Nucleosomes enter cells by clathrin- and caveolin-dependent endocytosis

Abstract

DNA damage and apoptosis lead to the release of free nucleosomes-the basic structural repeating units of chromatin-into the blood circulation system. We recently reported that free nucleosomes that enter the cytoplasm of mammalian cells trigger immune responses by activating cGMP-AMP synthase (cGAS). In the present study, we designed experiments to reveal the mechanism of nucleosome uptake by human cells. We showed that nucleosomes are first absorbed on the cell membrane through nonspecific electrostatic interactions between positively charged histone N-terminal tails and ligands on the cell surface, followed by internalization via clathrin- or caveolae-dependent endocytosis. After cellular internalization, endosomal escape occurs rapidly, and nucleosomes are released into the cytosol, maintaining structural integrity for an extended period. The efficient endocytosis of extracellular nucleosomes suggests that circulating nucleosomes may lead to cellular disorders as well as immunostimulation, and thus, the biological effects exerted by endocytic nucleosomes should be addressed in the future.

Figure 1.

Schematic diagram of the nucleosomes used in the present study.

Figure 2.

Cellular uptake of nucleosomes occurs by energy-dependent endocytosis. (A) Flow cytometric analysis of HeLa cells after incubation with FAM-145 nucleosomes (20 nM) under different conditions for 6 h. Quantitative results are shown in panels (B) and (C). Control, incubation of HeLa cells with FAM-145-dsDNA. MFI, mean fluorescence intensity. (D) Representative images showing the cellular location of FAM-145 nucleosomes in HeLa cells after incubation for 6 h. Scale bar, 20 μm. Error bars indicate the mean ± standard deviation of at least three independent experiments. Statistical significance was determined based on Student's t-test (ns, P > 0.05; *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P).

Figure 3.

Confocal fluorescence microscopy analyses of the binding of nucleosomes on the HeLa cell surface after incubation at 4°C for 30 min. (A) Representative images showing the binding of FAM-145 nucleosomes on the HeLa cell surface after different treatments. An acid wash of HeLa cells was applied after incubation with 145 nucleosomes. Trypsin treatment of HeLa cells was applied before incubation with 145 nucleosomes. The relative FAM fluorescence intensity is compared in panel (B). (C) Representative images showing the binding of different types of nucleosomes on the HeLa cell surface. The relative FAM fluorescence intensity is compared in panel (D). Scale bar, 20 μm. Error bars indicate the mean ± standard deviation of at least three independent experiments. Statistical significance was determined based on Student's t-test (ns, P > 0.05; *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P).

Figure 4.

Binding and cellular uptake of K/R-mutated 145-FAM nucleosomes in HeLa cells. (A) The sequences of wild-type (WT) and K/R mutated histones. In the mutated histones, all Lys residues in the N-terminal tails are mutated to Arg. (B) Representative images showing the binding of WT and K/R mutated 145-FAM nucleosomes on the HeLa cell surface at 4°C for 30 min. Scale bar, 20 μm. The relative FAM fluorescence intensity is compared in panel (C). (D) Flow cytometric analysis of HeLa cells after incubation with WT and mutated FAM-145 nucleosomes (20 nM) for 6 h. Quantitative results are shown in panel (E). Error bars indicate the mean ± standard deviation of at least three independent experiments. Statistical significance was determined based on Student's t-test (ns, P > 0.05; *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P).

Figure 5.

Flow cytometric analysis of cellular uptake of FAM-145 nucleosomes by HeLa cells in the presence of different inhibitors. (A, B) In the presence of chemical endocytosis inhibitors. (C, D) Knockdown of CHC and CAV by siRNA. MFI, mean fluorescence intensity. Error bars indicate the mean ± standard deviation of at least three independent experiments. Statistical significance was determined based on Student's t-test (ns, P > 0.05; *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P).

Figure 6.

Time-dependent colocalization of FAM-145 nucleosomes with Cy3-Tfn and AF555-CTB in HeLa cells. (A) Confocal microscopy analysis of the colocalization of FAM-145 nucleosomes with Cy3-Tfn. The time-dependent MCC of FAM-145 nucleosomes/Cy3-Tfn is shown in panel (C). (B) Confocal microscopy analysis of the colocalization of FAM-145 nucleosomes with AF555-CTB. The time-dependent MCC of FAM-145 nucleosomes/AF555-CTB is shown in panel (D). For all graphs, error bars indicate the mean ± standard deviation of at least three independent experiments. Scale bar, 20 μm.

Figure 7.

Time-dependent entrapment of FAM-145 nucleosomes in different cell compartments and cellular organelles after endocytosis. (A) MCC of FAM-145 nucleosomes and early endosome antigen 1 (EEA1). (B) MCC of FAM-145 nucleosomes and Lyso-Tracker Red. (C) MCC of FAM-145 nucleosomes and Golgi-Tracker Red. (D) MCC of FAM-145 nucleosomes and ER-Tracker Red. Error bars indicate the mean ± standard deviation of at least three independent experiments.

Figure 8.

Internalized nucleosomes maintain structural integrity after endosomal escape. (A) Preparation of FAM-Cy3-145-nucleosomes. (B) Agarose gel (1.5%) analysis of the structural integrity of FAM-Cy3-145 nucleosomes. (C) Confocal microscopy analysis of the intracellular distribution of FAM-Cy3-145 nucleosomes after incubation for 12 h. Scale bar, 20 μm. (D) The time-dependent MCC of FAM/Cy3 and Cy3/FAM. Error bars indicate the mean ± standard deviation of at least three independent experiments.

Figure 9.

Flow cytometric analysis of the cellular uptake of FAM-145 nucleosomes by THP1 cells in the presence of different inhibitors. (A) Chemical endocytosis inhibitors. (B) Knockdown of CHC and CAV by siRNA. Error bars indicate the mean ± standard deviation of at least three independent experiments. Statistical significance was determined based on Student's t-test (ns, P > 0.05; *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P).