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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00757

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Direct translocation of histone molecules across cell membranes

¹ Department of Organic Chemistry, Institute of Chemistry
² Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
³ Institut fuer Molekularbiologie und Biochemie, Free University of Berlin, Berlin 14195, Germany

View larger version (67K): [in a new window]   Fig. 1. Intracellular accumulation of rhodamine-labelled histone proteins in intact HeLa cells and human lymphocytes: fluorescent microscopy observations. HeLa cells were incubated for 1 hour in the presence of a mixture of rhodamine-labelled histones (2 µM) at 37°C. (A) After fixation by formaldehyde, cells were observed by fluorescent microscopy; (B-H) as in (A) but with the following conditions: (B) cells were incubated at 4°C; (C) incubation was performed in the presence of excess unlabelled histone mixture (x50 mole/mole); cells were pre-incubated for 30 minutes at 37°C before the addition of histones with the following: (D) incubated with NaF (2 mM) (ATP depletion); (E) cytochalasin D (5 µM); (F) BFA (10 µM); (G) nocodazole (20 µM); (H) nystatin (50 µg/ml) and sucrose (0.5 M); (I) as (A), (J) as (G) and (K) as (H) but using confocal microscopy; (L) as (A), (M) as (G) and (N) as (H) but with unfixed cells. (O) Human lymphocytes incubated for 1 hour in the presence of a mixture of rhodamine-labelled histones. All other experimental conditions were as described in Materials and Methods.

View larger version (17K): [in a new window]   Fig. 2. (A) Intracellular accumulation of biotinylated histone proteins (externally added) in cultured Colo-205 cells: quantitative estimation. Prior-neut (prior neutralization): biotinylated histones neutralized with avidin and biocytin before being added to the Colo-205 cells. All subsequent steps including cell permeabilization and estimation of biotinylated histones are as described in Materials and Methods. No detergent: estimation of cell-surface-bound biotin molecules. Biotinylated histones were incubated with Colo-205 cells and following neutralization of surface-bound biotin the remaining biotin was estimated on unlysed cells. Control 37°C and 4°C: the biotinylated histone mixture was incubated with Colo-205 cells at 37°C or at 4°C and after neutralizion with avidin and incubation with biocytin, the cells were treated with detergents. Prior fixation: cells were fixed by formaldehyde prior to the incubation period. Excess histone: as control 37°C but in the presence of excess unlabelled histone (x100 mole/mole). ATP-depleted cells: as control 37°C and ATP depletion was performed as described in Table 1. , nuclei; , cytosol. The amount of biotinylated histone present in the nuclei of cells incubated at 37°C was considered as 100% and was calculated to be 6.2 nmol histone/mg protein, which was estimated to be about 60% of the total added histones. (B) Kinetic studies of histone penetration. Biotinylated histones were incubated with Colo-205 cells at 37°C in the absence () or in the presence () of 0.5 M sucrose and at 4°C (). Pre-incubation with sucrose (0.5 M) was performed as described in Table 1. An optical density (OD) of 0.25 represents 4.7 nmol histone/mg protein. (C) Kinetic studies of histone and importin alpha penetration. Biotinylated histone () and importin alpha () were incubated with Colo-205 cells at 37°C. Penetration of histone and importin was estimated as described in Materials and Methods, except that the incubation was performed in PBS and not in TB as in B.

View larger version (22K): [in a new window]   Fig. 3. (A) Accumulation of biotinylated individual histones: quantitative estimation. All experimental conditions of estimation of cellular uptake are as described in Materials and Methods. An optical density of 0.2 represents 0.47 nmol histone/mg protein. (B) The effect of various inhibitors on the intracellular accumulation of H2A and H4. (a) Accumulation of biotinylated H2A () and H4 () into Colo-205 cells. Cells were pre-incubated before the addition of the histones for 30 minutes at 37°C with the various endocytic inhibitors at the concentrations described in Table 1. The amount of biotinylated H2A and H4 present in the nuclei of cells incubated in 37°C was considered to be 100% and was found to be 5.9 and 5.5 nmol histone/mg protein, respectively. (C) Intracellular accumulation of biotinylated H2A in Colo-205 cells: effect of ATP depletion and unlabelled H2A. Accumulation of biotinylated H2A within the cell cytosol and nuclei was estimated as described in Materials and Methods. ATP depletion was performed as described in Table 1. , nuclei; , cytosol. The amount of biotinylated H2A present in the nuclei of cells incubated in 37°C was considered to be 100% and represents 5.8 nmol/mg protein; it was estimated to be about 48% of the total added histones. The ratio given (in A and C) are in mole:mole.

View larger version (95K): [in a new window]   Fig. 4. Intracellular accumulation of the individual histones: fluorescent microscopy observations. HeLa cells were incubated for 1 hour at 37°C in the presence of rhodamine-labelled (A) H2A, (B) H2B, (C) labelled H2A + non-labelled H2B (1:1 mole/mole), (D) labelled H2B + non-labelled H2A (1:1 mole/mole), (E) H3, (F) H4, (G) labelled H3 + non labelled H4 (1:1 mole/mole) or (H) labelled H4 + non-labelled H3 (1:1 mole/mole), (I) lissamine rhodamine (2 mg/ml) was incubated with the Hela cells as described for incubation with the histone for 1 hour at 37°C. Timecourse studies (J-L) as in (A) but following 5 (J), 15 (K) or 30 (L) minutes of incubation. The amount of labelled-histone added as well as all other experimental conditions are as described in Materials and Methods.

View larger version (40K): [in a new window]   Fig. 5. Accumulation of externally added BSA-histone conjugates into HeLa cells. (A) HeLa cells were incubated for 1 hour in the presence of only rhodamine-labelled BSA. (B) Incubation of HeLa cells with Rho-BSA-histone mixture conjugate in the absence or in the presence (C) of excess unlabelled histone mixture (x50 mole/mole); (D) as in (B) but using confocal microscopy. (E) Incubation of HeLa cells with Rho-BSA-H2A conjugate.

View larger version (12K): [in a new window]   Fig. 6. Quantitative accumulation of externally added BSA-histone conjugates into Colo-205 cells. Bb-histone conjugates were prepared and their accumulation within the cell cytosol and nuclei was estimated as described in Materials and Methods. ATP depletion was performed as described in Table 1. , nuclei; , cytosol. The amount of Bb-histone present in the nuclei of cells incubated in 37°C was considered as 100% and represents 6.3 nmol/mg protein; it was estimated to be about 28% of the total added Bb-histone. The ratio given are in mole:mole.

View larger version (22K): [in a new window]   Fig. 7. Cellular and nuclear accumulation of Bb-histone conjugate within the Colo-205 cells – cytoplasm and nuclei. (A) The amount of Bb-H2A present in the nuclei of cells incubated in 37°C was considered to be 100% and represents 6.4 nmol/mg protein; it was estimated to be 58% of the total added Bb-H2A. (B) Bb-H2B conjugates. ATP depletion was performed as described in Table 1 and cells were fixed (prior fixation) as described in Fig. 2. 100% represents 6.2 nmol histone/mg protein and was estimated to be 62% of the total added Bb-H2B. *Bb-H2B incubated in 37°C, 4°C, ATP-depleted cells or after prior fixation gave the same results as biotinylated Bb-H2B alone. Biotinylated conjugates were prepared and their accumulation within the cell cytosol and nuclei was estimated as described in Materials and Methods. , nuclei; , cytosol. The ratio given (in A and B) are in mole:mole.

View larger version (8K): [in a new window]   Fig. 8. Effect of the histone mixture on transcription process in Colo-205 cells. Colo-205 cells were incubated in the presence and absence of the histone mixture as well as with [3H]-uridine as described in Materials and Methods. The degree of transcription was estimated by monitoring the amount of radioactivity in total RNA and in mRNA.