Probing Intranuclear Environments at the Single Molecule Level
David Grunwald 1, Robert M. Martin 2, Volker Buschmann 2, David P. Bazett-Jones 3, Heinrich Leonhardt 4, Ulrich Kubitscheck 1 and M. Cristina Cardoso 5*
1 Rheinische Friedrich-Wilhelms-University
2 Max Delbr
ck Center for Molecular Medicine
3 The Hospital for Sick Children
4 Ludwig Maximilians University Munich
5 Max Delbrueck Center for Molecular Medicine
* To whom correspondence should be addressed. E-mail: cardoso{at}mdc-berlin.de.
Submitted on June 14, 2007
Revised on July 23, 2007
Accepted on 16 November 2007
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Abstract |
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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 in 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 did neither exclude single molecules nor impede their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin lasting though, only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude the streptavidin like 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 rather 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.
Key Words:
electron spectroscopic imaging, heterochromatin, live-cell microscopy, nuclear structure and function, nucleolus, single molecule tracking microscopy
