During spindle assembly, microtubules are highly enriched near chromatin by a process which, in many systems, is driven by the GTPase Ran. The Ran pathway has been proposed to establish a reaction-diffusion network that generates gradients in the behaviors of soluble proteins around chromatin, but the manner in which this happens is poorly understood. To better characterize the behavior of the Ran pathway, we developed a novel form of fluorescence fluctuation spectroscopy capable of quantitatively measuring the concentration, diffusion, and interactions of soluble proteins simultaneously at hundreds of locations throughout cells. We use this technique to study the behaviors of soluble Ran, importin-alpha, importin-beta, RanBP1, RanGAP, and a variety of downstream cargo proteins throughout mitotic human tissue culture cells, and we investigate how the spatial organization of this network changes in response to perturbations. Our results suggest that a self-focusing of the Ran pathway is produced by an interplay between soluble gradients of upstream signaling molecules and the mechanics of the microtubule network they generate. This feedback has interesting implications for models of spindle assembly and the maintenance of spindle size.
Lunch @ 11:45, Seminar 12-1.