The PI proposes to design, build, and use a novel "optical trapping interferometer" which integrates the following functions: 1) real-time, computer-enhanced light microscopic imaging; 2) multiple laser light traps; 3) a dual-beam interferometer for measuring nm-scale displacements of microscopic beads; and 4) a feedback system for detection of force fluctuations in the pN range. The main use of this instrument will be to detect and characterize pN forces and nm movements generated by single microtubule motor proteins, including kinesin, kinesin-related proteins, and cytoplasmic dynein. In cells, such proteins carry intracellular cargos and walk in a particular direction along microtubules; in so doing they accomplish various forms of cell motility, such as vesicle transport and mitosis. The proposed instrumentation is required for the PI's research on microtubule motors and vesicle transport. A long-term goal of this research is to determine the molecular mechanism of motility and force production by kinesin, kinesin-related proteins, and cytoplasmic dynein. Using the proposed instrumentation, the fundamental molecular mechanical events which occur during movement will be defined and related to intermediates in the ATP hydrolytic cycle. These molecular mechanics will be related to the primary structure of the motor domains by analysis of mutant proteins. The proposed instrumentation is based on a prototype optical trapping interferometer developed collaboratively between the PI's lab and the lab of Dr. Steve Block at the Rowland Institute for Science (which funded the instrument). Dr. Block has recently moved to Princeton with the prototype instrumentation. The new optical trapping interferometer will perform the same functions as the earlier version, including manipulation of beads carrying single motor proteins onto microtubules (Block et al, 1990) and detection of 8 nm steps corresponding to single kinetic cycles of the kinesin motor (Svoboda et al, 1993). In addition, the new instrument will have fundamentally new features, the most important of which are: 1) Accomodation of an improved assay system which avoids direct irradiation of the motor protein; 2) feedback from the interferometer output to the laser trap, enabling measurement of isometric force fluctuations during the mechanochemical cycle of single motors, 3) increased stiffness to diminish Brownian noise and thereby reveal more details of the stepping process; and 4) three independent laser light traps to allow a variety of manipulations. The new instrument will be constructed around a custom optical bench light microscope, developed by the PI and capable of high resolution differential interference contrast (DIC) or fluorescence imaging. Single microtubules (or possibly single actin or intermediate filaments, or single strands of DNA or RNA) will be imaged in real time by digital enhancement of the light microscope image. Two laser light traps will be used to capture, apply tension to, and manipulate single microtubules (or possibly other filaments) via latex bead "handles" attached to the filament surface. Trapped microtubules will be manipulated onto fixed bead surfaces carrying single motor molecules. A third laser beam will be used as the source of coherent, polarized illumination for an interferometer that will track the position of the trapped microtubule (with precision better than 1 nm at 100 Hz) as it is driven by the motor on the fixed bead surface. In other assays, the third laser beam will be used simultaneously as the source of the interferometer and as a light trap to manipulate beads carrying single biological motors onto the surface of a trapped filament held in solution. The latter use of the trap will benefit the investigation of processive DNA enzymes using in vitro motility assays. The proposed research bears on several fields: 1) The molecular mechanism of cell motility, including muscle contraction and the operation of processive DN A enzymes; 2) the mechanism of enzymatic catalysis; 3) nanotechnology and surface chemistry.
