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dc.contributor.authorDange, Thomas
dc.contributor.authorGrunwald, David
dc.contributor.authorGrunwald, Antje
dc.contributor.authorPeters, Reiner
dc.contributor.authorKubitscheck, Ulrich
dc.date2022-08-11T08:08:00.000
dc.date.accessioned2022-08-23T15:38:54Z
dc.date.available2022-08-23T15:38:54Z
dc.date.issued2008-10-06
dc.date.submitted2014-09-04
dc.identifier.citation<p>Dange T, Grünwald D, Grünwald A, Peters R, Kubitscheck U. Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. J Cell Biol. 2008 Oct 6;183(1):77-86. doi: 10.1083/jcb.200806173. <a href="http://dx.doi.org/10.1083/jcb.200806173" target="_blank">Link to article on publisher's site</a></p>
dc.identifier.issn0021-9525 (Linking)
dc.identifier.doi10.1083/jcb.200806173
dc.identifier.pmid18824568
dc.identifier.urihttp://hdl.handle.net/20.500.14038/26037
dc.description<p>At the time of publication, David Grünwald was not yet affiliated with the University of Massachusetts Medical School.</p>
dc.description.abstractAll 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.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=18824568&dopt=Abstract">Link to Article in PubMed</a>
dc.rightsCopyright 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 <a href="http://www.jcb.org/misc/terms.shtml">http://www.jcb.org/misc/terms.shtml</a>). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">http://creativecommons.org/licenses/by-nc-sa/3.0/</a>).
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/3.0/
dc.subjectActive Transport, Cell Nucleus
dc.subjectBinding Sites
dc.subjectCell Nucleus
dc.subjectCytoskeleton
dc.subjectGreen Fluorescent Proteins
dc.subjectHeLa Cells
dc.subjectHumans
dc.subjectKinetics
dc.subjectMembrane Glycoproteins
dc.subjectMicroscopy, Fluorescence
dc.subjectMutation
dc.subjectNuclear Envelope
dc.subjectNuclear Localization Signals
dc.subjectNuclear Pore
dc.subjectProtein Binding
dc.subjectProtein Transport
dc.subjectRecombinant Fusion Proteins
dc.subjectSerum Albumin, Bovine
dc.subjectalpha Karyopherins
dc.subjectbeta Karyopherins
dc.subjectBiochemistry
dc.subjectBiochemistry, Biophysics, and Structural Biology
dc.subjectCell Biology
dc.subjectMolecular Biology
dc.titleAutonomy and robustness of translocation through the nuclear pore complex: a single-molecule study
dc.typeJournal Article
dc.source.journaltitleThe Journal of cell biology
dc.source.volume183
dc.source.issue1
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1172&amp;context=bmp_pp&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/bmp_pp/172
dc.identifier.contextkey6076995
refterms.dateFOA2022-08-23T15:38:54Z
html.description.abstract<p>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.</p>
dc.identifier.submissionpathbmp_pp/172
dc.contributor.departmentRNA Therapeutics Institute
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.source.pages77-86


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Copyright 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 <a href="http://www.jcb.org/misc/terms.shtml">http://www.jcb.org/misc/terms.shtml</a>). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">http://creativecommons.org/licenses/by-nc-sa/3.0/</a>).
Except where otherwise noted, this item's license is described as Copyright 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 <a href="http://www.jcb.org/misc/terms.shtml">http://www.jcb.org/misc/terms.shtml</a>). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">http://creativecommons.org/licenses/by-nc-sa/3.0/</a>).