Publications de l’équipe
Année de publication : 2020
Experimentally-Generated Ground Truth for Detecting Cell Types in an Image-Based Immunotherapy Screen
2020 IEEE 17th International Symposium on Biomedical Imaging (ISBI) : 886-890 En savoir plusRésumé
ReplierAnnée de publication : 2019
RAB6 and microtubules restrict protein secretion to focal adhesions.
The Journal of cell biology : DOI : 10.1083/jcb.201805002 En savoir plusRésumé
To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.
ReplierAnnée de publication : 2017
Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma Patient Derived Xenografts.
Pigment cell & melanoma research : DOI : 10.1111/pcmr.12577 En savoir plusRésumé
Monoclonal antibodies specific for biomarkers expressed on the surface of uveal melanoma (UM) cells would simplify the immune-capture and genomic characterization of heterogeneous tumor cells originated from patient derived xenografts (PDXs). Antibodies against four independent tumor antigens were isolated by panning a nanobody synthetic library. Such antibodies enabled flow-cytometry-based sorting of distinct cell sub-populations from UM PDXs and to analyze their genomic features. The complexity and specificity of the biochemical and genomic biomarker combinations mirrored the UM tumor polyclonality. The data showed that MUC18 is highly and universally displayed at the surface of UM cells with different genetic background and consequently represents a reliable pan-biomarker for their identification and purification. In contrast, the other three biomarkers were detected in very variable combinations in UM PDX cells. The availability of the identified nanobodies will be instrumental in developing clone-specific drug evaluation and rational clinical strategies based on accurate genomic profiling. This article is protected by copyright. All rights reserved.
ReplierAnnée de publication : 2016
Localized Mechanical Stress Promotes Microtubule Rescue.
Current biology : CB : 3399-3406 : DOI : S0960-9822(16)31274-X En savoir plusRésumé
Microtubule dynamics rely on the properties of tubulin and are regulated by microtubule-associated proteins. GTP-tubulin assembles into hollow polymers, which can depolymerize upon GTP hydrolysis. Depolymerizing microtubules may stop shrinking and resume growth. Such rescues are regulated by microtubule-associated proteins like CLIP-170 and the CLASPs [1, 2]. Microtubule domains prone to rescues contain discrete regions (previously termed « GTP islands ») that retain a GTP-tubulin-like conformation in the main body of the microtubule [3]. However, the exact nature of these domains and the mechanisms controlling their occurrence and distribution are largely unknown. Here we show that collisions between growing microtubules and mechanical obstacles (including other microtubules) in vitro result in the higher abundance of GTP-like islands in stressed microtubule regions. Furthermore, these islands were found to be efficiently generated by both lateral contacts and mechanical constraints applied to the main body of the microtubules. They were also particularly prominent where shifts in the number of protofilaments occur in the microtubule lattice. GTP-like islands and rescues frequently co-occurred at microtubule intersections in vitro and in living cells, both in crossing and in crossed microtubules. We also observed that CLIP-170 recognizes GTP-like islands in vivo and is retained at microtubule crossings. Therefore, we propose that rescues occur via a two-stage mechanism: (1) lattice defects determine potential rescue-promoting islands in the microtubule structure, and (2) CLIP-170 detects these islands to stimulate microtubule rescue. Our results reveal the interplay between rescue-promoting factors and microtubule architecture and organization to control microtubule dynamics.
ReplierThe endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR.
The Journal of cell biology : 543-558 En savoir plusRésumé
Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.
ReplierNaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.
eLife : DOI : 10.7554/eLife.16228 En savoir plusRésumé
In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.
ReplierMicrotubule-independent secretion requires functional maturation of Golgi elements.
Journal of cell science : DOI : jcs.188870 En savoir plusRésumé
The Golgi apparatus is responsible for processing and sorting of secretory cargos. Microtubules are known to accelerate the transport of proteins from the endoplasmic reticulum to the Golgi apparatus and from the Golgi to the plasma membrane. However, whether post-Golgi transport strictly requires microtubules is still unclear. Using the retention using selective hooks (RUSH) system to synchronize the trafficking of cargos, we show that anterograde transport of tumor necrosis factor (TNF) is strongly reduced without microtubules. We show that two populations of Golgi elements co-exist in these cells. A centrally located and giantin-positive Golgi complex sustains trafficking while newly formed peripheral Golgi mini-stacks accumulate cargos in cells without microtubules. Using a genome-edited GFP-giantin cell line, we observe that the trafficking-competent Golgi population corresponds to the pre-existing one that was present before removal of microtubules. All Golgi elements support trafficking after long-term microtubules depletion or after relocation of Golgi proteins in the endoplasmic reticulum using Brefeldin A. Our results demonstrate that functional maturation of Golgi elements is needed to ensure post-Golgi trafficking and that microtubule-driven post-Golgi transport is not strictly required.
ReplierControl of protein trafficking by reversible masking of transport signals.
Molecular biology of the cell : 1310-9 : DOI : 10.1091/mbc.E15-07-0472 En savoir plusRésumé
Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term « controlled unmasking of targeting elements » (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin-biotin system. The targeting signal is generated within or immediately after a 38-amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum.
ReplierAnnée de publication : 2015
A kinome siRNA screen identifies HGS as a potential target for liver cancers with oncogenic mutations in CTNNB1.
BMC cancer : 1020 : DOI : 10.1186/s12885-015-2037-8 En savoir plusRésumé
Aberrant activation of the Wnt/β-catenin pathway is a major and frequent event in liver cancer, but inhibition of oncogenic β-catenin signaling has proven challenging. The identification of genes that are synthetically lethal in β-catenin-activated cancer cells would provide new targets for therapeutic drug design.
ReplierThe plasma membrane repair shop: Fixing the damage.
Seminars in cell & developmental biology : 1 : DOI : 10.1016/j.semcdb.2015.11.008 En savoir plusRésumé
ReplierDiversifying the secretory routes in neurons.
Frontiers in neuroscience : 358 : DOI : 10.3389/fnins.2015.00358 En savoir plusRésumé
Nervous system homeostasis and synaptic function need dedicated mechanisms to locally regulate the molecular composition of the neuronal plasma membrane and allow the development, maintenance and plastic modification of the neuronal morphology. The cytoskeleton and intracellular trafficking lies at the core of all these processes. In most mammalian cells, the Golgi apparatus (GA) is at the center of the biosynthetic pathway, located in the proximity of the microtubule-organizing center. In addition to this central localization, the somatic GA in neurons is complemented by satellite Golgi outposts (GOPs) in dendrites, which are essential for dendritic morphogenesis and are emerging like local stations of membranes trafficking to synapses. Largely, GOPs participation in post-ER trafficking has been determined by imaging the transport of the exogenous protein VSVG. Here we review the diversity of neuronal cargoes that traffic through GOPs and the assortment of different biosynthetic routes to synapses. We also analyze the recent advances in understanding the role of cytoskeleton and Golgi matrix proteins in the biogenesis of GOPs and how the diversity of secretory routes can be generated.
ReplierPhysico-chemical and biological considerations for membrane wound evolution and repair in animal cells.
Seminars in cell & developmental biology : 2-9 : DOI : 10.1016/j.semcdb.2015.09.023 En savoir plusRésumé
Membrane damage is a daily threat to the life of a cell, especially cells from muscles, gut, epidermis and vasculature, tissues that are particularly subjected to mechanical stress. Damages can come from different sources and give rise to different holes in terms of size and nature. For example, while some holes are simply scratches in the lipid bilayer, others are delimited by pore forming proteins. It is thus expectable that these wounds will not evolve similarly in a cellular context, and that repair mechanisms will differ to a certain extent. It would therefore be misleading to fully generalize cell membrane damage and repair, and consider it as one universal phenomenon. Indeed, damage has been observed in cells ranging from the rather small mammalian cells (∼30μm) to the very big Urchin egg (∼100μm). Moreover, the wounds observed or artificially induced in eukaryotic cells range from some nanometers to several micrometers, and can be delimited by particular molecules as mentioned before. This chapter aims at reviewing the different physico-chemical and biological parameters that can influence wound evolution in cells and to conciliate the different repair mechanisms that have been described by evaluating them in their cellular and wound type context.
ReplierTECPR2 Cooperates with LC3C to Regulate COPII-Dependent ER Export.
Molecular cell : 89-104 : DOI : 10.1016/j.molcel.2015.09.010 En savoir plusRésumé
Hereditary spastic paraplegias (HSPs) are a diverse group of neurodegenerative diseases that are characterized by axonopathy of the corticospinal motor neurons. A mutation in the gene encoding for Tectonin β-propeller containing protein 2 (TECPR2) causes HSP that is complicated by neurological symptoms. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy, the exact function of TECPR2 is unknown. Here, we show that TECPR2 associates with several trafficking components, among them the COPII coat protein SEC24D. TECPR2 is required for stabilization of SEC24D protein levels, maintenance of functional ER exit sites (ERES), and efficient ER export in a manner dependent on binding to lipidated LC3C. TECPR2-deficient HSP patient cells display alterations in SEC24D abundance and ER export efficiency. Additionally, TECPR2 and LC3C are required for autophagosome formation, possibly through maintaining functional ERES. Collectively, these results reveal that TECPR2 functions as molecular scaffold linking early secretion pathway and autophagy.
ReplierResolving bundled microtubules using anti-tubulin nanobodies.
Nature communications : 7933 : DOI : 10.1038/ncomms8933 En savoir plusRésumé
Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.
ReplierArtificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport.
Journal of the American Chemical Society : 10464-7 : DOI : 10.1021/jacs.5b05672 En savoir plusRésumé
Artificial ligands of streptavidin (ALiS) with association constants of ∼10(6) M(-1) were discovered by high-throughput screening of our chemical library, and their binding characteristics, including X-ray crystal structure of the streptavidin complex, were determined. Unlike biotin and its derivatives, ALiS exhibits fast dissociation kinetics and excellent cell permeability. The streptavidin-ALiS system provides a novel, practical compound-dependent methodology for repeated reversible cycling of protein localization between intracellular organella.
Replier