We used fluorescence imaging with one nanometer accuracy (FIONA) to analyze organelle movement by conventional kinesin and cytoplasmic dynein in a cell. We located a green fluorescence protein (GFP)-tagged peroxisome in cultured Drosophila S2 cells to within 1.5 nanometers in 1.1 milliseconds, a 400-fold improvement in temporal resolution, sufficient to determine the average step size to be approximately 8 nanometers for both dynein and kinesin. Furthermore, we found that dynein and kinesin do not work against each other in vivo during peroxisome transport. Rather, multiple kinesins or multiple dyneins work together, producing up to 10 times the in vitro speed.

The centroid of a fluorophore can be determined within Ϸ1.5-nm accuracy from its focused image through fluorescence imaging with one-nanometer accuracy (FIONA). If, instead, the sample is moved away from the focus, the point-spread-function depends on both the position and 3D orientation of the fluorophore, which can be calculated by defocused orientation and position imaging (DOPI). DOPI does not always yield position accurately, but it is possible to switch back and forth between focused and defocused imaging, thereby getting the centroid and the orientation with precision. We have measured the 3D orientation and stepping behavior of single bifunctional rhodamine probes attached to one of the calmodulins of the light-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD rotates and then dwells in the leading and trailing position, respectively. The probe angle relative to the barbed end of the actin (␤) averaged 128°while the LCD was in the leading state and 57°in the trailing state. The angular difference of 71°r epresents rotation of LCD around the bound motor domain and is consistent with a 37-nm forward step size of myosin V. When ␤ changes, the probe rotates ؎27°azimuthally around actin and then rotates back again on the next step. Our results remove degeneracy in angles and the appearance of nontilting lever arms that were reported.3D orientation ͉ lever arm ͉ single molecule ͉ fluorescence imaging with one-nanometer accuracy C omplementary conformational changes can be measured on single motor proteins by use of fluorescence imaging with one-nanometer accuracy (FIONA) (1-3) and by single-molecule fluorescence polarization microscopy (SMFP) (4). FIONA is a method in which the emission distribution of a single fluorophore is detected by using a charge-coupled device and fitted to a 2D Gaussian function to determine the position of the probe. The positional accuracy of the measurement, typically Ϸ1.5 nm, is generally limited by the number of collected photons (1-3). In contrast, SMFP is sensitive to the 3D orientation of a single dye's transition dipole moments (4). In SMFP, the dye is excited by multiple polarized beams, incident from different directions. The resulting emission is split with respect to its polarization and detected with avalanche photodiodes (APDs). However, by slightly defocusing the microscope objective and by using appropriate fitting routines, the defocused image of the probe can be used to determine both its position and its orientation (5-7). We call this technique defocused orientation and position imaging (DOPI). When the sample is deliberately moved Ϸ500 nm away from the best focus position, combinations of lobes and fringes appear on the charge-coupled device. These images are compared with calculated model images to obtain the best estimates of both 3D orientation and position of the probe. Because the image is spread out over a greater number of pixels in DOPI versus FIONA, DOPI inherently has poorer signal-tonoise ratio...

Summary The molecular clock relies on a delayed negative feedback loop of transcriptional regulation to generate oscillating gene expression. Although the principal components of the clock are present in all circadian neurons, different neuronal clusters have varying effects on rhythmic behavior, suggesting that the clocks they house are differently regulated. Combining biochemical and genetic techniques in Drosophila, we identify a phosphorylation program native to the master pacemaker neurons that regulates the timing of nuclear accumulation of the Period/Timeless repressor complex. GSK-3/SGG binds and phosphorylates Period-bound Timeless, triggering a CK2-mediated phosphorylation cascade. Mutations that block the hierarchical phosphorylation of Timeless in vitro, also delay nuclear accumulation in both tissue culture and in vivo, and predictably change rhythmic behavior. This two-kinase phosphorylation cascade is anatomically restricted to the eight master pacemaker neurons, distinguishing the regulatory mechanism of the molecular clock within these neurons from the other clocks that cooperate to govern behavioral rhythmicity.

We studied the structural dynamics of chicken myosin V by combining the localization power of fluorescent imaging with one nanometer accuracy (FIONA) with the ability to detect angular changes of a fluorescent probe. The myosin V was labeled with bifunctional rhodamine on one of its calmodulin light chains. For every 74 nm translocation, the probe exhibited two reorientational motions, associated with alternating smaller and larger translational steps. Molecules previously identified as stepping alternatively 74-0 nm were found to actually step 64-10 nm. Additional tilting often occurred without full steps, possibly indicating flexibility of the attached myosin heads or probing of their vicinity. Processive myosin V molecules sometimes shifted from the top to the side of actin, possibly to avoid an obstacle. The data indicate marked adaptability of this molecular motor to a nonuniform local environment and provide strong support for a straight-neck model of myosin V in which the lever arm of the leading head is tilted backwards at the prepowerstoke angle.