Understanding complete transport cycles mediated by importin beta-type nuclear transport receptors in situ and in living cells
Final Report Abstract
The genome of eukaryotic cells is compartmentalized inside the nucleus. This organelle is delimited by the nuclear envelope (NE), which consists of two concentric membranes that are perforated by nuclear pore complexes (NPCs) — large protein assemblies that form aqueous channels and mediate all traffic between nucleus and cytoplasm. The goal of our studies here was to derive basic physico-chemical parameters of the constituent proteins of the NPC, the nucleoporins, and the nucleoplasm in intact cells to allow a quantitative understanding of nuclear transport cycles. Our studies yielded two major results. First, by determining the dissociation rate of 19 GFP tagged nucleoporins from the NPC, we could map the steady state dynamics of its structural organization. We found three kinetic classes of nucleoporins, immobile scaffold nucleoporins, moderately mobile adaptor nucleoporins and highly mobile dynamic nucleoporins. The residence times of nucleoporins at the NPC spanned four orders of magnitude, between 20 s and 70 h. However, even the most dynamic nucleoporin remained associated with the NPC at least one order of magnitude longer than a typical transport receptor. We can therefore exclude that nucleoporins participate in bulk translocation through the NPC, providing an important constraint to understand transport through the pore. Second, to address the transient and diffusion limited association of transporting molecules with the NPC, we established a method to dissect diffusion and chemical interactions in fluorescence perturbation experiments. Using fluorescence photoactivation and 3D kinetic modeling of a reaction-diffusion system we could show that nuclear viscosity is three times higher than water in live cells and that the mobility of many nuclear proteins is limited by both diffusive processes and binding interactions. The developed method now allows us to determine the rate constants of both processes in live cells, providing important parameters for a comprehensive model of nuclear transport.
Publications
- 2004. Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J Microsc. 216:131-7
Rabut, G., and J. Ellenberg
- 2004. Mapping the dynamic organization of the nuclear pore complex inside single living cells. Nature Cell Biology. 6:1114-21
Rabut, G., V. Doye, and J. Ellenberg
- 2004. The entire nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis. Mol Biol Cell. 15:3333-44
Loiodice, I., A. Alves, G. Rabut, M. Van Overbeek, J. Ellenberg, J.B. Sibarita, and V. Doye
