Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Medical Subject Headings
Active Transport, Cell Nucleus; Binding Sites; Cell Nucleus; Cytoskeleton; Green Fluorescent Proteins; HeLa Cells; Humans; Kinetics; Membrane Glycoproteins; Microscopy, Fluorescence; Mutation; Nuclear Envelope; Nuclear Localization Signals; Nuclear Pore; Protein Binding; Protein Transport; Recombinant Fusion Proteins; Serum Albumin, Bovine; alpha Karyopherins; beta Karyopherins
All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapalpha2, kapbeta1, kapbeta1DeltaN44, and kapbeta2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy with a time resolution of 5 ms, achieving a colocalization precision of 30 nm. These measurements allowed defining the interaction sites with the NPCs with an unprecedented precision, and the comparison of the interaction kinetics with previous in vitro measurements revealed new insights into the translocation mechanism.
Dange, Thomas; Grunwald, David; Grunwald, Antje; Peters, Reiner; and Kubitscheck, Ulrich, "Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study" (2008). Biochemistry and Molecular Pharmacology Publications and Presentations. 172.