vitro Glo kinase assay for screening inhibitors of FAK kinase activity. Screening a large library of small molecule chemicals using these assays identified at least twenty FAK inhibitors, including a new FAK inhibitor developed by Pfizer and undergoing human clinical trials, and the non-specific kinase inhibitor staurosporine. Molecular studies of selective FAK inhibitors are undergoing in my host laboratory. In addition to this in vitro assay, I established similar assays to examine FAK kinase and adapter function in intact cells. The latter consists of ErbB-transformed cells deficient in FAK, and their matched cells where wild-type or kinase-dead FAK was restored. Biological characterization of these models revealed that both FAK kinase and adaptor activities cooperate for the regulation of cell migration, cell invasion, and tumor formation.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 115-128).

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Bibliography : leaves 84-91.

Thesis (Ph. D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from thesis description page. Includes bibliographical references.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007. / Radhakrishna, Harish, Committee Member ; Zhu, Cheng, Committee Member ; LaPlaca, Michelle C., Committee Member ; Garca, Andrs J., Committee Chair ; Kowalczyk, Andrew P., Committee Member.

The ability to adhere to the extracellular matrix (ECM) is critical for numerous cell types and tissues including epithelia and muscle. Cell-ECM adhesion is primarily mediated by integrins which provide a direct link between the ECM and the actin cytoskeleton. Integrin adhesions are frequently associated with a core of 60 different proteins (integrin-associated proteins, IAPs). Integrins are required for muscle attachment and in Drosophila, loss of integrins and several IAPs results in embryonic lethality and muscle detachment. However, the IAPs FAK, RSU1, tensin, vinculin and zyxin are not required for viability or embryonic muscle attachment. Furthermore, FAK, RSU1, tensin and vinculin have been observed to localise to muscle attachment sites in Drosophila, indicating that they have some function in muscle attachment. Unlike FAK, RSU1, tensin and vinculin, it was not previously known whether zyxin is expressed in Drosophila muscles. To test this, I generated a genomic zyxin-GFP construct that should contain most of the endogenous zyxin promotor. The genomic zyxin-GFP construct was not observed at muscle attachment sites, suggesting that it is not normally expressed in muscle. I wished to know whether FAK, RSU1, tensin and vinculin are required for muscle function. Various behavioural assays were employed to test for muscle function in larvae and adult flies. The results suggest that larval muscle function was normal in flies lacking these IAPs, but that adult muscle function might be impaired, although it proved difficult to demonstrate a clear functional defect. I then tested whether the IAPs FAK, RSU1, tensin and vinculin are required for normal morphology of adult muscles, focusing on the adult indirect flight muscles (IFMs). The IFMs are fibrillar muscles which attach to the cuticle via specialised epithelial cells known as tendon cells. At the end of the myofibril, where the myofibril attaches to the tendon cell, is a dense region of actin and IAPs known as the modified terminal Z-band (MTZ). I have found that the MTZ is not a homogenous zone of proteins, but is instead organised into at least three distinct layers. Because of the similarity between the structure of the MTZ with that of a hand, I refer to the layers as ‘fingers’, ‘palm’ and ‘wrist’. I discovered that the IAPs FAK, RSU1, tensin and vinculin are each required for the proper structure of the MTZ in unique ways. The fingers were elongated in IFMs lacking FAK, RSU1, tensin or vinculin, while the palm was disrupted in IFMs lacking RSU1, tensin or vinculin. Finally, I was intrigued by the enrichment of the actin-binding protein filamin/Cheerio in the palm and wished to know if it is required for palm function. Deletion of the C-terminus of filamin/Cheerio resulted in a reduction in palm length. Filamin/Cheerio is a mechanosensitive protein which exists in a closed and open conformation. I found that filamin/Cheerio must be open in order to help form a normal palm. Furthermore, vinculin is required to convert filamin/Cheerio from and closed to an open filamin/Cheerio state so that it can perform its function in the palm.

Gliomas são tumores altamente invasivos, resistentes aos tratamentos disponíveis atualmente e com alta taxa de mortalidade. A superexpressão de RECK na linhagem de glioma humano T98G comprometeu a capacidade das células de migrar e invadir in vitro, com rearranjo do citoesqueleto e alteração na distribuição espacial de FAK fosforilado. Entretanto, o possível mecanismo envolvido na inibição da migração mediada por RECK não foi desvendado. Para estudarmos os mecanismos envolvidos nesta alteração da capacidade migratória, as células T98G foram transfectadas com o vetor plasmidial pCXN2-hRECK (RECK+). A via das integrinas, a atividade de alguns membros da família das RhoGTPases e elementos do citoesqueleto foram avaliados através de imunoblotting, imunomarcação e ensaios de pull-down para as células RECK+ em comparação com células T98G não-transfectadas (WT), células T98G transfectadas com vetor pCXN2 na ausência do gene RECK (vetor) e fibroblastos primários humanos (FF287). Nossos resultados mostram um aumento na expressão de integrina β1 e uma diminuição da fosforilação de FAK no sítio de auto-fosforilação Tyr397 que, juntamente com o aumento das fibras de estresse e a diminuição dos lamelipódios, sugerem um fenótipo menos migratório da célula. Porém, quando avaliada a atividade de Rac1, esta se mostrou aumentada, embora uma das vias de ativação de Rac1 seja através da fosforilação de FAK levando à formação dos lamelipódios. A hipótese é que RECK inibe a quebra das adesões focais que participam do processo de migração, dificultando a mobilidade celular. Como as células continuam recebendo o estímulo para migrar, estas ativam Rac1 através de uma via independente de FAK. Além disso, a imunomarcação de paxilina mostrou um aumento no tamanho das adesões focais nas células RECK+, indicando que RECK pode influenciar nas estruturas responsáveis pelo contato célula-matriz. / Gliomas are highly invasive, treatment-resistant and lethal tumors. Overexpression of RECK in human glioma cell line T98G decreased cell migration and invasion in vitro, lead to cytoskeleton rearrangement and caused changes in phospho-FAK distribution. However, the pathway involved in RECK-mediated inhibition of cell migration has not been elucidated yet. To study the mechanisms by which RECK affects cell motility, T98G cells were transfected with pCXN2-hRECK vector (RECK+). Some proteins involved in the integrin pathway, activity of some proteins of RhoGTPase family and cytoskeleton proteins were analyzed through immunoblotting, immunostaining and pull-down assay in RECK+ cells and compared with non-transfected T98G cells, T98G transfected with pCXN2 without RECK gene and human primary fibroblasts (FF287). Our results showed an increase in integrin β1 expression and a decrease in FAK phosphorylation in the Tyr397 site, which together with the increase of stress fibers and decrease of lamellipodia, suggest a less migratory phenotype. Despite this, Rac1 activity was increased even though one of Rac activation pathways is through phospho-FAK, leading to lamellipodium formation. Our hypotheses is that RECK affects focal adhesion turnover, diminishing cell motility. As cells are still receiving a positive signal to migrate, they activate Rac1 through a FAK-independent pathway. Besides that, paxillin immunostaining showed that focal adhesions are larger in RECK+ cells, indicating that RECK can influence structures related with cell-matrix contact.

La migration des cellules est nécessaire au cours du développement. Chez l’adulte, elle contribue au renouvellement des tissus, à la cicatrisation et à la circulation des cellules immunitaires. Les cellules cancéreuses acquièrent des capacités de migration qui échappent aux mécanismes normaux de régulation. Elles peuvent ainsi envahir les tissus environnants et, éventuellement, former des métastases. Les astrocytes représentent une majorité de cellules gliales du système nerveux central. Ils migrent en réponse à des facteurs inflammatoires et interviennent ainsi dans la cicatrisation et la régénération des tissus lésés. Les astrocytes peuvent être à l’origine de tumeurs appelées gliomes qui représentent la majorité des tumeurs cérébrales primaires. Les formes les plus agressives, appelées glioblastomes, sont des tumeurs extrêmement invasives, ce qui les rend particulièrement difficiles à traiter. Les microtubules jouent un role crucial dans la migration des astrocytes et des cellules de gliomes (Etienne-Manneville, 2013a). Au cours de la migration, le réseau de microtubules est totalement réorganisé pour permettre la polarisation de la cellule. Formés par association de dimères de tubuline, leur régulation intervient de multiples manières comme par exemple au niveau de leur dynamique de polymérisation à la périphérie de leurs interactions avec les composants cellulaires ou par leurs modifications post-traductionnelles (Etienne- Manneville, 2010). En effet, les dimères de tubulines polymérisées, inclues dans les microtubules, peuvent être, entre autres, détyrosinées, acétylées, mono ou poly-glutamylées, et mono ou poly-glycylées (Janke and Chloë Bulinski, 2011). Durant la migration cellulaire, ces modifications post-traductionnelles peuvent notamment jouer un rôle dans la régulation du trafic intracellulaire. L’objectif majeur de mon projet de thèse est d’étudier les mécanismesIii de régulation des microtubules au cours de la migration des astrocytes normaux et tumoraux, et plus particulièrement le rôle des modifications post-traductionnelles de la tubuline. Je me suis focalisée sur trois principales modifications post-traductionnelles des microtubules durant la migration : la polyglutamylation, la détyrosination et l’acétylation. En premier lieu, j’ai étudié le rôle potentiel de la polyglutamylation dans la mise en place de la polarité cellulaire chez les astrocytes. Je n’ai pas observé d’effet de cette modification durant la migration… / The migration of cells is necessary during the development. At the adult, she contributes to the renewal of fabrics, to the healing and to the traffic of immune cells. Cancer cells acquire capacities of migration which escape the normal mechanisms of regulation...