Dans les organismes multicellulaires, les cellules génèrent et subissent des forces mécaniques qui se propagent aux cellules voisines. Ces forces peuvent déterminer la forme des tissus et organes, et aussi être converties en signaux biochimiques. Dans un épithélium, les cellules forment un tissu en adhérant directement les unes aux autres grâce à des complexes d’adhérence, tels que les Jonctions Adhérentes. Ces Jonctions Adhérentes sont composées de protéines transmembranaires les E-cadhérines, dont la partie cytoplasmique est sous tension générée par le cytosquelette d’actomyosine par un lien assurée par la β-caténine. La β-caténine est aussi un cofacteur de transcription majeur qui régule l’activité de gènes impliqués dans la transition épithélio-mésenchymateuse une fois dans le noyau. L’accumulation nucléaire et l’activité transcriptionnelle de la β-caténine peuvent avoir lieu à la suite de stimulations mécaniques dans des situations physiologiques et pathologiques, et ont été proposées comme la conséquence d’une libération de la β-caténine des Jonctions Adhérentes suite à sa phosphorylation. Néanmoins, les preuves directes de ce phénomène et ses mécanismes manquent, et le rôle qu’y tient la tension des E-cadhérines n’est pas connu.Dans cette thèse, nous avons établi la relation entre la tension des E-cadhérines et la localisation nucléaire et l’activité de la β-caténine, prouvé l’existence d’une translocation de la membrane au noyau de la β-caténine, et caractérisé les mécanismes moléculaires sous-jacents dans des cellules en migration induite par un facteur de croissance ou par blessure sur un épithélium, deux conditions qui récapitulent au moins partiellement une transition épithélio-mésenchymateuse.Nous avons montré que l’accumulation nucléaire de la β-caténine est due à un départ substantiel de celle-ci de la membrane, spécifiquement dans les cellules en migration. Cette translocation a lieu en aval d’une voie de signalisation impliquant les kinases Src et FAK, et qui conduit à une relaxation de tension des E-cadhérines. Le mécanisme sous-jacent implique une réorganisation du cytosquelette d’actine, caractérisé par un enrichissement des fibres des stress ventrales, soutenant les protrusions, en phospho-myosine, au détriment du cortex d’actine des Jonctions Adhérentes. En revanche, les phosphorylations dans le complexe cadhérine/caténine ne sont pas requises. Ces résultats démontrent que les E-cadhérines ont un rôle de senseur de la mécanique intracellulaire, et que les adhésions focales sont impliquées dans l’activation de la voie de signalisation β-caténine / In multicellular organisms, cells generate and experience mechanical forces that propagate between and within cells. These forces may shape cells, tissues and organs, and also convert into biochemical signals. In a simple epithelium, cells form tissue sheets by directly adhering to one another through adhesion complexes, such as the Adherens Junctions. Adherens Junctions comprise transmembrane proteins E-cadherins, which are under actomyosin-generated tension via a link that contains β-catenin. β-catenin is also a major transcription cofactor that regulates gene activity associated with Epithelial-to-Mesenchyme Transition when translocated in the nucleus. β-catenin nuclear localization and transcriptional activity are mechanically inducible in a variety of healthy and disease models and were proposed to follow phosphorylation-induced -catenin release from E-cadherin. However, direct evidence for this translocation and these mechanisms are lacking, and whether E-cadherin tension is involved is unknown.In this thesis, we assess the relationship between E-cadherin tension and β-catenin nuclear localization and activity, determine the relevance of β-catenin shuttling between membrane and nucleus, and characterize the underlying molecular mechanisms in cells migrating in an at least partial EMT-like fashion upon hepatocyte growth factor (HGF) or wound stimulation. We showed that β-catenin nuclear activity follows a substantial release from the membrane that is specific to migrating cells. This translocation occurs downstream of the Src-FAK pathway, which targets E-cadherin tension relaxation. The underlying mechanisms sufficiently involve actomyosin remodeling, characterized by an enrichment of ventral stress fibers that capture phosphomyosin at the expense of the cortex at Adherens Junctions. In contrast, phosphorylations of the cadherin/catenin complex are not substantially required. These data demonstrate that E-cadherin acts as a sensor of intracellular mechanics in a crosstalk with cell-substrate adhesions that targets β-catenin signaling

Cell division requires accurate DNA replication and cells develop checkpoint mechanisms toensure the correct passage of the genetic material. Cells arrest by a checkpoint when DNAdamage is found. After the checkpoint is silenced, the cell cycle can be resumed. Polo-likekinase 1 (Plk1) and Aurora A kinase (AurA) are both important regulators for checkpointrecovery. The question how AurA is activated was studied by many researchers, but the exactmechanism stays unclear.We developed a new setup to study AurA activation during checkpoint recovery. Quantitativeimmunofluorescence of fixed cells as well as a FRET probe that monitors Plk1 activity intime-lapse filming were applied in this study as indirect readouts of Aurora A activation. Theresult suggests that a Plk1-AurA feedback loop exists during checkpoint recovery. It can alsobe concluded that the inhibition of Cdk1 reduces Plk1 and AurA activity during checkpointrecovery. We also investigated the effect of calcium interfering drugs on AurA activation butno conclusive result was obtained.

The objective of my PhD study was to develop and characterize new methods and sensors based on fluorescence resonance energy transfer (FRET) for bioanalysis. Chapter 3 describes the use of FRET between donor fluorophores and acceptor labeled murine macrophage cells. FRET microscopy was used to determine whether the donor molecules truly permeate through the cell membrane or only adsorb to the cell surface. This method was found to be partially successful since the donor red tail fluorescence overlapped with the sensitized acceptor fluorescence and led to false reading of FRET. We found that is easier to monitor delivery of acceptor molecules into donor-labeled cells. Using donor labeled cells it was possible to determine whether the acceptor molecules were actually delivered into cells. However, a relatively high acceptor concentration in the hundreds of micromolar level was needed to obtain measurable FRET signals in the 3-D cellular system. The results underscored the need to reduce the dimensionality of FRET systems in order to increase the FRET efficiency between donor and acceptor molecules. Chapter 4 describes the development of FRET sensing lipobeads labeled with donors and their use to evaluate the interactions of acceptor molecules with the phospholipid membrane of FRET sensing lipobeads. The change in the dimensionality of the system in which FRET occurs, improved the sensitivity of our measurements by 3-folds compared to FRET measurements in solution. We concluded that a molecular recognition component had to be added to the sensing particles to further increase their selectivity and sensitivity. Chapter 5 describes the development of FRET trap sensing beads and their use for screening nonfluorescent carbohydrates and glycoproteins. The FRET sensing technique was based on binding between dextran molecules labeled with Texas Red (Dextran-TR) and polystyrene microparticles labeled with Fluorescein tagged Concanavalin A (FITC-ConA). It was found that carbohydrates and glycoproteins inhibit the binding between dextran-TR and FITC-ConA labeled particles. The inhibition effect was concentration dependent thus enabled screening carbohydrates and glycoproteins based on their inhibition potency. The dissertation critically evaluates the performance of FRET microscopy and FRET based sensors in delivery and screening applications.

No description available.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.