The major aim of this Core is to provide a facility for novel optical instrumentation to the investigators of this Program Project. The primary function of the Core will be to assist in experiments that require expertise and specialized equipment not available in the individual sub-project laboratories. The members of the Core will provide guidance and expert help to the different members of the Program Project in using Optical Trapping, Total Internal Reflection Microscopy (TIRF) and Total Internal Reflection Polarized Fluorescence Microscopy (polTIRF). This state of the art shared facility provides methods for measuring the mechanical properties of individual motor proteins and the spatial orientation of fluorescent probes linked to subdomains of individual engineered proteins. The core will further develop these techniques by testing several methods for obtaining three dimensional data from TIRF images, by combining the trap with polTIRPF to measure the orientation of single motor molecules as they proceed through their enzymatic cycles under varying load, improve the long term spatial resolution and stiffness of the trap. In vitro analysis of single wild type and engineered motor molecules will unambiguously measure fundamental motor properties such as stiffness, orientation changes, unitary force, step length and kinetics as a function of strain and relate these properties to structural features of the protein.
Advanced light microscopy and optical trapping provide unique information at the single molecule level. These novel assays will be used here to understand the underlying mechanisms of cell motility and membrane trafficking, both essential processes for normal function in eukaryotic cells. Defects in either of these processes lead to a wide range of diseases.
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