La capacité à mesurer le volume cellulaire est essentielle pour l'étude de la régulation du volume cellulaire et ses nombreux processus. La perméabilité de la membrane des cellules de mammifères implique que les intrants ou les extrants de biomolécules causent des transports d'eau qui éprouvent l'homéostasie cellulaire. Dès lors, le volume d'une cellule, ainsi que d'autres paramètres biophysiques y étant reliés, sont modifiés aussi bien par des changements du milieu extracellulaire que par sa propre activité. Les cellules ont développé toute une série de mécanismes pour réguler leur volume afin de préserver leur homéostasie. Ainsi, la régulation de volume d'une cellule est un indicateur potentiel de son état. Dans le but de produire des mesures fiables de volume lors de l'étude de ces processus, il est à priori avantageux de développer des outils et des méthodes efficaces. À cet effet, l'imagerie quantitative de phase avec l'holographie numérique de phase peut donner d'utiles informations sur la morphologie et la composition de la cellule. Le signal quantitatif de phase d'un objet contient deux paramètres critiques pour l'étude de la régulation de volume et du transport d'eau : l'indice de réfraction et l'épaisseur cellulaire. L'ambiguïté de leur contribution dans le signal total peut être résolue par le découplage à deux liquides, mesurant deux fois le même échantillon dans deux milieux extracellulaires d'indices de réfraction différents et connus. La procédure sépare les paramètres en un système facilement soluble de deux équations et de deux inconnus. Avec ces deux paramètres, il est possible de déduire le volume cellulaire absolu ainsi que d'autres paramètres biophysiques, comme lamasse sèche, la concentration en biomolécule et la fraction d'eau. Pour mieux utiliser l'information obtenue par le changement de fluide, une chambre d'écoulement imprimable en 3d a été conçue et utilisée conjointement avec des lamelles rainurées permettant d'obtenir l'indice de réfraction du milieu d'immersion en temps réel. Cet ajout, avec une compréhension de la mécanique des fluides, ouvre de nouvelles possibilités pour résoudre des changements de volume dans le temps. Le découplage de l'indice de réfraction et de l'épaisseur cellulaire n'est désormais plus limité à l'état initial et final des changements de fluide, mais peut être effectué dans la période transitoire. Ces nouveaux essais fluidiques sont utilisés pour mesurer les changements de volume des cultures primaires d'astrocytes de cortex de rat lors de chocs au glutamate. / The ability to measure the absolute cell volume is essential to the study of cellular volume regulation and its many different pathways. The permeability of the cell membrane implies that biomolecule inputs and outputs cause transmembrane water transport challenging cell homeostasis. Thenceforth, acell's volume and a number of other biophysical parameters are modified by changes to the extracellular medium as well as its own activity. Thus, cells have developed a whole series of mechanisms toregulate their volume and maintain their homeostasis. Therefore, cell volume regulation is indicativeof the cell's state. In the interest of producing reliable volume measurements, it is first advantageous to develop effective tools and methods. For this purpose, quantitative-phase imaging with digital holographic microscopy can provide useful information on the morphology and composition of the cell.The quantitative-phase signal of any object contains two critical parameters for the study of volume regulation, the refractive index and the thickness. The ambiguity of their contribution in the total quantitative phase signal can be resolved by the two-liquid decoupling approach, measuring twice the same sample in two extracellular mediums with different but known refractive indices. The procedure results in two easily solvable phase shift equations with two unknown variables. With the two parameters, the whole-cell volume can be deduced in addition to the dry mass, intracellular concentrationand water content fraction. In order to make better use of the information acquired with the change of fluid, a custom 3d-printed imaging chamber was designed and optimized for stable laminar flow. Forfurther accuracy, grooves were etched on the sample coverslips to monitor in real-time the extracellular refractive index. The combination of both tools expands the utility of the decoupling procedure.It is no longer limited to the initial and final state of the liquid exchange, but can now be used inthe transition period. These novel fluidic essays are used to measure glutamate-induced swelling of primary rat cortical astrocytes

In the past fifteen years, ubiquitin has emerged as a central regulator of membrane protein trafficking. In this context, covalent attachment of this small protein to lysine residues of cargo proteins, a reversible modification termed ubiquitylation, provides a signal for their targeting to the vacuolar/lysosomal lumen where they are degraded, both in yeast and higher eukaryotes. Ubiquitylation is also used as a means of controlling the function of specific proteins in several trafficking machineries. The role of lipids - and in particular of membrane domains named lipid rafts - in controlling the intracellular trafficking of membrane proteins has also been the subject of intense investigation in recent years.<p>One of the membrane proteins of the yeast Saccharomyces cerevisiae whose intracellular trafficking has been extensively studied is the general amino acid permease Gap1. Yet some aspects of the function of ubiquitin in the nitrogen-dependent control of this protein remain controversial. Moreover, the potential role of lipid rafts in regulating the functional properties and traffic of the Gap1 permease had not been investigated before this thesis work. <p>The first part of our work readdresses the role of Gap1 ubiquitylation, and more precisely of the modification of the permease with polyubiquitin chains linked through the lysine 63 of ubiquitin, in controlling the fate of this protein in the secretory pathway. Our observations indicate that nitrogen-induced ubiquitylation of newly synthesised Gap1 occurs in the trans-Golgi complex. However, contrary to the generally accepted view, this modification is not necessary for the permease to exit this compartment en route to the endosome but only for its subsequent targeting to the vacuolar lumen via the multivesicular body (MVB) pathway. Our results also provide evidence that K63-linked polyubiquitylation is important mostly at the late endosomal level, for proper sorting of Gap1 into the MVB pathway, whether the permease comes from the cell surface by endocytosis or directly from the secretory pathway. <p>In the second part of this work, we present a set of data providing novel insights into the controversial question of the exact nature of lipid rafts in yeast. We first showed that the Gap1 permease is associated with detergent-resistant membranes (DRMs) - the proposed biochemical equivalent of lipid rafts - when it is located at the cell surface. Our data further suggest that this may be true for most if not all yeast plasma membrane proteins. Moreover, we found that Gap1 production must be coupled to de novo synthesis of sphingolipids (SLs), major constituents of rafts, in order for the newly synthesised permease to be correctly folded, active, associated with DRMs, and stable at the cell surface. We propose a model where Gap1 would associate with newly synthesised SLs during its biogenesis and/or secretion, this association shaping the permease into its native conformation and ensuring its incorporation and stabilisation in specific lipid domains at the plasma membrane. Failure of Gap1 to acquire this lipidic microenvironment in turns leads to its ubiquitin-dependent degradation by a quality-control mechanism. This model might be valid for many other plasma membrane proteins and might account for their lateral distribution between distinct membrane domains. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished