Studying in vitro membrane curvature recognition by proteins and its role in vesicular trafficking.
In recent years, the interest for proteins that exert key functions in vesicular trafficking through their ability to sense or induce positive membrane curvature has expanded. In this chapter, we first present simple protocols to determine whether a protein targets positively curved membranes with liposomes of well-defined size. Next we describe more sophisticated approaches based on the controlled deformation of giant liposomes. These approaches allow visualization and quantification of protein binding to membrane regions of high curvature by real-time fluorescence microscopy. Last we describe several functional assays to measure how membrane curvature controls the activation state of Arf1 via ArfGAP1 or the asymmetric tethering between flat and curved membranes via the golgin GMAP-210.
Unité Mixte de Recherche 144, CNRS and Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Jean-Baptiste
JB
Manneville
Cécile
C
Leduc
Benoit
B
Sorre
Guillaume
G
Drin
D020816:Amino Acid Motifs; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D048430:Cell Shape; D002478:Cells, Cultured; D003034:Coated Pits, Cell-Membrane / Q000737:chemistry / Q000378:metabolism*; D018699:Coated Vesicles / Q000737:chemistry / Q000378:metabolism; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D016547:Kinesins / Q000737:chemistry; D008027:Light; D008081:Liposomes / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification; D052898:Optical Tweezers; D010316:Particle Size; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D012542:Scattering, Radiation; D059010:Single-Cell Analysis; D013499:Surface Properties; D022161:Transport Vesicles / Q000737:chemistry / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000737:chemistry
C503239:Arfgap1 protein, rat; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D008081:Liposomes; D009687:Nuclear Proteins; D010446:Peptide Fragments; C109331:TRIP11 protein, human; D053835:Unilamellar Liposomes; D016547:Kinesins