Embryogenesis is one of the most delicate biological processes which requires precise control in various levels, including molecular distribution and gene expression, cellular orientation and specification, and tissue dynamics giving rise to proper morphology. The diverse animal morphology can be resulted from the difference during early embryonic cleavages. Spiral cleavage is a conserved embryonic patterning strategy used in the majority of the animal clade Spiralia. The specific cell positioning during cell division and quadrant-based clonal domain formation make the embryos with the blastomeres orientated in a spiral manner when viewing from the animal pole. Although spiral cleavage is conserved in many phyla, the detailed cellular, molecular and biophysical mechanisms for this left-right symmetry breaking event remain unclear. Here I studied the early development of the prototypic annelid spiral-cleaver Platynereis dumerilii, which performs two unequal embryonic cleavages followed by the first dextral spiral cleavages, and compared the mechanisms to other spiralians or to other cleavage types. First, I described the morphology of each cell cycle from the zygote until 64-cell stage by imaging the fluorescently labeled fixed embryos. Second, with mRNA injection, whole-embryo live-imaging with Selective Plane Illumination Microscopy (SPIM), and in silico cell tracking, I monitored these cleavages in 4-D, constructed the early cell lineages, and revealed the subtle asynchrony of the four quartets. Third, together with the spindle inclination angle measurement, I discovered the leading role of the D macromere during P. dumerilii spiral cleavage. I also confirmed that the dextral micromere orientation is neither affected by the eggshell nor the presence of all the neighbor macromeres, suggesting that this cellular property may be achieved by cell autonomous molecular mechanisms. In order to quantify the candidate cytoskeletal dynamics during spiral cleavage, I optimized the construction of the injected mRNAs and the injection protocol to achieve the highest translational level of the fluorescent protein within a given developmental time. Beside mRNA injection, I also established a protein expression and injection protocol for P. dumerilii protein injection in order to visualize the target gene as early as possible. Both techniques didn’t dramatically influence embryogenesis and allow for quantification of the protein dynamics. With these strategies, I discovered and measured the chiral counter rotational flow of cortical actomyosin in each spiral cleavage and revealed that it’s present in the first two spiral cleavages, especially of the macromeres. The biophysical force generated by actomyosin contributes in the cell deformation and spindle inclination, resulting in proper dextral micromere positioning, during the first spiral cleavage, confirmed by the chemical treatment to the P. dumerilii embryos. The asymmetric actomyosin distribution, nuclei migration, and the change of the cell axes during cytokinesis in the macromeres also suggests that the macromeres may play critical roles to lead spiral cleavage. This work is built on the knowledge of the spiral cleavage machinery and has extended it in multiple dimensions. The detailed phase-by-phase description of each cleavage increases the information of P. dumerilii embryogenesis. The established labeling and imaging techniques in this thesis are the important basis for investigation and comparisons of different spiralian development in the future. More broadly, the discovery of actomyosin dynamics shows conservation to the left-right symmetry breaking events of the animals which does not belong to Spiralia. These together bring insights to a global evolutionary speculation: a conserved mechanical force generation pathway, tuned by the upstream molecular signals, may be the key of the miscellaneous cleavage types, resulting in the astonishing variety of embryo patterning.

SammanfattningSyftet med denna studie var att jämföra NADH-kopplade mätsystem och in vitro motilitets-analys (IVMA) för att bestämma aktiviteten hos isolerat myosin. Från NADH-kopplade analysmätningar bestämdes tre parameter: den maximala hastigheten med vilken myosin hydrolyserar ATP i frånvaro av F-aktin (V0), den maximala ATPas-hastigheten för myosin i närvaro av mättande aktin (kcat) och den koncentration av aktin som behövs för att nå halv maximal aktivering av myosin ATPas-aktivitet (KATPas). Från in vitro-motilitets-analys (IVMA) bestämdes två parametrar: fraktion av rörliga filament (FMF) och totala antalet rörliga filament (TMF). Från detta kunde vi uppskatta den fraktion av aktiva huvuden i myosinpreparationer som behövs för en lyckad IVMA.Myosin är ett protein som tillsammans med aktin är ansvarigt för muskelkontraktionen. I denna studie används två myosin preparationer (HMM-fragment) som vi betecknade ”bra HMM” och ”dåligt HMM” på grund av deras kvalitet för aktin motilitet. Först mättes ATPas-aktiviteten hos myosinmotorer med hjälp av ett NADH-kopplat mätsystem som bygger på övervakning av förändringen i absorbans av NADH. Därefter bestämdes V0, kcat, och KATPas för aktin-beroende av myosin-ATPast genom att mäta myosinaktivitet vid olika aktinkoncentrationer, följt av anpassning av data till Michaelis-Menten ekvationen.Parallellt utfördes IVMA-studier genom att HMM immobiliserades på ett objektglas som derivatiserats med trimetylklorsilan. Sedan observerades när HMM flyttar fram fluorescensmärkta aktinfilament i närvaro av ATP. Under samma förhållanden gav resultaten för basalt myosin ATPas aktivitet V0 värden som var ~0,03 ATP s-1 myosinhuvud -1for både dåligt och bra HMM. I en jämförelse mellan de två HMM vid olika F-aktin-koncentrationer var hastighet i ATP-förbrukningen högre för bra än för dåligt HMM. Anpassning av data till Michaelis-Menten-ekvationen gav kcat på 7,18 ATP s-1myosinhuvud-1för dåligt HMM jämfört med 11,21 ATP s-1 myosinhuvud-1för bra HMM (35 % högre). KATPas (Km) för dåligt HMM var lite högre jämfört med den för bra HMM. Vid IVMA-studierna var FMF och TMF 80 % respektive 98 % lägre för dåligt än bra HMM. Slutsatsen var att de två metoderna karakteriserar HMM-funktionen på olika sätt och med olika känslighet. Om man antar att bra HMM har nästan 100 % aktiva huvuden och eftersom man vet att uppmätt kcat är direkt proportionellt mot antalet aktiva myosinhuvuden ser man från dessa mätningar att mycket mer än 65 % av totalt myosin måste vara aktivt för att ge god aktinmotilitet i en IVMA.

La rétinopathie diabétique (RD) se compose d’une part d’une ischémie rétinienne périphérique et d’autre part d’une exsudation rétinienne responsable d’un œdème maculaire diabétique, première cause de cécité chez les moins 55 ans. Les traitements utilisés actuellement sont non spécifiques et traitent les complications tardives de la RD. Les phases précoces de la RD ne sont donc pas ciblées. L’hyperglycémie chronique entraine un stress oxydant et une activation des PKC qui participent à l’altération des BHR. L’objectif de ce travail a été 1°) d’étudier l’implication de la PKCζ et de la voie Rho/ROCK/Myosine II sur la physiopathogénie de la RD et 2°) de montrer l’effet bénéfique de leurs inhibiteur sur les BHR et sur la reperfusion des capillaires rétiniens. Nous avons confirmé l’hyperactivation de la PKCζ et de la voie Rho/ROCK/Myosine II chez les rats diabétiques et leur participation à la rupture de la BHR externe. Le traitement par leurs inhibiteurs respectifs normalise l’activation des deux enzymes et restaure l’intégrité anatomique et fonctionnelle de la BHR externe. De plus l'hyperactivation de ROCK altère la perfusion rétinienne par 1) constriction focale artériolaire, 2) protrusions membranaires endoluminales des cellules endothéliales (blebbing) et 3) vasoconstriction capillaire diffuse. Nous avons montré que l'ensemble de ces phénomènes étaient réversibles par traitement intravitréen de son inhibiteur le Fasudil. De manière importante le traitement par Fasudil induit également une diminution du VEGF rétinien responsable de la perméabilité des barrières et témoin indirect de l’ischémie rétinienne. Ces travaux éclairent la physiopathogénie de la RD et ouvre des perspectives thérapeutiques permettant de cibler les événements précoces de la RD. / Diabetic retinopathy (DR) mainly results from peripheral retinal ischemia and exudation leading to sight threatening complications such as retinal neovascularization or macular edema. This latter represents the main cause of visual loss among working age individuals. Current treatments address late complications of DR and are non-specific. Therefore, early events are currently not addressed. Chronic hyperglycemia increases oxidative stress and activates PKC leading to blood retinal barrier (BRB) breakdown. The aims of the present work were two fold. First, to assess the implication of PKCζ and the Rho/ROCK/Myosin II pathway on the pathogenesis of DR and second, to assess whether their specific inhibitors have the potential to restore the phenotype. Herein we have demonstrated the pathogenic role of PCKζ and ROCK hyperactivation on the development of diabetes induced external BRB breakdown. Furthermore their inhibitors restored the morphologic and functional aspect of the external BRB. We also found that ROCK hyperactivation was responsible for altered retinal perfusion through several mechanism namely 1) focal constriction of retinal arterioles; 2) endoluminal protrusions of the endothelial cell membrane (blebs) and 3) capillary diffuse vasoconstriction. We were able to demonstrate that all this aspects were reversible by Fasudil, a ROCK inhibitor, administrated into the vitreous. Of importance this treatment decreased also retinal VEGF that is a well-known factor responsible for barrier breakdown and a surrogate marker for retinal ischemia. To conclude the present findings not only shed light on the mechanisms of DR but also open new therapeutic avenues addressing the early events of DR a current unmet medical need.

Im Zuge der frühen embryonalen Herzentwicklung entstehen ausgehend von einem einfachen Herzschlauch zwei deutlich voneinander getrennte Herzkammern. Die Kardiomyozyten des Atriums und Ventrikels weisen spezifische Eigenschaften auf, die sich morphologisch wie auch funktionell auf das Herz auswirken. Veränderungen in der Gewebsarchitektur werden hauptsächlich durch Zellinterkalation und kollektive Zellmigration erreicht. Viele Studien zeigen, dass der Wnt/PCP-Signalweg eine essentielle Rolle in der Regulation dieser Bewegungen einnimmt. Die Daten dieser Studie belegen, dass die nicht-kanonischen Liganden Wnt11 und Wnt5b sowie die Kernkomponenten des PCP Signalweges Fzd7, Vangl2, Dvl2 und Pk1 an der Steuerung der Reorganisation der Kardiomyozyten während der Kammerbildung beteiligt sind, was Einfluss auf die Architektur des frühen Myokardiums nimmt. Effektoren des PCP Signalweges umfassen das Zytoskelett sowie Adhäsions- und Migrationsprozesse. In dieser Studie wird gezeigt, dass die Komponenten dieses Signalweges im Myokardium hauptsächlich Prozesse der Actomyosin Modulation regulieren und damit unter anderem die Morphologie der Kardiomyozyten beeinflussen. Zusätzlich ist die frühe Kardiogenese durch eine Relokalisierung der phosphorylierten Form der Myosin Regulatory Light Chain (MRLC) vom Kern zur Membran gekennzeichnet. Hier wird gezeigt, dass die Phosphorylierung von MRLC sowie die Relokalisation von den Kernkomponenten des PCP Signalweges kontrolliert werden sowie dass es im Verlauf der frühen Herzentwicklung u.a. durch die Relokalisierung von pMRLC zu Änderungen in der Gewebespannung kommt, welche sich auf die nukleäre Spannung auswirken und damit Veränderungen in der Genregulation hervorrufen. Diese Veränderungen werden hauptsächlich durch Effekte auf die Lokalisation und Aktivität des Serum Response Factors (SRF) vermittelt, welche in diesem Kontext durch die PCP Kernkomponente Pk1 reguliert sind. / Formation of a complex multiple-chambered heart from the simple linear heart tube does not only require orchestrated morphogenesis of the myocardium, but also cardiac muscle differentiation and changes in intercellular electrical coupling. To date, the processes that lead to the formation of a functional syncytium are incompletely understood. One of the major pathways controlling multiple aspects of organogenesis and tissue morphogenesis is the planar cell polarity (PCP) pathway. Changes in tissue architecture are controlled by cell intercalation and collective cell migration. It is widely accepted that Wnt/PCP signaling plays a crucial role in guiding these cellular processes. This study provides evidence that morphogenesis of the heart is controlled by the non-canonical ligands Wnt11 and Wnt5b and the PCP core components Fzd7, Vangl2, Dvl2, and Pk1 through regulation of cell rearrangements during embryonic cardiac remodeling. Downstream effectors of the PCP pathway target adhesion processes, cytoskeleton, and migration. Here, it is revealed that PCP signaling in the heart affects cardiomyocyte morphology and actomyosin organization. Specifically, changes in the subcellular localization of the phosphorylated non-muscle myosin II regulatory light chain (pMRLC) at LHT stage are targeted by the PCP pathway core components. Furthermore, actomyosin relocalization concurs with changes in nuclear tension and SRF signal transduction within the myocardium. This study unravels a novel function of the PCP core component Pk1 in regulation of SRF translocation and target gene expression that is critical to cardiac maturation. Taken together, this study provides evidence that the PCP pathway is a major regulator of cardiac remodeling and organ maturation by modulating mechanosensitive SRF signal transduction involved in muscle differentiation.

Myosin II is the molecular motor responsible for muscle contraction. It transforms the chemical energy in ATP into mechanical work while interacting with actin filaments in so called cross-bridge cycles. Myosin II or its proteolytic fragments e.g., heavy meromyosin (HMM) can be adsorbed to moderately hydrophobic surfaces in vitro, while maintaining their ability to translocate actin filaments. This enables observation of myosin-induced actin filament sliding in a microscope. This “in vitro motility assay” (IVMA) is readily used in fundamental studies of actomyosin, including studies of muscle contraction. The degree of correlation of the myosin II function in the IVMA with its function in muscle depends on how the myosin molecules are arranged on the surface. Therefore a multi-technique approach, including total internal reflection spectroscopy, fluorescence interference contrast microscopy and quartz crystal microbalance with dissipation, was applied to characterize the HMM surface configurations. Several configurations with varying distributions were identified depending on the surface property. The most favorable HMM configurations for actin binding were observed on moderately hydrophobic surfaces.   The effects on actomyosin function of different cargo sizes and amount of cargo loaded on an actin filament were also investigated. No difference in sliding velocities could be observed, independent of cargo size indicating that diffusional processive runs of myosin II along an actin filament are not crucial for actomyosin function in muscle. Furthermore, a tool for accurate velocity measurements appropriate for IVMAs at low [MgATP] was developed by utilizing the actin filament capping protein CapZ. These improvements allowed an investigation of the [MgATP]-velocity relationship to study possible processivity in fast skeletal muscle myosin II.  It is shown that the [MgATP]–velocity relationship is well described by a Michaelis-Menten hyperbola.  In addition, statistical cross-bridge modeling showed that the experimental results are in good agreement with recent findings of actomyosin cross-bridge properties, e.g., non-linear cross-bridge elasticity. However, no effect of inter-head cooperativity could be observed.   In conclusion, the described results have contributed to in-depth understanding of the actomyosin cross-bridge cycle in muscle contraction.

Au sein d'un tissu épithélial la division cellulaire doit être couplée à la formation de nouvelles jonctions intercellulaires entre les futures cellules-filles, afin de préserver l'intégrité du tissu et maintenir son adhésion et polarité. Chez les vertébrés et les invertébrés, lors de la constriction de l'anneau contractile les jonctions assemblées entre la cellule en division et ses voisines est remodelé. Concomitamment, la myosine non-musculaire II (MyoII) s'accumule dans les cellules voisines y produit la force nécessaire pour juxtaposer les membranes de la cellule en division, définissant ainsi la longueur de la future jonction formée entre les cellules-filles. Dans le cadre de mes travaux de doctorat, j'ai cherché à comprendre les mécanismes moléculaires sous-jacents au dialogue entre les cellules épithéliales pendant la division. J'ai montré que chaque division cellulaire est associée à un processus de mécano-transduction qui contrôle la dynamique de la MyoII dans les cellules voisines. Les forces produites par l'anneau contractile allongent localement la membrane des voisines diluant ainsi la concentration d'E-Cadhérine (E-Cad). En retour, cette réduction locale d'E-Cad, couplée à la contractilité intrinsèque des cellules voisines, génère des flux auto-organisés d'actine et myosine, qui conduisent à l'accumulation de MyoII dans les cellules voisines. En montrant que la cytocinèse épithéliale est une source endogène de contraintes mécaniques, mon travail définit un nouveau mécanisme de mécano-transduction qui coordonne les dynamiques d'actine et myosine dans les cellules en division et leurs voisines, et qui est permet de plus le remodelage des jonctions adhérentes. / During epithelial cytokinesis, the remodelling of adhesive cell-cell contacts between the dividing cell and its neighbours has profound roles in the integrity, the arrangement and morphogenesis of proliferative tissues. In both vertebrates and invertebrates, this remodelling requires the activity of non-muscle Myosin II (MyoII) in the interphasic cells neighbouring the dividing cells. However, the mechanisms coordinating cytokinesis and MyoII activity in the neighbours are unknown. Here, we found that, in the Drosophila notum epithelium, each cell division is associated with a mechano-sensing and transmission event controlling MyoII dynamics in the neighbours. We established that the ring pulling forces promote local junction elongation, resulting in a decrease of E-Cadherin (E-Cad) concentration at the ingressing adherens junction (AJ). In turn, the local reduction of E-Cad concentration and the contractility of the neighbouring cells promote self-organized actomyosin flows, ultimately leading to MyoII accumulation at the base of the ingressing AJ. While mechano-sensing has been extensively studied in the context of AJ reinforcement to stabilize the adhesive cell-cell contacts, we propose an alternative mechano-sensing mechanism able to coordinate actomyosin dynamics between epithelial cells and to sustain AJ remodelling in response to mechanical forces.

Nucleus is the largest cellular organelle in animal cells. Due to its bulky nature and the stiffness of nuclear lamina the nucleus constitutes the substantial problem for migrating cells where nucleus has to move. The actomyosin generated forces and LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, that is composed of SUN and nesprin proteins, play key role in nuclear movement. LINC complex mechanically couples nuclear lamina to the cytoskeleton and allows the forces exerted by the cytoskeleton to move the nucleus. Perinuclear actin fibers, also termed actin cap, mechanically link focal adhesions with nucleus and they may generate forces that position the nucleus in a way that is optimal for cellular movement. However, molecular mechanism of how perinuclear actin fibers and LINC complex orchestrate the nuclear movement and functional significance of this process remain poorly understood. The specific aim was to determine the mechanisms by which perinuclear actin fibers are formed and how are these mechanisms employed to facilitate cell migration. The role of LPA-RhoA signaling axis and LINC complex in the formation of perinuclear actin fibers was also examined. It was confirmed that LPA is essencial stimulus during actin cap formation. On the other hand, FAK kinase was found necessary for...

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Forces requises pour les changements de forme au cours de la morphogenèse des tissus sont générés par d’actine et de myosine. Durant ma thèse, je étudié le rôle de la réglementation MyoII par la voie Rho1-Rok durant l’élongation de l’ectoderme ventro-latéral par intercalation cellulaire. Les pulsations de MyoII médio-apicale se déplacent de manière anisotrope vers les jonctions parallèles avec l’axe dorso-ventral (ou jonctions verticales). Ceci provoque le rétrécissement graduel des jonctions qui sont stabilisées par une population de MyoII polarisée dans le plan du tissu et enrichie au niveau de ces jonctions. Les mécanismes cellulaires qui régulent la pulsatilité, la stabilité et la polarité de la myosine II restent à élucider. J’ai identifié deux propriétés cruciales de la dynamique de la myosine II régie par phospho- à savoir la cinétique d’échange gouvernée par les cycles de phosphorylation-déphosphorylation des chaines légères régulatrices de la MyoII (RLC) et l’advection due à la contraction des moteurs sur le réseau de F-actine. Contrôle spatial sur le chiffre d'affaires MyoII établit 2 régimes stables des taux élevés et faibles dissociation résultant dans MyoII polarité. Pulsatilité est un comportement auto-organisé qui émerge à taux de dissociation intermédiaires permettant d'advection MyoII et les régulateurs en amont. Dans la deuxième partie de ma thèse, je l'ai montré que la protéine GPCR- GRsmog et la brume, et la voie G-protéines en aval permettent l'activation progressive des MyoII, établissant pulsatilité et de la stabilité pour produire des déformations de forme polarisées cours de la morphogenèse. / Forces required to power shape changes during tissue morphogenesis are generated by non-muscle MyosinII (MyoII) pulling filamentous actin. During my PhD, I investigated the role of MyoII regulation through the conserved Rho1-Rok pathway during Drosophila germband extension. The morphogenetic process is powered by cell intercalation involving shrinkage of junctions in the dorsal-ventral axis (‘vertical junctions’) followed by junction extension in the anterior-posterior axis. Advances in light microscopy revealed that the actomyosin networks exhibit pulsed contractions to power junction shrinkage, and alternate with steps of stabilization by MyoII enriched on vertical junctions (planar-polarity) to result in irreversible shape changes. Although described in many different contexts, the underlying mechanisms of this ratchet-like behavior remained unclear. Using genetic and biophysical tools, quantitative imaging and subtle perturbations, I identified 2 critical properties underlying MyoII dynamics- turnover governed by phospho-cycling of the MyoII Regulatory Light Chain, and advection due to contraction of the motors on actin networks. Spatial control over MyoII turnover establishes 2 stable regimes of high and low dissociation rates resulting in MyoII planar polarity. Pulsatility is a self-organized behavior that emerges at intermediate dissociation rates enabling advection of MyoII and upstream regulators. In the second part of my thesis, I showed that G protein coupled receptors- GRsmog and Mist, and the downstream G-protein pathway allow step-wise activation of MyoII, establishing pulsatility and stability, to drive polarized shape deformations during morphogenesis.

Myosin subfragment-2 (S2) is a coiled coil linker between myosin subfragment-1 and light meromyosin (LMM). This dissertation examines whether the myosin S2 coiled coil could regulate the amount of myosin S1 heads available to bind actin thin filaments by modulating the stability of its coiled coil. A stable myosin S2 coiled coil would have less active myosin S1 heads compared to a more flexible myosin S2 coiled coil, thus causing increased force production through acto-myosin interaction. The stability of the myosin S2 coiled coil was modulated by the binding of a natural myosin S2 binding protein, myosin binding protein C (MyBPC), and synthetic myosin S2 binding proteins, stabilizer and destabilizer peptide, to myosin S2. Competitive enzyme linked immunosorbent assay (cELISA) experiments revealed the cross specificity and high binding affinity of the synthetic peptides to the myosin S2 of human cardiac and rabbit skeletal origins. Gravitational force spectroscopy (GFS) was performed to test the stability of myosin S2 coiled coil in the presence of these myosin S2 binding proteins. GFS experiments demonstrated the stabilization of the myosin S2 coiled coil by the binding of MyBPC and stabilizer peptide to myosin S2, while the binding of destabilizer peptide to the same resulted in a flexible myosin S2 coiled coil. The binding of MyBPC and stabilizer peptide respectively, resulted in 3.35 and 1.5 times increase in force required to uncoil the myosin S2, while the binding of destabilizer peptide resulted in 1.6 times decrease in force required to uncoil the myosin S2. The myofibrillar contractility assay was performed to test the effect of synthetic myosin S2 binding proteins on the sarcomere shortening in myofibrils. The stabilizer peptide resulted in decreased sarcomere shortening of myofibrils as a result of decreased acto-myosin interaction, on the other hand, the binding of destabilizer peptide caused an increase in sarcomere shortening. The in vitro motility assay was performed to test the effect of altered stability of myosin S2 by binding of these myosin S2 binding proteins on the motility of actin filaments sliding over myosin. The motility of actin filaments was hindered by treating myosin thick filaments with whole length skeletal MyBPC or by treating heavy meromyosin with stabilizer peptide, while the motility of actin filaments was enhanced when heavy meromyosin was treated with destabilizer peptide. This study demonstrates that the myosin S2 coiled coil stability influences the force produced by acto-myosin interaction in striated skeletal muscle. The myosin S2 coiled coil when stabilized by MyBPC and stabilizer peptide resulted in decreased force production by reduced acto-myosin interaction. While the binding of destabilizer resulted in a flexible myosin S2 coiled coil and increased force production by enhanced acto-myosin interaction. The potentially cooperative response of contractility to the instability of the S2 coiled coil promises that this biological mechanism may be the target of drugs to modulate muscle performance.