Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic.

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups.

Reconstitution of Clathrin Coat Disassembly for Fluorescence Microscopy and Single-Molecule Analysis.

The disassembly of the clathrin lattice surrounding coated vesicles is the obligatory last step in their life cycle. It is mediated by the coordinated recruitment of auxilin and Hsc70, an ATP-driven molecular clamp. Here, we describe the preparation of reagents and the single-particle fluorescence microscopy imaging assay in which we visualize directly the Hsc70-driven uncoating of synthetic clathrin coats or clathrin-coated vesicles.

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic.

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes.

Intradimer/Intermolecular interactions suggest autoinhibition mechanism in endophilin A1.

Endophilin A1 is a homodimeric membrane-binding endocytic accessory protein with a high dimerization affinity. Its function has been hypothesized to involve autoinhibition. However, the autoinhibition mechanism, as well as the physicochemical basis for the high dimerization affinity of endophilin in solution, have remained unclear.

Exo70 generates membrane curvature for morphogenesis and cell migration.

Dynamic shape changes of the plasma membrane are fundamental to many processes, ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here, we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations toward the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism.

Kinetics of endophilin N-BAR domain dimerization and membrane interactions.

The recruitment to plasma membrane invaginations of the protein endophilin is a temporally regulated step in clathrin-mediated endocytosis. Endophilin is believed to sense or stabilize membrane curvature, which in turn likely depends on the dimeric structure of the protein. The dynamic nature of the membrane association and dimerization of endophilin is thus functionally important and is illuminated herein.

Curvature sorting of peripheral proteins on solid-supported wavy membranes.

Cellular membrane deformation and the associated redistribution of membrane-bound proteins are important aspects of membrane function. Current model membrane approaches for studying curvature sensing are limited to positive curvatures and often require complex and delicate experimental setups. To overcome these challenges, we fabricated a wavy substrate by imposing a range of curvatures onto an adhering lipid bilayer membrane.

Thermodynamics and mechanics of membrane curvature generation and sensing by proteins and lipids.

Research investigating lipid membrane curvature generation and sensing is a rapidly developing frontier in membrane physical chemistry and biophysics. The fast recent progress is based on the discovery of a plethora of proteins involved in coupling membrane shape to cellular membrane function, the design of new quantitative experimental techniques to study aspects of membrane curvature, and the development of analytical theories and simulation techniques that allow a mechanistic interpretation of quantitative measurements. The present review first provides an overview of important classes of membrane proteins for which function is coupled to membrane curvature.

Quantifying Membrane Curvature Generation of Amphiphysin N-BAR Domains.

Biological membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic helix-containing BIN/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization.

Curvature sensing by the epsin N-terminal homology domain measured on cylindrical lipid membrane tethers.

The protein epsin is believed to play important roles in clathrin-mediated endocytosis, including generation of the high membrane curvature necessary for vesicle formation. Here we assess the basis for this hypothesis by systematically quantifying the curvature dependence of the area density of epsin N-terminal homology (ENTH) domain on cylindrical membranes of controlled curvature. In cylindrical tethers pulled from micropipet-aspirated giant unilamellar vesicles, repartitioning of membrane-bound ENTH from vesicles onto highly curved membranes was observed by fluorescence microscopy.

Bending stiffness depends on curvature of ternary lipid mixture tubular membranes.

Lipid and protein sorting and trafficking in intracellular pathways maintain cellular function and contribute to organelle homeostasis. Biophysical aspects of membrane shape coupled to sorting have recently received increasing attention. Here we determine membrane tube bending stiffness through measurements of tube radii, and demonstrate that the stiffness of ternary lipid mixtures depends on membrane curvature for a large range of lipid compositions.

Intramolecular cross-linking evaluated as a structural probe of the protein folding transition state.

We examine the utility of intramolecular covalent cross-linking to identify the structure present in the folding transition state. In mammalian ubiquitin, cysteine residues located across two beta-strands are cross-linked with dichloroacetone. The kinetic effects of these covalent cross-links in ubiquitin, and engineered disulfide bonds in src SH3 (Grantcharova, V.