Biomedical subjects

Bruno Goud

Publications and source records attributed to Bruno Goud.

At least 19 recordsLinked to original sources

PubMed2006

Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.

Cytokinesis is the final step of cell division and leads to the physical separation of the daughter cells. After the ingression of a cleavage membrane furrow that pinches the mother cell, future daughter cells spend much of the cytokinesis phase connected by an intercellular bridge. Rab proteins are major regulators of intracellular transport in eukaryotes, and here, we report an essential role for human Rab35 in both the stability of the bridge and its final abscission. We find that Rab35, whose function in membrane traffic was unknown, is localized to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway. Consistent with a key requirement for Rab35-regulated recycling during cell division, inhibition of Rab35 function leads to the accumulation of endocytic markers on numerous cytoplasmic vacuoles in cells that failed cytokinesis. Moreover, Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid and the septin SEPT2. We propose that the Rab35-regulated pathway plays an essential role during the terminal steps of cytokinesis by controlling septin and PIP2 subcellular distribution during cell division.

Animals↗

PubMed2006

Rab1 defines a novel pathway connecting the pre-Golgi intermediate compartment with the cell periphery.

The function of the pre-Golgi intermediate compartment (IC) and its relationship with the endoplasmic reticulum (ER) and Golgi remain only partially understood. Here, we report striking segregation of IC domains in polarized PC12 cells that develop neurite-like processes. Differentiation involves expansion of the IC and movement of Rab1-containing tubules to the growth cones of the neurites, whereas p58- and COPI-positive IC elements, like rough ER and Golgi, remain in the cell body. Exclusion of Rab1 effectors p115 and GM130 from the neurites further indicated that the centrifugal, Rab1-mediated pathway has functions that are not directly related to ER-to-Golgi trafficking. Disassembly of COPI coats did not affect this pathway but resulted in missorting of p58 to the neurites. Live cell imaging showed that green fluorescent protein (GFP)-Rab1A-containing IC elements move bidirectionally both within the neurites and cell bodies, interconnecting different ER exit sites and the cis-Golgi region. Moreover, in nonpolarized cells GFP-Rab1A-positive tubules moved centrifugally towards the cell cortex. Hydroxymethylglutaryl-CoA reductase, the key enzyme of cholesterol biosynthesis, colocalized with slowly sedimenting, Rab1-enriched membranes when the IC subdomains were separated by velocity sedimentation. These results reveal a novel pathway directly connecting the IC with the cell periphery and suggest that this Rab1-mediated pathway is linked to the dynamics of smooth ER.

Animals↗

PubMed2006

A role for the Rab6A' GTPase in the inactivation of the Mad2-spindle checkpoint.

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A', regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A' function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150(Glued), a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A' likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A', through its interaction with p150(Glued) and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition.

Blotting, Western↗

PubMed2006

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking.

The closely related Rab6 isoforms, Rab6A and Rab6A', have been shown to regulate vesicular trafficking within the Golgi and post-Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A' biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A' hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B-subunit along the endocytic pathway, and causes defects in Golgi- associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A' is required for cell cycle progression through mitosis and identify Ile(62) as a key residue for uncoupling Rab6A' functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A' perform different functions within the cell and suggests a novel role for Rab6A' as the major Rab6 isoform regulating previously described Rab6-dependent transport pathways.

Alternative Splicing↗

PubMed2005

Role of curvature and phase transition in lipid sorting and fission of membrane tubules.

We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes.

Brain Chemistry↗

PubMed2005

Protein interaction mapping: a Drosophila case study.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

Animals↗

PubMed2005

Selection and application of recombinant antibodies as sensors of rab protein conformation.

The existence of a conformational switch of Rabs and other small GTPases involved in intracellular transport regulation has been known for many years. This switch is superimposed on the membrane association/dissociation cycle for most of these GTPases. While these processes are key features of the dynamics of intracellular transport events, surprisingly very few previous studies have focused on the dynamics of the GDP/GTP cycle of Rab proteins in time and space. The main reason for this is the lack of tools available to dynamically probe for Rab GTPases conformation switches and membrane association/dissociation, in particular in vivo. We recently reported the in vitro selection of conformation-specific recombinant antibodies specific to the GTP-bound conformation of Rab6 proteins. These antibodies were obtained in vitro by phage display, a rather simple, rapid, and cheap technique. We additionally showed that these conformation-specific antibodies can be expressed in living cells to follow endogenous Rab6 in its activated conformation in vivo. The same strategy could be used to study other conformation switching mechanisms and, in general, to study the switching between states that antibodies can distinguish (e.g., phosphorylation, ubiquitination).

Antibodies↗

PubMed2005

Purification and properties of rab6 interacting proteins.

A crucial step in the characterization of novel partners of Rab proteins is the confirmation that they indeed interact together by techniques other than the yeast two-hybrid assay used to discover them. Some methods and clues that would help to discriminate between putative interactors are summarized. Pull-down, co-immunoprecipitation, and gel filtration experiments are described as ways of checking protein-protein interaction in vitro and in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Accelerated recycling of transferrin receptor in Theileria-transformed B cells.

We found that phoshatidylinositol-3 kinase (PI3-K) markedly contributes to the increased surface expression of bovine transferrin receptor (TfR) on Theileria-infected lymphocytes. We observed that all aspects of TfR turnover are upregulated in parasitized B cells and we were able to detect TfR colocalizing with EEA1 (early endosome antigen 1) and Rab11 at the ultrastructure level in Theileria-infected B cells. We demonstrated recycling of TfR through Rab5- and Rab11-positive compartments by transfection of dominant negative guanosine diphosphate (GDP)-on mutants of the GTPases. Therefore, in Theileria-transformed B cells constitutive PI3-K activity leads to accelerated TfR recycling through Rab5- and Rab11-positive compartments.

Animals↗

PubMed2005

The cellular pathway of CD1e in immature and maturing dendritic cells.

Dendritic cells (DCs) present antigens to T cells via CD1, HLA class I or class II molecules. During maturation, HLA class II-restricted presentation is optimized. The relocalization of CD1e from Golgi to endosomal compartments during DC maturation suggests also an optimization of the antigen-presentation pathway via CD1 molecules. We here detail the biosynthesis and cellular pathway of CD1e in immature and maturing DCs. Unlike the other CD1 molecules, CD1e was found to reach late endosomes through sorting endosomes, without passing through the plasma membrane in either immature or maturing cells. After induction of DC maturation, CD1e disappeared rapidly from the Golgi and was transiently localized in HLA-DR+ vesicles, while the number of CD1e+/CD1b+ compartments increased for at least 20 h. High-resolution light microscopy showed that, in immature DCs, CD1e+ vesicles were often in close apposition to EEA1+ or HLA-DR+ compartments, while CD1e displayed a nearly exclusive distribution in the lysosomes of mature DCs, a finding corroborated by immunoelectron microscopy. During maturation, CD1e synthesis progressively declined, while the endosomal cleavage of CD1e still occurred. Thus, CD1e displays peculiar properties, suggesting an unexpected role among the family of CD1 antigen-presenting molecules.

Antigens, CD1↗

PubMed2005

Rab6 interacts with the mint3 adaptor protein.

The Rab6 GTPase regulates a retrograde transport route connecting endosomes and the endoplasmic reticulum (ER) via the Golgi apparatus. Recently it was shown that active (GTP-loaded) Rab6A regulates intracellular processing of the amyloid precursor protein (APP). To characterize the role of Rab6A in APP trafficking and to identify effector proteins of the active Rab6A protein, we screened a human placenta cDNA library using the yeast two-hybrid system. We isolated an interacting cDNA clone encoding part of the adaptor protein mint3. The interaction between Rab6A and mint3 is GTP-dependent and requires the complete phosphotyrosine-binding (PTB) domain of the mint protein, which also mediates the association with APP. By confocal microscopy we show that Rab6A, mint3 and APP co-localize at Golgi membranes in HeLa cells. Density gradient centrifugation of cytosolic extracts confirms a common distribution of these three proteins. Our data suggest that mint3 links Rab6A to APP traffic.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Cooperative extraction of membrane nanotubes by molecular motors.

In eukaryotic cells, nanotubes represent a substantial fraction of transport intermediates between organelles. They are extracted from membranes by molecular motors walking along microtubules. We previously showed that kinesins fixed on giant unilamellar vesicles in contact with microtubules are sufficient to form nanotubes in vitro. Motors were attached to the membrane through beads, thus facilitating cooperative effects. Koster et al. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. We demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. We determine the motor density along a tube by using fluorescence intensity measurements. We also perform a theoretical analysis describing the dynamics of motors and tube growth. The only adjustable parameter is the motor binding rate onto microtubules, which we measure to be 4.7 +/- 2.4 s(-1). In addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. We find that the number of motors pulling a tube can range from four at threshold to a few tens away from it. The threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traffic regulation in cells.

Animals↗

PubMed2004

TSAP6 facilitates the secretion of translationally controlled tumor protein/histamine-releasing factor via a nonclassical pathway.

Translationally controlled tumor protein (TCTP) is cytoplasmic and structurally related to guanine-nucleotide free chaperones. TCTP (also called histamine-releasing factor) has been described previously as a secreted protein that participates in inflammatory responses by promoting the release of histamine. How TCTP is eventually exported out of the cell to promote such activities is unknown. Here we show that TCTP secretion was insensitive to either brefeldin A or monensin, suggesting that it proceeds via an endoplasmic reticulum/Golgi-independent or nonclassical pathway. Moreover, our analyses also suggest that secreted TCTP originates from pre-existing pools. TSAP6, a p53-inducible 5-6 transmembrane protein, was found to interact with TCTP in a yeast two-hybrid hunt. GST pull down assays confirmed their direct interaction, and immunofluorescence analysis revealed their partial co-distribution to vesicular-like structures at the plasma membrane and around the nucleus. Functionally, the overexpression of TSAP6 consistently leads to enhanced secretion of both endogenously and exogenously expressed TCTP. Finally, we found TCTP in preparations of small secreted vesicles called exosomes, which have been suggested as a possible pathway for nonclassical secretion. Overexpression of TSAP6 also increased TCTP levels in exosome preparations. Altogether, these data identify a novel role for TSAP6 in the export of TCTP and indicate that this multipass membrane protein could have a general role in the regulation of vesicular trafficking and secretion.

Biomarkers, Tumor↗

PubMed2004

CLIPR-59 is a lipid raft-associated protein containing a cytoskeleton-associated protein glycine-rich domain (CAP-Gly) that perturbs microtubule dynamics.

We recently have identified a new cytoplasmic linker protein (CLIP), CLIPR-59, which is involved in the regulation of early endosome/trans-Golgi network dynamics. In contrast with CLIP-170, CLIPR-59 is not localized to microtubules at steady state but is associated with the trans-Golgi network and the plasma membrane. Here we show that the last 30 amino acids (C30) are sufficient for membrane targeting and that two cysteines in the C30 domain are palmitoylated. We demonstrate that CLIPR-59 is associated with lipid rafts via its C-terminal palmitoylated domain. In vitro experiments suggest that CLIPR-59 and its microtubule-binding domain alone have a better affinity for unpolymerized tubulin or small oligomers than for microtubules. In contrast with the CLIP-170 microtubule-binding domain, the CLIPR-59 microtubule-binding domain diminishes microtubule regrowth after nocodazole washout in vivo, showing that this domain can prevent microtubule polymerization. In contrast with the role of linker between membranes and microtubules that was proposed for CLIP function, CLIPR-59 thus may have an "anti-CLIP" function by preventing microtubule-raft interactions.

Amino Acid Sequence↗

PubMed2004

Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network.

An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.

Animals↗

PubMed2004

Reproduction of Langerin/CD207 traffic and Birbeck granule formation in a human cell line model.

Birbeck granules (BG) are organelles specific to Langerhans cells (LCs), which form where the C-type lectin Langerin accumulates. Their function remains obscure due to morphologic and dynamic alterations induced by maturation of isolated LC. In this study, we attempted to reconstitute Langerin traffic and BG formation in the endosomal pathway of a human melanoma cell line. In the selected Langerin-transfected cell line, M10-22E, Langerin is distributed between the early recycling endosomal compartment and the plasma membrane, as in LC. Whereas mainly concentrated in membranes related to the Rab11(+) endosomal recycling compartment at the steady state, Langerin also recycles in M10-22E cells and drives BG biogenesis in the endosomal recycling compartment. Interruption of endocytosis or recycling induces redistribution of intracellular Langerin with an associated alteration in BG location and morphology. We have, therefore, generated a stable, Langerin-transfected cell line in which Langerin traffic and distribution and BG morphology replicate that seen in freshly isolated LC. This practical model can now be used to further delineate the nature and function of BG.

Antigens, CD↗

PubMed2004

Golgi inheritance under a block of anterograde and retrograde traffic.

In mitosis, the Golgi complex is inherited following its dispersion, equal partitioning and reformation in each daughter cell. The state of Golgi membranes during mitosis is controversial, and the role of Golgi-intersecting traffic in Golgi inheritance is unclear. We have used brefeldin A (BFA) to perturb Golgi-intersecting membrane traffic at different stages of the cell cycle and followed by live cell imaging the fate of Golgi membranes in those conditions. We observed that addition of the drug on cells in prometaphase prevents mitotic Golgi dispersion. Under continuous treatment, Golgi fragments persist throughout mitosis and accumulate in a Golgi-like structure at the end of mitosis. This structure localizes at microtubule minus ends and contains all classes of Golgi markers, but is not accessible to cargo from the endoplasmic reticulum or the plasma membrane because of the continuous BFA traffic block. However, it contains preaccumulated cargo, and intermixes with the reforming Golgi upon BFA washout. This structure also forms when BFA is added during metaphase, when the Golgi is not discernible by light microscopy. Together the data indicate that independent Golgi fragments that contain all classes of Golgi markers (and that can be isolated from other organelles by blocking anterograde and retrograde Golgi-intersecting traffic) persist throughout mitosis.

Brefeldin A↗

PubMed2003

The structural GDP/GTP cycle of Rab11 reveals a novel interface involved in the dynamics of recycling endosomes.

The small GTP-binding protein Rab11 is an essential regulator of the dynamics of recycling endosomes. Here we report the crystallographic analysis of the GDP/GTP cycle of human Rab11a, and a structure-based mutagenesis study that identifies a novel mutant phenotype. The crystal structures show that the nucleotide-sensitive switch 1 and 2 regions differ from those of other Rab proteins. In Rab11-GDP, they contribute to a close packed symmetrical dimer, which may associate to membranes in the cell and allow Rab11 to undergo GDP/GTP cycles without recycling to the cytosol. The structure of active Rab11 delineates a three-dimensional site that includes switch 1 and is separate from the site defined by the Rab3/Rabphilin interface. It is proposed to form a novel interface for a Rab11 partner compatible with the simultaneous binding of another partner at the Rabphilin interface. Mutation of Ser(29) to Phe in this epitope resulted in morphological modifications of the recycling compartment that are distinct from those induced by the classical dominant-negative and constitutively active Rab11 mutants. Recycling endosomes condensed in the perinuclear region where they retained recycling transferrin, and they clustered Rab11- and EEA1-positive membranes. Altogether, our study suggests that this mutation impairs a specific subset of Rab11 interactions, possibly those involved in cytoskeleton-based movements driving the slow recycling pathway.

Amino Acid Sequence↗