New Super-Accurate and Super-Resolved Fluorescence Microscopy Techniques and Probes for Studying Single Molecular Motors - The long-range goal of my laboratory is to measure the conformations of biomolecules at the single molecule level with nanometer and (sub)-millisecond resolution, ultimately in a cell. Here our focus is mostly on conforma- tional changes within the cytoplasmic molecular motors—kinesin and dynein and eventually myosin— and on a new “limping” mechanism that may lead to short (in distance and time) substeps. We also investigate motors’ response when multiple ones operate on a cargo simultaneously and how they respond to force, which may underpin many clinical abnormalities. Both in vitro and in vivo (cellular) measurements are proposed. On single kinesins, for example, in vitro conformational changes involving sub-steps have recently been revealed and extending this to other motors and as a function of force is a major goal. This work requires MIN- FLUX (Project 1), a fluorescence technique that can achieve extraordinary resolution of 1–2 nm at a rate of 0.1– 1 msec, while extending the photobleaching time to many minutes. Doing this under force with nanometer-size fluorescent probes means that almost any site can be monitored—but without the limitation of optical traps which require large beads to monitor only the center-of-mass. MINFLUX will also be used in vivo, looking at fluorescent peroxisomes moving radially away (via kinesin) and towards (via dynein) the nucleus of a cell. Combined with recent drugs, this approach will enable us to determine whether the speed of the cargo is a function of the type and number of motors, as we (controversially) proposed. The Univ. of Illinois has recently purchased a MINFLUX microscope that we will use in this work. It will also be extended to have an applied force. MINFLUX, however, can look at only one fluorescent molecule at a time, making acquiring statistics slow and the simultaneous conformations of many motors difficult. In contrast, Fluorescence Imaging with One Na- nometer Accuracy (FIONA), which we invented in 2003, can look at a whole ensemble of molecules with na- nometer- and msec resolution. But to see biomolecules with such fast resolution, an unusually bright fluorophore that doesn’t bleach is required. In Project 2, quantum dots (QDs) and fluorescence nanodiamonds (FND)— particularly within 50 nm of a photonic crystal (PC)—give (an amazing) 900––3000-fold increase in fluorescence with little blinking. For the first time, PCs will be applied in vitro to molecular motors (by FIONA and possibly by MINFLUX, all with an applied force) and on living cells (to EGFR and AMPAR membrane proteins). In Project 3, we have a new technique for intracellular, live cell labeling that will enable (organic) fluorophores to penetrate the cell membrane, sometimes in the presence of native oxygen. We can use either new fluoro- phores (from Janelia Farms) combined with a new transfected protein (from E. coli dihydrofolate reductase, eDHFR), or we can use Streptolysin O (SLO) that temporarily permeabilizes the cell membrane and allows essentially any fluorophore to enter the cell. We show that three different fluorophores may be used simultane- ously, extending our previously published technique.
