Cargando…
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 kapα2, kapβ1, kapβ1ΔN44, and kapβ2, and the model transport substrate, BSA-NLS, with NPCs to determ...
Autores principales: | , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
The Rockefeller University Press
2008
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557044/ https://www.ncbi.nlm.nih.gov/pubmed/18824568 http://dx.doi.org/10.1083/jcb.200806173 |
_version_ | 1782159625424994304 |
---|---|
author | Dange, Thomas Grünwald, David Grünwald, Antje Peters, Reiner Kubitscheck, Ulrich |
author_facet | Dange, Thomas Grünwald, David Grünwald, Antje Peters, Reiner Kubitscheck, Ulrich |
author_sort | Dange, Thomas |
collection | PubMed |
description | 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 kapα2, kapβ1, kapβ1ΔN44, and kapβ2, 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. |
format | Text |
id | pubmed-2557044 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-25570442009-04-06 Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study Dange, Thomas Grünwald, David Grünwald, Antje Peters, Reiner Kubitscheck, Ulrich J Cell Biol Research Articles 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 kapα2, kapβ1, kapβ1ΔN44, and kapβ2, 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. The Rockefeller University Press 2008-10-06 /pmc/articles/PMC2557044/ /pubmed/18824568 http://dx.doi.org/10.1083/jcb.200806173 Text en © 2008 Dange et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). |
spellingShingle | Research Articles Dange, Thomas Grünwald, David Grünwald, Antje Peters, Reiner Kubitscheck, Ulrich Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study |
title | Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study |
title_full | Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study |
title_fullStr | Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study |
title_full_unstemmed | Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study |
title_short | Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study |
title_sort | autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557044/ https://www.ncbi.nlm.nih.gov/pubmed/18824568 http://dx.doi.org/10.1083/jcb.200806173 |
work_keys_str_mv | AT dangethomas autonomyandrobustnessoftranslocationthroughthenuclearporecomplexasinglemoleculestudy AT grunwalddavid autonomyandrobustnessoftranslocationthroughthenuclearporecomplexasinglemoleculestudy AT grunwaldantje autonomyandrobustnessoftranslocationthroughthenuclearporecomplexasinglemoleculestudy AT petersreiner autonomyandrobustnessoftranslocationthroughthenuclearporecomplexasinglemoleculestudy AT kubitscheckulrich autonomyandrobustnessoftranslocationthroughthenuclearporecomplexasinglemoleculestudy |