Single ovalbumin molecules exploring nucleoplasm and nucleoli of living cell nuclei.
The nucleus is the center of direction and coordination of the cell's metabolic and reproductive activities and contains numerous functionally specialized domains. These subnuclear structures are not delimited by membranes like cytoplasmic organelles and their function is only poorly understood. Here, we studied the most prominent nuclear domains, nucleoli and the remaining nucleoplasm. We used fluorescently labeled ovalbumin-ATTO647N, an inert protein, to examine their physical properties. This inert tracer was microinjected into the cytoplasm of HeLa cells, and after diffusion into the nucleus the tracer distribution and mobility in the two nuclear compartments was examined. Like many macromolecular probes ovalbumin was significantly less abundant in nucleoli compared to the nucleoplasm. High-speed fluorescence microscopy allowed visualizing and analyzing single tracer molecule trajectories within nucleoli and nucleoplasm. In accordance with previous studies we found that the viscosity of the nucleus is sevenfold higher than that of aqueous buffer. Notably, nucleoplasm and nucleoli did not significantly differ in viscosity, however, the fraction of slow or trapped molecules was higher in the nucleoplasm than in nucleoli (6% versus 0.2%). Surprisingly, even a completely inert molecule like ovalbumin showed at times short-lived binding events with a decay time of 8 ms in the nucleoplasm and even shorter-6.3 ms-within the nucleoli.
Speil
Jasmin
J
Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms University Bonn, Wegelerstrasse 12, 53115 Bonn, Germany. speil@pc.uni-bonn.de
Kubitscheck
Ulrich
U
D000818:Animals; D002466:Cell Nucleolus / Q000378:metabolism* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D010047:Ovalbumin / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D010047:Ovalbumin