Two-state displacement by the kinesin-14 Ncd stalk.

The nonprocessive kinesin-14 Ncd motor binds to microtubules and hydrolyzes ATP, undergoing a single displacement before releasing the microtubule. A lever-like rotation of the coiled-coil stalk is thought to drive Ncd displacements or steps along microtubules. Crystal structures and cryoelectron microscopy reconstructions imply that stalk rotation is correlated with ADP release and microtubule binding by the motor. Here we report FRET assays showing that the end of the stalk is more than ~9nm from the microtubule when wild-type Ncd binds microtubules without added nucleotide, but the stalk is within ~6nm of the microtubule surface when the microtubule-bound motor binds an ATP analogue, matching the rotated state observed in crystal structures. We propose that the stalk rotation is initiated when the motor binds to microtubules and releases ADP, and is completed when ATP binds.

Mature Drosophila meiosis I spindles comprise microtubules of mixed polarity.

New information has been obtained recently regarding microtubule organization in Xenopus extract spindles. These spindles assemble in vitro by chromatin-mediated microtubule nucleation and consist of randomly interspersed long and short microtubules with minus ends distributed throughout the spindle. Fluorescence speckle microscopy has led to the proposal that the Xenopus steady-state spindles contain two overlapping arrays of parallel or antiparallel microtubules with differing poleward-flux velocities. Although some of these features have also been reported for C. elegans female meiotic spindles, it is not clear whether they are representative of microtubule organization and dynamics in oocyte meiotic spindles. Here we examine anastral meiosis I spindles of live Drosophila oocytes expressing the microtubule plus end-tracking protein, EB1, fused to GFP, and find fluorescent particles throughout the spindle and movement toward both the poles and the equator. EB1 particle velocities, corresponding to microtubule growth rates, are similar in both directions, but slower than growth from the poles in mitotic spindles of early embryos. Meiosis I spindles yielded data from photobleaching analysis showing similar microtubule growth rates and dynamics at the poles and the equator, consistent with spindle microtubules of mixed polarity, differing from early-embryo mitotic spindles.

Ncd motor binding and transport in the spindle.

The Ncd kinesin-14 motor is required for meiotic spindle assembly in Drosophila oocytes and produces force in mitotic spindles that opposes other motors. Despite extensive studies, the way the motor binds to the spindle to perform its functions is not well understood. By analyzing Ncd deleted for the conserved head or the positively charged tail, we found that the tail is essential for binding to spindles and centrosomes, but both the head and tail are needed for normal spindle assembly and function. Fluorescence photobleaching assays to analyze binding interactions with the spindle yielded data for headless and full-length Ncd that did not fit well to previous recovery models. We report a new model that accounts for Ncd transport towards the equator revealed by fluorescence flow analysis of early mitotic spindles and gives rate constants that confirm the dominant role the Ncd tail plays in binding to the spindle. By contrast, the head binds weakly to spindles based on analysis of the tailless fluorescence recovery data. Minus-end Ncd thus binds tightly to spindles and is transported in early metaphase towards microtubule plus-ends, the opposite direction to that in which the motor moves, to produce force in the spindle later in mitosis.

Real-time detection of alpha1A-AR movement stimulated by phenylephrine in single living cells.

AIM: To investigate the movement of alpha(1A)-adrenergic receptors(alpha(1A)-AR) stimulated by agonist, phenylephrine (PE), and the dynamics of receptor movement in real time in single living cells with millisecond resolution. METHODS: We labeled alpha(1A)-AR using the monoclonal, anti-FLAG (a kind of tag) antibody and Cy3-conjugated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. RESULTS: The specific detection of alpha(1A)-AR on the surface of living HEK293A-alpha(1A) cells was achieved. alpha(1A)-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20 min, apparent colocalization was found between alpha(1A)-AR and F-actins. After 40 min stimulation of PE, trajectories of approximate linear motion in HEK293A-alpha(1A) cells were recorded, and their velocity was calculated. CONCLUSION: The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect alpha(1A)-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.

The transport of alpha(1A)-adrenergic receptor with 33-nm step size in live cells.

We used the technique of single particle tracking (SPT) with high tempo-spatial resolution to efficiently explore the route and mechanism for the transport of alpha(1A)-adrenergic receptor (alpha(1A)-AR) in real time in living cells. We found that the initial transport of alpha(1A)-AR in cells depended on actin filaments with the velocity of 0.2 microm/s and exhibited discrete 33-nm steps. It was noted that the step size, the rate constant, and the velocities were in accordance with the character of single myosin in vitro, implying that while transporting each endosome myosins did not work in the "tug-of-war" mode and that they did not adopt the strategy to boost up transporting speed by working coordinately. These results provided insight into the mechanism of GPCR transport in vivo.