Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study.
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
T
Institute for Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms-University Bonn, D-53115 Bonn, Germany.
Grünwald
David
D
Grünwald
Antje
A
Peters
Reiner
R
Kubitscheck
Ulrich
U
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics; D022022:Nuclear Pore / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000235:genetics; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
C424166:KPNB1 protein, human; D008562:Membrane Glycoproteins; D019913:Nuclear Localization Signals; C094157:POM121 protein, human; D011993:Recombinant Fusion Proteins; C108121:TNPO1 protein, human; D028901:alpha Karyopherins; D028961:beta Karyopherins; C087978:karyopherin alpha 2; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine