Biomedical subjects

Franck Perez

Publications and source records attributed to Franck Perez.

14 recordsLinked to original sources

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

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

Kinesin is involved in protecting nascent microtubules from disassembly after recovery from nocodazole treatment.

Upon recovery from nocodazole treatment, microtubules from cultured epithelial cells exhibit unusual properties: they re-grow as fast as any highly dynamic microtubule, but they are also protected against disassembly when challenged with nocodazole like the stable microtubules of steady-state cells. Exploring the mechanism that underlies this protection, we found that it was sensitive to ATP treatment and that it involved conventional kinesin. Kinesin localized at the growing end or along nascent microtubules. Its inhibition using a dominant-negative construct for cargo binding, or by micro-injecting an anti-kinesin heavy chain antibody that impairs motor activity, resulted in the partial or total loss of microtubule protection. Finally, in an ex vivo elongation assay, we found that kinesin also participates in the control of microtubule re-growth. Altogether, our findings suggest that kinesin is involved in an early microtubule protection process that is linked to the control of their dynamics during their early growth phase.

Adenosine Triphosphate↗

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↗

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

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

Recombinant antibodies to the small GTPase Rab6 as conformation sensors.

Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.

Antibodies↗

PubMed2003

A plus-end raft to control microtubule dynamics and function.

Cells require a properly oriented and organised microtubule array to transmit positional information. Recent data have revealed a heterogeneous population of microtubule-binding proteins that accumulates mainly at distal ends of polymerising microtubules. Two mechanisms may account for this concentration: transient immobilisation, which involves association of proteins with growing ends, followed by release more proximally; and deposition at ends via a molecular motor. As with lipid rafts, protein concentration at distal ends may allow a cascade of interactions in the restricted area of a microtubule plus end. This may, in turn, control the dynamic behaviour of this cytoskeletal network and its anchoring to other structures.

Animals↗

PubMed2003

Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo.

Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo. Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.

Animals↗

PubMed2002

Hodgkin and Reed-Sternberg cell-associated autoantigen CLIP-170/restin is a marker for dendritic cells and is involved in the trafficking of macropinosomes to the cytoskeleton, supporting a function-based concept of Hodgkin and Reed-Sternberg cells.

Little is known about the distribution in normal cells of CLIP-170, a linkage mediator between endocytic vesicles and microtubules, and restin, a splice variant encoded by the same gene and marker for Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin disease. Although only trace amounts of CLIP-170/restin are present in peripheral blood mononuclear cell subpopulations, monocyte-derived dendritic cells (DCs) and interleukin-4 (IL-4) + CD40L-activated B cells express high levels of CLIP-170/restin. CLIP-170/restin colocalizes preferentially with membranes of intermediate macropinocytic vesicles, suggesting a new function of CLIP-170/restin in the trafficking of macropinosomes to the cytoskeleton, which is a crucial step in antigen presentation. The strong expression of CLIP-170/restin in HRS cells, DCs, and activated B cells underscores their functional similarities supporting a function-based concept of HRS cells as professional antigen-presenting cells.

Antigen-Presenting Cells↗

PubMed2002

Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170.

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.

Actins↗

PubMed2002

CLIPR-59, a new trans-Golgi/TGN cytoplasmic linker protein belonging to the CLIP-170 family.

The microtubule cytoskeleton plays a fundamental role in cell organization and membrane traffic in higher eukaryotes. It is well established that molecular motors are involved in membrane-microtubule interactions, but it has also been proposed that nonmotor microtubule-binding (MTB) proteins known as CLIPs (cytoplasmic linker proteins) have basic roles in these processes. We report here the characterization of CLIPR-59, a CLIP-170-related protein localized to the trans-most part of the Golgi apparatus. CLIPR-59 contains an acidic region followed by three ankyrin-like repeats and two CLIP-170-related MTB motifs. We show that the 60-amino acid-long carboxy-terminal domain of CLIPR-59 is necessary and sufficient to achieve Golgi targeting, which represents the first identification of a membrane targeting domain in a CLIP-170-related protein. The MTB domain of CLIPR-59 is functional because it localizes to microtubules when expressed as a fragment in HeLa cells. However, our results suggest that this domain is normally inhibited by the presence of adjacent domains, because neither full-length CLIPR-59 nor a CLIPR-59 mutant missing its membrane-targeting region localize to microtubules. Consistent with this observation, overexpression of CLIPR-59 does not affect the microtubule network. However, CLIPR-59 overexpression strongly perturbs early/recycling endosome-TGN dynamics, implicating CLIPR-59 in the regulation of this pathway.

Amino Acid Sequence↗