Detection of DNA in the nucleoids of chloroplasts and mitochondria in Euglena gracilis by immunoelectron microscopy

Size: px

Start display at page:

Download "Detection of DNA in the nucleoids of chloroplasts and mitochondria in Euglena gracilis by immunoelectron microscopy"

Lynn Porter
5 years ago
Views:

1 Journal of Cell Science 105, (1993) Printed in Great Britain The Company of Biologists Limited Detection of DNA in the nucleoids of chloroplasts and mitochondria in Euglena gracilis by immunoelectron microscopy Yasuko Hayashi-Isimaru, Katsumi Ueda* and Mitsuko Nonaka Biological Laboratory, Nara Women s University, 630 Nara, Japan *Author for correspondence SUMMARY DNA in the nucleoids of chloroplasts and of mitochondria in Euglena gracilis was detected with anti-dna antibodies by immunoelectron microscopy. After treatment with the antibodies, DNA in these organelles combined with gold particles that had been coated with anti- IgM antibodies such that it was possible to trace the outlines of the nucleoids. Nucleoids in chloroplasts appeared to be composed of twisted threads nm in diameter. The twisted threads were entangled to form thicker nodes of nm diameter. Most nucleoids in mitochondria were spherical or ovoid, nm in diameter. Nucleoids both in chloroplasts and in mitochondria contained cores with which DNA threads were in tight contact. The structure of the nucleoids was very different from those previously observed by conventional electron microscopy. Key words: chloroplast nucleoid, immunocytochemistry, immunoelectron microscopy, mitochondrial nucleoid, organellar DNA INTRODUCTION Chloroplasts and mitochondria contain DNA which appears to be combined with proteins to form nucleoids in the organelles (Kuroiwa, 1982; Nemoto et al., 1989). In general, the DNA content of these organelles is far lower than that of nuclei. Thus, it was previously difficult to detect the DNA in situ and to visualize the shapes and distribution patterns of the nucleoids in the organelles. Use of the dye DAPI for detection of nucleoids by fluorescence microscopy has increased the information that is available about the shape and the distribution pattern of nucleoids. Coleman (1978) was the first to succeed in visualizing the nucleoids in chloroplasts, and later she classified the chloroplast nucleoids in various eucaryotic algae into 6 groups (Coleman, 1985). Kuroiwa et al. (1981) classified the nucleoids in various plants into 5 groups. Changes in the number, size, and distribution pattern of chloroplast nucleoids during the life cycle or development of several algae have been investigated in some detail (Nakamura et al., 1986; Hatano and Ueda, 1987). Relatively little has been reported about the behavior of mitochondrial nucleoids during the cell cycle except in Physarum (Kuroiwa, 1982), Saccharomyces (Miyakawa et al., 1984), and Euglena (Hayashi and Ueda, 1989, 1992). Studies of chloroplast and mitochondrial nucleoids by fluorescence microscopy are subject to the limits of the resolving power of the light microscope. Detailed morphological studies should be done by electron microscopy. By conventional electron microscopy, Kellenberger s (1958) or related fixatives have often been used for the visualization of nucleoids or DNA fibrils. In most published reports, nucleoids appear as electron-transparent regions containing fine fibrils (Nass and Nass, 1963a,b; Nass et al., 1965; Yokomura, 1967a,b). In most cases, small numbers of DNA fibrils are found in relatively large electron-transparent regions. It has not yet been determined whether entire transparent regions, without clear boundaries, actually correspond to the nucleoids. Moreover, no evidence has been presented from which we can infer that only the fibrous regions correspond to the nucleoids in both chloroplasts and mitochondria. That is, even the outer boundaries of the nucleoids in chloroplasts and mitochondria have yet to be determined. Accordingly, detailed morphological studies on organellar nucleoids are necessary using methods other than conventional electron microscopy. Immunoelectron microscopy provides reliable information about the distribution pattern of specific antigens at the ultrastructural level. Using immunogold electron microscopy, we have visualized the outer boundaries of the nucleoids and have detected the possible distribution of DNA in the nucleoids of chloroplasts and mitochondria in Euglena gracilis as described below. MATERIALS AND METHODS Cells of Euglena gracilis Z strain were cultured at 25 C in Cramer-Myers medium (Cramer and Myers, 1952), and the culture was illuminated by fluorescent light with a photon flux density of 150 µmol m 2 s 1 for 12 h per day.

1160 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka For visualization of nucleoids in the mitochondria and chloroplasts, cells were treated with 5 µg/ml DAPI (4 6-diamidino-2- phenylindole; Sigma, St.

2 1160 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka For visualization of nucleoids in the mitochondria and chloroplasts, cells were treated with 5 µg/ml DAPI (4 6-diamidino-2- phenylindole; Sigma, St. Louis, MO, USA) and were observed by fluorescence microscopy as described previously (Hayashi and Ueda, 1989, 1992). For immunoelectron microscopy, cells were fixed for 1 h at 4 C in phosphate buffer (ph 7.4) that contained 3% paraformaldehyde and 2% glutaraldehyde. After washing with water, they were dehydrated with ethanol and embedded in Lowicryl resin at 0 C. Polymerization of the resin was carried out at 25 C under UV light. Serial sections, each of 70 nm thickness, were attached on Formvar coated nickel glids. Sections were treated successively with 3% H2O2 for 20 min, 3% bovine serum albumin (BSA) for 30 min and 30 µg/ml anti-dna antibodies (Boehringer Mannheim Biochemica, Mannheim, Germany) for 2 h at room temperature. After washing with Tween-PBS (20 mm PBS plus 0.5% Tween- 20), sections were treated for 1 h at room temperature with colloidal gold (5 nm in diameter) conjugated anti-mouse IgM antibodies (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA). The gold particles were conjugated with the antibodies by the method of De May and Moeremans (1986). Sections were stained with an aqueous solution of 1% uranyl acetate and with lead citrate. To examine the specificity of the binding between DNA and the anti-dna antibodies, treatment of sections with the antibodies was omitted but the rest of the procedure for detection of DNA was performed. For digestion of DNA, sections were immersed for 1 h at 30 C in a buffered solution (ph 5.6) that contained 0.1% DNase I (Worthington Biochem. Corp, NJ, USA) and 1 mm MgSO 4. After washing with water, sections were treated for detection of DNA with the antibodies. RESULTS Nucleoids in the chloroplasts and in the mitochondria, visualized by fluorescence microscopy after staining with DAPI, are shown in Fig. 1. Nucleoids in chloroplasts appeared as rods of µm in length. Their diameters cannot be accurately measured because they are so fine. Nucleoids in the mitochondria appeared as dots. Most nucleoids emitted fluorescence of similar intensity, but one third of the nucleoids emitted stronger fluorescence. Fig. 2A shows part of a nucleus, a chloroplast and a mitochondrion that were treated by the immunocytochemical procedure for detection of DNA. As was anticipated, prominent deposits of gold particles could be seen on the chromosomes in the nucleus. Similar deposits of gold particles were also visible at places both in the chloroplast and the mitochondrion. Treatment of sections with DNase during the immunocytochemical procedure eliminated depositions of gold particles in defined groups in nuclei, chloroplasts (Fig. 2B), and mitochondria (Fig. 2C). In the absence of treatment with the anti-dna antibodies during the immunocytochemical procedure, no gold particles were also deposited in groups in nuclei, chloroplasts, or mitochondria (Fig. 2D). Seven sectional profiles of chloroplasts are seen in Fig. 3. In each chloroplast, 5-8 small electron-dense regions of irregular shape are visible, which are the nucleoids. The high electron-density results from the deposits of gold particles on the nucleoids. The total area of the 7 chloroplasts is 27.5 µm 2 and that of nucleoids 0.51 µm 2. Thus the Fig. 1. Fluorescence of nucleoids in chloroplasts (ch) and mitochondria (arrows). n, nucleus. Bar, 10 µm. nucleoids occupy about 1.9% of the cross-sectional area of the chloroplasts. Three serial sections of a portion of two chloroplasts are shown in Fig. 4 (A, B, C). In each panel, there are 6 to 8 regions in which gold particles are distinctly localized. Deposits of gold particles are seen in similar regions in the chloroplasts in all three panels, strongly suggesting that the particles were not deposited randomly. We can conclude that DNA was present in the three serial sections and that the gold particles were deposited as a result of the immunological reaction. The sites of the gold particles in the specific regions of the chloroplasts in the three panels were superimposed on one another to give Fig. 5. The nucleoid regions a, b, and c overlap completely with the respective corresponding regions in the three panels. Region d shifts to the left gradually as the sections advance from Fig. 4A to C. There are three regions designated e in Fig. 4A, and they shift to combine finally as region e in Fig. 4C. Region f appears in two sections (Fig. 4A,B), while regions g and h appear in only one section each. Nucleoids a-h are not completely included within the three sections, and parts of them may be contained in the sections adjacent to those shown in Fig. 4. Fig. 5 shows that the nucleoids are twisted threads of nm in diameter. The diameters of these threads or the nucleoids cannot be accurately measured due to undetermined factors concerning the binding pattern between antigens, antibodies, and gold particles at specific regions, so that the figures are put as approximations. The left halves of nucleoids a, b, d, and e in Fig. 5 seem to be twisted spirally, and the central portions seem to be wound more than twice into thicker nodes of nm in thickness. Gold particles are not distributed randomly in the nucleoids but are arranged along wavy or irregularly folded lines. These particle-lines may reflect the arrangement of DNA fibrils in the nucleoids. Transverse sections of the nucleoids reveal DNA-free central cores and surrounding DNA areas (Fig. 4, arrows). The cores appeared to be slightly more electrondense than the stroma of the chloroplast. Central cores were nm in diameter and the total diameter of the

Detection of DNA in the nucleoids 1161 Fig. 3. Distribution of nucleoids in 7 chloroplasts. Electron-dense regions with irregular outlines represent the nucleoids. Bar, 1 µm. Fig. 2.

No defined groups of gold particles are visible. Bar, 1 µm. (D) Treatment with the anti-dna antibodies was omitted. No gold-particles were deposited in groups in organelles. Bar, 1 µm. nucleoids, including the DNA areas, was 60-100 nm.

3 Detection of DNA in the nucleoids 1161 Fig. 3. Distribution of nucleoids in 7 chloroplasts. Electron-dense regions with irregular outlines represent the nucleoids. Bar, 1 µm. Fig. 2. (A) Deposits of gold particles on the chromosomes and on the nucleoids in a chloroplast (ch) and a mitochondrion (m). Bar, 1 µm. DNase treated nucleus and chloroplast (B) and mitochondria (C). No defined groups of gold particles are visible. Bar, 1 µm. (D) Treatment with the anti-dna antibodies was omitted. No gold-particles were deposited in groups in organelles. Bar, 1 µm. nucleoids, including the DNA areas, was nm. The presence of central cores could also be recognized in oblique sections (Fig. 4, arrowheads). The central cores seemed not to be completely covered by DNA. Some profiles of mitochondria were partially covered with deposits of gold particles. As in the cases of chloroplasts, deposits appeared in the same regions of mitochondria in 2 or 3 successive sections (Figs 6-9). The regions corresponding to the nucleoids were generally spherical or ovoid, nm in diameter; the nucleoid in Fig. 7 is 100 nm and the nucleoids in Figs 6 and 8 are 120 nm in diameter. Therefore, the regions corresponding to nucleoids were seen in two successive sections in Fig. 7, and in three successive sections in Figs 6 and 8. Sometimes, large regions covered with gold particles were seen (Fig. 9). When sites of gold particles in two successive sections (Fig. 9B,C) were superimposed, Fig. 9D was obtained. The nucleoid in Fig. 9D appears to contain DNA threads, nm in diameter, that are twisted spirally. DNA threads surrounded cores in several profiles (Fig. 9B,C, arrows). Cores of mitochondrial nucleoids could also be seen in nucleoids of average size (Fig. 8, arrow). Nucleoids were slightly more electron-dense than other regions of the mitochondria (Fig. 10A,B). Regions with groups of particles located side by side or end to end were sometimes observed (Fig. 11A,B). Dumbbell-shaped nucleoids and paired nucleoids have been observed by fluorescence microscopy in mitochondria, and they have been interpreted as nucleoids in the process of division (Hayashi and Ueda, 1989). The groups of particles located side by side or end to end may correspond to dividing nucleoids. DISCUSSION Several reports have described investigations of the distribution of DNA in nuclei and chromosomes with DNAspecific antibodies (Hansmann and Falk, 1986; Thiry and Thiry-Blaise, 1989; Martin et al., 1992; Thiry, 1992). The presence of regions in chloroplasts and mitochondria in which antibody-conjugated gold particles were precipitated has been reported as an incidental observation in research processes on the nuclei. For example, Hansmann and Falk (1986) reported, in addition to their observations of nuclei and nucleomorphs, that DNA-containing regions were vis-

1162 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka Fig. 5. Sites of gold particles in the three successive sections in Fig. 4 were superimposed to generate the respective profiles (see text for details).

4 1162 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka Fig. 5. Sites of gold particles in the three successive sections in Fig. 4 were superimposed to generate the respective profiles (see text for details). 45,000. Fig. 4. Nucleoids in chloroplasts in three successive sections (A through C). Arrows, cores transversely cut; arrowheads, cores obliquely cut (see text for details). Bar, 0.2 µm. ible around the center of chloroplasts and that isolated DNA-containing regions were visible in the mitochondria of Cryptomonas. In Thiry s paper (1992), a photograph showing the deposition of gold particles on chloroplast and a similar photograph on the mitochondria of Chlamy - domonas are included among photographs concerning the main topic of the detection of DNA within the nucleolus. One of the essential procedures for immunocytochemical detection of extremely small structures is the examination of serial sections, which is necessary to confirm that deposits of gold particles appear at the same positions in organelles in successive sections. This analysis eliminates the possibility that non-specific or artifactual deposits will be misinterpreted as specific deposits. Without an examination of serial sections, deposits of antibody-coated gold particles do not provide convincing evidence for the location of DNA. Previous studies on the detection of organellar DNA failed to include this procedure. In the present study, gold particles in each serial section were examined and compared with those in adjacent sections. The deposits in one section matched those in the adjacent sections. Thus, the possibility that the particles were deposited artifactually is clearly excluded, and the distribution pattern of antigen DNA in the two kinds of organelles appears convincing. The figure constructed from 3 serial sections (Fig. 5) shows that the nucleoids in chloroplasts are rather evenly distributed in the inter-thylakoid regions and are constructed with twisted threads of nm in diameter much more twisted than can be recognized by fluorescence microscopy (Fig. 1). The twisted threads were entangled to form thicker nodes of nm in diameter. The way in which the threads are entangled in the nodes could not be clarified. As judged from the reconstructed images, nucleoids in the chloroplasts appeared to have no standard shape. Most nucleoids in the mitochondria were spherical or ovoid and they were smaller than those in the chloroplasts. However, large nucleoids (Fig. 9) appeared to be composed of twisted threads of nm in diameter, being fundamentally similar in structure to the nucleoids in the chloroplasts. Mitochondrial nucleoids have been isolated from the mitochondria of Physarum (Suzuki et al., 1982). They are µm in diameter and µm in length, and they are composed of DNA filaments that are packed compactly into three-dimensional rod-shaped structures. They are larger than those in Euglena and similar in size to the nucleoids in the chloroplasts of Euglena. The twisting or the folding of DNA-containing threads seems to be more complicated in the nucleoids of the chloroplasts of Euglena than in the isolated mitochondrial nucleoids of Physarum. One of the important findings in the present study is the presence of cores in the nucleoids. These cores were found in the nucleoids of both chloroplasts and mitochondria. There are two possibilities about the nature of such core; they may be real cores with some specific chemical con-

Detection of DNA in the nucleoids 1163 Fig. 6. Two mitochondria, each containing a nucleoid with gold deposits, in three successive sections (A through C). Bar, 0.2 µm. Fig. 7.

5 Detection of DNA in the nucleoids 1163 Fig. 6. Two mitochondria, each containing a nucleoid with gold deposits, in three successive sections (A through C). Bar, 0.2 µm. Fig. 7. Mitochondrial nucleoid with gold deposits that appear in two of four successive sections (A through D). Bar, 0.2 µm. Fig. 8. A mitochondrial nucleoid that appeared in three of five successive sections (A through E). Arrow, nucleoid core. Bar, 0.2 µm. Fig. 9. A large mitochondrial nucleoid in three successive sections (A through C). Arrows, nucleoid cores. (D) Gold particles in (B) and (C) are superimposed. Bar, 0.2 µm. Fig. 10. (A,B) Nucleoids of mitochondria with higher electron-density than matrix. Bar, 0.2 µm. Fig. 11. Pairs of nucleoids arranged end to end (A) and side by side (B). Bar, 0.2 µm.

6 1164 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka stituents or they may be spaces surrounded by DNA threads. Since most cores were more electron-dense than matrixsubstances, it seems likely that they were real cores. We plan to examine whether the cores contain histones, RNA, or enzymes, such as RNA polymerases. Around the nucleoid regions, which were specified by the deposition of gold particles, no special regions could be recognized that correspond to the electron-transparent zones of nucleoids seen in organelles fixed by Kellenberger s or related fixatives (Nass and Nass, 1963a,b; Yokomura, 1967a,b). Moreover, the distribution of gold particles in the present work was very different from profiles of DNA fibrils seen in organelles by those fixations. DNA fibrils appeared in some cases as thick fibrils and in other cases as fine networks without any fundamental organization by such fixations. Kellenberger s fixative may introduce artifacts by leaching out certain substances from the nucleoids and causing aggregation that results in the formation of DNA fibrils and the electron transparent zones around them. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES Coleman, A. W. (1978). Visualization of chloroplast DNA with two fluorochromes. Exp. Cell Res. 114, Coleman, A. W. (1985). Diversity of DNA configuration among classes of eukaryote algae. J. Phycol. 21, Cramer, M. and Myers, J. (1952). Growth and photosynthetic characteristics of Euglena gracilis. Arch. Microbiol. 17, De May, J. and Moeremans, M. (1986). The preparation of colloidal gold probes and their use as marker in electron microscopy. In Advanced Techniques in Biological Electron Microscopy, vol. 3 (ed. G. Koehler), pp Berlin, Heidelberg, New York, Tokyo: Springer. Hansmann, P. and Falk, H. (1986). Ultrastructural localization of DNA in two Cryptomonas species by use of a monoclonal DNA antibody. Eur. Cell Biol. 42, Hatano, K. and Ueda, K. (1987). Changes in number of chloroplastnucleoids in the asexual reproduction cycle of Hydrodictyon reticulatum. Cell Biol. Int. Rep. 11, Hayashi, Y. and Ueda, K. (1989). The shape of mitochondria and the number of mitochondrial nucleoids during the cell cycle of Euglena gracilis. J. Cell Sci. 93, Hayashi, Y. and Ueda, K. (1992). Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis. Physiologia Pl. 86, Kellenberger, E., Ryter, A. and Sechaud, J. (1958). Electron microscope study of DNA-containing plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J. Biophys. Biochem. Cytol. 4, Kuroiwa, T., Suzuki, T., Ogawa, K. and Kawano, S. (1981). The chloroplast nucleus: distribution, number, size, and shape, and a model for the multiplication of the chloroplast genome during chloroplast development. Plant Cell Physiol. 22, Kuroiwa, T. (1982). Mitochondrial nuclei. Int. Rev. Cytol. 75, Martin, M., Moreno Diaz de la Espina, Jimenez-Garcia, L. F., Fernandez-Gomez, M. E. and Medina, F. J. (1992). Further investigations on the functional role of two nuclear bodies in onion cells. Protoplasma 167, Miyakawa, I., Aoi, H., Sando, N. and Kuroiwa, T. (1984). Fluorescence microscopic studies of mitochondrial nucleoids during meiosis and sporulation in the yeast Saccharomyces cerevisiae. J. Cell Sci. 66, Nakamura, S., Ito, S. and Kuroiwa, T. (1986). Behavior of chloroplast nucleus during chloroplast development and degeneration in Chlamydomonas reinhardtii. Plant Cell Physiol. 27, Nass, M. M. K. and Nass, S. (1963a). Intramitochondrial fibers with DNA characteristics. I. Fixation and electron staining reactions. J. Cell Biol. 19, Nass, M. M. K. and Nass, S. (1963b). Intramitochondrial fibers with DNA characteristics. II. Enzymatic and other hydrolytic treatments. J. Cell Biol. 19, Nass, M. M. K., Nass, S. and Afzelius, B. A. (1965). The general occurrence of mitochondrial DNA. Exp. Cell Res. 37, Nemoto, Y., Nagata, T. and Kuroiwa, T. (1989). Studies on plastid-nuclei (nucleoids) in Nicotiana tabacum L. II. Disassembly and reassembly of proplastid-nuclei isolated from cultured cells. Plant Cell Physiol. 30, Suzuki, T., Kawano, S. and Kuroiwa, T. (1982). Structure of threedimensionally rod-shaped mitochondrial nucleoids isolated from the slime mould Physarum polycephalum. J. Cell Sci. 58, Thiry, M. and Thiry-Blaise, L. (1989). In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA. Eur. J. Cell Biol. 50, Thiry, M. (1992). Ultrastructural detection of DNA within the nucleolus by sensitive molecular immunocytochemistry. Exp. Cell Res. 200, Yokomura, E. (1967a). An electron microscopic study of DNA-like fibrils in Chloroplasts. Cytologia 32, Yokomura, E. (1967b). An electron microscopic study of DNA-like fibrils in plant mitochondria. Cytologia 32, (Received 16 February Accepted 14 April 1993)

(Ficus cayica ) Changes of Chloroplast Ultrastructure. Greening under Light in Etiolated. Fig Leaves

(Ficus cayica ) Changes of Chloroplast Ultrastructure. Greening under Light in Etiolated. Fig Leaves J. Japan. Soc. Hort. Sci. 59 (2) : 333-340. 1990. Changes of Chloroplast Ultrastructure and Plastid Nucleoids during Greening under Light in Etiolated Fig Leaves (Ficus cayica ) Naosuke N1i1 and Tsuneyoshi