36 research outputs found
Sparse denoising and adaptive estimation enhances the resolution and contrast of fluorescence emission difference microscopy based on array detector
International audienceArray detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high-resolution image by linearly combining sub-detector images, especially the fluorescence emission difference (FED) technique. To improve the resolution and contrast of FED microscopy based on array detector, we propose to asso-ciate sparse denoising with spatial adaptive estimation. We show on both calibration slides and real data that our approach applied to the full stack of spatially reassigned detector signals, enables to achieve a higher reconstruction performance in terms of resolution, image contrast, and noise reduction
Regulation of RhoGTPases family : implication in cell migration and invasion
Les GTPases de la famille Rho sont les principaux rĂ©gulateurs du remodelage du cytosquelette dâactine lors de la migration et lâinvasion cellulaire. En particulier, deux membres de cette famille sont importants dans ce processus : les GTPases RhoA et Rac1. En effet, il existe une balance dâactivitĂ© de ces GTPases, responsables respectivement de la contraction cellulaire et de la formation dâextensions cytoplasmiques, des Ă©tapes clefs de la migration. Lâobjectif de ce travail de thĂšse a Ă©tĂ© dâĂ©tudier la rĂ©gulation de ces protĂ©ines dans la migration et lâinvasion cellulaire. Pour cela, plusieurs stratĂ©gies ont Ă©tĂ© entreprises. Tout dâabord, une Ă©tude structure/fonction de la protĂ©ine p190RhoGAP-A (p190A), un des rĂ©gulateurs majeurs de la GTPase RhoA, a Ă©tĂ© rĂ©alisĂ©e. Cette Ă©tude a permis de mettre en Ă©vidence un domaine, appelĂ© PLS pour « protrusion localization sequence », permettant Ă cette protĂ©ine de se localiser au niveau des extensions membranaires appelĂ©es « replis membranaires » et « lamellipodes » oĂč RhoA est rĂ©gulĂ©e localement. Dâautre part, un mutant dĂ©lĂ©tĂ© de ce domaine, appelĂ© PLSp190A, ne peux pas se localiser au niveau de ces structures et a un impact nĂ©gatif sur leur formation et la migration cellulaire. De plus, lâanalyse de ce mutant a rĂ©vĂ©lĂ© que le domaine PLS Ă©tait impliquĂ© dans la rĂ©gulation nĂ©gative de p190A. Ainsi, nous avons mis en Ă©vidence un nouveau domaine de p190A responsable de sa localisation intracellulaire et de sa fonction. La deuxiĂšme partie de ce travail de thĂšse a Ă©tĂ© consacrĂ©e Ă la mise en place dâun outil de mesure de lâactivitĂ© des GTPases Rho par la technologie Alphascreen. Ce test a permis de mesurer lâactivitĂ© de Rac1 in vitro et in cellulo mais a Ă©galement Ă©tĂ© appliquĂ© Ă un crible en vue dâidentifier de nouvelles molĂ©cules rĂ©gulatrices de Rac1. Ainsi, ce travail de thĂšse, en abordant par plusieurs angles la rĂ©gulation des GTPases de la famille Rho, a permis dâapporter des informations et des outils pour la comprĂ©hension des mĂ©canismes complexes rĂ©gissant la capacitĂ© des cellules Ă se mouvoir dans leur environnement.RhoGTPases are major regulators of the actin cytoskeleton during cell migration and invasion. Particularly, the two members of the RhoGTPase family, RhoA and Rac1 play important roles in these processes. Indeed, a reciprocal balance between these GTPasesâactivity that leads to cell contraction and cell protrusion formation, determines cell movement. The aim of this PhD thesis was to study the regulation of RhoA and Rac1 during cell migration and invasion. To this end, various strategies were undertaken.We first performed a structure/function analysis of p190RhoGAP-A (p190A), a major negative regulator of RhoA. This led to the identification of a protrusion localization sequence (âPLSâ) necessary and sufficient for p190A targeting to actin-based structures. A p190A mutant deleted of the PLS domain (PLS), does not localize to ruffles and lamellipodia, where RhoA is locally regulated during cell migration. This analysis also revealed that the PLS is required for the negative regulation of p190A activity. Finally, p190APLS expression has a dominant negative effect on the formation of actin protrusions and cell migration. Thus, we identified a novel functional domain of p190A required for its proper subcellular localization and functions. The second part of this PhD thesis was focused on the design of an Alphascreen technology-based assay to study GTPases activity. This assay allowed the measurement of Rac1 activity in vitro and in cellulo. Moreover, we used this assay to screen for new regulators of Rac1 activity. In conclusion, this work provides new insights and new tools for the understanding of RhoGTPase involvement in cell migration
Regulation of RhoGTPases family : implication in cell migration and invasion
Les GTPases de la famille Rho sont les principaux rĂ©gulateurs du remodelage du cytosquelette dâactine lors de la migration et lâinvasion cellulaire. En particulier, deux membres de cette famille sont importants dans ce processus : les GTPases RhoA et Rac1. En effet, il existe une balance dâactivitĂ© de ces GTPases, responsables respectivement de la contraction cellulaire et de la formation dâextensions cytoplasmiques, des Ă©tapes clefs de la migration. Lâobjectif de ce travail de thĂšse a Ă©tĂ© dâĂ©tudier la rĂ©gulation de ces protĂ©ines dans la migration et lâinvasion cellulaire. Pour cela, plusieurs stratĂ©gies ont Ă©tĂ© entreprises. Tout dâabord, une Ă©tude structure/fonction de la protĂ©ine p190RhoGAP-A (p190A), un des rĂ©gulateurs majeurs de la GTPase RhoA, a Ă©tĂ© rĂ©alisĂ©e. Cette Ă©tude a permis de mettre en Ă©vidence un domaine, appelĂ© PLS pour « protrusion localization sequence », permettant Ă cette protĂ©ine de se localiser au niveau des extensions membranaires appelĂ©es « replis membranaires » et « lamellipodes » oĂč RhoA est rĂ©gulĂ©e localement. Dâautre part, un mutant dĂ©lĂ©tĂ© de ce domaine, appelĂ© PLSp190A, ne peux pas se localiser au niveau de ces structures et a un impact nĂ©gatif sur leur formation et la migration cellulaire. De plus, lâanalyse de ce mutant a rĂ©vĂ©lĂ© que le domaine PLS Ă©tait impliquĂ© dans la rĂ©gulation nĂ©gative de p190A. Ainsi, nous avons mis en Ă©vidence un nouveau domaine de p190A responsable de sa localisation intracellulaire et de sa fonction. La deuxiĂšme partie de ce travail de thĂšse a Ă©tĂ© consacrĂ©e Ă la mise en place dâun outil de mesure de lâactivitĂ© des GTPases Rho par la technologie Alphascreen. Ce test a permis de mesurer lâactivitĂ© de Rac1 in vitro et in cellulo mais a Ă©galement Ă©tĂ© appliquĂ© Ă un crible en vue dâidentifier de nouvelles molĂ©cules rĂ©gulatrices de Rac1. Ainsi, ce travail de thĂšse, en abordant par plusieurs angles la rĂ©gulation des GTPases de la famille Rho, a permis dâapporter des informations et des outils pour la comprĂ©hension des mĂ©canismes complexes rĂ©gissant la capacitĂ© des cellules Ă se mouvoir dans leur environnement.RhoGTPases are major regulators of the actin cytoskeleton during cell migration and invasion. Particularly, the two members of the RhoGTPase family, RhoA and Rac1 play important roles in these processes. Indeed, a reciprocal balance between these GTPasesâactivity that leads to cell contraction and cell protrusion formation, determines cell movement. The aim of this PhD thesis was to study the regulation of RhoA and Rac1 during cell migration and invasion. To this end, various strategies were undertaken.We first performed a structure/function analysis of p190RhoGAP-A (p190A), a major negative regulator of RhoA. This led to the identification of a protrusion localization sequence (âPLSâ) necessary and sufficient for p190A targeting to actin-based structures. A p190A mutant deleted of the PLS domain (PLS), does not localize to ruffles and lamellipodia, where RhoA is locally regulated during cell migration. This analysis also revealed that the PLS is required for the negative regulation of p190A activity. Finally, p190APLS expression has a dominant negative effect on the formation of actin protrusions and cell migration. Thus, we identified a novel functional domain of p190A required for its proper subcellular localization and functions. The second part of this PhD thesis was focused on the design of an Alphascreen technology-based assay to study GTPases activity. This assay allowed the measurement of Rac1 activity in vitro and in cellulo. Moreover, we used this assay to screen for new regulators of Rac1 activity. In conclusion, this work provides new insights and new tools for the understanding of RhoGTPase involvement in cell migration
The clathrin adaptor AP-1B independently controls proliferation and differentiation in the mammalian intestine
International audienceMaintenance of the polarity of the epithelial cells facing the lumen of the small intestine is crucial to ensure the vectorial absorption of nutrients as well as the integrity of the apical brush border and the intestinal barrier. Polarized vesicular trafficking plays a key role in this process, and defective transport due to mutations in apical trafficking-related genes has been shown to affect nutrient absorption. Interestingly, it has been demonstrated that downregulation of the polarized sorting clathrin adaptor AP-1B led to both epithelial polarity and proliferation defects in the mouse intestine. This enlightened a new function of polarized trafficking in the gut epithelium and a novel link between trafficking, polarity, and proliferation. Here, using CRISPR-Cas9-mediated mutation of the AP-1B coding gene Ap1m2 in mouse intestinal organoids, we uncovered a novel proliferation pathway controlled by AP-1B. We showed that the polarity defects induced by Ap1m2 mutations led to a defective apical targeting of both Rab11 + apical recycling endosomes and of the polarity determinant Cdc42. Moreover, we showed that these polarity defects were accompanied by an induction of YAP and EGFR/mTOR-dependent proliferation pathways. Finally, we showed that AP-1B additionally controlled a proliferation-independent differentiation pathway towards the secretory lineage. Overall, our results highlighted the pleiotropic roles played by AP-1B in the homeostasis of the gut epithelium
A V0-ATPase-dependent apical trafficking pathway maintains the polarity of the intestinal absorptive membrane
International audienceIntestine function relies on the strong polarity of intestinal epithelial cells and the array of microvilli forming a brush border at their luminal pole. Combining a genetic RNA interference (RNAi) screen with super-resolution imaging in the intestine, we found that the V0 sector of the vacuolar ATPase (V0-ATPase) controls a late apical trafficking step, involving Ras-related protein 11 (RAB-11) endosomes and the -ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) synaptosome-associated protein 29 (SNAP-29), and is necessary to maintain the polarized localization of both apical polarity modules and brush border proteins. We show that the V0-ATPase pathway also genetically interacts with glycosphingolipids and clathrin in enterocyte polarity maintenance. Finally, we demonstrate that silencing of the V0-ATPase fully recapitulates the severe structural, polarity and trafficking defects observed in enterocytes from individuals with microvillus inclusion disease (MVID) and use this new MVID model to follow the dynamics of microvillus inclusions. Thus, we describe a new function for V0-ATPase in apical trafficking and epithelial polarity maintenance and the promising use of the intestine as an model to better understand the molecular mechanisms of rare genetic enteropathies
A comparison of high resolution image reconstruction methods from confocal array detector with a new deconvolution algorithm
Array detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high resolution image by linearly combining sub-detector images. To overcome the limitations of these techniques, we propose a new reconstruction method that takes the full stack of spatially reassigned detector signals as input. We show on both calibration slides and real data that our deconvolution method allows to achieve a better reconstruction performance in terms of resolution, image contrast, and spatial intensity homogeneity. The tested algorithms are available in an open source software
Transcytosis maintains CFTR apical polarity in the face of constitutive and mutation-induced basolateral missorting
International audienceApical polarity of cystic fibrosis transmembrane conductance regulator (CFTR) is essential for solute and water transport in secretory epithelia and can be impaired in human diseases. Maintenance of apical polarity in the face of CFTR non-polarized delivery and inefficient apical retention of mutant CFTRs lacking PDZ-domain protein (NHERF1, also known as SLC9A3R1) interaction, remains enigmatic. Here, we show that basolateral CFTR delivery originates from biosynthetic (âŒ35%) and endocytic (âŒ65%) recycling missorting. Basolateral channels are retrieved via basolateral-to-apical transcytosis (hereafter denoted apical transcytosis), enhancing CFTR apical expression by two-fold and suppressing its degradation. In airway epithelia, CFTR transcytosis is microtubule-dependent but independent of Myo5B, Rab11 proteins and NHERF1 binding to its C-terminal DTRL motif. Increased basolateral delivery due to compromised apical recycling and accelerated internalization upon impaired NHERF1âCFTR association is largely counterbalanced by efficient CFTR basolateral internalization and apical transcytosis. Thus, transcytosis represents a previously unrecognized, but indispensable, mechanism for maintaining CFTR apical polarity that acts by attenuating its constitutive and mutation-induced basolateral missorting. © 2019. Published by The Company of Biologists Ltd
High resolution dynamic mapping of the C. elegans intestinal brush border
International audienceThe intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption, and host defense. Studies on mammalian cultured epithelial cells uncovered some of the molecular players and physical constrains required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not been investigated in detail yet. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode C. elegans to build a high-resolution and dynamic localization map of known and new brush border markers. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation but become highly stable once microvilli are built. This new toolbox will be instrumental to understand the molecular processes of microvilli growth and maintenance in vivo as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity
V<sub>0</sub>-ATPase downregulation induces MVID-like brush border defects independently of apical trafficking in the mammalian intestine
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Downregulation of V-ATPase V(0) Sector Induces Microvillus Atrophy Independently of Apical Trafficking in the Mammalian Intestine
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