Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 degrees C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

Multiple pathways contribute to nuclear import of core histones

Nuclear import of the four core histones H2A, H2B, H3 and H4 is one of the main nuclear import activities during S-phase of the cell cycle. However, the molecular machinery facilitating nuclear import of core histones has not been elucidated. Here, we investigated the pathways by which histone import can occur. First, we show that core histone import can be competed by the BIB (β-like import receptor binding) domain of ribosomal protein L23a suggesting that histone import is an importin mediated process. Secondly, affinity chromatography on immobilized core histones revealed that several members of the importin β family of transport receptors are able to interact with core histones. Finally, we demonstrate that at least four known and one novel importin, importin 9, can mediate nuclear import of core histones into the nuclei of permeabilized cells. Our results suggest that multiple pathways of import exist to provide efficient nuclear uptake of these abundant, essential proteins.     

Internalisation of cell-penetrating peptides into tobacco protoplasts

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.     

Metabolic behaviors of the core histones in proliferating Friend cells

This study examines the turnover of the core histones in proliferating Friend cells. It was calculated that these proteins turn over with half-lives of 21.6 days for H2A, 13.8 days for H2B, 43.3 days for H3, and 138.6 days for H4. The significant differences in the half-lives of the four core histones indicate that the protein moiety of the nucleosome is not replaced as one entire unit but as a "mosaic" in which each component follows its own rate of replacement. In some experiments the turnover rates of the variants of H2A, H2B, and H3 were compared. The results did not indicate any differences among these histone variants, suggesting that they are not excluded from the mechanisms controlling histone turnover. Metabolic heterogeneity was discovered, however, when the turnover rates of the acetylated and nonacetylated molecules of histone H4 were followed: it appeared that the acetylated molecules are replaced 2.5 times faster. The comparison of the rate of replacement of the histones in proliferating and differentiated cells from one site and their level of acetylation from another suggests that this postsynthetic modification might be involved in the control of histone metabolism. Such a conclusion is supported also by a number of model experiments.     

Nonenzymatic glycation of histones in vitro and in vivo

Purified histones in solution, purified nuclei, or whole endothelial cells in cell culture were used to study the reactivity of histones with various sugars. The sugar incubation of purified histones produced nonenzymatic glycation and formation of histone cross-links showing disappearance of individual histone molecules and appearance of dimers and polymers in SDS-PAGE. In solution, core histones react considerably faster with sugars as compared to H1 histones. In sugar-incubated nuclei where histones are nucleosomally organized, H1 histones, which are located at the periphery of the nucleosome, and H2A-H2B dimers, which are associated with the central H3(2)-H4(2) tetramer, are more reactive as compared to H3 and H4 histones, which are most protected from the glycation reaction. Our in vivo experiments using endothelial cells show that high concentrations of ribose are able to generate protein cross-links paralleled by apoptotic cell death. High concentrations of glucose or fructose do not increase histone glycation or cell death, even after 60 days of incubation of endothelial cells. In long-time glucose- or fructose-treated cells, under nondenaturing and nonreducing SDS-PAGE conditions part of the H3 histones shifted away from their normal location. Because it is known that the mitochondrial production of reactive oxygen species (ROS) increases after hyperglycaemia, we hypothesize that ROS could be responsible for the formation of a disulphide bridge between the side chain of the cysteine residues of H3 molecules.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Histone deacetylation is required for progression through mitosis in tobacco cells

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.     

Prothymosin alpha interacts with free core histones in the nucleus of dividing cells

The acidic protein prothymosin alpha (ProTalpha), with a broad presence in mammalian cells, has been widely considered to have a role in cell division, through an unrevealed mechanism in which histones may be involved in view of their ability to interact with ProTalpha in vitro. Results of co-immunoprecipitation experiments presented here demonstrate that ProTalpha interacts in vivo with core histones in proliferating B-lymphocytes (NC-37 cells). This interaction occurs with histones H3, H2A, H2B and H4 located free in the nucleoplasm, whereas no interaction was detected with histone H1, mono-nucleosome particles or chromatin. Moreover, the core histones form part of a nuclear multiprotein complex of about 700 kDa separated by ProTalpha-Sepharose affinity, with components including H3 and H4 acetyltranferases, H3 methyltransferases, hnRNP isotypes A3, A2/B1 and R, ATP-dependent and independent DNA helicases II, beta-actin and vimentin, all co-purifying by gel filtration. This indicates that the interaction of ProTalpha with core histones in the nucleus may be related to the structural modification of histones H3 and H4, and hence to chromatin activity, raising the possibility that the other proteins in the nuclear complex may play a role in this process.     

Internalisation of cell-penetrating peptides into tobacco protoplasts

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.     

Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5-15) in G0/G1- and G2/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G0/G1-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G0/G1 into the S and G2/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.     

Different mechanism for in vitro formation of nucleosome core particles

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.     

Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light

Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14-3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts.     

Infrared picosecond laser for perforation of single plant cells

The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. In this report, we demonstrate the use of an infrared (1,064 nm) picosecond laser for the perforation of tobacco cell protoplasts. A single pulse was sufficient to perforate the plasma membrane enabling the uptake of dye from the surrounding medium into the cytosol. Moreover, the procedure was shown to be suitable for the efficient delivery of DNA expression constructs to the nucleus, as demonstrated by the subsequent expression and correct targeting of a recombinant fluorescent protein. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.     

Histone acetylation and heterochromatin content of cultured Peromyscus cells

In order to explore the relationship between unacetylated arginine-rich histones and condensed chromatin structure, the extent of histone acetylation was examined in cultured cell lines derived from three species of deer mice. These species differ considerably in their genomic content of heterochromatin but contain essentially the same euchromatin content. Cells of Peromyscus eremicus, containing 34–36% more constitutive heterochromatin than Peromyscus boylii or Peromyscus crinitus cells were found to contain 28–35% more unacetylated histone H4, 22–29% more unacetylated histone H3, and 18–22% more unacetylated histone H2B. This relationship between unacetylated histones and heterochromatin content was further explored by inducing hyperacetylation of P. eremicus and P. boylii histones through treatment of cells with 15 mM sodium butyrate for 24 h. It was found that the percentages of unacetylated histones H3 and H4 remaining after butyrate treatment were proportional to the amount of constitutive heterochromatin in the genome. These data support the concept that a small core of histones in constitutive heterochromatin is inaccessible to acetylation. It was also found that the acetylated state of isolated histones was sensitive to the method of histone extraction. Thus concern must be given to preparative procedures when studying histone acetylation in order to minimize these acetate losses.     

Binding of linker histones to the core nucleosome

Binding of chicken erythrocyte linker histones H1/H5 to the core nucleosome has been studied. Histones H1/H5 bind very efficiently to the isolated core nucleosome in vitro. The binding of linker histones to the core nucleosome is associated with aggregation of the particles. Approximately one molecule of linker histone binds per core nucleosome in the aggregates, irrespective of the concentration of the linker histones and the salt used. Histone H5 shows greater binding affinity to the core nucleosome as compared to H1. The carboxyl-terminal fragment of the linker histones binds strongly to the core nucleosome while the binding of the central globular domain is weak. Each core nucleosome is capable of binding two molecules of carboxyl-terminal fragment of linker histone. The core nucleosome containing one molecule of carboxyl-terminal fragment of linker histone requires higher salt concentration for aggregation while the core nucleosome containing two molecules of carboxyl-terminal fragment of linker histone can self-associate even at lower salt concentrations. On the basis of these results we are proposing a novel mechanism for the condensation of chromatin by linker histones and other related phenomena.     

Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry

The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G1, S, and G2/M. For all core histones, the modifications in the G1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G2/M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G2/M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G2/M, H3 Lys27 and Lys36 were decreased, whereas H4 Lys20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.     

Histones of terminally differentiated cells undergo continuous turnover

In contrast to the widely accepted idea of the nearly absolute metabolic stability of histones, our experiments support the view that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement. This conclusion is based on the incorporation of labeled amino acids into the histones of mouse kidney and liver cells after their intraperitoneal introduction. We have found that the intranuclear uptake of the histones made in the absence of replicative synthesis and their integration into chromatin proceed with striking delay. The metabolic rates of individual histones measured by calculating their half-lives suggest that each histone turns over at a specific rate. With regard to the basic chromatin structure, the nucleosome, such unequal turnover should mean that the histone core does not participate in this process as a single unit but rather as a protein mosaic in which each partner follows its own rate of removal. Additional experiments suggested that intact nucleosomes take part in the replacement, but the relative proportion of the nucleosomes involved should be limited. The nonnucleosomal H1A and H1 degree histones have been found to undergo faster replacement than the core histones. Moreover, in comparison to each other, these two histone subfractions are also replaced at a different rate. The results of autoradiography of isolated kidney and liver nuclei after continuous labeling with [3H]-thymidine suggest that the histone replacement is not associated with the repair of DNA.