Publications by authors named "Bruno Goud"

130 Publications

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterisation in different cell types.

Biol Cell 2021 Mar 5. Epub 2021 Mar 5.

Institut de Pharmacologie Moléculaire et Cellulaire, Université Côte d'Azur et CNRS, UMR7275, Valbonne, F-06560, France.

Background Information: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins.

Results: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria.

Conclusions And Significance: This library should facilitate further cellular investigations, notably by imaging techniques.
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http://dx.doi.org/10.1111/boc.202000158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014678PMC
March 2021

RAB6 GTPase regulates mammary secretory function by controlling the activation of STAT5.

Development 2020 10 8;147(19). Epub 2020 Oct 8.

Department of Cell Biology and Cancer, Institut Curie, PSL Research University, Sorbonne Université, CNRS, UMR144, Paris F-75005, France

The Golgi-associated RAB GTPases, RAB6A and RAB6A', regulate anterograde and retrograde transport pathways from and to the Golgi. , RAB6A/A' control several cellular functions including cell division, migration, adhesion and polarity. However, their role remains poorly described Here, we generated BlgCre; mice presenting a specific deletion of in the mammary luminal secretory lineage during gestation and lactation. loss severely impaired the differentiation, maturation and maintenance of the secretory tissue, compromising lactation. The mutant epithelium displayed a decreased activation of STAT5, a key regulator of the lactogenic process primarily governed by prolactin. Data obtained with a mammary epithelial cell line suggested that defective STAT5 activation might originate from a perturbed transport of the prolactin receptor, altering its membrane expression and signaling cascade. Despite the major functional defects observed upon deletion, the polarized organization of the mammary epithelial bilayer was preserved. Altogether, our data reveal a crucial role for RAB6A/A' in the lactogenic function of the mammary gland and suggest that the trafficking pathways controlled by RAB6A/A' depend on cell-type specialization and tissue context.
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http://dx.doi.org/10.1242/dev.190744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561474PMC
October 2020

[Armand Tavitian (1931-2020): from oncogenes to the Ras superfamily].

Med Sci (Paris) 2020 Aug-Sep;36(8-9):810-812. Epub 2020 Aug 21.

Directeur de recherche au CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex, France.

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http://dx.doi.org/10.1051/medsci/2020136DOI Listing
November 2020

[Pierre Chardin, a pioneer in the discovery of the genes and proteins of the Ras superfamily].

Med Sci (Paris) 2020 Apr 1;36(4):394-398. Epub 2020 May 1.

Centre de Psychiatrie et Neurosciences, 102 rue de la Santé, 75014 Paris, France.

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http://dx.doi.org/10.1051/medsci/2020058DOI Listing
April 2020

Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to Studies.

J Med Chem 2020 05 7;63(10):5568-5584. Epub 2020 May 7.

Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, F-75005 Paris, France.

Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, () (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of was then scrutinized and , and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00431DOI Listing
May 2020

Synthesis, Characterization, Cytotoxic Activity, and Metabolic Studies of Ruthenium(II) Polypyridyl Complexes Containing Flavonoid Ligands.

Inorg Chem 2020 Apr 19;59(7):4424-4434. Epub 2020 Mar 19.

Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, F-75005 Paris, France.

Four novel monocationic Ru(II) polypyridyl complexes were synthesized with the general formula [Ru(DIP)flv]X, where DIP is 4,7-diphenyl-1,10-phenanthroline, flv stands for the flavonoid ligand (5-hydroxyflavone in [Ru(DIP)(5-OHF)](PF), genistein in [Ru(DIP)(gen)](PF), chrysin in [Ru(DIP)(chr)](OTf), and morin in [Ru(DIP)(mor)](OTf)), and X is the counterion, PF, and OTf ̅ (triflate, CFSO̅), respectively. Following the chemical characterization of the complexes by H and C NMR, mass spectrometry, and elemental analysis, their cytotoxicity was tested against several cancer cell lines. The most promising complex, [Ru(DIP)(gen)](PF), was further investigated for its biological activity. Metabolic studies revealed that this complex severely impaired mitochondrial respiration and glycolysis processes, contrary to its precursor, Ru(DIP)Cl, which showed a prominent effect only on the mitochondrial respiration. In addition, its preferential accumulation in MDA-MB-435S cells (a human melanoma cell line previously described as mammary gland/breast; derived from metastatic site: pleural effusion), which are used for the study of metastasis, explained the better activity in this cell line compared to MCF-7 (human, ductal carcinoma).
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http://dx.doi.org/10.1021/acs.inorgchem.9b03562DOI Listing
April 2020

Rationally Designed Long-Wavelength Absorbing Ru(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy.

J Am Chem Soc 2020 04 25;142(14):6578-6587. Epub 2020 Mar 25.

Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, 75005 Paris, France.

The utilization of photodynamic therapy (PDT) for the treatment of various types of cancer has gained increasing attention over the last decades. Despite the clinical success of approved photosensitizers (PSs), their application is sometimes limited due to poor water solubility, aggregation, photodegradation, and slow clearance from the body. To overcome these drawbacks, research efforts are devoted toward the development of metal complexes and especially Ru(II) polypyridine complexes based on their attractive photophysical and biological properties. Despite the recent research developments, the vast majority of complexes utilize blue or UV-A light to obtain a PDT effect, limiting the penetration depth inside tissues and, therefore, the possibility to treat deep-seated or large tumors. To circumvent these drawbacks, we present the first example of a DFT guided search for efficient PDT PSs with a substantial spectral red shift toward the biological spectral window. Thanks to this design, we have unveiled a Ru(II) polypyridine complex that causes phototoxicity in the very low micromolar to nanomolar range at clinically relevant 595 nm, in monolayer cells as well as in 3D multicellular tumor spheroids.
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http://dx.doi.org/10.1021/jacs.9b13620DOI Listing
April 2020

Increasing the Cytotoxicity of Ru(II) Polypyridyl Complexes by Tuning the Electronic Structure of Dioxo Ligands.

J Am Chem Soc 2020 04 17;142(13):6066-6084. Epub 2020 Mar 17.

Department of Chemistry, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany.

Due to the great potential expressed by an anticancer drug candidate previously reported by our group, namely, Ru-sq ([Ru(DIP)(sq)](PF) (DIP, 4,7-diphenyl-1,10-phenanthroline; sq, semiquinonate ligand), we describe in this work a structure-activity relationship (SAR) study that involves a broader range of derivatives resulting from the coordination of different catecholate-type dioxo ligands to the same Ru(DIP) core. In more detail, we chose catechols carrying either an electron-donating group (EDG) or an electron-withdrawing group (EWG) and investigated the physicochemical and biological properties of their complexes. Several pieces of experimental evidences demonstrated that the coordination of catechols bearing EDGs led to deep-red positively charged complexes - in which the preferred oxidation state of the dioxo ligand is the uninegatively charged semiquinonate. Complexes and , on the other hand, are blue/violet neutral complexes, which carry an EWG-substituted dinegatively charged catecholate ligand. The biological investigation of complexes - led to the conclusion that the difference in their physicochemical properties has a strong impact on their biological activity. Thus, complexes - expressed much higher cytotoxicities than complexes and . Complex constitutes the most promising compound in the series and was selected for a more in depth biological investigation. Apart from its remarkably high cytotoxicity (IC = 0.07-0.7 μM in different cancerous cell lines), complex was taken up by HeLa cells very efficiently by a passive transportation mechanism. Moreover, its moderate accumulation in several cellular compartments (i.e., nucleus, lysosomes, mitochondria, and cytoplasm) is extremely advantageous in the search for a potential drug with multiple modes of action. Further DNA metalation and metabolic studies pointed to the direct interaction of complex with DNA and to the severe impairment of the mitochondrial function. Multiple targets, together with its outstanding cytotoxicity, make complex a valuable candidate in the field of chemotherapy research. It is noteworthy that a preliminary biodistribution study on healthy mice demonstrated the suitability of complex for further in vivo studies.
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http://dx.doi.org/10.1021/jacs.9b12464DOI Listing
April 2020

A Maltol-Containing Ruthenium Polypyridyl Complex as a Potential Anticancer Agent.

Chemistry 2020 Apr 26;26(22):4997-5009. Epub 2020 Mar 26.

Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, 75005, Paris, France.

Cancer is one of the main causes of death worldwide. Chemotherapy, despite its severe side effects, is to date one of the leading strategies against cancer. Metal-based drugs present several potential advantages when compared to organic compounds and they have gained trust from the scientific community after the approval on the market of the drug cisplatin. Recently, we reported the ruthenium complex ([Ru(DIP) (sq)](PF ) (where DIP is 4,7-diphenyl-1,10-phenantroline and sq is semiquinonate) with a remarkable potential as chemotherapeutic agent against cancer, both in vitro and in vivo. In this work, we analyse a structurally similar compound, namely [Ru(DIP) (mal)](PF ), carrying the flavour-enhancing agent approved by the FDA, maltol (mal). To possess an FDA approved ligand is crucial for a complex, whose mechanism of action might include ligand exchange. Herein, we describe the synthesis and characterisation of [Ru(DIP) (mal)](PF ), its stability in solutions and under conditions that resemble the physiological ones, and its in-depth biological investigation. Cytotoxicity tests on different cell lines in 2D model and on HeLa MultiCellular Tumour Spheroids (MCTS) demonstrated that our compound has higher activity than cisplatin, inspiring further tests. [Ru(DIP) (mal)](PF ) was efficiently internalised by HeLa cells through a passive transport mechanism and severely affected the mitochondrial metabolism.
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http://dx.doi.org/10.1002/chem.201904877DOI Listing
April 2020

Fe -Salen-Based Probes for the Selective and Sensitive Detection of E450 in Foodstuff.

Chemistry 2020 May 15;26(25):5717-5723. Epub 2020 Apr 15.

Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.

Inorganic pyrophosphate (PPi) is considered as a diagnostic marker for various diseases such as cancer and vascular calcification. PPi also plays an important preservative role as an additive E450 in foodstuff. In this work, a selective Fe -salen-based probe for PPi is described; this probe disassembles in the presence of the target analyte into its molecular blocks, 1,2-propanediamine and 3-chloro-5-formyl-4-hydroxybenzenesulfonic acid. The latter signaling unit leads to a fluorometric response. Compared with a related prototype, the new complex shows a 2.3-times stronger emission at 500 nm and a 155-times better selectivity of PPi over adenosine triphosphate (ATP). Importantly, the new probe was successfully applied for detecting E450 in foodstuff.
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http://dx.doi.org/10.1002/chem.201905686DOI Listing
May 2020

The Golgi apparatus and cell polarity: Roles of the cytoskeleton, the Golgi matrix, and Golgi membranes.

Curr Opin Cell Biol 2020 02 18;62:104-113. Epub 2019 Nov 18.

Institut Curie, PSL Research University, CNRS, UMR 144, 26 rue d'Ulm F-75005, Paris, France; Sorbonne Université, UPMC University Paris 06, CNRS, UMR 144, 26 rue d'Ulm F-75005, Paris, France. Electronic address:

Membrane trafficking plays a crucial role in cell polarity by directing lipids and proteins to specific subcellular locations in the cell and sustaining a polarized state. The Golgi apparatus, the master organizer of membrane trafficking, can be subdivided into three layers that play different mechanical roles: a cytoskeletal layer, the so-called Golgi matrix, and the Golgi membranes. First, the outer regions of the Golgi apparatus interact with cytoskeletal elements, mainly actin and microtubules, which shape, position, and orient the organelle. Closer to the Golgi membranes, a matrix of long coiled-coiled proteins not only selectively captures transport intermediates but also participates in signaling events during polarization of membrane trafficking. Finally, the Golgi membranes themselves serve as active signaling platforms during cell polarity events. We review here the recent findings that link the Golgi apparatus to cell polarity, focusing on the roles of the cytoskeleton, the Golgi matrix, and the Golgi membranes.
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http://dx.doi.org/10.1016/j.ceb.2019.10.003DOI Listing
February 2020

Synthesis, characterization, kinetic investigation and biological evaluation of Re(i) di- and tricarbonyl complexes with tertiary phosphine ligands.

Dalton Trans 2020 Jan 19;49(1):35-46. Epub 2019 Nov 19.

University of the Free State, Department of Chemistry, Nelson Mandela Drive, Bloemfontein, South Africa.

Rhenium(i) di- and tri-carbonyl complexes of the form fac-[Re(CO)(L,L'-Bid)X] and [Re(CO)(L,L'-Bid)X], where X = aqua (HO), methanol (CHOH), triphenylphosphine (PPh), 1,3,5-triaza-7-phosphaadamantane (PTA), tricyclohexylphosphine (PCy) and L,L'-Bid = O,O' bidentate ligands (tropolone = TropH and 3-hydroxyflavone = FlavH) and N,O bidentate ligands (8-hydroxyquinoline = QuinH, 5,7-chloro-8-hydroxyquinoline = diCl-QuinH and quinoline-2,4-dicarboxylic acid = QuinH), were synthesized and unambiguously characterized by H-, C-and P-NMR, IR, UV/Vis and micro-analysis. The crystal structures of four complexes, namely fac-[Re(CO)(QuinH)(HO)]·HO (5), fac-[Re(CO)(Quin)(PPh)] (11), fac-[Re(CO)(diCl-Quin)(PPh)] (12) and [Re(CO)(Trop)(PPh)]·2CHCH (20) were obtained. Re-P bonding distances for 11 and 12 are 2.4948(8) and 2.4908(8) Å, respectively, indicating the effect of the electron-withdrawing substituents of the diCl-Quin ligand. The second-order rate constants for the substitutions of methanol at 25.1 °C in fac-[Re(CO)(L,L'-Bid)(CHOH)] (L,L'-Bid = Trop, Flav and QuinH) type complexes by different entering phosphine ligands (PPh, PCy, and PTA) varied between 7.23(7) × 10 and 1.32(3) × 10 M s and were found to depend on the coordinated bidentate ligand (in general k (QuinH) < k (Trop) < k (Flav)). The toxicity of fac-[Re(CO)(QuinH)(PTA)], fac-[Re(CO)(Trop)(PTA)], fac-[Re(CO)(Trop)(PPh)] and fac-[Re(CO)(Flav)(PPh)] on the cervical cancer HeLa and epithelial RPE-1 cell lines was then evaluated. Complex fac-[Re(CO)(Flav)(PPh)] (16) and fac-[Re(CO)(Trop)(PPh)] (13) displayed the highest cytotoxicity with IC values of 12.21 ± 0.17 μM and 13.35 ± 0.94 μM, respectively in HeLa cells. Interestingly, a small selectivity towards cancer over non-cancerous cells was observed for these compounds (IC = 18.41 ± 3.16 μM and >25 μM in RPE-1 cells).
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http://dx.doi.org/10.1039/c9dt04025kDOI Listing
January 2020

Role of a Kinesin Motor in Cancer Cell Mechanics.

Nano Lett 2019 11 7;19(11):7691-7702. Epub 2019 Oct 7.

Institut Curie, PSL Research University, CNRS, UMR 144 , 26 rue d'Ulm , Paris Cedex 05 75248 , France.

Molecular motors play important roles in force generation, migration, and intracellular trafficking. Changes in specific motor activities are altered in numerous diseases. KIF20A, a motor protein of the kinesin-6 family, is overexpressed in bladder cancer, and KIF20A levels correlate negatively with clinical outcomes. We report here a new role for the KIF20A kinesin motor protein in intracellular mechanics. Using optical tweezers to probe intracellular mechanics and surface AFM to probe cortical mechanics, we first confirm that bladder urothelial cells soften with an increasing cancer grade. We then show that inhibiting KIF20A makes the intracellular environment softer for both high- and low-grade bladder cancer cells. Upon inhibition of KIF20A, cortical stiffness also decreases in lower grade cells, while it surprisingly increases in higher grade malignant cells. Changes in cortical stiffness correlate with the interaction of KIF20A with myosin IIA. Moreover, KIF20A inhibition negatively regulates bladder cancer cell motility irrespective of the underlying substrate stiffness. Our results reveal a central role for a microtubule motor in cell mechanics and migration in the context of bladder cancer.
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http://dx.doi.org/10.1021/acs.nanolett.9b02592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737127PMC
November 2019

Synthesis and Characterization of an Epidermal Growth Factor Receptor-Selective Ru Polypyridyl-Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy.

Chembiochem 2020 02 22;21(4):531-542. Epub 2019 Oct 22.

Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, 75005, Paris, France.

There is a current surge of interest in the development of novel photosensitizers (PSs) for photodynamic therapy (PDT), as those currently approved are not completely ideal. Among the tested compounds, we have previously investigated the use of Ru polypyridyl complexes with a [Ru(bipy) (dppz)] and [Ru(phen) (dppz)] scaffold (bipy=2,2'-bipyridine; dppz=dipyrido[3,2-a:2',3'-c]phenazine; phen=1,10-phenanthroline). These complexes selectively target DNA. However, because DNA is ubiquitous, it would be of great interest to increase the selectivity of our PDT PSs by linking them to a targeting vector in view of targeted PDT. Herein, we present the synthesis, characterization, and in-depth photophysical evaluation of a nanobody-containing Ru polypyridyl conjugate selective for the epidermal growth factor receptor (EGFR) in view of targeted PDT. Using ICP-MS and confocal microscopy, we could demonstrate that our conjugate has high selectivity for the EGFR receptor, which is a crucial oncological target because it is overexpressed and/or deregulated in a variety of solid tumors. However, in contrast to expectations, this conjugate was found to not produce reactive oxygen species (ROS) in cancer cells and is therefore not phototoxic.
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http://dx.doi.org/10.1002/cbic.201900419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065149PMC
February 2020

Systematic investigation of the antiproliferative activity of a series of ruthenium terpyridine complexes.

J Inorg Biochem 2019 09 17;198:110752. Epub 2019 Jun 17.

Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, 75005 Paris, France. Electronic address:

Due to acquired resistance or limitations of the currently approved drugs against cancer, there is an urgent need for the development of new classes of compounds. Among others, there is an increasing attention towards the use of Ru(II) polypyridyl complexes. Most studies in the literature were made on complexes based on the coordination of N-donating bidentate ligands to the ruthenium core whereas studies on 2,2':6', 2″-terpyridine (terpy) coordinating ligands are relatively scare. However, several studies have shown that [Ru(terpy)2] derivatives are able bind to DNA through various binding modes making these compounds potentially suitable as chemotherapeutic agents. Additionally, light irradiation of these compounds was shown to enable DNA cleavage, highlighting their potential use as photosensitizers (PSs) for photodynamic therapy (PDT). In this work, we present the systematic investigation of the potential of 7 complexes of the type [Ru(terpy)(terpy-X)]2+ (X = H (1), Cl (2), Br (3), OMe (4), COOH (5), COOMe (6), NMe2 (7)) as potential chemotherapeutic agents and PDT PSs. Importantly, six of the seven complexes were found to be stable in human plasma as well as photostable in acetonitrile upon continuous light irradiation (480 nm). The determination of the distribution coefficient logP values for the 7 complexes revealed their good water solubility. Complex 7 was found to be cytotoxic in the micromolar range in the dark as well as to have some phototoxicity upon light exposure at 480 nm in non-cancerous retinal pigment epithelium (RPE-1) and cancerous human cervical carcinoma (HeLa) cells. SYNOPSIS: The systematic investigation of the potential of 7 complexes of the type [Ru(terpy)(terpy-X)] (terpy: 2,2':6', 2″-terpyridine; X = H (1), Cl (2), Br (3), OMe (4), COOH (5), COOMe (6), NMe2 (7)) as potential chemotherapeutic agents and photosensitizers for photodynamic therapy is presented.
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http://dx.doi.org/10.1016/j.jinorgbio.2019.110752DOI Listing
September 2019

RAB6 and microtubules restrict protein secretion to focal adhesions.

J Cell Biol 2019 07 29;218(7):2215-2231. Epub 2019 May 29.

Molecular Mechanisms of Intracellular Transport Laboratory, Institut Curie, PSL Research University, Sorbonne Université, Centre National de la Recherche Scientifique, UMR 144, Paris, France

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.
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http://dx.doi.org/10.1083/jcb.201805002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605799PMC
July 2019

MYO1C stabilizes actin and facilitates the arrival of transport carriers at the Golgi complex.

J Cell Sci 2019 04 26;132(8). Epub 2019 Apr 26.

Institut Curie, PSL Research University, Molecular Mechanisms of Intracellular Transport group, 75248 Paris, France

In this study, we aimed to identify the myosin motor proteins that control trafficking at the Golgi complex. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identified MYO1C as a novel player at the Golgi in a human cell line. We demonstrate that depletion of induces Golgi complex fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi complex that colocalize with Golgi-associated actin dots. depletion leads to loss of cellular F-actin, and Golgi complex decompaction is also observed after inhibition or loss of the actin-related protein 2/3 complex, Arp2/3 (also known as ARPC). We show that the functional consequence of depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes actin at the Golgi complex, facilitating the arrival of incoming transport carriers at the Golgi.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.225029DOI Listing
April 2019

Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity.

iScience 2019 Mar 19;13:138-153. Epub 2019 Feb 19.

INSERM, Unité 1193, Villejuif 94800, France; Université Paris-Sud, UMR-S 1193, Villejuif 94800, France. Electronic address:

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology.
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http://dx.doi.org/10.1016/j.isci.2019.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403118PMC
March 2019

Mechanisms of action of Ru(ii) polypyridyl complexes in living cells upon light irradiation.

Chem Commun (Camb) 2018 Nov;54(93):13040-13059

Chimie ParisTech, PSL University, Laboratory for Inorganic Chemical Biology, Paris, France.

The unique photophysical properties of Ru(ii) polypyridyl complexes make them very attractive candidates as photosensitisers in Photodynamic Therapy (PDT). However, to date, there are not many studies exploring in detail the mechanism(s) of action of such compounds in living systems upon light irradiation. This feature article provides an overview of the most in-depth biological studies on such compounds.
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http://dx.doi.org/10.1039/c8cc05928dDOI Listing
November 2018

Detecting phospholipase activity with the amphipathic lipid packing sensor motif of ArfGAP1.

Biochem Biophys Res Commun 2018 10 22;505(1):290-294. Epub 2018 Sep 22.

Departamento de Química Biológica-Ranwel Caputto, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende S/n, Córdoba, X5000HUA, Argentina; CONICET, Universidad Nacional de Córdoba, Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Haya de la Torre y Medina Allende S/n, Córdoba, X5000HUA, Argentina. Electronic address:

The amphipathic lipid packing sensor (ALPS) motif of ArfGAP1 brings this GTPase activating protein to membranes of high curvature. Phospholipases are phospholipid-hydrolyzing enzymes that generate different lipid products that alter the lateral organization of membranes. Here, we evaluate by fluorescence microscopy how in-situ changes of membrane lipid composition driven by the activity of different phospholipases promotes the binding of ALPS. We show that the activity of phospholipase A2, phospholipase C and phospholipase D drastically enhances the binding of ALPS to the weakly-curved membrane of giant liposomes. Our results suggest that the enzymatic activity of phospholipases can modulate the ArfGAP1-mediated intracellular traffic and that amphiphilic peptides such as the ALPS motif can be used to study lipolytic activities at lipid membranes.
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http://dx.doi.org/10.1016/j.bbrc.2018.09.116DOI Listing
October 2018

ProLIF - quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes.

J Cell Sci 2018 08 20;132(4). Epub 2018 Aug 20.

Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520 Turku, Finland

Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719).
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http://dx.doi.org/10.1242/jcs.214270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398480PMC
August 2018

Lipid packing defects and membrane charge control RAB GTPase recruitment.

Traffic 2018 07 6;19(7):536-545. Epub 2018 Apr 6.

Institut Curie, Paris Sciences et Lettres Research University, Sorbonne Université, CNRS UMR144, Paris, France.

Specific intracellular localization of RAB GTPases has been reported to be dependent on protein factors, but the contribution of the membrane physicochemical properties to this process has been poorly described. Here, we show that three RAB proteins (RAB1/RAB5/RAB6) preferentially bind in vitro to disordered and curved membranes, and that this feature is uniquely dependent on their prenyl group. Our results imply that the addition of a prenyl group confers to RAB proteins, and most probably also to other prenylated proteins, the ability to sense lipid packing defects induced by unsaturated conical-shaped lipids and curvature. Consistently, RAB recruitment increases with the amount of lipid packing defects, further indicating that these defects drive RAB membrane targeting. Membrane binding of RAB35 is also modulated by lipid packing defects but primarily dependent on negatively charged lipids. Our results suggest that a balance between hydrophobic insertion of the prenyl group into lipid packing defects and electrostatic interactions of the RAB C-terminal region with charged membranes tunes the specific intracellular localization of RAB proteins.
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http://dx.doi.org/10.1111/tra.12568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032855PMC
July 2018

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

J Exp Med 2018 04 12;215(4):1245-1265. Epub 2018 Feb 12.

Crosstalk between T Cells and Dendritic Cells Group, Institut Curie, Paris Sciences and Lettres Research University, INSERM U932, Paris, France

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble -ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4 T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.
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http://dx.doi.org/10.1084/jem.20162042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881459PMC
April 2018

[Cell complexity should be placed at the heart of cancer research].

Med Sci (Paris) 2018 Jan 31;34(1):63-71. Epub 2018 Jan 31.

Institut Curie, université de recherche Paris sciences et lettres (PSL), CNRS, UMR 144, 26, rue d'Ulm, 75248 Paris Cedex 05, France.

Genetic and most likely epigenetic alterations occurring during tumor progression and metastatic process lead to a broad deregulation of major cellular functions. However, the molecular mechanisms involved are still poorly understood. To understand them, the cell, the basic unit of life, remains more than ever the essential level to integrate the functional impact of genetics and epigenetics processes in the light of the global economy of the normal and cancerous cell, and of its interactions with its microenvironment.
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http://dx.doi.org/10.1051/medsci/20183401015DOI Listing
January 2018

Rab proteins as major determinants of the Golgi complex structure.

Small GTPases 2018 03 29;9(1-2):66-75. Epub 2018 Jan 29.

b Department of Physiology and Biophysics , University of Arkansas for Medical Sciences , Little Rock , USA.

GTP-ases of the Rab family (about 70 in human) are key regulators of intracellular transport and membrane trafficking in eukaryotic cells. Remarkably, almost one third associate with membranes of the Golgi complex and TGN (trans-Golgi network). Through interactions with a variety of effectors that include molecular motors, tethering complexes, scaffolding proteins and lipid kinases, they play an important role in maintaining Golgi architecture.
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http://dx.doi.org/10.1080/21541248.2017.1384087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902205PMC
March 2018

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

Nat Commun 2017 11 1;8(1):1254. Epub 2017 Nov 1.

Institut Curie, PSL Research University, CNRS, UMR 144, Molecular Mechanisms of Intracellular Transport, F-75005, Paris, France.

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6-Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.
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http://dx.doi.org/10.1038/s41467-017-01266-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665954PMC
November 2017

Constitutive resistance to viral infection in human CD141 dendritic cells.

Sci Immunol 2017 Jul;2(13)

Immunity and Cancer Department, Institut Curie, PSL Research University, INSERM U932, 75005 Paris, France.

Dendritic cells (DCs) are critical for the launching of protective T cell immunity in response to viral infection. Viruses can directly infect DCs, thereby compromising their viability and suppressing their ability to activate immune responses. How DC function is maintained in light of this paradox is not understood. By analyzing the susceptibility of primary human DC subsets to viral infections, we report that CD141 DCs have an innate resistance to infection by a broad range of enveloped viruses, including HIV and influenza virus. In contrast, CD1c DCs are susceptible to infection, which enables viral antigen production but impairs their immune functions and survival. The ability of CD141 DCs to resist infection is conferred by RAB15, a vesicle-trafficking protein constitutively expressed in this DC subset. We show that CD141 DCs rely on viral antigens produced in bystander cells to launch cross-presentation-driven T cell responses. By dissociating viral infection from antigen presentation, this mechanism protects the functional capacity of DCs to launch adaptive immunity against viral infection.
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http://dx.doi.org/10.1126/sciimmunol.aai8071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749640PMC
July 2017

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.

Nat Commun 2017 06 13;8:15835. Epub 2017 Jun 13.

Institut Curie, PSL Research University, CNRS, UMR 144, Structure and Membrane Compartments, Paris F-75005, France.

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function.
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http://dx.doi.org/10.1038/ncomms15835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474736PMC
June 2017

Are cancer cells really softer than normal cells?

Biol Cell 2017 May 6;109(5):167-189. Epub 2017 Apr 6.

Institut Curie, PSL Research University, CNRS, UMR 144, Paris, France.

Solid tumours are often first diagnosed by palpation, suggesting that the tumour is more rigid than its surrounding environment. Paradoxically, individual cancer cells appear to be softer than their healthy counterparts. In this review, we first list the physiological reasons indicating that cancer cells may be more deformable than normal cells. Next, we describe the biophysical tools that have been developed in recent years to characterise and model cancer cell mechanics. By reviewing the experimental studies that compared the mechanics of individual normal and cancer cells, we argue that cancer cells can indeed be considered as softer than normal cells. We then focus on the intracellular elements that could be responsible for the softening of cancer cells. Finally, we ask whether the mechanical differences between normal and cancer cells can be used as diagnostic or prognostic markers of cancer progression.
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http://dx.doi.org/10.1111/boc.201600078DOI Listing
May 2017