Dissertationen zum Thema „Actomyosine – Contraction“

Au cours du développement préimplantatoire, l'embryon de mammifère forme le blastocyste, qui est la structure fixant l'embryon dans l'utérus. La formation du blastocyste repose en grande partie sur les forces contractiles générées par le cortex d'actomyosine. Chez la souris, sur des échelles de temps de quelques secondes, nous observons des impulsions de contractions d'actomyosine voyageant périodiquement autour du périmètre cellulaire. Nous appelons ce phénomène vagues de contraction corticale périodique (PeCoWaCo), une manifestation fascinante et mal comprise de la contractilité. Dans cette étude, nous profitons du développement lent de l'embryon de souris pour étudier des milliers d'impulsions de contraction. Nous mettons également à profit la robustesse de ce développement pour explorer les propriétés biophysiques des PeCoWaCo pendant les étapes de clivage précédant la morphogenèse précoce des mammifères.Nous constatons que, lors des étapes de clivage, des mouvements périodiques apparaissent occasionnellement au stade zygote et 2-cellules puis deviennent systématiques après le 2ème cycle de divisions de clivage. Fait intéressant, la période des oscillations diminue progressivement de 200s au stade zygote à 80s au stade 8-cellules. Étant donné que les cellules deviennent de plus en plus petites avec des divisions de clivage successives, la taille des cellules pourrait être un déterminant important dans l'initiation et la régulation des PeCoWaCo. Nous manipulons la taille des cellules sur une large gamme de rayons cellulaires (10-40 µm) en utilisant la fragmentation et la fusion des cellules et constatons que l'initiation, la persistance ou les propriétés des PeCoWaCo ne dépendent pas de la taille des cellules. Après la période des PeCoWaCo, on découvre que les tensions de surface des blastomères diminuent progressivement jusqu'au stade de 8-cellules et que l’assouplissement artificiel des cellules augmente prématurément les PeCoWaCo. Par conséquent, lors des étapes de clivage, l’assouplissement cortical réveille la contractilité zygotique avant la morphogenèse préimplantatoire. En plus, en manipulant la contractilité de l'actomyosine à l'aide de mutants et des drogues, nous avons montré que la période des PeCoWaCo peut être contrôlée par la régulation du taux de polymérisation de l'actine filamenteuse et de l'activité motrice de la myosine.Dans l'ensemble, nos résultats sur les aspects biophysiques et moléculaires du PeCoWaCo nous aident à comprendre comment la contractilité de l'actomyosine s'éveille avant la morphogenèse préimplantatoire et comment elle est régulée au niveau mécanique et moléculaire
During pre-implantation development, the mammalian embryo forms the blastocyst, which is the structure embedding the embryo into the uterus. The shaping of the blastocyst relies in large part on contractile forces generated by the actomyosin cortex. In the mouse, on timescales of seconds, we observe pulses of actomyosin contractions traveling periodically around the cell perimeter. We call this phenomenon periodic cortical waves of contraction (PeCoWaCo), a fascinating and poorly understood manifestation of contractility. In this study, we take advantage of the slow development of the mouse embryo to study thousands of contraction pulses and of the robustness of the mouse embryo to size manipulation to explore the biophysical properties of PeCoWaCo during the cleavage stages preceding early mammalian morphogenesis. We find that, during cleavage stages, periodic movements appear occasionally at the zygote and the 2-cell stage and become systematic after the 2nd round of cleavage divisions. Interestingly, the period of oscillations progressively decreases from 200 s at the zygote stage to 80 s at the 8-cell stage. Since cells becomes successively smaller with successive cleavage divisions, cell size could be an important determinant in the initiation and regulation of PeCoWaCo. We manipulate cell size on a broad range of cell radii (10-40 µm) using fragmentation and fusion of cells and find that the initiation, persistence or properties of PeCoWaCo do not depend on cell size. Following the period of PeCoWaCo, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis. In addition, by manipulating actomyosin contractility using mutants and drugs, we showed that the period of PeCoWaCo can be tuned by F-actin polymerization rate and myosin motor activity. Altogether our results on biophysical and molecular aspects of PeCoWaCo help us understand how actomyosin contractility awakens before preimplantation morphogenesis and how it is regulated both mechanically and molecularly
İmplantasyon öncesi gelişim sırasında, memeli embriyosu, embriyoyu rahim içineyerleştiren yapı olan blastosisti oluşturur. Blastosistin şekillendirilmesi büyük ölçüdeaktomiyozin korteks tarafından oluşturulan kasılma kuvvetlerine dayanır. Farede, saniyelikzaman ölçeklerinde, hücre çevresinde periyodik olarak dolaşan aktomiyozin kasılmalarınındarbeleri gözlemlenebilir. Bu fenomene, kasılmanın büyüleyici ve yeterince anlaşılmamış birtezahürü olan periyodik kortikal kasılma dalgaları (periodic cortical waves of contraction:PeCoWaCo) diyoruz. Bu çalışmada, erken memeli morfogenezinden önceki bölünmeaşamaları sırasında PeCoWaCo'nun biyofiziksel özelliklerini keşfetmek ve binlerce kasılmadarbesini inceleyebilmek için fare embriyosunun yavaş gelişiminden ve fare embriyosununboyut manipülasyonuna dayanıklılığından faydalandık.Bölünme aşamaları sırasında, zigotta ve 2 hücreli aşamada periyodik hareketlerinzaman zaman ortaya çıktığını ve ikinci tur bölünmeden sonra bu hareketlerin sistematik halegeldiğini bulduk. İlginç bir şekilde, salınım periyodunun zigot aşamasında 200 saniyeden, 8hücreli aşamada 80 saniyeye sistematik olarak azaldığını gözlemledik. Hücreler ardışıkbölünmeleriyle sürekli küçüldüğünden, hücre boyutu PeCoWaCo'nun başlatılmasında vedüzenlenmesinde önemli bir belirleyici olabilir. Hücreleri geniş bir hücre yarıçapı aralığında(10-40 μm) küçük parçalara bölerek veya birbirleriyle birleştirerek PeCoWaCo'nunbaşlatılmasının, kalıcılığının veya genel özelliklerinin hücre boyutuna bağlı olmadığını bulduk.PeCoWaCo periyodunu takiben, embriyo hücrelerinin zigottan 8 hücreli aşamaya kadar yüzeygerilimini kademeli olarak azalttığını ve yapay olarak korteksleri yumuşatılan hücrelerinPeCoWaCo'yu zamanından önce geliştirdiğini keşfettik. Bu sonuçlarla bölünme aşamalarısırasında, kortikal yumuşama, ilke implantasyon öncesi morfogenezinden önce zigotikkasılmaları uyandırdığını gösterdik. Ayrıca, genetik mutantlar ve kimyasallar kullanarakaktomiyozin kasılmasını manipüle ederek, PeCoWaCo periyodunun F-aktin polimerizasyonhızı ve miyozin motor aktivitesinin düzenlenmesi ile ayarlanabileceğini gösterdik.Sonuç olarak, PeCoWaCo'nun biyofiziksel ve moleküler yönleriyle ilgili bulgularımız,aktomiyosin kontraktilitesinin implantasyon öncesi morfogenezinden önce nasıl uyandığını,ayrıca hem mekanik hem de moleküler olarak nasıl düzenlendiğini anlamamıza yardımcı olur

L'etude utilise principalement trois techniques differentes: premierement, la proteolyse specifique suivie de la purification et de la caracterisation des fragments peptiques obtenus; deuxiemement, l'introduction de pontages intra et inter-moleculaires et finalement, des etudes en cinetique rapide. Nous avons pu ainsi identifier une region de la tete de myosine contenant un site de liaison de la partie polyphosphate de l'atp et un site d'interaction de l7actine. La liaison des deux ligants (actine et atp) a ce peptide induisent une mobiite interne. Ces mouvements intramoleculaires reveles egalement dans d'autres regions de la tete de myosine fut mise en evidence grace a des experiences de resonance magnetique nucleaire du proton.

La contractilité cellulaire, un phénomène orchestrée par le système d'actomyosine, est un régulateur critique d'une large gamme de processus cellulaires, y compris l'établissement de la polarité cellulaire, la migration cellulaire, l'intégrité des tissus au cours de la morphogenèse ou du développement. Une simple perturbation de la génération de la force et des propriétés mécaniques des cellules peut affecter leurs fonctions physiologiques et par conséquent peut conduire à des défauts pathologiques y compris le cancer.Cependant, les mécanismes qui contrôlent la production de la force par le système acto-myosine et leurs modes de régulation dans les cellules ne sont pas pleinement compris. Au cours de ma thèse, j'ai utilisé un système biomimétique fait d'un ensemble minimal de protéines purifiées pour étudier les propriétés contractiles du système actomyosin.L'objectif était de comprendre comment l'architecture des filaments d'actine peut modifier la réponse contractile. A cet effet, j'étais d'abord intéressée par la construction d'une variété d'organisation de l'actine qui servira après comme substrat pour les moteurs moléculaires (la myosine) lors de la contraction.Afin de comprendre les principes généraux qui dictent l'assemblage de l'actine, nous avons développé un modèle numérique qui nous a permis d'identifier les paramètres clés, y compris l'interaction entre les filaments d'actine, les propriétés mécaniques de ces filaments et l'activation par contact entre une région de nucléation et les filaments d'actine qui poussent à partir d'un motif adjacent. Ce modèle a été utilisé en premier lieu pour implémenter les propriétés reliées à l'actine et en second lieu pour évaluer la réponse contractile des structures d'actine induite par la myosine.Durant ma thèse, j'ai pu démontrer que le niveau de connectivité module la déformation du réseau d'actine induite par la myosine, selon leur architecture. J'ai montré aussi que les protéines de pontages des filaments d'actine sont nécessaires pour effectuer une déformation et générer des forces au niveau des réseaux d'actine dynamiques en présence de la myosine. De plus, nous avons développé les simulations numériques dans le but de relier la déformation macroscopique des structures d'actines due à la myosine avec le mécanisme microscopique sous-jacent.Ce travail a révélé comment la variété des réseaux d'actine contracte d'une façon différente même en respectant les mêmes conditions biochimiques et a démontré l'importance de l'effet du réarrangement dynamique des structures d'actine sur la modulation de sa contractilité
Cellular contractility – the internal generation of force by a cell orchestrated by theactomyosin machinery – is a critical regulator of a wide range of cellular processes includingthe establishment of cell polarity, cell migration, tissue integrity or morphogenesis duringdevelopment. Disruptions of the force generation and of mechanical properties of living cellsaffect their physiological functions and consequently can lead to pathological defectsincluding cancer. However, the parameters or mechanisms that drive force production by theactin-myosin system and their mode of regulation in cells are not fully understood. During myPhD, I used biomimetic system made of a minimum set of proteins to study the properties ofactomyosin contractile systems. The goal was to understand how/if the actin architecture canmediate the contractile response. For this purpose, I was first interested in building a varietyof actin organization that will serve next as substrate for myosin during contraction. Tounderstand the general principles that dictate geometrically-controlled actin assembly, wedeveloped a model that allowed us to identify key parameters including filaments/filamentsinteraction, filament mechanical property and contact activation between actin filamentsgrowing from the adjacent pattern and the nucleation area. These actin templates were usedthen to evaluate the response of oriented actin structures to myosin-induced contractility. Idemonstrated that crosslinking level modulates the myosin-induced deformation of actinnetworks according to their architecture. I showed also that crosslinkers are necessary tosustain myosin-driven deformation and force production of dynamic actin networks. Inaddition, we developed numerical simulation in order to relate the observed myosin-drivenactin deformation with the underlying microscopic mechanism. This work revealed howdiverse cellular actin networks contract differently to a define set of biochemical conditionsand hence how dynamic rearrangements can modulate network contractility

Dans les tissus, les cellules génèrent et sont soumises en permanence à des forces mécaniques. Les perturbations biochimiques à l'intérieur des cellules, ainsi que les altérations de leur environnement mécanique peuvent modifier l'équilibre physiologique et mener à des pathologies, comme le cancer. Bien que les propriétés mécaniques puissent être modifiées à l'échelle du tissus, la compréhension de la mécanique au niveau de la cellule unique demeure importante. En particulier, la différenciation, la migration des cellules immunitaires et le caractère invasif d'un cancer dépendent fortement des propriétés mécaniques des cellules uniques. Les déformations mécaniques peuvent induire un changement de la surface et du volume cellulaires. Nous nous intéressons particulièrement à la régulation du volume cellulaire chez les cellules mammifères dans le contexte de déformations à différentes échelles de temps. Jusqu'à présent, la régulation du volume dans ce contexte n'a été que très peu étudiée, en raison de la difficulté d'obtention de mesures précises, et du fait que le volume de la cellule est généralement considéré comme constant. Nous avons développé une méthode de mesure du volume cellulaire reposant sur l'exclusion de fluorescence, qui nous permet d'effectuer des mesures de volume précise au niveau de la cellule unique. Dans cette étude, nous nous sommes concentrés sur la régulation du volume cellulaire au cours de l'étalement dynamique sur un substrat (échelle de temps : minutes). Nous avons démontré qu'il existe différents régimes de régulation du volume lors de l'étalement : les cellules réduisent, augmentent ou ne modifient pas leur volume, en fonction de l'état du cortex d'actomyosine et de la vitesse d'étalement. Nous avons constaté que les cellules s'étalant plus vite ont tendance à perdre davantage de volume. Notre hypothèse est que lors d'une extension rapide de lamellipode dépendante d'Arp2/3, l'actine tire sur la membrane et génère une tension et l'activation de transport ionique, s'accompagnant d'une perte de volume compensatoire. L'inhibition de la polymérisation de l'actine ou de sa ramification dépendante d'Arp2/3 réduit la vitesse d'étalement et ainsi la perte de volume. Nous avons ensuite montré que l'inhibition de la contractilité augmente la vitesse d'étalement et la perte de volume. Cependant, l'inhibition d'Arp2/3 dans des cellules à faible contractilité conduit à un étalement rapide sans perte de volume. En effet, l'inhibition d'Arp2/3 induit des bulles de membranes, une déformation rapide n'induirait donc pas de perte de volume car la cellule peut relâcher la tension en dépliant la membrane. Nous avons également montré que la régulation du volume en réponse à une compression mécanique rapide (échelle de temps : millisecondes) indépendante de l'adhérence dépend également de l'état du cortex d'actomyosine. Les cellules perdent jusqu'à 30% de leur volume lorsqu'elles sont confinées, car la membrane plasmique est attachée au cortex et ne peux pas être dépliée en réponse à l'augmentation de la tension. La perturbation du cortex d'actine induit le détachement de la membrane et limite la perte de volume. Enfin, nous avons montré que la réponse du volume à un choc osmotique (échelle de temps : secondes) est plus que complexe que décrite dans la littérature. Nos données indiquent qu'au niveau de la cellule unique, la réponse initiale du volume au changement de l'osmolarité extérieure n'est pas un processus passif uniforme. En utilisant la technique du choc osmotique, nous avons également confirmé que les cellules ont un large excès de membrane repliée dans des réservoirs. Nos résultats montrent que le volume et l'aire cellulaires sont couplés par l'homéostasie de la tension de surface, et, étant donné que les déformations induisent une augmentation de la tension de surface, elles conduisent à des modifications du volume et de l'aire de la cellule
The field of biomechanics significantly progressed in the last two decades. The importance of the feedback between biochemical signaling and physical properties was revealed in many studies. Cells within tissues constantly generate and experience mechanical forces. Biochemical perturbations inside the cells as well as alterations in the mechanical environment can shift the tiny balance of normal physiological state and lead to pathologies, e.g. cancer. Although the mechanical properties of individual cells can alter when they are within the tissues, the understanding of single cell mechanics is still important. Differentiation, immune cell migration, and cancer invasion strongly depend on the mechanical properties of individual cells. Mechanical deformations can lead to a change in cell surface area and volume. We are particularly interested in single mammalian cell volume regulation in the context of deformations of different timescales. For the moment, volume regulation in this context was out from the research interest, probably due to the difficulties of accurate measurements, and cell volume often considered as a constant parameter. We developed a method for cell volume measurements based on a fluorescent exclusion that allowed us to perform precise volume measurements of individual live cells. In the present study, we mainly focused on cell volume regulation while dynamic spreading on a substrate (timescale – minutes). We demonstrated that there are different regimes for volume regulation while spreading: cells decrease, increase or do not change volume, and a type of the regime depends on the state of the actomyosin cortex and spreading speed. We obtained that faster-spreading cells tend to lose more volume. Our hypothesis is that during fast Arp2/3-driven lamellipodia extension actin pull on the membrane that generates tension and activation of ion transport and regulatory volume loss. Inhibition of actin polymerization or Arp2/3-dependent actin branching decreases spreading speed and volume loss. Next, we showed that inhibition of contractility increases spreading speed and volume loss. However, inhibition of Arp2/3 complex in cells with low contractility leads to fast spreading without volume loss. Our explanation is that inhibition of Arp2/3 induces cell blebbing and even fast deformation does not lead to volume loss as a cell can relax tension by membrane unfolding. We also showed that volume regulation in response to fast mechanical compression (timescale – milliseconds) independent of adhesion also depends on the actomyosin cortex state. Control cells lose up to 30% of volume under confinement, as the cell membrane is attached to the cortex and cannot be unfolded in response to the tension increase. Disruption of actin cortex leads to membrane detachment and prevents volume loss under confinement. Additionally, we showed that cell volume response to the osmotic shock (timescale – seconds) is more complex than it used to be known in the literature. For instance, our data indicate that at the level of individual cells initial volume response to the change of external osmolarity is not a uniform passive process. Using osmotic shock technique, we also confirmed that cells have a large excess of membrane folded in reservoirs. Taken together, our data show that cell volume and surface area are coupled through surface tension homeostasis and as deformations induce surface tension increase, they lead to change volume and surface area

Dissertation presented to obtain the Ph.D degree in Molecular Medicine
Animal and yeast cells use a contractile ring that is attached to the plasma membrane to create a cleavage furrow that partitions a cell into two in the latest step of cell division. The contractile ring is a network of actin and myosin-II motor filaments embedded in a complex and compact protein core structure at the cell division site. In the absence of myosin-II, cells fail to assemble the contractile ring pursuing death or rapidly evolving divergent pathways to restore growth and cytokinesis, an event associated to aneuploidy, a common trait in cancer development and progression. The molecular mechanisms underlying myosin-II localization and function at the cell division site with actin ring assembly and contraction remain poorly understood. Based on analogy to the striated muscle, it has been classically proposed that contractile stress in the actomyosin ring is generated via a “sliding filament” mechanism in which bipolar myosin-II motor filaments walk along actin filaments, within organized sarcomere-like arrays. However, ultra-structural and genetic studies in different cellular systems have shown that contractile rings are more complex than striated muscles, and in some examples the motor activity can actually be dispensable for the contractibility of the cytokinetic ring.(...)
PhD fellowship awarded by the Rong Li laboratory and a previous awarded fellow of the GABBA PhD program at the Faculty of Medicine, University of Porto, Portugal and the Portuguese Foundation for Science and Technology, Portugal. Apoio financeiro da Fundação para a Ciência e Tecnologia e do Fundo Social Europeu no âmbito do Quadro Comunitário de Apoio, BD n°SFRH/BD/11760/2003.

Epithelial tissues undergo extensive remodeling during embryonic development. Recent studies have revealed that, in a number of developmental processes, epithelial remodeling is associated with pulsations of individual cell surface areas and cortical actomyosin flows. During Drosophila dorsal closure, the amnioserosa (AS), a contractile tissue covering the dorsal region of the embryo, shows contractile pulsations and regular actomyosin flows during the reduction of its apical surface area. The biophysical mechanism driving these shape pulsations as well as the role of contractile actomyosin waves in epithelial contraction and dorsal closure still remains unclear. In this project, we developed a biophysical model for cell shape oscillations that is based on intrinsic properties of the cell: cortex turnover, active contractility by force producing molecules and cell elasticity. We show that coupling these three key ingredients is sufficient for generating stable oscillations. Further, within this framework we were also able to generate waves by coupling the oscillating units and introducing a diffusion term to account for exchange of force producing molecules between the units. Next, we investigated the role for these contractile actomyosin waves in tissue remodeling. We developed a novel technique that allowed us to apply mechanical stretch on the AS tissue and study the response of cells to such stress. With this method, we were able to arrest the pulsatile contractions and actomyosin flows in AS cells. We show that this arrest is associated with the relocalisation of actin and myosin from the medial region of the cells towards the adherens junctions to maintain junction integrity upon stretch. This relocalisation of myosin directly correlates with the junctional strain and does not occur in cells that have excessive membrane material as a consequence of endocytosis inhibition. In the latter case, cells continue pulsing and seem to be “insensitive” to stretch. Upon stretch release, myosin relocalises to the medial area of the cell and pulsations resume. This indicates that cells can switch between two states depending on tension: one in which cells exhibit shape oscillations associated with contractile actomyosin pulses and waves, and the other where cell shape is stabilised with myosin preferentially localised at the cell junctions. Further, following release from long duration ( >10mins ) stretch application, cell junctions were highly wrinkled. Strong and consistent localisation of myosin waves at these regions led to straightening and reduction of junctional lengths. Moreover, during dorsal closure, AS cells constantly reduce their areas while maintaining junctions of consistent thickness and length relative to area. This is not the case where endocytosis is blocked or myosin activity is down-regulated. Our results not only shed light on fundamental physical properties of the actomyosin cortex, in particular they also indicate a role of myosin contractile waves in junctional remodeling during AS cell constriction.
Los tejidos epiteliales llevan a cabo una remodelación extensiva durante el desarrollo embrionario. Estudios recientes han revelada que, en un sinnumero de procesos de desarrollo embrionario, la remodelación epitelial se asocia con pulsaciones de áreas en células individuales y con flujos corticales de actomiosina. Durante el cierre dorsal de Drosophila, la amnioserosa (AS), un tejido contractil que cubre la región dorsal del embrión, se observan pulsaciones contráctiles en células individuales y flujos regulares de actomiosina durante la reducción de la superficial apical celular. Al día de hoy, no se conoce el mecanismo biofísico que produce estas pulsaciones celulares ni y el papel que tienen las oscilaciones contráctiles de actomiosina en el epitelio del cierre dorsal embrionario. En este proyecto, se desarrolló un modelo biofísico para entender estas oscilaciones celulares. El modelo se basa en propiedades intrínsecas de la célula como la rotación de la corteza celular, la contractilidad activa mediante moléculas productoras de fuerza y la elasticidad celular. Utilizando éste modelo, se muestra que acoplando estas tres propiedades clave es suficiente para generar oscilaciones celulares estables. Además, dentro de este marco, se han generado oscilaciones mediante el acoplamiento de varias unidades oscilantes y la introducción de un término de difusión para considerar el intercambio de moléculas productoras de fuerza entre las unidades. A continuación, se investigó el papel de estas oscilaciones contráctiles de actomiosina en la remodelación de tejidos. Como resultado, se desarrolla una técnica innovadora que permite aplicar extensión mecánica al tejido de AS y estudiar la respuesta celular ante tal estrés. Con este método, se pueden detener las pulsaciones contráctiles y los flujos de actomiosina en células de la AS. Se muestra que este arresto celular está asociado con la relocalización de actina y miosina de la región central de las células hacia las uniones adherentes intercelulares para mantener su integridad durante la extension epitelial. Esta relocalización de miosina se correlaciona directamente con la tensión en uniones intercelulares y no ocurre en células en las que el reciclaje cellular a través de endocitosis se ha bloqueado. El resultado es un exceso en la acumulación de membrana plasmática en células oscilantes que no responden a la extension epitelial. Tras liberar al tejido de la extension epithelial, la miosina se relocaliza a la área central de las células y las pulsaciones continuan. Esto indica que las células pueden cambiar entre dos estados según la tension aplicada: uno dónde las células muestran oscilaciones asociadas con pulsaciones contráctiles de actomiosina, y otra donde la forma celular se establece con la localización preferente de miosina en las uniones intercelulares. Además, tras liberar el tejido de una extensión de alta duración (>10mins), las uniones intercelulares sufrieron corrugaciones. La localización consistente de oscilaciones de miosina en las regions corrugadas, resulta en una extension y reducción en la longitud de las uniones intercelulares. Además, durante el cierre dorsal, las células de la AS reducen sus areas constantemente, mientras mantienen uniones intercelulares de espesor consistente y longitud relativa a su área. Esto no es el caso cuando la endocitosis se bloquea o la actividad de miosina se reduce. Nuestros resultados no solo muestran las propiedades fundamentales de la corteza cellular de actomiosina, también indican el papel de oscilaciones contráctiles de miosina en la remodelación de uniones intercelulares durante la constricción de la AS.

The cytoskeleton is an intrinsic aspect of all cells, and is essential for many cellular events including cell motility, endocytosis, cell division and wound healing. Remodeling of the cytoskeleton in response to these cellular activities leads to significant alterations in the morphology of the cell. One such alteration is the formation of an actomyosin contractile array required for cytokinesis, wound healing and embryonic development.
Cellular structure and shape depends upon tensional prestress brought about by the organization of cytoskeletal components. Using the Xenopus laevis oocyte wound healing model, it is first described how diminished cellular tension affects the balance of the Rho family of GTPases, and subsequently prevents the formation of actomyosin contractile arrays. This suggests that cellular tension in the cell is not created at the level of the cytoskeletal elements but rather via the upstream signaling molecules: RhoA and Cdc42.
The role of N-WASP (Neural-Wiscott Aldrich Syndrome Protein), a mediator of Arp2/3 based actin polymerization, is next examined for its putative role in cellular wound healing. Xenopus laevis oocytes injected with mutant N-WASP constructs reveals in vivo evidence that functional N-WASP is required for appropriate contractile array formation and wound closure.
Lastly, it is revealed that the cellular structures involved with single cell wound healing in other model systems are also important for the initial repair of severed muscle cells. Actin, non-muscle myosin-II, microtubules, sarcomeric myosin and Cdc42 are all recruited and reorganized at the edge of damaged C2C12 myotubes. This data promotes the possibility that an actomyosin array may be established in injured muscle cells as well.

Epithelial tissues undergo extensive remodeling during embryonic development. Recent studies have revealed that, in a number of developmental processes, epithelial remodeling is associated with pulsations of individual cell surface areas and cortical actomyosin flows. During Drosophila dorsal closure, the amnioserosa (AS), a contractile tissue covering the dorsal region of the embryo, shows contractile pulsations and regular actomyosin flows during the reduction of its apical surface area. The biophysical mechanism driving these shape pulsations as well as the role of contractile actomyosin waves in epithelial contraction and dorsal closure still remains unclear. In this project, we developed a biophysical model for cell shape oscillations that is based on intrinsic properties of the cell: cortex turnover, active contractility by force producing molecules and cell elasticity. We show that coupling these three key ingredients is sufficient for generating stable oscillations. Further, within this framework we were also able to generate waves by coupling the oscillating units and introducing a diffusion term to account for exchange of force producing molecules between the units. Next, we investigated the role for these contractile actomyosin waves in tissue remodeling. We developed a novel technique that allowed us to apply mechanical stretch on the AS tissue and study the response of cells to such stress. With this method, we were able to arrest the pulsatile contractions and actomyosin flows in AS cells. We show that this arrest is associated with the relocalisation of actin and myosin from the medial region of the cells towards the adherens junctions to maintain junction integrity upon stretch. This relocalisation of myosin directly correlates with the junctional strain and does not occur in cells that have excessive membrane material as a consequence of endocytosis inhibition. In the latter case, cells continue pulsing and seem to be “insensitive” to stretch. Upon stretch release, myosin relocalises to the medial area of the cell and pulsations resume. This indicates that cells can switch between two states depending on tension: one in which cells exhibit shape oscillations associated with contractile actomyosin pulses and waves, and the other where cell shape is stabilised with myosin preferentially localised at the cell junctions. Further, following release from long duration ( >10mins ) stretch application, cell junctions were highly wrinkled. Strong and consistent localisation of myosin waves at these regions led to straightening and reduction of junctional lengths. Moreover, during dorsal closure, AS cells constantly reduce their areas while maintaining junctions of consistent thickness and length relative to area. This is not the case where endocytosis is blocked or myosin activity is down-regulated. Our results not only shed light on fundamental physical properties of the actomyosin cortex, in particular they also indicate a role of myosin contractile waves in junctional remodeling during AS cell constriction.
Los tejidos epiteliales llevan a cabo una remodelación extensiva durante el desarrollo embrionario. Estudios recientes han revelada que, en un sinnumero de procesos de desarrollo embrionario, la remodelación epitelial se asocia con pulsaciones de áreas en células individuales y con flujos corticales de actomiosina. Durante el cierre dorsal de Drosophila, la amnioserosa (AS), un tejido contractil que cubre la región dorsal del embrión, se observan pulsaciones contráctiles en células individuales y flujos regulares de actomiosina durante la reducción de la superficial apical celular. Al día de hoy, no se conoce el mecanismo biofísico que produce estas pulsaciones celulares ni y el papel que tienen las oscilaciones contráctiles de actomiosina en el epitelio del cierre dorsal embrionario. En este proyecto, se desarrolló un modelo biofísico para entender estas oscilaciones celulares. El modelo se basa en propiedades intrínsecas de la célula como la rotación de la corteza celular, la contractilidad activa mediante moléculas productoras de fuerza y la elasticidad celular. Utilizando éste modelo, se muestra que acoplando estas tres propiedades clave es suficiente para generar oscilaciones celulares estables. Además, dentro de este marco, se han generado oscilaciones mediante el acoplamiento de varias unidades oscilantes y la introducción de un término de difusión para considerar el intercambio de moléculas productoras de fuerza entre las unidades. A continuación, se investigó el papel de estas oscilaciones contráctiles de actomiosina en la remodelación de tejidos. Como resultado, se desarrolla una técnica innovadora que permite aplicar extensión mecánica al tejido de AS y estudiar la respuesta celular ante tal estrés. Con este método, se pueden detener las pulsaciones contráctiles y los flujos de actomiosina en células de la AS. Se muestra que este arresto celular está asociado con la relocalización de actina y miosina de la región central de las células hacia las uniones adherentes intercelulares para mantener su integridad durante la extension epitelial. Esta relocalización de miosina se correlaciona directamente con la tensión en uniones intercelulares y no ocurre en células en las que el reciclaje cellular a través de endocitosis se ha bloqueado. El resultado es un exceso en la acumulación de membrana plasmática en células oscilantes que no responden a la extension epitelial. Tras liberar al tejido de la extension epithelial, la miosina se relocaliza a la área central de las células y las pulsaciones continuan. Esto indica que las células pueden cambiar entre dos estados según la tension aplicada: uno dónde las células muestran oscilaciones asociadas con pulsaciones contráctiles de actomiosina, y otra donde la forma celular se establece con la localización preferente de miosina en las uniones intercelulares. Además, tras liberar el tejido de una extensión de alta duración (>10mins), las uniones intercelulares sufrieron corrugaciones. La localización consistente de oscilaciones de miosina en las regions corrugadas, resulta en una extension y reducción en la longitud de las uniones intercelulares. Además, durante el cierre dorsal, las células de la AS reducen sus areas constantemente, mientras mantienen uniones intercelulares de espesor consistente y longitud relativa a su área. Esto no es el caso cuando la endocitosis se bloquea o la actividad de miosina se reduce. Nuestros resultados no solo muestran las propiedades fundamentales de la corteza cellular de actomiosina, también indican el papel de oscilaciones contráctiles de miosina en la remodelación de uniones intercelulares durante la constricción de la AS.

Three novel mutations (G159D, L29Q and E59D/D75Y) in cardiac troponin C (CTnC) associate their clinical outcomes with a given cardiomyopathy. Current paradigms propose that sarcomeric mutations associated with dilated cardiomyopathy (DCM) decrease the myofilament calcium sensitivity while those associated with hypertrophic (HCM) cardiomyopathy increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the calcium regulation of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. This required the development of new calculator programs to solve complex ionic equilibria to more accurately buffer and expand the free calcium range of our test solutions. In accordance with the DCM paradigms, our result show that G159D and E59D/D75Y CTnC decrease the myofilament calcium sensitivity and force generating capabilities which would likely increase the rate of muscle relaxation and weaken the contractile force of the myocardium. Alternatively, the lack of myofilament change from L29Q CTnC (associated with HCM) may explain why the only proband is seemingly unaffected. Notably, the changes in the calcium sensitivity of tension (in fibers) do not necessarily occur in the isolated CTnC and vice versa. These counter-intuitive findings are justified through a transition in calcium affinity occurring at the level of cardiac troponin (CTn) and higher, implying that the true effects of these mutations become apparent as the hierarchal level of the myofilament increases. Despite these limitations, the regulated thin filament (RTF) retains its role as the calcium regulatory unit and best indicates a mutation's ability to sensitize (+) or desensitize (-) the muscle to calcium. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse (+ or -) these aberrant changes in myofilament sensitivity. Therefore, we have developed an RTF mediated High Throughput Screening assay to identify compounds in libraries of molecules that can specifically modulate the calcium sensitivity of cardiac contraction. The knowledge gained from these studies will help us and others to uncover new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases.

Le complexe actomyosine, formé de l’association de la myosine II avec les filaments d’actine, stabilise le cytosquelette d’actine et génère la contraction cellulaire nécessaire à plusieurs processus comme la motilité et l’apoptose dans les cellules non-musculaires. La myosine II est un hexamère formé d’une paire de chaînes lourdes (MHCs) et de deux paires de chaînes légères MLC20 et MLC17. La régulation de l’activité de la myosine II, c'est-à-dire son interaction avec les filaments d’actine, est directement liée à l’état de phosphorylation des MLC20, mais il reste beaucoup à découvrir sur l’implication des MHCs. Il existe trois isoformes de MHCs de myosine II, MHCIIA, MHCIIB et MHCIIC qui possèdent des fonctions à la fois communes et distinctes. Notre but est de mettre en évidence les différences de fonction entre les isoformes de myosine II, au niveau structurale, dans la stabilisation du cytosquelette d’actine, et au niveau de leur activité contractile, dans la génération des forces de tension. Nous nous sommes intéressés au rôle des isoformes des MHCs dans l’activité du complexe actomyosine qui est sollicité durant le processus de contraction cellulaire de l’apoptose. Dans quatre lignées cellulaires différentes, le traitement conjoint au TNFα et à la cycloheximide causait la contraction et le rétrécissement des cellules suivi de leur détachement du support de culture. Par Western blot, nous avons confirmé que la phosphorylation des MLC20 est augmentée suite au clivage de ROCK1 par la caspase-3, permettant ainsi l’interaction entre la myosine II et les filaments d’actine et par conséquent, la contraction des cellules apoptotiques. Cette contraction est bloquée par l’inhibition des caspases et de ROCK1. MHCIIA est dégradée suite à l’activation de la caspase-3 alors que MHCIIB n’est pas affectée. En utilisant une lignée cellulaire déficiente en MHCIIB, ou MHCIIB (-/-), nous avons observé que la contraction et le détachement cellulaires durant l’induction de l’apoptose se produisaient moins rapidement que dans la lignée de type sauvage (Wt) ce qui suggère que l’isoforme B est impliquée dans la contraction des cellules apoptotiques. Parallèlement, la kinase atypique PKCζ, qui phosphoryle MHCIIB et non MHCIIA, est activée durant l’apoptose. PKCζ joue un rôle important puisque son inhibition bloque la contraction des cellules apoptotiques. Par la suite, nous nous sommes intéressés à la modulation de la morphologie cellulaire par la myosine II. Les fibroblastes MHCIIB (-/-), présentent un large lamellipode dont la formation semble dû uniquement à l’absence de l’isoforme MHCIIB, alors que les fibroblastes Wt ont une morphologie cellulaire étoilée. La formation du lamellipode dans les fibroblastes MHCIIB (-/-) est caractérisée par l’association de la cortactine avec la membrane plasmique. L’observation en microscopie confocale nous indique que MHCIIA interagit avec la cortactine dans les fibroblastes Wt mais très peu dans les fibroblastes MHCIIB (-/-). Le bFGF active la voie des MAP kinases dans les fibroblastes Wt et MHCIIB (-/-) et induit des extensions cellulaires aberrantes dans les fibroblastes MHCIIB (-/-). Nos résultats montrent que l’implication de l’isoforme B de la myosine II dans la modulation de la morphologie cellulaire. L’ensemble de nos résultats participe à distinguer la fonction structurale et contractile de chacune des isoformes de myosine II dans la physiologie cellulaire.
We are interested in studying the modulation of the actomyosin complex which is involved in different cellular processes such as cell locomotion and apoptosis. The actomyosin complex is formed by the association of actin filaments and myosin II. The non-muscle myosin II is a hexamer formed by one pair of heavy chains (MHCs) and two pairs of light chain (MLC20 and MLC17). The actomyosin activity is dependent on MLC20 and MHCs phosphorylation. There are three isoforms of MHCs (MHCIIA, MHCIIB and MHCIIC) which have common but also distinctive roles in several cellular processes. Our aim is to clarify the structural and contractile functions of each isoforme of myosin II in different cellular processes, in particular, cell contraction and cell morphology. First, we studied the implication of myosin II isoforms in cell shrinkage and detachment during apoptosis which are both dependent on actomyosin contractility. We treated four different cell lines with TNFα in combination with cycloheximide (CHX) to trigger apoptosis. We confirmed that TNFα induced caspase-3 activation, ROCK1 cleavage and increased MLC20 phosphorylation. We showed that TNFα/CHX induced the caspase-dependent MHCIIA degradation, whereas MHCIIB levels and association with the actin cytoskeleton remained virtually unchanged. Cell shrinkage and detachment were blocked by caspase and ROCK1 inhibitors. Using the MHCIIB (-/-) cell line, we observed that the absence of MHCIIB did not affect cell death rate. However, MHCIIB (-/-) fibroblasts showed more resistance to TNFα-induced actin disassembly, cell shrinkage and detachment than wild type (Wt) fibroblasts, indicating the participation of MHCIIB in these events. PKCζ, which only phosphorylates MHCIIB, was cleaved during apoptosis, co-immunoprecipitated preferentially with MHCIIB and, interestedly, PKCζ inhibition blocked TNFα-induced shrinkage and detachment. Our results demonstrate that MHCIIB, together with MLC phosphorylation and actin, constitute the actomyosin cytoskeleton that mediates contractility during apoptosis. Second, we studied the involvement of myosin II isoforms in cell shape modulation. Fibroblasts MHCIIB (-/-) spontaneously formed lamellipodia whereas Wt fibroblasts presented a stellate shape. Cortactin was associated with the leading edge of lamellipodia in MHCIIB (-/-) fibroblasts, but it localised diffusely in the cytoplasm or at the end of fine cellular projections in Wt fibroblasts. The levels of cortactin and cortactin phosphorylated in Tyr421 associated with membrane in MHCIIB (-/-) fibroblasts were higher than in Wt fibroblasts. Confocal microscopy showed cortactin/MHCIIA colocalization in wild type but not in MHCIIB (-/-) fibroblasts. bFGF activates Erk1/2 in wild type and MHCIIB (-/-) fibroblasts and induces the formation of aberrant membrane projections in MHCIIB (-/-) fibroblasts. In conclusion, our results contribute to characterize the structural and contractile role of each myosin II isoforms in the physiology of the cell.