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Fig. 5. Nucleoplasmic DQ-OVA foci are protein degradation centres. HEp-2 cells were microinjected with 0.5 mg/ml DQ-OVA (cell b) or 0.5 mg/ml predigested DQ-OVA (predig DQ-OVA, DQ-OVA peptides, cell a), and observed by confocal microscopy over time. (A) Time lapse micrographs show strong fluorescence, and diffuse distribution of microinjected DQ-OVA peptides throughout the nucleoplasm and cytoplasm that decreases rapidly, and disappears within 30 minutes (cell a). Microinjected DQ-OVA shows weaker fluorescence in the cell nucleus that concentrates in distinct foci, and persists over 60 minutes (cell b). The insets show blow ups of a representative DQ-OVA degradation centre (arrows) that forms within 10 minutes, persists for about 20 minutes, and has disappeared after 60 minutes (insets). Note, for time-lapse observation of living cells fluorescent signals were acquired with low laser intensity (5%) and high confocal aperture to minimize cellular stress and photobleaching effects, and to enable simultaneous observation of fluorescent signals in different focal planes. (B) After the time-lapse observation cell b was scanned with routine confocal settings (20% laser intensity, low confocal aperture). (C) Confocal microscopy (settings as in B) of a HEp-2 cell microinjected with predigested DQ-OVA shows that DQ-OVA peptides distribute diffusely throughout the nucleoplasm. Bars, 10 µm.
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