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First published online 25 October 2005
doi: 10.1242/jcs.02642

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Proteasomes degrade proteins in focal subdomains of the human cell nucleus

¹ Institut für umweltmedizinische Forschung at Heinrich-Heine-University, Auf'm Hennekamp 50, 40225 Düsseldorf, Germany
² Institute for Biochemistry and Molecular Biology I, Heinrich-Heine-University, 40225 Düsseldorf, Germany

View larger version (42K): [in a new window]   Fig. 1. Subcellular localization of proteasomes. (A) Differential interference contrast (DIC) of a representative HEp-2 cell during interphase. (B) Confocal immunofluorescence micrograph of the cell seen in (A) with mouse monoclonal antibody PW8195 against 20S proteasomes shows cytoplasmic localization (cy), and a speckled staining pattern in the nucleoplasm (nu). Large unlabelled areas correspond to nucleoli (no) and the nuclear envelope (ne). (C) Immunoblot of biochemically fractionated HEp-2 cells with antibody PW8155 confirms that proteasomes occur in the cytoplasm (cy) and in the nucleoplasm (nu), but neither in the nucleolar (no), nor the nuclear envelope (ne) fraction. 20, 30, 40, 50, 60, 70 molecular mass in kDa. NB: the same subcellular fractions were used in Fig. 1C, Fig. 2A-D. (D) Subcellular distribution of proteasomes was analysed by confocal immunofluorescence with antibody PW8155 in a variety of mammalian cell types, such as human epidermoid cells (KB), primary fibroblasts (F38), murine fibroblasts (L929), human keratinocytes (HaCaT) and in HEp-2 cells treated with proteasome-inhibitors (E). LC, lactacystin. Scale bars, 5 µm.

View larger version (37K): [in a new window]   Fig. 2. Proteasomal activity in subcellular fractions. HEp-2 cells were grown to subconfluence, followed by biochemical purification of cytoplasmic (A), nucleoplasmic (B), nucleolar (C) and nuclear envelope (D) cell fractions as detailed in Materials and Methods. The purity of cell fractions was monitored by immunoblotting. Proteins from preparations of cellular fractions were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose and immunolabelled with signature proteins of subcellular compartments, such as ß-tubulin (cytoplasm), SC35 (splicing speckles, nucleoplasm), fibrillarin (nucleolus) and lamin A/C (nuclear envelope). Proteasomal activity of subcellular fractions was determined by their incubation with fluorogenic precursor Suc-LLVY-AMC at 37°C and fluorometric detection of cleaved substrates. The resulting product curves (filled circles) were observed over time. Fluorescence intensity values represent mean values of three independent experiments with standard errors between 0.00 and 1.25 (fluorescence intensity, arbitrary units). To control substrate specificity proteasome-inhibitor lactacystin (10 µM) was added after 2 hours (open arrowheads) to double determination assays (open circles). Biochemically purified 20S proteasomes were included as positive controls (grey curves). 20, 30, 40, 50, 60, 70 molecular mass in kDa.

View larger version (43K): [in a new window]   Fig. 3. Proteasomal activity in the nucleus of living cells. (A) Fluorogenic precursor substrate Suc-LLVY-AMC was microinjected into the nucleus of a HEp-2 cell and proteasomal cleavage measured by detection of fluorescence intensity increase over time. Images were acquired every 5 minutes by means of epifluorescent light microscopy. After 115 minutes the same cell was microinjected with DAPI to detect DNA and to confirm localization of the cell nucleus. (B) Fluorometric quantification of time-lapse experiments. Comparison between proteasomal activity of cell nucleus-microinjected Suc-LLVY-AMC (filled triangles), caspase-specific substrate Ac-DEVD-AMC (filled squares), and predigested Suc-LLVY-AMC (filled circles), with Suc-LLVY-AMC that was digested in vitro by cytoplasmic (CY, open circles), or nucleoplasmic (NU, open triangles) cell fractions. Note the similar proteasomal activity in the nucleus of the living cell and in nucleoplasmic cell fractions. Scale bar, 5 µm.

View larger version (125K): [in a new window]   Fig. 4. In situ localization of proteasome-dependent protein degradation. Panels (A-C) and (F,G) show confocal fluorescence micrographs of HEp-2 cells with the corresponding differential interference contrast (DIC) image on the left. (A) HEp-2 cells were microinjected with 0.5 mg/ml fluorogenic substrate protein DQ-OVA into the cytoplasm and cell nucleus (cell a) or into the cell nucleus (cell b) or into the cytoplasm (cell c). Microinjected DQ-OVA shows a granular cytoplasmic localization. In the cell nucleus DQ-OVA localizes to distinct nucleoplasmic foci. Unlabelled areas correspond to nucleoli. Note, DQ-OVA is excluded from the nucleus in cells that were exclusively microinjected into the cytoplasm. (B) A representative HEp-2 cell that was microinjected with DQ-OVA and 10 µM proteasome inhibitor lactacystin shows a diffuse localization of DQ-OVA in the nucleus without formation of nucleoplasmic foci. (C) Equal numbers of HEp-2 cells in highlighted areas were microinjected with DQ-OVA (–LC) or DQ-OVA and 10 µM lactacystin (+LC). Corresponding fluorescence micrographs show decreased DQ-OVA fluorescence in proteasome inhibitor-treated (+LC) versus untreated (–LC) cells. (D) Fluorescence-quantification of (C). (E) HEp-2 cells were treated as in (C), and fluorescence of DQ-OVA quantified after 5, 10 and 15 minutes (min). (F) HEp-2 cells were co-incubated for 30 minutes with 200 µg/ml DQ-OVA, which localizes to endosomal vesicles in the cytoplasm, whereas the nucleus is unlabelled. (G) Confocal immunofluorescence of a representative HEp-2 cell that was microinjected with 200 µg/ml plain ovalbumin, fixed and immunolabelled with anti-ovalbumin antibodies. The ectopic protein ovalbumin distributes diffusely throughout the cytoplasm, and cell nucleus. Scale bars, 10 µm (A,B,F,G); 150 µm (C).

View larger version (52K): [in a new window]   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.

View larger version (104K): [in a new window]   Fig. 6. Proteasome-dependent degradation of DQ-OVA occurs in distinct subnuclear domains. Panels (A-H) show a confocal fluorescence micrograph of a representative HEp-2 cell microinjected with DQ-OVA (green), and a corresponding overview of untreated and microinjected cells (arrowheads) on the left. HEp-2 cells were double labelled (red) with ß-tubulin (A), lamin A/C (B), fibrillarin (C), SC35 (D), PML (E), RNA polymerase II (pol II, F), ubiquitin (G), and proteasomes (H). Microinjected DQ-OVA co-localizes partially with SC35, PML, pol II, ubiquitin and proteasomes in nucleoplasmic foci (yellow). (I) Quantification of co-localization between DQ-OVA and ß-tubulin or nuclear proteins in microinjected cells. Co-localization of DQ-OVA with ß-tubulin was quantified by defining whole cells as regions of interest (asterisk). Co-localization with nuclear proteins and nucleoplasmic proteasomes (two asterisks) was determined using cell nuclei as regions of interest. Note, exclusion of DQ-OVA from the nucleus in cells that were exclusively microinjected into the cytoplasm (open arrowheads). Scale bars, 5 µm.