The GL protein has also been attached directly to the amino-terminal end of MVP. Even though this GL-MVP fusion protein is ~30 kD larger than MVP, it still assembles into vault particles with surprising structural integrity. The GL-MVP vaults are highly fluorescent and thus can be visualized by fluorescence microscopy. Purified GL-MVP vaults have been added to cultured cells and their uptake monitored by examining fluorescence in the cells by confocal microscopy. An adherent cell line (HeLa cells) was used to aid in the analysis. Following a one hour incubation at 37oC the media containing the fluorescent vaults was removed followed by rinsing the cells and monitoring for fluorescence. Using this approach, it was shown that HeLa cells can readily take up vaults from the culture media. An example of HeLa cells that engulfed GL-MVP vaults is shown in the Figure. Most likely the vaults seen inside these cells entered via the endocytic pathway.
Figure (below). HeLa cells grown on cover slips were incubated with GL-MVP vaults (10 micrograms) for 1h at 37oC, washed with PBS, incubated with CT-B 594 (Cholera toxin-B Alexa 594 from Molecular Probes to stain cell membranes) for 10 min at 12oC, washed, mounted on slides and visualized by confocal microscopy (Green HeNe 594nm, Argon 488nm, 0.2mm slices). (a) DiC showing the HeLa cell; (b) Mostly membrane staining (red) seen with CT-B; (c) Green fluorescent vaults seen on the membrane and inside the cells; (d) a merge of b and c showing vaults inside the cells (arrows). Fluorescent microscopy was performed at the UCLA/CNSI Advanced Light Microscopy/Spectroscopy Shared Facility. Figure is from Kickhoefer et al. (2005).
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