Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type

Détournement de fonctions cellulaires par Chlamydia trachomatis

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Transfer learning for versatile and training free high content screening analyses

Abstract High content screening (HCS) is a technology that automates cell biology experiments at large scale. A High Content Screen produces a high amount of microscopy images of cells under many conditions and requires that a dedicated image and data analysis workflow be designed for each assay to select hits. This heavy data analytic step remains challenging and has been recognized as one of the burdens hindering the adoption of HCS. In this work we propose a solution to hit selection by using transfer learning without additional training. A pretrained residual network is employed to encode each image of a screen into a discriminant representation. The deep features obtained are then corrected to account for well plate bias and misalignment. We then propose two training-free pipelines dedicated to the two main categories of HCS for compound selection: with or without positive control. When a positive control is available, it is used alongside the negative control to compute a linear discriminant axis, thus building a classifier without training. Once all samples are projected onto this axis, the conditions that best reproduce the positive control can be selected. When no positive control is available, the Mahalanobis distance is computed from each sample to the negative control distribution. The latter provides a metric to identify the conditions that alter the negative control’s cell phenotype. This metric is subsequently used to categorize hits through a clustering step. Given the lack of available ground truth in HCS, we provide a qualitative comparison of the results obtained using this approach with results obtained with handcrafted image analysis features for compounds and siRNA screens with or without control. Our results suggests that the fully automated and generic pipeline we propose offers a good alternative to handcrafted dedicated image analysis approaches. Furthermore, we demonstrate that this solution select conditions of interest that had not been identified using the primary dedicated analysis. Altogether, this approach provides a fully automated, reproducible, versatile and comprehensive alternative analysis solution for HCS encompassing compound-based or downregulation screens, with or without positive controls, without the need for training or cell detection, or the development of a dedicated image analysis workflow

Production of reactive oxygen species is turned on and rapidly shut down in epithelial cells infected with Chlamydia trachomatis.

International audienceReactive Oxygen Species (ROS) are many-faceted compounds involved in cell defense against pathogens as well as in cell signaling. Their involvement in the response to infection in epithelial cells remains poorly documented. Here we investigated the production of ROS during infection with Chlamydia trachomatis, a strict intracellular pathogen, in HeLa cells. C. trachomatis induced a transient increase in the ROS level within a few hours, followed by a return to basal level nine hours after infection. At this time point, the host enzyme dedicated to ROS production, the NADPH oxidase, could no longer be activated by external stimuli such as interleukin-1ss. In addition, Rac, a regulatory subunit of the NADPH oxidase complex, was relocated to the membrane of the compartment in which the bacteria develop, the inclusion, while other subunits were not. Altogether, these results indicate that C. trachomatis infection elicits the production of ROS, and that the bacteria rapidly target the activity of the NADPH oxidase to shut it down. Prevention of ROS production at the onset of the bacterial developmental cycle might delay the host response to infection

BML-265 and Tyrphostin AG1478 Disperse the Golgi Apparatus and Abolish Protein Transport in Human Cells

International audienceThe steady-state localization of Golgi-resident glycosylation enzymes in the Golgi apparatus depends on a balance between anterograde and retrograde transport. Using the Retention Using Selective Hooks (RUSH) assay and high-content screening, we identified small molecules that perturb the localization of Mannosidase II (ManII) used as a model cargo for Golgi resident enzymes. In particular, we found that two compounds known as EGFR tyrosine kinase inhibitors, namely BML-265 and Tyrphostin AG1478 disrupt Golgi integrity and abolish secretory protein transport of diverse cargos, thus inducing brefeldin A-like effects. Interestingly, BML-265 and Tyrphostin AG1478 affect Golgi integrity and transport in human cells but not in rodent cells. The effects of BML-265 are reversible since Golgi integrity and protein transport are quickly restored upon washout of the compounds. BML-265 and Tyrphostin AG1478 do not lead to endosomal tubulation suggesting that, contrary to brefeldin A, they do not target the trans-Golgi ARF GEF BIG1 and BIG2. They quickly induce COPI dissociation from Golgi membranes suggesting that, in addition to EGFR kinase, the cis-Golgi ARF GEF GBF1 might also be a target of these molecules. Accordingly, overexpression of GBF1 prevents the effects of BML-265 and Tyrphostin AG1478 on Golgi integrity

Rab7-harboring vesicles are carriers of the transferrin receptor through the biosynthetic secretory pathway

International audienceThe biosynthetic secretory pathway is particularly challenging to investigate as it is underrepresented compared to the abundance of the other intracellular trafficking routes. Here, we combined the retention using selective hook (RUSH) to a CRISPR-Cas9 gene editing approach (eRUSH) and identified Rab7-harboring vesicles as an important intermediate compartment of the Golgi-to-plasma membrane transport of neosynthesized transferrin receptor (TfR). These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles. Rab7A was transiently associated to neosynthetic TfR-containing post-Golgi vesicles but dissociated before fusion with the plasma membrane. Together, our study reveals a role for Rab7 in the biosynthetic secretory pathway of the TfR, highlighting the diversity of the secretory vesicles' nature

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

International audienc

The Intracellular Ion flux mediated by TMEM176A and TMEM176B is involved in the regulation of Dendritic Cell Functions

International audienceThe regulation of immune cells by intracellular ion channels remains poorly explored. Here we investigated the role of two cation channels encoded by Tmem176a and Tmem176b that are highly expressed in myeloid cells in the immune system. To avoid any functional compensation, we generated double knock-out (DKO) mice, allowing simultaneous deletion of these two highly redundant and coregulated genes using the CRISPR-Cas9 system. The absence of Tmem176a/b significantly impacted antigen processing and presentation to CD4+ T cells in vivo and selectively altered cytokine production by dendritic cells (DCs). Using a novel real-time fluorescence-based system to analyze intracellular trafficking we found that both channels co-localized in highly dynamic post-Golgi vesicles preferentially interacting with, but not accumulating in, acidic organelles. Thus, these results highlight the importance of TMEM176A/B-mediated cation flux for the fine regulation of DC biology

The Intracellular Ion flux mediated by TMEM176A and TMEM176B is involved in the regulation of Dendritic Cell Functions

International audienceThe regulation of immune cells by intracellular ion channels remains poorly explored. Here we investigated the role of two cation channels encoded by Tmem176a and Tmem176b that are highly expressed in myeloid cells in the immune system. To avoid any functional compensation, we generated double knock-out (DKO) mice, allowing simultaneous deletion of these two highly redundant and coregulated genes using the CRISPR-Cas9 system. The absence of Tmem176a/b significantly impacted antigen processing and presentation to CD4+ T cells in vivo and selectively altered cytokine production by dendritic cells (DCs). Using a novel real-time fluorescence-based system to analyze intracellular trafficking we found that both channels co-localized in highly dynamic post-Golgi vesicles preferentially interacting with, but not accumulating in, acidic organelles. Thus, these results highlight the importance of TMEM176A/B-mediated cation flux for the fine regulation of DC biology

Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type