Molecular motors - kinesin, dynein, and myosins - play a crucial role in the maintenances and development of the organization, motility, and cell signaling, of healthy cells. Central nervous-system disorders such as Alzheimer's, Huntington's and Parkinson's Disease, and muscular diseases such as heart disease, high blood pressure, and uterine problems, all arise from molecular motors gone awry. At its most basic level, a single, isolated """"""""head"""""""", which gives motors its ATP-dependent motility, has been fairly well studied. However, in general, motors have two heads, and there is a growing body of evidence that multiple motors can interact. Perhaps the most extreme example is our Science paper (Kural, 2005), where kinesin and dynein interact in vivo, to yield up to ~12 times their in vitro speed. The basic biology we want to address is how these motors are affected by interaction between heads within one molecular motor, via in vitro studies, and how multiple motors of different species interact, through in vivo studies. We wish to study: ? ? a) Smooth muscle myosin: To continue the study of the conformational changes within the actomyosin complex using lanthanide-based resonance energy transfer (LRET), single molecule FRET. We will utilize an improved """"""""cysteine-light"""""""" smooth muscle myosin II. The goal is to understand the effect of actin and phosphorylation on the structure of S1, and particularly, heavymeromyosin (HMM). Does actin cause a cleft composure in myosin? Is the model of Wendt et al., involving the structure of HMM with actin, correct? Does actin undergo conformational changes upon myosin binding? (With L. Sweeney, UPenn, and I. Rayment, U. Wisconsin). ? ? b) Kinesin, Dynein, and Myosin V, Dynamics: To study the in vivo dynamics of kinesin, dynein, and myosin V in peroxisomes, melanosomes, PC12 cells, and other cell lines, using single molecule FIONA (Fluorescence Imaging with One Nanometer Accuracy), and optical trapping. This follows up on our results using FIONA in vivo, where we have achieved 1 msec and 1.5 nm temporal and spatial resolution. We found that kinesin and dynein are not engaged in a """"""""tug-of-war"""""""", despite moving peroxisomes in opposite directions. The idea is to now optically trap a peroxisome inside a cell (which we have shown is possible), and determine, among other things: 1) the stall force of the peroxisomes in both the dynein and kinesin direction, therefore definitively showing how many of them are pulling at a time; 2) go from in vivo to in vitro studies to understand where the transformation occurs that enables the motors to pull so fast; 3) to test other systems, e.g. PC12 neurites, to see if the kinetics are similar; 4) to measure the movement of melanosomes (which are non-fluorescent), using bright-Field Imaging with One Nanometer Accuracy (bFIONA), a knock-off of FIONA, and test the kinetics under various conditions: e.g., no actin, no microtubules, no intermediate filaments..., to show how melanosomes make the transition between one motor system and another. (With V. Gelfand, Northwestern). ? ? c) Myosin VI: To study myosin VI in vivo, and to understand the transition between an anchor (monomer) and processive motor (dimer) as a function of: position within the cell; as a function of accessory proteins (Dab2, GIPC...); as a function of insertion length of the cargo-binding domain of myosin VI (with L. Sweeney, UPenn.). ? ? d) Technological developments: Optics/microscopy needs to be achieved to fulfill the above goals. ? ? ?
