Elaine Del Nery
Institut Curie, Translational Research, Department Member
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Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function... more
Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent fla...
Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- and HER2-negative phenotype and in most cases, a relatively aggressive clinical behaviour. The lack of specific targeted therapies and low... more
Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- and HER2-negative phenotype and in most cases, a relatively aggressive clinical behaviour. The lack of specific targeted therapies and low efficiency of currently available chemotherapies spurred several clinical trials in the last few years. Despite encouraging results, TNBC still remains a major unmet medical need that prompted us to explore the role of 863 epigenetic modulators in TNBC cell survival.Methods: A comprehensive siRNA library was screened to explore the role of known epigenetic modulators in TNBC cell viability and growth. The knock-down effect was evaluated for 863 epigenetic genes using 4 siRNAs/gene in two TNBC and a non-TNBC cell lines using ATP-based luminescence and nuclei count image-based assays. Considering siRNA off-target effects, four analysis methods including a classical threshold-based analysis and three ranking methods were applied to determine on-target hit...
Efficient cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumour immune responses. Yet, several steps of antigen intracellular traffic during cross-presentation are incompletely understood: in... more
Efficient cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumour immune responses. Yet, several steps of antigen intracellular traffic during cross-presentation are incompletely understood: in particular, the molecular mechanisms and the relative importance of antigen import from endocytic compartments into the cytosol. Here, we asked whether antigen import into the cytosol is rate-limiting for cross-presentation and anti-tumour immunity. By screening 700 FDA-approved drugs, we identified 37 import enhancers. We focused on prazosin and tamoxifen, and generated proteomic organellar maps of drug-treated DCs, covering the subcellular localisations of over 2000 proteins. By combining organellar mapping, quantitative proteomics, microscopy, and bioinformatics, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release into the cytosol. Enhancing antigen import facilitates cross-presentation of...
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1,... more
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.
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Motivation High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, High Content drug screens are performed on one single cell line. Yet, a single cell line cannot be thought of as a perfect disease... more
Motivation High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, High Content drug screens are performed on one single cell line. Yet, a single cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterise drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process. Results The contribution of this paper is twofold. First, we investigate whether we can predict drug mechanism of action (MOA) at the molecular level without optimisation of the MOA classes to the screen specificities. To this end, we benchmark a set of algorithms within a conventional pipeline, and evaluate their MOA prediction performance according to a statistically rigorous framework. Second...
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We aim at the identification of myosin motor proteins that control trafficking at the Golgi apparatus. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identify MYO1C as a novel player at the Golgi.... more
We aim at the identification of myosin motor proteins that control trafficking at the Golgi apparatus. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identify MYO1C as a novel player at the Golgi. We demonstrate that depletion of MYO1C induces Golgi apparatus fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi apparatus that colocalize with Golgi-associated actin dots. Interestingly, MYO1C depletion leads to loss of cellular F-actin, and Golgi apparatus decompaction is also observed after the inhibition or loss of the Arp2/3 complex. We show that the functional consequences of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes branched actin at the Golgi apparatus that facilitates the arrival of incoming transport at the Golgi.
Proteins destined to the cell surface are conveyed through membrane-bound compartments using the secretory pathway. Multiple secretory routes exist in cells, which paves the way to the development of inhibitory molecules able to... more
Proteins destined to the cell surface are conveyed through membrane-bound compartments using the secretory pathway. Multiple secretory routes exist in cells, which paves the way to the development of inhibitory molecules able to specifically perturb the transport of a chosen cargo. We used differential high-content screening of chemical libraries to identify molecules reducing the secretion of CCR5, the major co-receptor for HIV-1 entry. Three molecules strongly affected the anterograde transport of CCR5, without inhibiting the transport of the related G protein-coupled receptors CCR1 and CXCR4. These three molecules perturb the transport of endogenous CCR5 and decrease the entry of HIV in human primary target cells. Two molecules were found to share the same mode of action, inhibiting palmitoylation of CCR5. Our results demonstrate that secretory routes can be specifically targeted which allows to envisage novel strategies to provoke the intracellular retention or rerouting of secr...
High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, drug screens are performed in one single cell line. Yet, a single cell line cannot be thought of as a perfect disease model. Many diseases... more
High Content Screening (HCS) is an important tool in drug discovery and characterisation. Often, drug screens are performed in one single cell line. Yet, a single cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterise drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process. The contribution of this paper is twofold. First, we investigate whether we can predict drug mechanism of action (MOA) at the molecular level without optimisation of the MOA classes to the screen specificities. To this end, we benchmark a set of algorithms within a conventional pipeline, and evaluate their MOA prediction performance according to a statistically rigorous framework. Second, we extend this conventional pi...
Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis... more
Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance. We established four chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by and silencing, and mass spectrometry analysis was used to identify its downstream effectors. We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulate...
Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeutic drugs mainly because they allow for scanning a wide spectrum of possible targets at once. However, despite compelling methodological... more
Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeutic drugs mainly because they allow for scanning a wide spectrum of possible targets at once. However, despite compelling methodological advances, posterior identification of a compound's mechanism of action (MOA) has remained difficult and highly refractory to automated analyses. Methods such as the cell painting assay and multiplexing fluorescent dyes to reveal broadly relevant cellular components were recently suggested for MOA prediction. We demonstrated that adding fluorescent dyes to a single assay has limited impact on MOA prediction accuracy, as monitoring only the nuclei stain could reach compelling levels of accuracy. This observation suggested that multiplexed measurements are highly correlated and nuclei stain could possibly reflect the general state of the cell. We then hypothesized that combining unrelated and possibly simple cell-based assays could bring a solution that ...
Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved... more
Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These result...
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Research Interests: Kinetics, Fluorescent Dyes and Reagents, Comparative Study, Biological Sciences, Animals, and 14 moreBiochemical, High Pressure Liquid Chromatography, CHEMICAL SCIENCES, Trypanosoma Cruzi, Trypanosoma brucei, Trypanosoma brucei brucei, Substrate Specificity, Carboxypeptidases, Amino Acid Sequence, Recombinant Proteins, Fluorescence quenching, Molecular Sequence Data, fluorescent dyes, and Medical and Health Sciences
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Research Interests: Kinetics, Protein, Crystal structure, Humans, Protein Chemistry, and 14 morePeptides, Enzyme, High Pressure Liquid Chromatography, Amino Acids, Substrate Specificity, Protein Conformation, Amino Acid Sequence, Hydrolysis, Kinetic Parameter, Biochemistry and cell biology, binding sites, Papain, fluorescent dyes, and cathepsin B
We have determined the kinetic parameters for the hydrolysis by cathepsin B of peptidyl-coumarin amide and intramolecularly quenched fluorogenic peptides with the general structures epsilonNH2-Cap-Leu-X-MCA and... more
We have determined the kinetic parameters for the hydrolysis by cathepsin B of peptidyl-coumarin amide and intramolecularly quenched fluorogenic peptides with the general structures epsilonNH2-Cap-Leu-X-MCA and Abz-Lys-Leu-X-Phe-Ser-Lys-Gln-EDDnp, respectively. Abz (orthoaminobenzoic acid) and EDDnp (2,4-dinitrophenyl-ethylenediamine) are the fluorescent donor-acceptor pair, and X was Cys(SBzl), Ser(OBzl), and Thr(OBzl) containing benzyl group (Bzl) at the functional side chain of Cys, Ser, and Thr. The peptidyl-coumarin-containing Cys(SBz1), Ser(OBzl), and Thr(OBzl) have higher affinity cathepsin B, supporting the interpretation of the crystal structure of rat cathepsin B complexed with the inhibitor Z-Arg-Ser(OBzl)-CH2Cl that the benzyl group attached to Ser hydroxyl side chain occupies the enzyme S'(1) subsite [Jia et al. (1995), J. Biol. Chem. 270, 5527]. A similar effect of benzyl group was also detected in the internally quenched peptides. Finally, the benzyl group in subs...
Research Interests: Kinetics, Protein, Crystal structure, Humans, Protein Chemistry, and 14 morePeptides, Enzyme, High Pressure Liquid Chromatography, Amino Acids, Substrate Specificity, Protein Conformation, Amino Acid Sequence, Hydrolysis, Kinetic Parameter, Biochemistry and cell biology, binding sites, Papain, fluorescent dyes, and cathepsin B
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Research Interests: Multidisciplinary, Signal Transduction, Toll like receptor signaling, RNA interference, Cell line, and 10 moreHumans, Tandem Mass Spectrometry, Immunoprecipitation, Membrane transport proteins, Analysis of Variance, Epitopes, Endosomes, Immunoblotting, reverse transcriptase polymerase chain reaction, and cathepsin B
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We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like... more
We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like enzymes, isolated from the same source, reported as CL1 and CL2. Radioimmunoassay experiments have shown that these CL1- (25 kDa) and CL2-like (26 kDa) cysteine proteinases mediated kinin release from high molecular weight kininogen (HMWK). Lys-bradykinin (KRPPGFSPFR) was characterized as the kinin released from a synthetic fragment of HMWK from Leu373 to Ile393 (Abz-LGMISLMKRPPGFSPFRSSRI-NH2) labeled with the fluorescent group Abz (ortho-aminobenzoic acid). We examined the activity of CL1- and CL2-like on internally quenched fluorescent peptides containing HMWK sequences, in which Met379-Lys380 or Arg389-Ser390 bonds were present in the middle of the molecules. These peptides were flanked by the fluorescent donor-acceptor pair Abz and EDDnp (N-[2,4-dinitrophenyl] ethylenediamine). Peptidyl-methylcoumarin amides (MCA) were used to study the substrate specificity requirements. The enzymes presented significantly lower Km values at pH 8.0. The inverse was observed with the kcat values, which were higher at pH 5.0.
Research Interests: Biological Sciences, Molecular, Animals, Time of Flight, Fasciola hepatica, and 12 moreHigh Performance Liquid Chromatography, Enzyme, Amides, Substrate Specificity, Cysteine endopeptidases, Molecular weight, Amino Acid Sequence, Hydrolysis, Radioimmunoassay, Kinins, Molecular Sequence Data, and Medical and Health Sciences
Research Interests: Kinetics, Peptide Science, Enzyme Inhibitors, Fluorescent Dyes and Reagents, Mammals, and 15 moreHumans, Animals, Peptides, Amino Acids, Trypanosoma Cruzi, Substrate Specificity, Cysteine endopeptidases, Amino Acid Profile, Protein Conformation, Protozoan Proteins, Amino Acid Sequence, Fluorescence quenching, binding sites, fluorescent dyes, and cathepsin B
Development of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE). We synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz... more
Development of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE). We synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinitrophenyl]-ethylenediamine) at their N- and C-terminal groups, respectively, and these were assayed as ACE substrates. We examined two series of peptides, Abz-GFSPFRX-EDDnp and Abz-GFSPFXQ-EDDnp (X, various amino acids). Hydrolysis of the fluorogenic substrates by ACE was followed by continuous recording of the rising fluorescence (lambda(em) = 420 nm and lambda(ex) = 320 nm). The peptides were obtained by solid-phase synthesis or by classical solution methods. Despite of the blocked C-terminal sequences, the internally quenched bradykinin-related peptides were hydrolysed by ACE. The best substrates for plasma guinea pig ACE were Abz-GFSPFRA-EDDnp and Abz-GFSPFFQ-EDDnp, in which the fluorescence appeared after the first cleavage that occurred at R-A and F-Q bond, respectively. This ACE activity was sensitive to NaCl concentration and the optimum pH is greater than 8.0. Measurements of ACE activity with Hip-His-Leu and Abz-GFSPFFQ-EDDnp in the serum of 20 healthy patients correlated closely (r = 0.959). Complete inhibition of the hydrolysis of Abz-GFSPFFQ-EDDnp by human serum was observed with captopril and lisinopril. We describe internally quenched fluorogenic substrates for ACE devoid of free C-terminal carboxyl group. They are convenient tools for ACE studies as they permit continuous fluorimetric measurements of the enzymatic activity, even in human serum.
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A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box... more
A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box statistical testing. Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape and distribution of intracellular compartments avoiding strong cell-to-cell variation that is a major limitation of classical culture conditions. To detect changes in cell morphology induced by compound treatment, fluorescently labeled intracellular structures from several tens of micropatterned cells were transformed into probabilistic density maps. Then, the similarity or difference between two given density maps was quantified using statistical testing that evaluates differences directly from the data without additional analysis or any subjective decision. The versatility of this organelle mapping approach for different magnifications and i...
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Research Interests: Kinetics, Biological Chemistry, Biological Sciences, Humans, Animals, and 11 moreBiological, Glycoproteins, CHEMICAL SCIENCES, Trypanosoma Cruzi, Substrate Specificity, Cysteine endopeptidases, Amino Acid Sequence, Recombinant Proteins, Hydrolysis, Molecular Sequence Data, and Medical and Health Sciences
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We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor,... more
We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.
Research Interests: Analytical Chemistry, Fluorescent Dyes and Reagents, Thrombin, Humans, Liver, and 15 moreAnalytical, Trypsin, Enzyme, Substrate Specificity, Fluorescent probes, Cysteine endopeptidases, Amino Acid Profile, Analytical Biochemistry, Hydrolysis, Serine Protease, Biochemistry and cell biology, Papain, fluorescent dyes, Cysteine Protease, and cathepsin B
Research Interests: Cell Cycle, Membrane Proteins, Membrane Trafficking, RNA interference, Mitosis, and 13 moreHumans, Endoplasmic Reticulum, Alternative splicing, Traffic, Phenotype, HeLa cells, Shiga Toxin, Alternative Splicing, Protein isoforms, Base Sequence, Golgi Apparatus, Biochemistry and cell biology, and Intracellular Transport
The Ras family G-proteins RalA and RalB make critical non-overlapping contributions to the generation of a tumorigenic regulatory network, supporting bypass of the normal restraints on both cell proliferation and survival. The Sec6/8... more
The Ras family G-proteins RalA and RalB make critical non-overlapping contributions to the generation of a tumorigenic regulatory network, supporting bypass of the normal restraints on both cell proliferation and survival. The Sec6/8 complex, or exocyst, has emerged as a principal direct effector complex for Ral GTPases. Here, we show that RalA and RalB support mitotic progression through mobilization of the exocyst for two spatially and kinetically distinct steps of cytokinesis. RalA is required to tether the exocyst to the cytokinetic furrow in early cytokinesis. RalB is then required for recruitment of the exocyst to the midbody of this bridge to drive abscission and completion of cytokinesis. The collaborative action of RalA and RalB is specified by discrete subcellular compartmentalization and unique pairs of RalGEF proteins that provide inputs from both Ras-family protein-dependent and protein-independent regulatory cues. This suggests that Ral GTPases integrate diverse upstream signals to choreograph multiple roles for the exocyst in mitotic progression.
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Research Interests: Biochemistry, Genetics, Biophysics, Molecular Biology, Stem Cells, and 23 moreMigration, MicroRNA, Cancer, Cell Cycle, Cell Biology, DNA damage, Apoptosis, Adhesion, Nature, Cytoskeleton, Signal Transduction, Transcription, Biological Sciences, Humans, Animals, Endoplasmic Reticulum, Molecular motor proteins, Signalling, Chromatin, HeLa cells, Polarity, Zebrafish, and Golgi Apparatus
The small GTPase rab6A but not the isoform rab6A' has previously been identified as a regulator of the COPI-independent recycling route that carries Golgi-resident... more
The small GTPase rab6A but not the isoform rab6A' has previously been identified as a regulator of the COPI-independent recycling route that carries Golgi-resident proteins and certain toxins from the Golgi to the endoplasmic reticulum (ER). The isoform rab6A' has been implicated in Golgi-to-endosomal recycling. Because rab6A but not A', binds rabkinesin6, this motor protein is proposed to mediate COPI-independent recycling. We show here that both rab6A and rab6A' GTP-restricted mutants promote, with similar efficiency, a microtubule-dependent recycling of Golgi resident glycosylation enzymes upon overexpression. Moreover, we used small interfering RNA mediated down-regulation of rab6A and A' expression and found that reduced levels of rab6 perturbs organization of the Golgi apparatus and delays Golgi-to-ER recycling. Rab6-directed Golgi-to-ER recycling seems to require functional dynactin, as overexpression of p50/dynamitin, or a C-terminal fragment of Bicaudal-D, both known to interact with dynactin inhibit recycling. We further present evidence that rab6-mediated recycling seems to be initiated from the trans-Golgi network. Together, this suggests that a recycling pathway operates at the level of the trans-Golgi linking directly to the ER. This pathway would be the preferred route for both toxins and resident Golgi proteins.