The atomic force microscope (AFM) is becoming increasingly useful for studying ultra-structure and functional properties of biological molecules and tissue. The DBEPS Instrumentation Research and Development Resource is strengthening the AFM capabilities at NIH to support the diverse needs of IC scientific projects, in particular, adapting the AFM platform to allow measurement of biological samples. A perfusion chamber has been developed to enable a single cell to be retained for examination and permitting manipulation of the extra-cellular environment. A temperature controlled environmental chamber has also been developed to allow samples that exhibit temperature dependent properties to be examined. The chamber is capable of 0.1 degree stability within a temperature range from 10 to 40 degrees Celsius. Using DBEPS expertise in optics, electronics, mechanical design, and other areas, associated instrumentation and quantitative analysis methods are being pursued to advance AFM technology and apply it to solve novel biomedical problems. Collaborative intramural biological projects include the investigation of the viscoelastic energetics of the protein clathrin and its assemblies that are important to subcellular protein trafficking (NICHD), organization of rhodopsin on the surface of native disk membranes (NIAAA), surface modified protein interaction dynamics (NIDDK), DNA-protein interactions in gene regulation pathways (NCI), cytochrome c adsorption to anionic supporterd bilayers (NIDDK), and measured mechanical properties of the tectorial membrane in the mammalian cochlea (NIDCD). New AFM components are being acquired and integrated to expand our facility capabilities towards meeting the varying needs of intramural researchers from across NIH. This technology is being applied to studying the mechanical properties associated with vesicle formation and vesicular trafficking. In this particular project we examine the assembly of clathrin triskelions into polyhedral coats of about 100-nanometer diameter that is believed to play a central role in receptor-mediated endocytosis and intracellular trafficking from the trans-Golgi network. Knowledge of the mechanical properties of the clathrin coat is needed in order to fully understand the function of the coat in the dynamical control of vesicle formation. The objective here is to measure the mechanical properties of clathrin-coated and uncoated vesicles in biological fluid environments. We have developed a scheme by which the nanomechanics of both vesicle types can be quantitatively explored by AFM, employing a deformation force in the 50-100 pN range. The measurements are being refined via better instrument parameter calibrations and vesicle sample preparations. The environmental chambers have been applied to studying the organization of rhodopsin in the native state to gain an understanding of protein-protein interactions in the signal transduction pathway. AFM images have been captured at a range of temperatures to permit a modeling of translational diffusion of rhodopsin and its affect on AFM imaging. This study shows that atomic force microscopy can resolve both static rhodopsin organization to sub-nanometer resolution and receptor dynamics in sub-millisecond time periods. Another example of ancillary technology development for enhancing the AFM capabilities is the use of elastomeric microchannels to temporarily create a microdevice on an AFM substrate. Because flow in such microchannels is laminar, all mixing is diffusive, which allows us to create a well-controlled buffer gradient, either in ionic strength or pH, over a distance of a few hundred microns. This permits rapid sampling of buffer conditions for sample deposition on AFM substrates and offers the potential of reducing substantially the time required to optimize adsorption conditions for AFM imaging.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Intramural Research (Z01)
Project #
1Z01OD011043-06
Application #
7146062
Study Section
(BEPS)
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2005
Total Cost
Indirect Cost
Name
Office of the Director, NIH
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Jin, Albert J; Prasad, Kondury; Smith, Paul D et al. (2006) Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy. Biophys J 90:3333-44
Wang, K; Forbes, J G; Jin, A J (2001) Single molecule measurements of titin elasticity. Prog Biophys Mol Biol 77:1-44