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Table of contents
From cell discovery to membrane binding properties of RAB GTPases
1 Biology and physics of membranes
1.1 The Lipid bilayer
1.1.1 From a lipid molecule to a bilayer
1.1.2 Different classes of lipids
1.1.3 Lipid synthesis and distribution in cells
1.2 Membrane domain formation
1.2.1 Different states of membranes
1.2.2 Phase state of cellular membranes
1.3 Membrane deformations
1.3.1 Membrane curvature in cells
1.3.2 Mechanisms of membrane deformation
1.3.3 Protein curvature sensing
1.4 In vitro experimental approaches
1.4.1 Model membranes for in vitro experiments
1.4.2 Phase separation from living cells to model membranes
1.4.3 Curvature sensing on model membranes
2 RAB GTPases
2.1 RAB discovery and evolution
2.2 RAB sequence and structure
2.2.1 G-domain
2.2.2 RAB specific sequence motifs
2.2.3 RAB C-terminal region
2.3 RAB posttranslational modifications
2.3.1 RAB activation cycle
2.3.2 RAB membrane insertion and extraction
2.4 Membrane targeting of RAB GTPases
2.5 RAB GTPases and vesicular transport
2.5.1 General mechanism of intracellular transport
2.5.2 RABs and membrane tethering
2.6 Focus on the RAB proteins used in this study
2.6.1 RAB1 and the ER-Golgi intermediate compartment
2.6.2 RAB6 and the Golgi
2.6.3 RAB4 / RAB5 / RAB11 and the endosomal system
2.6.4 RAB35 and the plasma membrane
3 Materials and Methods
3.1 Protein synthesis and modification
3.1.1 Protein expression and purification
3.1.2 In vitro modifications of RAB and GST proteins
3.2 Experimental studies with GUVs
3.2.1 Synthesis of giant unilamellar vesicles
3.2.2 Generalities of the experimental approach
3.2.3 Curvature sensing experiments with GUVs
3.3 Experimental studies with purified Golgi membranes
3.3.1 Purification of Rat Liver Golgi stacks
3.3.2 Experimental chamber
3.3.3 Pulling tubes with kinesins
3.3.4 Immunofluorescence on Golgi membranes
4 Article: RAB proteins bind lipid packing defects
5 RAB4 and RAB11 binding requirements
5.1 Description of the in vitro approach
5.2 RAB4 and RAB11 recruitment to GUV membranes
5.2.1 RAB4 and RAB11 are not recruited to PC-containing membranes
5.2.2 RAB4 and RAB11 are not recruited to GUVs of various lipid composition
5.2.3 Membrane curvature has no effect on the recruitment of RAB4 and RAB11
5.3 RAB4 and RAB11 recruitment to purified Golgi fractions
5.3.1 RAB4 and RAB11 are positively recruited through their prenyl group
5.3.2 RAB4/RAB11 membrane recruitment does not depend on the presence of effector proteins
5.4 Monoprenylated RAB proteins are mislocalized to the same membrane structures
5.4.1 Monoprenylated RAB proteins localize to the same membrane structures
5.4.2 Monoprenylated RAB proteins do not localize to Golgi or recycling endosomal structures
5.5 Discussion
6 RAB6-induced membrane tethering
6.1 Specificities of RAB6-induced membrane tethering
6.1.1 Vesicle tethering is a RAB6-specific effect
6.1.2 Vesicle tethering is nucleotide and concentration dependent
6.1.3 RAB6-induced vesicle tethering is mediated by a RAB-RAB dimerization in trans
6.1.4 The RAB-RAB interaction is dynamic
6.2 Involvement of the Switch regions
6.2.1 RAB6A mutant induces vesicle tethering
6.2.2 Unprenylated RAB6A does not interact with membrane-bound RAB6A
6.2.3 Bivalent αRAB:GTP antibodies promote vesicle tethering
6.2.4 Effect of monovalent RAB6 effector proteins
6.3 Discussion
Concluding remarks
References
