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. An AFM instrument system has been obtained and is being adapted to allow measurements to be made 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 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) and surface modified protein interaction dynamics (NIDDK). Integration of AFM with near-field scanning optical microscopy (NIAID) is being pursued through a collaborative agreement with NIST to develop and adapt existing instrumentation for application to biological problems. Preliminary implementation of our novel methodology has been successful when applied to standard samples for calibration and validation. 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 which 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.
| 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 |
