Dynamique de l’organisation intra-cellulaire

Publications de l’équipe

Année de publication : 2015

Anne Chevrel, Agathe Urvoas, Ines Li de la Sierra-Gallay, Magali Aumont-Nicaise, Sandrine Moutel, Michel Desmadril, Franck Perez, Alexis Gautreau, Herman van Tilbeurgh, Philippe Minard, Marie Valerio-Lepiniec (2015 Jul 17)

Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications.

Bioscience reports : DOI : 10.1042/BSR20150080 En savoir plus
Résumé

A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes.

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Valentina Millarte, Gaelle Boncompain, Kerstin Tillmann, Franck Perez, Elizabeth Sztul, Hesso Farhan (2015 Apr 24)

Phospholipase C γ1 regulates early secretory trafficking and cell migration via interaction with p115.

Molecular biology of the cell : 2263-78 : DOI : 10.1091/mbc.E15-03-0178 En savoir plus
Résumé

The role of early secretory trafficking in the regulation of cell motility remains incompletely understood. Here we used a small interfering RNA screen to monitor the effects on structure of the Golgi apparatus and cell migration. Two major Golgi phenotypes were observed-fragmented and small Golgi. The latter exhibited a stronger correlation with a defect in cell migration. Among the small Golgi hits, we focused on phospholipase C γ1 (PLCγ1). We show that PLCγ1 regulates Golgi structure and cell migration independently of its catalytic activity but in a manner that depends on interaction with the tethering protein p115. PLCγ1 regulates the dynamics of p115 in the early secretory pathway, thereby controlling trafficking from the endoplasmic reticulum to the Golgi. Our results uncover a new function of PLCγ1 that is independent of its catalytic function and link early secretory trafficking to the regulation of cell migration.

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Alejo E Rodríguez-Fraticelli, Jennifer Bagwell, Minerva Bosch-Fortea, Gaelle Boncompain, Natalia Reglero-Real, Maria J García-León, Germán Andrés, Maria L Toribio, Miguel A Alonso, Jaime Millán, Franck Perez, Michel Bagnat, Fernando Martín-Belmonte (2015 Feb 24)

Developmental regulation of apical endocytosis controls epithelial patterning in vertebrate tubular organs.

Nature cell biology : 241-50 : DOI : 10.1038/ncb3106 En savoir plus
Résumé

Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.

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Nina Dupuis, Assia Fafouri, Aurélien Bayot, Manoj Kumar, Tifenn Lecharpentier, Gareth Ball, David Edwards, Véronique Bernard, Pascal Dournaud, Séverine Drunat, Marie Vermelle-Andrzejewski, Catheline Vilain, Marc Abramowicz, Julie Désir, Jacky Bonaventure, Nelly Gareil, Gaelle Boncompain, Zsolt Csaba, Franck Perez, Sandrine Passemard, Pierre Gressens, Vincent El Ghouzzi (2015 Feb 6)

Dymeclin deficiency causes postnatal microcephaly, hypomyelination and reticulum-to-Golgi trafficking defects in mice and humans.

Human molecular genetics : 2771-83 : DOI : 10.1093/hmg/ddv038 En savoir plus
Résumé

Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve-Melchior-Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation.

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Franck Perez (2015 Jan 7)

Cell biology: Organelles under light control.

Nature : 41-2 : DOI : 10.1038/nature14086 En savoir plus
Résumé

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Année de publication : 2014

Gaelle Boncompain, Franck Perez (2014 Jun 21)

Synchronization of secretory cargos trafficking in populations of cells.

Methods in molecular biology (Clifton, N.J.) : 211-23 : DOI : 10.1007/978-1-4939-0944-5_14 En savoir plus
Résumé

In mammalian cells, secretory proteins are transported to their destination compartment via the secretory pathway. Cargos start their journey in the endoplasmic reticulum and then reach the Golgi complex where they are processed and sorted to be delivered to their target intracellular compartment. To analyze and visualize this flow of proteins, one needs to synchronize their transport. We recently developed the retention using selective hooks (RUSH) system enabling simultaneous and synchronous release of secretory cargos from the endoplasmic reticulum in a population of cells. Here, we describe how to subclone the gene coding for a cargo of interest into a RUSH plasmid and to monitor its synchronized transport along the secretory pathway in fixed samples and in living cells.

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Andrea L J Marschall, Congcong Zhang, André Frenzel, Thomas Schirrmann, Michael Hust, Franck Perez, Stefan Dübel (2014 May 23)

Delivery of antibodies to the cytosol: debunking the myths.

mAbs : 943-56 : DOI : 10.4161/mabs.29268 En savoir plus
Résumé

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG 1 Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells.

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Ana Joaquina Jimenez, Paolo Maiuri, Julie Lafaurie-Janvore, Séverine Divoux, Matthieu Piel, Franck Perez (2014 Jan 30)

ESCRT machinery is required for plasma membrane repair.

Science (New York, N.Y.) : 1247136 : DOI : 10.1126/science.1247136 En savoir plus
Résumé

Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.

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Anaïs Bouissou, Christel Vérollet, Hélène de Forges, Laurence Haren, Yohanns Bellaïche, Franck Perez, Andreas Merdes, Brigitte Raynaud-Messina (2014 Jan 15)

γ-Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning.

The EMBO journal : 114-28 : DOI : 10.1002/embj.201385967 En savoir plus
Résumé

γ-Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ-Tubulin Ring Complexes (γ-TuRCs). While the subunits that constitute γ-Tubulin Small Complexes (γ-TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ-TuRC-specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ-TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ-TuRCs on astral MTs. γ-TuRCs locate along the length of astral MTs, and depletion of γ-TuRC-specific proteins increases MT dynamics and causes the plus-end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down-regulation rescues spindle orientation defects induced by γ-TuRC depletion. Therefore, we propose a role for γ-TuRCs in regulating spindle positioning by controlling the stability of astral MTs.

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Année de publication : 2013

Gaelle Boncompain, Franck Perez (2013 Dec 4)

Fluorescence-based analysis of trafficking in mammalian cells.

Methods in cell biology : 179-94 : DOI : 10.1016/B978-0-12-417164-0.00011-2 En savoir plus
Résumé

Proteins destined for the secretory pathway start their journey in the endoplasmic reticulum and transit through the Golgi complex to be delivered to their destination compartment. Over the last decades, several fluorescence-based assays were developed to analyze the transport of proteins along the secretory pathway. In this review, we briefly introduce the existing tools. We provide detailed protocols to allow the reader to use the newly developed secretory assay termed the RUSH system (Retention Using Selective Hooks). This assay enables the synchronous release of one to three cargos of interest from a donor compartment (the endoplasmic reticulum). Analysis of the transport steps of the cargos from the donor compartment to the acceptor compartment is accomplished by fluorescence-based methods.

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Sardar Faisal Mahmood, Nadège Gruel, Elodie Chapeaublanc, Aurianne Lescure, Thouis Jones, Fabien Reyal, Anne Vincent-Salomon, Virginie Raynal, Gaëlle Pierron, Franck Perez, Jacques Camonis, Elaine Del Nery, Olivier Delattre, François Radvanyi, Isabelle Bernard-Pierrot (2013 Oct 22)

A siRNA screen identifies RAD21, EIF3H, CHRAC1 and TANC2 as driver genes within the 8q23, 8q24.3 and 17q23 amplicons in breast cancer with effects on cell growth, survival and transformation.

Carcinogenesis : 670-82 : DOI : 10.1093/carcin/bgt351 En savoir plus
Résumé

RNA interference has boosted the field of functional genomics, by making it possible to carry out ‘loss-of-function’ screens in cultured cells. Here, we performed a small interfering RNA screening, in three breast cancer cell lines, for 101 candidate driver genes overexpressed in amplified breast tumors and belonging to eight amplicons on chromosomes 8q and 17q, investigating their role in cell survival/proliferation. This screening identified eight driver genes that were amplified, overexpressed and critical for breast tumor cell proliferation or survival. They included the well-described oncogenic driver genes for the 17q12 amplicon, ERBB2 and GRB7. Four of six other candidate driver genes-RAD21 and EIF3H, both on chromosome 8q23, CHRAC1 on chromosome 8q24.3 and TANC2 on chromosome 17q23-were confirmed to be driver genes regulating the proliferation/survival of clonogenic breast cancer cells presenting an amplification of the corresponding region. Indeed, knockdown of the expression of these genes decreased cell viability, through both cell cycle arrest and apoptosis induction, and inhibited the formation of colonies in anchorage-independent conditions, in soft agar. Strategies for inhibiting the expression of these genes or the function of the proteins they encode are therefore of potential value for the treatment of breast cancers presenting amplifications of the corresponding genomic region.

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Hélène de Forges, Antoine Pilon, Christian Poüs, Franck Perez (2013 Aug 27)

Imaging GTP-bound tubulin: from cellular to in vitro assembled microtubules.

Methods in cell biology : 139-53 : DOI : 10.1016/B978-0-12-407757-7.00010-4 En savoir plus
Résumé

Microtubules display a very dynamic behavior, and the presence of the guanosine-triphosphate (GTP) cap at the plus ends of microtubules is essential to regulate microtubule dynamics. Dimitrov et al. (2008) showed that GTP-tubulin is present not only at the plus ends but also in discrete locations along the microtubule lattice. These GTP islands were proposed to contribute to rescue events. Studying the localization of GTP-tubulin in microtubules is essential to better comprehend some core aspects in the regulation of microtubule dynamics. In this chapter, we recapitulate essential tools to study the GTP-tubulin using the recombinant antibody MB11 from permeabilized cells to in vitro assays.

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Gaelle Boncompain, Franck Perez (2013 Jul 13)

The many routes of Golgi-dependent trafficking.

Histochemistry and cell biology : 251-60 : DOI : 10.1007/s00418-013-1124-7 En savoir plus
Résumé

The Golgi apparatus lies at the center of intracellular trafficking, controlling secretory and retrograde routes. Its role is essential both to ensure housekeeping cellular functions and to sustain highly differentiated ones. While Golgi dynamics and function have been studied by many groups for several decades now, and intensely for more than 40 years, the mechanisms of Golgi-dependent secretion, how anterograde and retrograde intra-Golgi transports are controlled, as well as the regulation of the Golgi stacked structure and function are some of the many questions that are still open. We will review here the main characteristics of intra-Golgi trafficking discuss the different models that were built to explain the diversity of intra-Golgi routes and its dynamics and propose an integrative model.

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Yuko Fukata, Ariane Dimitrov, Gaelle Boncompain, Ole Vielemeyer, Franck Perez, Masaki Fukata (2013 Jul 10)

Local palmitoylation cycles define activity-regulated postsynaptic subdomains.

The Journal of cell biology : 145-61 : DOI : 10.1083/jcb.201302071 En savoir plus
Résumé

Distinct PSD-95 clusters are primary landmarks of postsynaptic densities (PSDs), which are specialized membrane regions for synapses. However, the mechanism that defines the locations of PSD-95 clusters and whether or how they are reorganized inside individual dendritic spines remains controversial. Because palmitoylation regulates PSD-95 membrane targeting, we combined a conformation-specific recombinant antibody against palmitoylated PSD-95 with live-cell super-resolution imaging and discovered subsynaptic nanodomains composed of palmitoylated PSD-95 that serve as elementary units of the PSD. PSD-95 in nanodomains underwent continuous de/repalmitoylation cycles driven by local palmitoylating activity, ensuring the maintenance of compartmentalized PSD-95 clusters within individual spines. Plasma membrane targeting of DHHC2 palmitoyltransferase rapidly recruited PSD-95 to the plasma membrane and proved essential for postsynaptic nanodomain formation. Furthermore, changes in synaptic activity rapidly reorganized PSD-95 nano-architecture through plasma membrane-inserted DHHC2. Thus, the first genetically encoded antibody sensitive to palmitoylation reveals an instructive role of local palmitoylation machinery in creating activity-responsive PSD-95 nanodomains, contributing to the PSD (re)organization.

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Aurélie Couesnon, Nicolas Offner, Véronique Bernard, Nathalie Chaverot, Stéphanie Backer, Ariane Dimitrov, Franck Perez, Jordi Molgó, Evelyne Bloch-Gallego (2013 Mar 14)

CLIPR-59: a protein essential for neuromuscular junction stability during mouse late embryonic development.

Development (Cambridge, England) : 1583-93 : DOI : 10.1242/dev.087106 En savoir plus
Résumé

CLIPR-59 is a new member of the cytoplasmic linker proteins (CLIP) family mainly localized to the trans-Golgi network. We show here that Clipr-59 expression in mice is restricted to specific pools of neurons, in particular motoneurons (MNs), and progressively increases from embryonic day 12.5 (E12.5) until the first postnatal days. We generated a Clipr-59 knockout mouse model that presents perinatal lethality due to respiratory defects. Physiological experiments revealed that this altered innervation prevents the normal nerve-elicited contraction of the mutant diaphragm that is reduced both in amplitude and fatigue-resistance at E18.5, despite unaffected functional muscular contractility. Innervation of the mutant diaphragm is not altered until E15.5, but is then partially lost in the most distal parts of the muscle. Ultrastructural observations of neuromuscular junctions (NMJs) in the distal region of the diaphragm reveal a normal organization, but a lower density of nerve terminals capped by terminal Schwann cells in E18.5 mutant when compared with control embryos. Similar defects in NMJ stability, with a hierarchy of severity along the caudo-rostral axis, are also observed in other muscles innervated by facial and spinal MNs in Clipr-59 mutant mice. Clipr-59 deficiency therefore affects axon maintenance but not axon guidance toward muscle targets. Thus, CLIPR-59 is involved in the stabilization of specific motor axons at the NMJ during mouse late embryogenesis and its role is crucial for mouse perinatal development.

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