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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.

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