Dynamic localization and the associated translocation mechanism of HMGBs in response to GCRV challenge in CIK cells

Abstract

High-mobility group box (HMGB) proteins, a family of chromatin-associated nuclear proteins, play amazingly multifaceted roles in the immune system of mammals. Thus far, little is known about the nucleocytoplasmic distribution of HMGBs in teleosts. The present study systematically investigated the dynamic localization of all six HMGB proteins in Ctenopharyngodon idella kidney (CIK) cells. Under basal conditions, all HMGBs exclusively localized to the nucleus. Grass carp reovirus (GCRV), polyinosinic-polycytidylic (poly(I∶C)) potassium salt and lipopolysaccharide (LPS) challenge evoked the nuclear export of HMGBs to various degrees: GCRV challenge induced the highest nuclear export of CiHMGB2b, and poly(I∶C) and LPS evoked the highest nucleocytoplasmic shuttling of CiHMGB1b. Overall, the nucleocytoplasmic shuttling of CiHMGB2a and CiHMGB3b was rarely induced by these challenges. Dynamic imaging uncovered that the nucleocytoplasmic GCRV-induced relocation of CiHMGB2b occurred in cells undergoing karyotheca rupture, apoptosis or proliferation. Western blot analyses were used to examine HMGB-EGFP fusion proteins in whole cell lysates, cytosol, nuclear fractions and culture medium. Further investigation demonstrated the nuclear retention of N-terminal HMG-boxes and the nucleocytoplasmic distribution of the C-terminal acidic tails. Comparative analyses of the dynamic relocation of full-length, truncated or chimeric HMGBs confirmed that the intramolecular interaction between HMG-boxes and C-tail domains mediated the nucleocytoplasmic translocation of HMGBs. These results not only provide an overall understanding of the subcellular localization of HMGBs, but also reveal the induction mechanism of the nucleocytoplasmic translocation of HMGBs by GCRV challenge, which lays a foundation for further studies on the interactions among pathogens, HMGBs and pattern recognition receptors in the innate immunity of teleosts.

Figure 1

Subcellular localization of HMGB proteins in CIK cells. (a) Nucleocytoplasmic distribution of EGFP in CIK cells. (b) Nuclear localization of all six grass carp HMGB proteins in CIK cells. Cells expressing CiHMGB-EGFP fusion proteins were seeded on microscope coverglasses in 12-well plates for 24 h for 50% confluency, then fixed with 4% (v/v) paraformaldehyde and stained with Hoechst 33342. Finally, all the samples were visualized using a confocal microscope. (c) NLSs predicted in HMGBs: amino acids 27–43 of CiHMGB1a, CiHMGB1b and CiHMGB2a, amino acids 28–44 of CiHMGB2b and CiHMGB3a, and amino acids 29–44 and 82–96 of CiHMGB3b. CIK, Ctenopharyngodon idella kidney; HMGB, high-mobility group box; NLS, nuclear localization signal.

Figure 2

Nucleocytoplasmic distribution of grass carp HMGB proteins induced by GCRV challenge. Stably transfected cells seeded on microscope coverglasses in 12-well plates were challenged with GCRV for 6 h and were then fixed, stained and photographed as described above. The green indicates EGFP or HMGB-EGFP fusion proteins, and the blue represents nuclei. (a) GCRV infection had no influence of the distribution of EGFP in CIK cells. (b) GCRV infection induced the nucleocytoplasmic translocation of HMGBs in CIK cells. (c) HMGBs were detected by western blotting in different cell fractions. A monoclonal antibody against EGFP was used to detect HMGB-EGFP proteins. Under basal conditions, all six HMGBs were only detected in nuclear fractions. Post-GCRV infection, CiHMGB1a, CiHMGB1b, CiHMGB2b and CiHMGB3a were detected in both nuclear and cytoplasmic fractions, but CiHMGB2a and CiHMGB3b were presented in only nuclear fractions. CIK, Ctenopharyngodon idella kidney; GCRV, grass carp reovirus; HMGB, high-mobility group box.

Figure 3

Statistic analysis of the cells where HMGB proteins relocated from the nucleus to the cytoplasm. Stably transfected cells (approximately 80% EGFP-positive) were challenged with (a) GCRV for 6 h or stimulated with (b) poly(I∶C) or (c) LPS for 24 h. Control groups were treated with DMEM. Afterward, all of the samples were examined using a fluorescence microscope. HMGBs in the control groups did not migrate from the nucleus to the cytoplasm (data not shown). The percentage was calculated as the quantity of the cells displaying a nucleocytoplasmic distribution of HMGBs as a percentage of all the EGFP-positive cells. Error bars indicate standard deviations. GCRV, grass carp reovirus; HMGB, high-mobility group box; LPS, lipopolysaccharide; poly(I∶C), polyinosinic–polycytidylic.

Figure 4

Dynamic translocation of CiHMGB2b induced by GCRV challenge in CIK cells. (a) Nucleocytoplasmic translocation of CiHMGB2b accompanied with karyotheca rupture. Above: nuclear export of CiHMGB2b induced by GCRV challenge was continuously monitored and shown at the indicated time points. Below: cells with karyotheca rupture display uniform nucleocytoplasmic distribution of CiHMGB2b. (b) Dynamic export of CiHMGB2b in dying cells. Left: EGFP signals indicate the export of CiHMGB2b from the nucleus to the cytoplasm. Right: bright-field image of the same visual field, with an arrow highlighting the dying cell. (c) Nucleocytoplasmic shuttling of CiHMGB2b occurs in dividing cells. Above: nuclear export of CiHMGB2b takes place in the process of cell division. Below: relocation of CiHMGB2b occurs in dividing cells observed by confocal microscopy. (d) CiHMGB2b-EGFP fusion protein was detected at 6 h and 12 h in the culture medium post-GCRV infection by western blotting. (e) Extracellular release of CiHMGB1a, CiHMGB1b and CiHMGB3a but not CiHMGB2a and CiHMGB3b were examined in the medium at 6 h post-GCRV infection. At 24 h, CiHMGB1a, CiHMGB1b, CiHMGB3a, CiHMGB2a and CiHMGB3b were detected in the culture medium by western blotting. CIK, Ctenopharyngodon idella kidney; GCRV, grass carp reovirus; HMGB, high-mobility group box.

Figure 5

Subcellular localization of truncated and chimeric HMGB proteins. (a) Schematic diagram of the truncated and chimeric HMGB proteins. (b) Subcellular localization of corresponding truncated and chimeric HMGB proteins. CiHMGB2bN+box, CiHMGB2bN+box-CiHMGB3bC+tail (2N3C) and CiHMGB3bN+box-CiHMGB2bC+tail (3N2C) displayed nuclear localization, but CiHMGB2bC+tail showed a uniform nucleocytoplasmic distribution. Cells were transfected, fixed, stained and visualized as outlined in Figure 1. HMGB, high-mobility group box.

Figure 6

Nuclear export of truncated and chimeric HMGB proteins induced by GCRV challenge in CIK cells. (a) Nucleocytoplasmic distribution of truncated and chimeric HMGBs in CIK cells post-GCRV challenge. CiHMGB2bN+box, 2N3C and 3N2C were released from the nucleus to the cytoplasm to different degrees. The accumulation of CiHMGB2bN+box and 2N3C in the nucleus was higher than in the cytoplasm, but the localization of 3N2C showed a uniform distribution in both the nucleus and the cytoplasm. GCRV challenge did not influence the localization of CiHMGB2bC+tail. (b) The comparative analyses of ratios of the cells displaying the cytoplasmic localization of the full-length, truncated and chimeric HMGB proteins. Left: schematic diagram of the corresponding proteins, with dashed boxes highlighting the differences among the diverse proteins. Right: comparison of the percentage of the full-length, truncated or chimeric cells with cytoplasmic localizations. Error bars indicate standard deviations, and asterisks (*) indicate significant differences. CIK, Ctenopharyngodon idella kidney; GCRV, grass carp reovirus; HMGB, high-mobility group box.