4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell-derived intestinal organoids.

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues

Remodelage des lipides et de l'actine-F au cours des étapes finales de la division cellulaire (rôle de la GTPase Rab35 et de son effecteur OCRL, une PI(4,5)P2 phosphatase impliquée dans le syndrome de Lowe)

La cytocinèse est la dernière étape de la division cellulaire permettant la séparation de deux cellules filles topologiquement distinctes. L étape finale de la cytocinèse, coupure physique des deux cellules filles ou abscission, est encore à élucider. Les études de ces dernières années ont permis de mettre en évidence que le trafic intracellulaire ainsi que la composition lipidique de la membrane du pont intercellulaire jouent un rôle essentiel pendant la cytocinèse. Néanmoins, la régulation des lipides par le trafic intracellulaire reste une question ouverte et nécessaire à la compréhension de ce processus clé de la division cellulaire. Nous avons montré que la GTPase Rab35 est requise pour la localisation au pont de cytocinèse de son effecteur, OCRL, une PI(4,5)P2 5-phosphatase impliquée dans une maladie récessive liée à l X, le syndrome de Lowe. L inhibition de cette voie entraine une accumulation de PI(4,5)P2 et d actine filamenteuse au pont de cytocinèse ce qui conduit à l inhibition de l abscission. Ainsi, nous avons montré que l hydrolyse du PI(4,5)P2 est nécessaire à la complétion de la cytocinèse et est régulée par Rab35 et OCRL. En parallèle, nous avons montré que l inhibition de l abscission observée après déplétion de Rab35 ou d OCRL ou dans des cellules issues de patients atteints du syndrome de Lowe peut être corrigée par l addition de faibles doses de latrunculine A, un agent dépolymérisant l actine-F. L ensemble de ces travaux a permis de mettre en évidence un lien nouveau entre la régulation locale, par une GTPase associée à une phosphatase, des niveaux de PI(4,5)P2, du cytosquelette d actine et des mécanismes régulant les étapes finales de la cytocinèsePARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell-derived intestinal organoids.

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues

Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation.

We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation

Unraveling the intracellular trafficking mechanism of monocarboxylate transporter 1 in mammalian cells

Plasma membrane (PM) proteins, such as nutrient transporters and receptors, have a determinant role in the regulation of the overall cellular metabolism. They contribute to sensing, adhesion, signaling and nutrient uptake, allowing the cell to adapt and respond to distinct environmental cues. Rapid and dynamic regulation of PM proteins is achieved by means of selective endocytosis, where target proteins are internalized into endosomes and then they are either degraded or recycled back to the PM. This study focuses on monocarboxylate transporters (MCTs), which play essential metabolic roles in most tissues. These proteins are found to be upregulated in several cancer cell lines, displaying an enhanced glycolytic activity. MCTs contribute to fuel the metabolism of tumor cells, so reducing their expression can somehow starve cancer cells and make them more vulnerable to chemotherapy, opening new pathways for future therapies. We have previously generated several gene-edited cancer cell lines, known to express differently MCT transporters, using the CRISPR-Cas9 system (1). These cells will be applied to a combined microscopy platform (2) in the attempt to characterize the internalization dynamics of MCT transporters upon distinct environmental stimuli. This will be achieved by utilizing fluorescence optical sectioning microscopy obtained through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). An overview of the most significant results will be presented

Characterization of intracellular trafficking of nutrient transporters using combined fluorescence optical sectioning and nanomechanical mapping atomic force microscopy in mammalian cells

Plasma membrane (PM) proteins, such as nutrient transporters and receptors, have a determinant role in the regulation of the overall cellular metabolism, contributing to sensing, adhesion, signaling and nutrient uptake, allowing the cell to adapt and respond to distinct environmental cues. Rapid and dynamic regulation of PM proteins is achieved by means of selective endocytosis, where target proteins are internalized into endosomes and then they are either degraded or recycled back to the PM. This study focuses on monocarboxylate transporters (MCTs), which play essential metabolic roles in most tissues. These proteins are found to be upregulated in several cancer cell lines, displaying an enhanced glycolytic activity. MCTs contribute to fuel the metabolism of tumor cells, so reducing their expression can somehow starve cancer cells and make them more vulnerable to chemotherapy, opening new pathways for future therapies. We have previously generated several gene-edited cancer cell lines, known to express differently MCT transporters, using the CRISPR-Cas9 system (1). These cells will be applied to a combined microscopy platform (2) in the attempt to characterize the internalization dynamics of MCT transporters upon distinct environmental stimuli. This will be achieved by utilizing fluorescence optical sectioning microscopy obtained through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM).info:eu-repo/semantics/publishedVersio

Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation

We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation