Le bon fonctionnement des réseaux neuronaux dépend des interactions entre les neurones et les cellules gliales. Alors que de nombreux efforts ont été faits pour comprendre les interactions entre les neurones, moins est connu sur la nature des interactions entre les cellules gliales ; ceci est due à la complexité du système nerveux des vertébrés, qui comprend plus de cellules gliales que de neurones. Cependant, le système nerveux de la drosophile à un rapport neurones-cellules gliales faible, ce qui fait de cet animal simple un modèle idéal pour évaluer ce concept. J’ai utilisé des approches génétiques à résolution cellulaire pour disséquer les mécanismes cellulaires et moléculaires de la migration collective des cellules gliales in vivo. En résumé, mes données révèlent les bases du mécanisme contrôlant la migration cellulaire collective : 1) les cellules du front de migration interagissent entre elles en amont et en aval et 2) N-cad est nécessaire pour une migration optimal de la glie. / The functionality of the complex neural network depends on the interactions between neurons and glia. While many efforts have been made to understand the neuron-neuron interactions, less is known about those amongst glial cells. Due to the complexity of the vertebrate nervous system, which comprises manifold more glia than neurons, it is hard to tackle the role of glia-glia interactions. The nervous system of Drosophila, however, has a lower glia-neuron ratio, which makes this simple animal an ideal model. I use genetic approaches at cellular resolution to dissect the cellular and molecular mechanisms of glial collective migration in vivo. In Sum, I have shown some basic mechanism controlling collective cell migration: 1) cells at the front of the collective interact with each other through anterograde and retrograde bidirectional interaction. 2) N-cad appears necessary for timely movement of glial community.

O excesso de tecido cicatricial (quelóides e cicatrizes hipertróficas) é um defeito do processo de cicatrização das feridas, caracterizado por um aumento na produção da matriz extracelular. Neste estudo, fibroblastos dérmicos humanos tratados com 50 <font face=\"symbol\">mM verapamil apresentaram discreta modificação na distribuição dos microfilamentos e alteraram sua morfologia de fusiformes para estrelados/arredondados. Estes efeitos poderiam estar associados a baixos níveis de cálcio citosólico. Esta hipótese foi confirmada através marcação de fibroblastos tratados com calcium green. Observamos também, que o verapamil inibiu a proliferação celular em 64,4%, aumentou a secreção de MMP1 e diminuiu o colágeno sintetizado pelos fibroblastos, sem aparentes efeitos citotóxicos. O metabolismo celular do cálcio está aparentemente relacionado a produção da matriz extracelular e portanto as patologias hipertróficas da cicatrização (quelóides e cicatrizes hipertróficas) podem responder ao tratamento com bloqueadores do canal de cálcio (verapamil). / Excessive scar tissue (keloids and hypertrophic scars) is a defective wound healing process characterized by overproduction of extracellular matrix. In the present study human dermal fibroblasts treated with 50 <font face=\"symbol\">mM verapamil changed their normal spindle-shaped morphology to stellate/rounded and showed discrete reorganization of microfilaments We hypothesized that these effects would be associated to lower levels of cytosolic Ca2+. Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels. We also observed that verapamil decrease cellular proliferation by 64.4%, increase the secretion of MMP1 and decrease synthesis of collagen in cultured fibroblasts. This alterations induced by verapamil are not associated with cytotoxic effects. The cellular calcium metabolism appears to regulate extracellular matrix production and so those hypertrophic disorders of wound healing (keloids and hypertrophic scars) may respond to therapy with calcium antagonist drugs (verapamil).

La stabilité structurale et l'intégrité mécanique sont des éléments clés pour le bon fonctionnement et la préservation des systèmes vivants complexes. Étant en interaction constante avec leur environnement et en ce qui concerne les intrants externes, de tels systèmes doivent pouvoir faire face aux changements afin de prospérer. Ces entrées peuvent affecter le système dans son ensemble. Toute perturbation qui ne peut pas être supportée mécaniquement par le système vivant entraînera un dysfonctionnement crucial ou, en fin de compte, sa mort. Le mécanisme responsable du maintien des conditions physiologiques du système à l'état correct, malgré les variations environnementales, est identifié comme étant l'homéostasie. Plus précisément, le processus connu en mécanobiologie pour préserver l'équilibre mécanique approprié d'un système vivant est appelé homéostasie tensionnelle.Il est important de noter que tout ce qui précède est vrai à la fois à l'échelle du comportement collectif des organismes complexes et jusqu'au niveau de la cellule unique. En fait, c'est en fait cette dernière petite échelle qui nous intéresse. Les cellules font face à des perturbations mécaniques constantes de leur environnement et sont capables de répondre au maintien d'un état mécanique interne relativement stable. L'existence de cet équilibre tensionnel interne est liée à un processus très dynamique avec des boucles de rétroaction constantes entre les machines contractiles biochimiques internes et les forces actives externes générées.Notre intérêt est de comprendre ce mécanisme dynamique en perturbant dynamiquement le système homéostatique tensionnel en étudiant son retour à l'équilibre. / The structural stability and mechanical integrity are key elements for the proper functioning and preservation of complex living systems. Being in constant interaction with their surroundings and subjected to external inputs, such systems need to be able to face changes in order to thrive. These inputs can affect the system both in a localized way or disturb it as a whole. Any perturbations that cannot be mechanically withstand by the living system will result in a crucial malfunctioning or, ultimately, in its death. The mechanism responsible for maintaining the system’s physiological conditions at the proper state, despite environmental variations, is identified as homeostasis. More specifically, the process known in mechanobiology to preserve the appropriate mechanical equilibrium of a living system is called tensional homeostasis.It is important to note that all of the above stated holds true both at the scale of collective behaviour of complex organisms, and all the way down to the single cell level. In fact, it is actually this last small scale which draws our interest. Cells face constant mechanical perturbations from their surrounding and are able to respond accordingly maintaining a relatively stable internal mechanical state. The existence of this internal tensional equilibrium relies on a very dynamic process with constant feedback loops between the internal biochemical contractile machinery and the external active generated forces.Our interest is to understand better this active mechanism by dynamically perturbing the tensional homeostatic system while studying its return to equilibrium.

Actin polymerization facilitated by the Arp2/3 complex plays a critical role in a wide range of cellular processes such as motility, endocytosis and cargo recycling. Activation and appropriate localization of the Arp2/3 complex is mediated by an interaction with the nucleation-promoting factor (NPF). WASH complex is the major endosomal NPF which plays a crucial role in the cargo recycling back to the trans-Golgi network (TGN) or plasma membrane. It is composed of five subunits: WASH1, SWIP, FAM21, CCDC53 and strumpellin. While WASH1 and FAM21 have been extensively studied, much less is known about strumpellin, a protein causally implicated in the onset of hereditary spastic paraplegia (HSP). This work focuses on strumpellin function in the cells, showing that only full-length protein incorporates into the WASH complex. In a strumpellin knock out cell line, we demonstrated that loss of strumpellin resulted in destabilization of the other WASH complex subunits. Still, an incomplete WASH complex without strumpellin was assembled. Cells also displayed enlarged endosomal subdomains and WASH complex nucleation activity on endosomes was largely diminished as assessed by loss of the actin patches. Finally, the absence of strumpellin was also accompanied by the accumulation of glucose transporter 1 (GLUT1)...

Les cellules épithéliales sont des cellules polarisées qui forment l’un des types cellulaires le plus abondant dans le corps humain. Leur polarité apico-basale (A/B) est établie et maintenue par la ségrégation asymétrique de protéines adaptatrices hautement conservées. Cette polarité est essentielle pour de nombreuses fonctions cellulaires clés comme l’adhésion (jonctions intercellulaires) ou la signalisation et la prolifération par la localisation et la concentration des complexes de signalisation. Durant la cancérogénèse, un grand nombre de ces processus est dérégulé aboutissant à la sur-prolifération, la migration et/ou l’invasion des cellules cancéreuses. Une meilleure compréhension des mécanismes à l’origine de ces processus est indispensable pour trouver de nouvelles cibles thérapeutiques pour le traitement du cancer. Dans l’équipe, nous sommes particulièrement intéressés par les protéines adaptatrices à domaines PDZ (domaine de liaison protéine-protéine). De par leur structure modulaire et la diversité de leurs partenaires, ces protéines adaptatrices sont impliquées dans la régulation de très nombreuses fonctions et fournissent des plateformes où différents processus peuvent être intégrés. Durant mon doctorat, j’ai étudié deux protéines adaptatrices dans le système animal modèle Drosophila melanogaster, Bbg et Magi, impliquées dans deux processus cellulaires essentiels : la dynamique des jonctions et la prolifération. Grâce aux molécules d’adhésion, les cellules non seulement restent cohésives dans un tissu, mais c’est aussi à ce niveau qu’elles peuvent obtenir une information concernant la densité cellulaire d'un tissu. Cette information est alors relayée au cytosquelette d’actine via des protéines adaptatrices spécialisées dans le but de réguler la prolifération et la voie Hippo. Cependant, le contrôle de la voie de signalisation Hippo par certaines protéines adaptatrices et par le cytosquelette d’actine n'est que partiellement compris à ce jour. Dans le laboratoire, nous étudions notamment le rôle d'une nouvelle protéine adaptatrice apicale nommée Big Bang (Bbg) dans le disque d’aile de la drosophile. Nous nous sommes intéressés à Bbg car c’est une cible de la voie Notch chez la drosophile et son homologue humain PDZD2 (pour PDZ domain-containing 2 protein) est sur-exprimé dans plusieurs cancers (sein et prostate).Mes résultats montrent que Bbg est un nouveau régulateur du cytosquelette d’actine et de la voie Hippo. Une étude détaillée de la fonction de Bbg et de ses partenaires permet de mieux comprendre les relations existantes entre dynamique de l’actine et prolifération. Bbg induit une accumulation d’actine filamenteuse en augmentant l’activité d’Enabled et la phosphorylation de Myosin Light Chain (MLC). Cette régulation résulte en une augmentation de l’activité de Yorkie, effecteur final de la voie Hippo, pour soutenir la prolifération cellulaire.La régulation des jonctions adhérentes est une étape cruciale lors de l’évolution d'une tumeur solide. Malgré les récentes avancées dans le domaine, de nombreux aspects clés de la dynamique des jonctions restent peu caractérisés.Dans le laboratoire, nous recherchons de nouveaux régulateurs de jonctions et grâce au modèle de remodelage des AJs lors du développement de l’œil de pupe de drosophile. Nous avons identifié Magi en tant que protéine adaptatrice recrutant le complexe formé de RASSF8 et ASPP. Magi régule le recrutement de Bazooka à la membrane, le dépôt d’E-Cadhérine et des Caténines et finalement le remodelage des jonctions pendant la morphogénèse. J’ai identifié Echinoid, une protéine de type immunoglobuline impliquée dans l’adhésion cellulaire et la régulation de la voie Hippo, comme un nouveau partenaire responsable du recrutement de Magi aux futures zones de jonctions. / Epithelial cells are polarised cells that form one of the most abundant cell types in the human body. Their apico-basal (A/B) polarity is established and maintained by the asymmetric segregation of highly conserved scaffold proteins. Proper A/B polarity is critical for many key cellular functions such as intercellular junctions and therefore adhesion, or signalling and proliferation by localising and concentrating signalling complexes. During carcinogenesis, many of these processes are mis-regulated leading to the over-proliferation, migration and/or invasion of cancer cells. A better understanding of the mechanisms underlying these processes is really needed to find new therapeutic targets in cancer treatment.In the team, we are particularly interested in scaffold proteins with PDZ domains (protein-protein interaction domains). Due to their modular structure, the high number of interactions they engage in, and the variety of their binding partners, these scaffold proteins are implicated in the regulation of many key cellular functions and processes. During my PhD, I have studied two scaffold proteins in the Drosophila melanogaster animal model, Bbg and Magi, which are involved in two important cell processes: adherens junctions (AJs) dynamic and cell proliferation.Through adhesion molecules, epithelial cells not only remain cohesive, but can also sense cellular density in a tissue and relay this information through dedicated scaffolds to the actin cytoskeleton to ultimately regulate the Hippo pathway and proliferation. However, many aspects of the control of Hippo signalling by apical scaffolds and the actin cytoskeleton are still poorly understood. In the laboratory, we are interested in the study of a new conserved apical scaffold, Big Bang (Bbg). Bbg is a new and quite unknown protein expressed in a variety of Drosophila epithelia, and appears as a potential Notch target in Drosophila. Its’ human homolog called PDZD2 (PDZ domain-containing 2 protein) has been shown to be over-expressed in several types of cancers (breast and prostate cancers).My results show that Bbg is a new regulator of the actin cytoskeleton and of the Hippo pathway in Drosophila. A detailed study of Bbg function and of its associated partners have helped to better understand the intricate relationships between actin dynamics and proliferation. My results suggest that Bbg promotes accumulation of filamentous actin (F-Actin) through the increase of the activity of Enabled (Ena) and the phosphorylation of the molecular motor Myosin Light Chain (MLC). This regulation leads to the increase of Yorkie activity, the final effector of the Hippo pathway, to promote cell proliferation.The regulation of adhesion, and in particular of Adherens Junctions (AJs), is a critical step during the evolution of solid tumours. A better understanding of how these structures are regulated will provide valuable insights into different phases of the disease. Despite the recent advances, many key aspects of AJ dynamics remain poorly understood. In the laboratory, we are interested in the identification of new AJs regulators. Using the remodelling of AJs during the development of the Drosophila pupal eye as a model, we have identified Magi as a scaffold recruiting a complex formed by RASSF8 and ASPP, regulating Bazooka membrane recruitment, E-Cadherin and catenins deposition, and ultimately AJs remodelling during morphogenesis. I uncovered Echinoid, an immunoglobulin-like protein involved in cell adhesion and in Hippo pathway regulation, as a new binding partner responsible for the recruitment of Magi at future AJ sites.

Lytic effector function of Natural Killer (NK) cells and CD8+ T cells occurs through discrete and regulated cell biological steps triggered by recognition of diseased cells. Recent studies of the NK cell synapse support the idea that dynamic nanoscale rearrangements in cortical filamentous (F)-actin are a critical cell biological checkpoint for lytic granule access to NK cell membrane. Loss of function mutations in the LYST gene, a well-characterised cause of Chediak- Hegashi syndrome (CHS), result in the formation of giant lysosomal organelles including lytic granules. Here, we report a mismatch between the extent of cortical F-actin remodelling and enlarged lytic granules that limits the functionality of LYST- deficient NK cells in a human model of CHS. Using super-resolution stimulated emission depletion (STED) microscopy we found that LYST-deficient NK cells had nanoscale rearrangements in the organisation of cortical actin filaments that were indistinguishable from control cells- despite a 2.5-fold increase in the size of polarised granules. Importantly, treatment of LYST-deficient NK cells with actin depolymerising drugs increased the formation of small secretory domains at the synapse and restored their ability to lyse target cells. These data establish that sub-synaptic F-actin is the major factor limiting the release of enlarged lytic granules from CHS NK cells, and reveal a novel target for therapeutic interventions. While the importance of cortical actin filaments in NK cell cytotoxicity have been established, its persistence at the early stages of T cell synapse formation is disputed. We studied the organisation of cortical actin filaments in synapses formed by primary human T cells using STED microscopy and detected intact cortical actin filaments in key T cell effector subsets including memory CD8+ T cells as early as 5-minutes post-activation. Quantitative analysis revealed that activation specific rearrangements in cortical actin filaments at both CD4+ and CD8+ T cell synapses serve to increase the space between filaments. Additionally, comparison of cytolytic T cells with freshly isolated and IL-2 activated primary NK cells revealed that rapid maturation of the cortical actin meshwork is a specific feature of CD8+ T cell lytic synapses. Using chemical inhibition of actin nucleators, we show that increased cortical relaxation is mediated primarily by the activity of actin related proteins (Arp) -2/3. Taken together, these data establish the critical requirement for dynamic rearrangements in cortical actin filaments at lytic synapses but underscore cell-specific differences in its regulation.

While actin was discovered in the nucleus over 50 years ago, research lagged for decades due to strong skepticism. The revitalization of research into nuclear actin occurred after it was found that cellular stresses both induce the nuclear localization and alter the structure of nuclear actin. These studies provided the first hints that actin has a nuclear function. Subsequently, it was established that the nuclear import and export of actin is highly regulated. While the structures of nuclear actin remain unclear, it can function as monomers, polymers, and even rods. Furthermore, even within a given structure, distinct pools of nuclear actin that can be differentially labeled have been identified. Numerous mechanistic studies have uncovered an array of functions for nuclear actin. It regulates the activity of RNA polymerases, as well as specific transcription factors. Actin also modulates the activity of several chromatin remodeling complexes and histone deacetylases, to ultimately impinge on transcriptional programing and DNA damage repair. Further, nuclear actin mediates chromatin movement and organization. It has roles in meiosis and mitosis, and these functions may be functionally conserved from ancient bacterial actin homologs. The structure and integrity of the nuclear envelope and sub-nuclear compartments are also regulated by nuclear actin. Furthermore, nuclear actin contributes to human diseases like cancer, neurodegeneration, and myopathies. The work presented in this thesis aims to describe the nuclear localization and functions of actin during Drosophila oogenesis. Drosophila oogenesis, i.e. follicle development, provides a developmental system with which to study nuclear actin. Follicles are composed of roughly 1000 somatic follicle cells and 16 germline cells, including 15 nurse or support cells and a single oocyte. Follicles progress through a series of 14 morphological stages, from the germanium to Stage 14 (S14). Ovary staining using the anti-actin C4 antibody reveals one pool of nuclear actin during early oogenesis (germarium through S9), including in the germline and somatic stem cells, a subset of mitotic follicles cells, and a subset of nurse cells during S5-S9. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged actin results in nuclear actin rod formation. These findings indicate that nuclear actin is tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of C4 nuclear actin, but does not alter the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate C4 nuclear actin. Evidence indicates that Fascin positively regulates C4 nuclear actin through Cofilin. Indeed, loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high C4 nuclear actin levels. Thus, through Cofilin, Fascin positively regulates C4 nuclear actin. These studies identified Fascin as a novel means of nuclear actin regulation. Having established Drosophila oogenesis as an in vivo, developmental system to study nuclear actin, I sought to identify the functions of nuclear actin. To uncover the functions of nuclear actin, I manipulate nuclear actin levels by blocking its nuclear import (Importin 9) and export (Exportin 6). Knockdown of Importin 9, results in female sterility and defects within the germarium, supporting a role for nuclear actin in stemness. Additionally, reduced Importin 9 levels cause chromatin organization defects. Loss or knockdown of Exportin 6 causes reduced female fertility, abnormal nucleolar morphology, alterations in the nuclear envelope, and aberrant heterochromatin status. These data suggest several functions for nuclear actin in the ovary: nuclear actin is essential for stem cell differentiation, proper chromatin organization and dispersal, nucleolar structure and likely function, nuclear envelope morphology, heterochromatin status and likely gene expression. Ultimately, nuclear actin is absolutely required for the highly conserved process of follicle development. These studies provide insight into the regulation and function of nuclear actin in Drosophila oogenesis. The data presented here indicate that nuclear actin is critical for chromatin organization, nucleolar morphology, nuclear envelope shape, and heterochromatin status and suggest that nuclear actin ultimately impacts transcription, a process essential for all cells. Considering the high level of sequence and functional conservation of actin, studies in Drosophila oogenesis will provide insight into the conserved functions of nuclear actin in follicle development across higher organisms. The study of nuclear actin in the many cell types of the Drosophila ovary provide insight into the functions of nuclear actin for all cell types across evolution. Further, aberrant nuclear actin regulation has been implicated in several disease states. The studies in Drosophila provide insight into the regulation of nuclear actin and how misregulation contributes to disease states. Together, the data presented in this thesis advance our understanding of the nuclear localization and functions of actin.

Hearing requires proper function of the auditory hair cell, which is critically dependent upon its actin-based cytoskeletal structure. Eleven point mutations in gamma (γ) nonmuscle actin have been identified as causing progressive autosomal dominant nonsyndromic hearing loss (DFNA20/26); however, exactly why these mutations lead to deafness is unclear. Organization, stability, and repair of the hair cell cytoskeleton are highly regulated by actin binding proteins (ABPs), and two of the mutations, K118M and K118N, are located near an area of the actin monomer believed to be important in actin-ABP interactions. A third mutation, D51N, is located in a region of the actin monomer believed to be important for polymerization dynamics and stability in filamentous actin. I therefore hypothesized that the K118M/N mutations cause hearing loss due to impaired regulation of the actin cytoskeleton within the hair cell, whereas the D51N mutation likely interferes with polymerization dynamics and actin filament stability or flexibility. The goal of my thesis was to investigate the effects of these three mutations, K118M, K118N, and D51N, on actin dynamics and regulation. I show in Chapter 2 that the K118M/N mutations differentially affect regulation of actin by the Arp2/3 complex, but also, surprisingly, that the K118N mutation accelerates polymerization dynamics. Chapter 3 details a continued investigation of the K118M/N mutations to ascertain their effects on actin structure and dynamics, particularly with regard to how they may affect polymerization. Chapter 4 provides both an in vivo and in vitro characterization of the D51N mutation, which revealed that not only does the mutation significantly accelerate actin polymerization, it also causes significant effects on yeast mitochondrial morphology and cytoskeletal regulation. The work detailed within this thesis provides new insight into how the K118M/N and D51N mutations affect actin structure and dynamics and how these effects could lead to deafness. More importantly, this work provides a strong foundation for many future studies, ranging from structural investigation of the K118N and D51N actins as F-actin mimics, to the potential role of mitochondria in actin-based disease.

Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need. To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system.

Durant mon travail de thèse, j'ai étudié le rôle central de l'ADF/cofiline, une protéine qui se lie au cytosquelette d'actine, décore spécifiquement les parties ‘âgées' des filaments d'actine, diminue localement par un facteur 5 la rigidité du filament et provoque la fragmentation du filament à l'interface entre les sections nues et décorées. Dans ma première étude (Suarez et al., Current Biology, 2011), j'ai utilisé la microscopie à onde évanescente et une ADF/cofiline fluorescente pour démontrer que l'ADF/cofiline est un marqueur de l'état nucléotidique (ATP, ADP-Pi ou ADP) des sous-unités d'un filament d'actine en cours de polymérisation. De plus, l'ADF/cofiline, en accélérant la dissociation du phosphate inorganique (Pi), limite la taille du cap ATP/ADP-Pi du filament d'actine, sans toutefois le réduire à une taille zéro. Des analyses statistiques sur filaments isolés établissent une corrélation parfaite entre la densité de fixation de l'ADF/cofiline et son efficacité de fragmentation. Paradoxalement, l'efficacité de fragmentation est maximale pour une densité d'ADF/cofiline de 0.5. Ceci est confirmé par des analyses supplémentaires qui montrent que les sites de fragmentation du filament coïncident avec la position des frontières entre zones décorées et zones nues. Les conséquences de ce dernier résultat paradoxal sont l'objet de ma seconde étude (McCullough et al., 2011, Biophysical Journal). En combinant différentes sources d'ADF/cofilines (vertébré et levure) et d'actines (vertébré et levure), nous montrons, sur les quatre couples actine-ADF/cofiline possibles, qu'il existe une très forte corrélation entre (1) l'efficacité de fragmentation (qui dépend de la combinaison entre actine et ADF/cofiline) et (2) la déformation du filament, mesurée à la frontière entre zone décorée et zone nue. Au cours de ma troisième étude (Reymann et al., Molecular Biology of the Cell, 2011), nous montrons que le mécanisme de fragmentation ADF/cofiline-dépendant, établi à l'échelle d'un filament isolé, peut s'appliquer aussi à l'échelle d'une comète d'actine qui comporte un réseau complexe de filaments. Mon travail de thèse a montré que le mode d'action de l'ADF/cofiline se situe à l'intersection entre mécanismes microscopiques et macroscopiques, d'une part, et entre chimie et physique, d'autre part. Les caractéristiques microscopiques des interactions de cette protéine avec un filament d'actine isolé sont fondamentales pour expliquer des évènements macroscopiques, comme la fragmentation de filaments ou de structures complexes. D'autre part, nous avons montré comment les propriétés chimiques de l'ADF/cofiline modifient les propriétés physiques locales du filament et conduisent à la fragmentation. L'ADF/cofiline a un rôle central pour l'intégration de mécanismes physico-chimiques, à l'échelle microscopique, afin d'assurer un comportement cohérent à l'échelle de la cellule. / During my thesis, I have studied the pivotal role of ADF/cofilin, a protein that binds to the actin cytoskeleton, specifically decorates ‘old' actin filament parts, decreases by a factor of 5 the local filament rigidity and triggers filament fragmentation at boundaries between decorated and non-decorated filament sections. In my first study (Suarez et al., Current Biology, 2011), I have used evanescent wave microscopy and labeled ADF/cofilin to demonstrate that ADF/cofilin is a marker of the nucleotide state (i.e. ATP, ADP-Pi or ADP) associated with the actin sub-units in actively polymerizing filaments. In addition, because ADF/cofilin accelerates inorganic phosphate (Pi) release, the size of the ATP/ADP-Pi cap is diminished, although it cannot be reduced to zero. Fragmentation events frequency, determined from a thorough analysis of a population of single filaments decorated with labeled ADF/cofilin, is perfectly correlated with the binding density of ADF/cofilin on filaments. However, the maximal severing efficiency is obtained for half ADF/cofilin density. This paradoxical result is confirmed by analysis showing that severing sites are mainly associated with boundaries between decorated and bare actin filament sections. In consequence, in a second paper (McCullough et al., Biophysical Journal, 2011), I have took part in the study of actin filament deformation in relation with severing efficiency. Using different ADF/cofilin (vertebrate and yeast) and actin (vertebrate and yeast), we have shown that filament deformation at the boundary between bare and ADF/cofilin-decorated filament sections (which depends on the ADF/cofilin/actin combination) and severing are highly correlated. During my third study, (Reymann et al., Molecular Biology of the Cell, 2011), we established that stochastic dynamics, discovered at the molecular level for single filaments (or bundles of them), is also relevant to describe the macroscopic fragmentation of a comet tail consisting of hundreds of thousands filaments. I have shown that ADF/cofilin activity is at the crossroad between macroscopic and microscopic systems, on one hand, and physics and chemistry, on the other hand. The characteristics of microscopic interactions of ADF/cofilin with a single filament are fundamental to understand the macroscopic dynamics of a fragmenting comet. In addition, we have established how the binding of ADF/cofilin (chemistry) controls the mechanical properties of the filament (physics) before fragmentation. ADF/cofilin is essential in the integration of physical and chemical mechanisms at the microscopic level, to ensure consistent behavior at the cell scale.