Visualization and Tracking of Single Protein Molecules in the Cell Nucleus
Identifieur interne : 000982 ( Main/Exploration ); précédent : 000981; suivant : 000983Visualization and Tracking of Single Protein Molecules in the Cell Nucleus
Auteurs : Thorsten Kues [Allemagne] ; Reiner Peters [Allemagne] ; Ulrich Kubitscheck [Allemagne]Source :
- Biophysical Journal [ 0006-3495 ] ; 1999.
Abstract
A recently developed laser fluorescence videomicroscopy method was used to determine for the first time the intranuclear trajectories of single protein molecules. Using the recombinant Escherichia coli β-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in the nuclei of permeabilized 3T3 cells at a spatial accuracy of ∼30nm and a time resolution of 18ms. Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller immobile fraction. The present study supported this observation but revealed a much larger variety of mobility classes. Thus, a fraction of P4K molecules appeared to be truly immobile while another fraction was mobile but confined to very small areas. In addition, a large fraction of the P4K molecules appeared to be mobile and to move over extended distances by diffusion. However, a quantitative analysis showed that at least two subpopulations were present differing widely in diffusion coefficients. Importantly, both the diffusion coefficients and the fractions of these subpopulations were time-dependent. Our results suggest that proteins can move inside the nucleus over extended distances by diffusion. However, intranuclear protein diffusion is severely restricted, most likely by multiple association-dissociation events and/or impermeable obstacles.
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DOI: 10.1016/S0006-3495(01)76261-3
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Using the recombinant Escherichia coli β-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in the nuclei of permeabilized 3T3 cells at a spatial accuracy of ∼30nm and a time resolution of 18ms. Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller immobile fraction. The present study supported this observation but revealed a much larger variety of mobility classes. Thus, a fraction of P4K molecules appeared to be truly immobile while another fraction was mobile but confined to very small areas. In addition, a large fraction of the P4K molecules appeared to be mobile and to move over extended distances by diffusion. However, a quantitative analysis showed that at least two subpopulations were present differing widely in diffusion coefficients. Importantly, both the diffusion coefficients and the fractions of these subpopulations were time-dependent. Our results suggest that proteins can move inside the nucleus over extended distances by diffusion. However, intranuclear protein diffusion is severely restricted, most likely by multiple association-dissociation events and/or impermeable obstacles.</div></front></TEI><affiliations><list><country><li>Allemagne</li></country><region><li>District de Münster</li><li>Rhénanie-du-Nord-Westphalie</li></region><settlement><li>Münster</li></settlement></list><tree><country name="Allemagne"><region name="Rhénanie-du-Nord-Westphalie"><name sortKey="Kues, Thorsten" sort="Kues, Thorsten" uniqKey="Kues T" first="Thorsten" last="Kues">Thorsten Kues</name></region><name sortKey="Kubitscheck, Ulrich" sort="Kubitscheck, Ulrich" uniqKey="Kubitscheck U" first="Ulrich" last="Kubitscheck">Ulrich Kubitscheck</name><name sortKey="Kubitscheck, Ulrich" sort="Kubitscheck, Ulrich" uniqKey="Kubitscheck U" first="Ulrich" last="Kubitscheck">Ulrich Kubitscheck</name><name sortKey="Peters, Reiner" sort="Peters, Reiner" uniqKey="Peters R" first="Reiner" last="Peters">Reiner Peters</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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