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Publications de l’équipe
Dynamique de l’organisation intra-cellulaire
Année de publication : 2011
Gaelle Boncompain, Severine Divoux, Nelly Gareil, Helene de Forges, Aurianne Lescure, Lynda
Latreche, Valentina Mercanti, Florence Jollivet, Graça Raposo, Franck Perez (2011 Aug 2)
Synchronization of secretory protein traffic in populations of cells.
Nature methods : 493-8 : DOI : 10.1038/nmeth.1928
Résumé
To dissect secretory traffic, we developed the retention using selective hooks (RUSH)
system. RUSH is a two-state assay based on the reversible interaction of a hook protein
fused to core streptavidin and stably anchored in the donor compartment with a reporter
protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a
synchronous release of the reporter from the hook. Using the RUSH system, we analyzed
different transport characteristics of various Golgi and plasma membrane reporters at
physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of
two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with
observation in other systems. We show preliminarily that the RUSH system is usable for
automated screening. The system should help increase the understanding of the
mechanisms of trafficking and enable screens for molecules that perturb pathological protein
transport.
Takao Nakata, Shinsuke Niwa, Yasushi Okada, Franck Perez, Nobutaka Hirokawa (2011 Jul 20)
Preferential binding of a kinesin-1 motor to GTP-tubulin-rich microtubules
underlies polarized vesicle transport.
The Journal of cell biology : 245-55 : DOI : 10.1083/jcb.201104034
Résumé
Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor
domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are
enriched in EB1 binding sites, and selectively accumulates at the tips of axons. However, it
remains unknown what cue KIF5 recognizes to result in this selective accumulation. We
found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody
hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that
hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to
guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding
sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted
the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed
approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than
to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in
axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular
transport.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1
Publications de l’équipe
Dynamique de l’organisation intra-cellulaire
Année de publication : 2010
Valentina Mercanti, Anna Marchetti, Emmanuelle Lelong, Franck Perez, Lelio Orci, Pierre Cosson
(2010 Sep 10)
Transmembrane domains control exclusion of membrane proteins from clathrincoated pits.
Journal of cell science : 3329-35 : DOI : 10.1242/jcs.073031
Résumé
Efficient sorting of proteins is essential to allow transport between intracellular
compartments while maintaining their specific composition. During endocytosis, membrane
proteins can be concentrated in endocytic vesicles by specific interactions between their
cytoplasmic domains and cytosolic coat proteins. It is, however, unclear whether they can be
excluded from transport vesicles and what the determinants for this sorting could be. Here,
we show that in the absence of cytosolic sorting signals, transmembrane domains control the
access of surface proteins to endosomal compartments. They act in particular by
determining the degree of exclusion of membrane proteins from endocytic clathrin-coated
vesicles. When cytosolic endocytosis signals are present, it is the combination of cytosolic
and transmembrane determinants that ultimately controls the efficiency with which a given
transmembrane protein is endocytosed.
Ole Vielemeyer, Clément Nizak, Ana Joaquina Jimenez, Arnaud Echard, Bruno Goud, Jacques
Camonis, Jean-Christophe Rain, Franck Perez (2010 Aug 24)
Characterization of single chain antibody targets through yeast two hybrid.
BMC biotechnology : 59 : DOI : 10.1186/1472-6750-10-59
Résumé
Due to their unique ability to bind their targets with high fidelity, antibodies are used widely
not only in biomedical research, but also in many clinical applications. Recombinant
antibodies, including single chain variable fragments (scFv), are gaining momentum because
they allow powerful in vitro selection and manipulation without loss of function. Regardless of
the ultimate application or type of antibody used, precise understanding of the interaction
between the antibody’s binding site and its specific target epitope(s) is of great importance.
However, such data is frequently difficult to obtain.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2