AFM visualization of clathrin triskelia under fluid and in air.

Atomic force microscopy (AFM) is used to characterize the structure and interactions of clathrin triskelia. Time sequence images of individual, wet triskelia resting on mica surfaces clearly demonstrate conformational fluctuations of the triskelia. AFM of dried samples yields images having nanometric resolution comparable to that obtainable by electron microscopy of shadowed samples.

Clathrin triskelia show evidence of molecular flexibility.

The clathrin triskelion, which is a three-legged pinwheel-shaped heteropolymer, is a major component in the protein coats of certain post-Golgi and endocytic vesicles. At low pH, or at physiological pH in the presence of assembly proteins, triskelia will self-assemble to form a closed clathrin cage, or "basket". Recent static light scattering and dynamic light scattering studies of triskelia in solution showed that an individual triskelion has an intrinsic pucker similar to, but differing from, that inferred from a high resolution cryoEM structure of a triskelion in a clathrin basket.

Conformation of a clathrin triskelion in solution.

A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral cages principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends upon the pH, with cages forming more readily at low pH and less readily at high pH. We have developed procedures, on the basis of static and dynamic light scattering, to determine the radius of gyration, R(g), and hydrodynamic radius, R(H), of isolated triskelia, under conditions where cage assembly occurs.

Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy.

Using a new scheme based on atomic force microscopy (AFM), we investigate mechanical properties of clathrin-coated vesicles (CCVs). CCVs are multicomponent protein and lipid complexes of approximately 100 nm diameter that are implicated in many essential cell-trafficking processes. Our AFM imaging resolves clathrin lattice polygons and provides height deformation in quantitative response to AFM-substrate compression force.

Structural basis of interdomain communication in the Hsc70 chaperone.

Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone.

Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling.

Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly.

Structure-function analysis of the auxilin J-domain reveals an extended Hsc70 interaction interface.

J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date.