TU Darmstadt / ULB / TUbiblio

Probing intranuclear environments at the single-molecule level.

Grünwald, David and Martin, Robert M. and Buschmann, Volker and Bazett-Jones, David P. and Leonhardt, Heinrich and Kubitscheck, Ulrich and Cardoso, M Cristina (2008):
Probing intranuclear environments at the single-molecule level.
In: Biophysical journal, pp. 2847-58, 94, (7), ISSN 1542-0086,
[Online-Edition: http://www.cardoso-lab.org/publications/Grunwald%202008.pdf],
[Article]

Abstract

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites.

Item Type: Article
Erschienen: 2008
Creators: Grünwald, David and Martin, Robert M. and Buschmann, Volker and Bazett-Jones, David P. and Leonhardt, Heinrich and Kubitscheck, Ulrich and Cardoso, M Cristina
Title: Probing intranuclear environments at the single-molecule level.
Language: English
Abstract:

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites.

Journal or Publication Title: Biophysical journal
Volume: 94
Number: 7
Divisions: 10 Department of Biology > Cell Biology and Epigenetics
?? fb10_zoologie ??
10 Department of Biology
Date Deposited: 06 Mar 2010 15:56
Official URL: http://www.cardoso-lab.org/publications/Grunwald%202008.pdf
Related URLs:
Export:
Suche nach Titel in: TUfind oder in Google

Optionen (nur für Redakteure)

View Item View Item