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_??_1992 The Japan Mendel society Cytologia 57: 515 -523 , 1992

Fine Structure of Centrosome Complex and its Connection with Nucleus in the Slime Mould, Physarum polycephalum

T. Ohta, S. Kawano and T. Kuroiwa

Department of Biology , Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan

Accepted September 30, 1992

Nuclear migration plays a fundamental role in many developmental processes in both higher and lower (for review see Meindl 1983, Raff and Glover 1989). This migra tion is also observed during the amoebo-flagellate transformation of the slime mould , Physarum spp. (Schuster 1965, Aldrich 1968, Goodman 1980, Uyeda and Furuya 1985). The is located at the center of the cell in amoeba cells but at the cell periphery , bound tightly to the b ase of flagella in swarm cells . During the amoebo-flagellate transformation of P. polycephalum, the cell nucleus first forms a sharp projection in the direction of movement . The amoeba cell then transforms into a comma-shaped swarm cell with flagella . The centrosome is first located on the tip of the projected cell nucleus and always moves ahead of it. This centrosome migra tion was shown to play an essential role in cell-nuclear migration during the amoebo-flagellate transformation (Ohta et al. 1991). Nuclear migration mediated by the centrosome has also been observed druing the embryogenesis of (Zolakar and Erk 1976, Raff and Glover 1989). Since nuclear migration was shown to be mediated by the centrosome, the centrosome would be expected to be strongly connected to the cell nucleus (Ohta et al. 1991). In most eukaryotes, the centrosome can be found in close proximity to cell nucleus. To maintain this spatial relationship, some type of connecting structure should exist universally between the centrosome and the cell nucleus. Since the centrosome functions as a organizing center (MTOC) (McIntosh 1983, Wheatley 1982) and several have indeed been observed to run between the centrosome and the cell nucleus, microtubules are generally believed to function as the connecting structure which anchor the centrosome. In P. polycephalum it has been suggested that a structure called mtoc, which is the microtubule organizing center as sociated with one of the two , is connected to the cell nucleus through microtubules (Wright et al. 1979, 1980a). However, both anchorage of the centrosome to the cell nucleus and cell-nuclear migration mediated by the centrosome were observed even after destruction of microtubules with a specific inhibitor, nocodazole (Ohta et al. 1991). This result suggests the presence of some connecting structure other than microtubules between the centrosome and the cell nucleus in P. polycephalum. Electron micrography with thin sections of intact amoeba cell is not suitable for observing fine details of electron opaque material near the centrosome, since around the cen trioles is stained uniformly by the conventional staining method. It is well known that cellular structures, such as the , centrioles or microtubules, are conserved after treatment with some detergents. The different concentrations of NaCl were also expected to gradually destroy the bond between so that debris of the cytoplasm would be removed while stubborn cytoskeletal structures would remain intact. Therefore, to remove the cytoplasm

Correspondence and requests for reprints: T. Ohta, Department of Biology, Faculty of Science, Uni versity of Tokyo, Hongo, Tokyo 113, Japan. 516 T. Ohta, S. Kawano and T. Kuroiwa Cytologia 57 near the centrosome, we treated amoeba cells of P. polycephalum histochemically with Triton X-100 and NaCl, and analyzed the fine structure of the centrosome and its connection with the cell nucleus using light and electron microscopy. Ca2+ was added to depolymerize micro tubules. It was shown that structures observed by Wright et al. (1979, 1980a), namely, mtoc, link, ppks (para-posterior kinetosomal structure) and extension, composed a single structural unit together with structures newly found in our observation, crown and lattice. In this report we observed the fine structure of the connection and showed that it is maintained even after microtubules are depolymerized by Ca2+.

Materials and methods

The amoeba strain NG15 (Kawano et al. 1987) of Physarum polycephalum was used. Amoebae were prepared as described in the previous paper (Ohta et al. 1991). For the per meabilization of amoebae, an equal volume of double-strength permeabilization solution (0.2 % Triton X-100, 4mM MgCl2, 0-1000mM NaCl, 2mM EGTA or 4mM CaCl2) was added to the suspension of amoebae in 10mM KPB. After 1 min, the suspension was fixed either with 3.7 % formaldehyde for 10 min (fluorescence staining) or with 2.0 % glutaraldehyde for 1 hour (electron microscopy). Double-fluorescence staining of microtubules and DNA was conducted as described in the

previous paper (Ohta et al. 1991). Triton permeabilized cells were prepared for whole mount electron microscopy by a modification of the method of Miller et al. (1970), originally used for studies of DNA transcription. 20-50 ƒÊl of sample suspension was sedimented at 1,000 g for 10 min directly onto carbon-coated grids. The grids were then negatively stained with 3

phosphotungstic acid, pH 7.0. Triton permeabilized cells in the presence of Call were pro cessed for thin section electron microscopy. After permeabilization, the cell suspension was

pre-fixed by 2.0 % glutaraldehyde in 25mM sodium cacodylate buffer, pH 7.2, for 2 hour, post fixed in 1.0 % osmium tetroxide in 25mM sodium cacodylate buffer, pH 7.2, for 2 hours and then stained in 1 % uranyl acetate solution for 2 hours at room temperature. The sample was dehydrated with graded ethanol and embedded in Spurr's epoxy resin. Ultra-thin sections were contrasted successively with 2 % uranyl acetate and Reynolds' lead nitrate. Alternatively, amoeba cells were briefly exposed to 8 % osmium tetroxide vapor and treated as above there after.

Results

An electron micrograph of the amoeba cell of P. polycephalum is shown in Fig. 1. The centrosome is located next to the cell nucleus, and a round-shaped electron opaque mtoc can be observed between the and the cell nucleus. In this electron micrograph, the mtoc appears to be directly connected to the outer membrane of the cell nucleus without intervening microtubules. This suggests that microtubules are not involved in this connection. How ever, a conventional thin-section technique such as in Fig. 1, does not produce a clear enough image of the region around the mtoc to demonstrate this possibility . To analyze the fine details of the perimeter of the centrosome, and especially the space between the cell nucleus and mtoc, we used Triton X-100 and different concentrations of NaCl to remove obstructive cytoplasm around the centrosome and the cell nucleus. The effects of 100mM and 1000mM NaCl on the amoeba cells permeabilized with Triton X-100 were ex amined by light and electron microscopy . In this lysis solution, we always used 2mM EGTA to exclude Ca2+ ion which depolymerizes microtubules. The treated cells were stained with 1992 Centro some Is Connected to Cell Nucleus 517

DAPI, anti- antibody and observed under equipped with phase-contrast (Fig. 2). Cells fixed with formaldehyde without treatment with NaCl and Triton X-100 were also observed as control. Control cells retained their cyto plasm and cellular microtubules. Mito chondrial nuclei () could still be observed around the cell nuclei (Fig. 2a, b, c). With 100mM NaCl, microtubular flu orescence was mostly degraded (Fig. 2e). Mitochondrial nuclear fluorescence was lost from around cell nucleus (Fig. 2f). In the phase contrast image, a filamentous appen dage could be seen extruding from the cell nucleus (Fig. 2d) and microtubular fluores cence originated from this structure. No sign of cytoplasm other than this appendage was visible under this condition. With 1000 mM NaCl, the amoeba cell nucleus became dispersed (Fig. 2g, i). The phase contrast image showed two black dots with a short tail and microtubular fluorescence was ob Fig. 1. Electron micorgraph of thin-sectioned amoeba cell showing the connection between the served as paired dots (Fig. 2h), correspond cell nucleus and the centrosome. The outer mem ingly, which indicates that these paired dots brane of the cell nucleus and mtoc of the centro represent centrioles visible at light microsco some seem to be directly connected to each other (indicated by arrows). Microtubules are barely pic level. distinguishable. N; cell nucleus, M; mtoc, L; link, AC; anterior centriole. Bar indicates 200 nm.

Fig. 2. Effect of NaCl on Triton X-100-treated amoeba cell. Cells were observed under phase contrast light microscope (a, d, g) after staining with anti-tubulin antibody (b, e, h), DAPI (c, f, i). Control cells (a, b, c) were treated in the absence of NaCl. Cells were first treated with Triton X -100 and EGTA in the presence of 100mM (d, e, f) or 1000mM (g, h, i) NaCl. Arrowheads in dicate the location of the centrosome. Bar indicates 2 ƒÊm. 518 T. Ohta, S. Kawano and T. Kuroiwa Cytologia 57

These treated amoeba cells were examined by whole mount electron microscopy (Fig. 3). The cell-nuclear appendage and microtubules were now visible in detail because the obscuring cytoplasm had been degraded with 100mM NaCl (Fig. 3a). Two components of the cell nuclear appendage which had been revealed by light microscopy, i.e., two dots and a short tail, were confirmed to be centrioles and a filamentous structure composed of two parallel filaments,

Fig. 3. Whole-mounted, negative-stained electron micrographs showing the effect of NaCl on amoebae. Amoebae were treated with Triton X-100 and EGTA in the presence of 100mM (a) or 1000mM (b) NaCl. M; mtoc, L; link, AC; anterior centriole, PC; posterior centriole, Cr; crown, P; para-posterior kinetosomal structure, E; extension, 4; microtubular array 4, Lt; lattice and N; cell nucleus. Bar indicates 200 nm. 1992 C entrosome Is Connected to Cell Nucleus 519

respectively. One of the two centrioles lo cated near the cell nucleus is called the anterior centriole and the other the posterior centriol e. Thin fibers were seen to connect the fila mentous structure like rungs in a ladder . A microtubular network was also observed . These res ults show that the connection between the centrosome and the cell nucleus persists under conditions that exclude other cytoplasmic structures such as mito chondria, membrane bound and so on (Fig . 2b). With 1000mM NaCl, the cell nucleus itself was disrupted and the connection between

Fig. 4. Effects of Ca2+ on amoeba cells permeabilized in the presence of 0mM (a-c) or 100mM

(d-f) NaCl. Cells were observed under phase contrast light microscope (a, d) after staining with anti-tubulin antibody (b, e), DAPI (c, f). Arrowheads indicate the location of centrosomes. Although most microtubular fluorescence was lost, paired spots of flourescence were observed over centrosomes. Whole-mounted negative-stained electron micrograph (g) and thin section

(h) of amoeba cell permeabilized in the presence of Ca2+ and 100mM NaCl. Arrows indicate the linkage site between the centrosome and the cell nucleus. Abbreviations are the same in Fig. 3. Bars indicate 2 ƒÊm (a-f) and 200 nm (g, h), respectively. 520 T. Ohta, S. Kawano and T. Kuroiwa Cytologia 57 the centrosome and the cell nucleus was not maintained (Figs. 2g, h, i and 3b). Therefore, the centrosome was released from the cell nucleus and isolated as a single unit. Its structure was more clearly visible since the cytoplasmic microtubules and other cytoplasmic debris around the centrosome were mostly removed at this high salt concentration. The centrosome was composed of 2 centrioles, mtoc, link, ppks and extension, which were already reported by Wright et al. (1979, 1980a). In addition, crown and lattice also participated in the composi tion of centrosome. Since the centrosome of amoeba of Physarum polycephalum consists of so many structures, it will be called the centrosome complex for simplicity hereafter. The mtoc was a globular, somewhat hexagonal amorphous material and was connected to the cell nucleus on one side. The link was a rectangular sharp-contoured structure, with one end buried into the mtoc and the other end attached to the anterior centriole. The ppks is located on the side of the posterior centriole, and the extension protrudes from this structure. They were two different ladder-like structures entangled with each other. One had long, ir regular rung intervals and indistinct rungs, while the other had short, regular intervals between clearly visible rungs. Microtubules were also observed to emanate from the centrosome and some ran in the direction of the cell nucleus. The crown was on the distal end of each centriole and was composed of two layers of thin filaments. The lattice was composed of 6-7 rows and was only seen with 1000mM NaCl condition. It started from the posterior centriole and ran along the ladder in the area where microtubular array 4 (Wright et al. 1980a) formerly existed. To examine the possibility that the centrosome complex is connected directly to the cell nucleus without microtubules, Ca2+ ion was added to the lysis solution to depolymerize and eliminate the effects of microtubules on the cell nucleus-centrosome linkage. The amoeba cells treated with Triton X-100 were simultaneously rinsed in 0mM (Fig. 4a-c) or 100mM (Fig. 4d-f) NaCl solutions, both containing 2mM Ca2+. The treated cells were stained with DAPI (Fig. 4c, f), anti-tubulin antibody (Fig. 4b, e) and observed under fluorescence micro scope equipped with phase-contrast (Fig. 4a, d). Under these conditions, microtubular fluo rescence was only detected on the centrosome as two spots (Fig. 4b, e) and the cellular micro tubular network was no longer visible, indicating the depolymerization of microtubules, except those which composed the two centrioles. Even after the depolymerization of microtubules with 100mM NaCl and 2mM Ca2+, the cell nucleus-centrosome linkage remained intact. It should be noted that microtubules which were previously believed to maintain the connec tion between the centrosome and the cell nucleus could not be detected between them even though their connection was still maintained. The treated cells were observed by whole mount electron micrography (Fig. 4g). The components of the centrosome complex and their structures were almost identical with those under conditions chelating Ca2+ (Fig. 3a), except for the absence of cytoplasmic microtubules. This electron micrograph clearly shows that the structure which connects the centrosome com plex to the cell nucleus is mtoc. On the other hand, thin section electron micrography of the treated cells also showed the linkage site between the mtoc and the cell nucleus, as well as other structures of the centrosome complex, in detail (Fig. 4h). The cell nucleus still retained traces of its outer and inner membranes. The mtoc of the centrosome complex was directly con nected to the outer membrane of the cell nucleus without any intervening microtubules.

Discussion

The treatment of amoeba cells with Triton X-100 and NaCl was very useful in revealing the fine structure of the centrosome complex at the electron microscopic level. The centrosome complex is composed of several structures: two centrioles, mtoc, link, ppks, extension, crown and lattice. Our observations of the fine structures of the centrosome complex support the 1992 Centrosome Is Connected to Cell Nucleus 521 earlier findings (Wright et al. 1979, 1980a, 1980b, 1985)and also provide some new information regarding this , which is listed below. (1) The crown was located on the distal end of two centrioles and had a two-layered structure. From its location, the flagella presumably extends from this structure. This structure has not been observed in the amoeba cell of Physarum polycephalum. (2) The lattice was a net-like structure and composed of 6-7 rows of meshes. It was only observed after cytoplasmic microtubules had been destructed. Since a bundle of microtubules, microtubular array 4 (Wright et al. 1979, 1980a), was present in the same region before depoly merization, the lattice may support this microtubular array. This is the first report of this structure. (3) Since microtubules composing centrioles were preserved after Call treatment which de polymerized other cytoplasmic microtubules, some additional entity(ies) must support this structure. (4) The link seemed to provide flexibility to centriole orientation. (5) The ppks and its extension was a combination of two ladder-like structures with different rung intervals. It was not clear where the distal end of the extension connected, and this in formation might be critical for an understanding of its function. These structures form a single unit in the cell and are resistant to 1 M NaCl which indicates the high stability of this complex. This property must be important in their function since cell-nuclear migration is mediated by the entire centrosome complex, and not by its separate parts. It has long been known that the centrosome exists consistently near the cell nucleus (Wheatley 1982). However, there has been no satisfactory explanation for this observation other than the suggestion that microtubules play some role in this anchorage. It this report using P. polycephalum, we demonstrated that the centrosome complex and the cell nucleus are tightly connected to each other and that this linkage is maintained by the binding of the cell nuclear membrane and the distal end of mtoc from the centriole. This does not deny the former suggestion that microtubules are connecting these two structures. Since cytoplasmic microtubules originating from mtoc were actually surrounding the cell nucleus and running very close to the nuclear membrane (Fig. 3), it is possible that these microtubules also par ticipate in the maintenance of the connection as described by Wright et al. (1985). It should be emphasized in our experiment, however, that the connection is preserved even after the de polymerization of microtubules with Ca2+ ion. This shows that the connection between the centrosome and the cell nucleus is mediated not by the microtubules but rather by direct linkage of mtoc to the membrane of the cell nucleus in this organism. It is intriguing that this amor phous globular mtoc has two simultaneous functions. It serves as both the microtubule or ganizing centrer and also as the connection between the centrosome complex and the cell nucleus. These two functions seem to be performed on different sites of mtoc: connection with the cell nucleus occurs on the distal end from the centriole and microtubule organization occurs on the side. The fact that there is no clear structural difference between these two sites raises a question concerning the organization of mtoc, which in turn leads to the further ques tion of what actually connects the mtoc to the cell nucleus. Some material(s) must connect these two structures. Our next challenge will be to pursue the morphological and molecular character of mtoc and some connecting material(s).

Summary

Cell-nuclear migration during the amoebo-flagellate transformation of Physarum poly cephalum is mediated by the centrosome. The centrosome is expected to be strongly connected 522 T. Ohta, S. Kawano and T. Kuroiwa Cytologia 57

to the cell nucleus. An electron micrograph of a thin section of intact amoeba cell suggested that one of the structures consisting centrosome complex, mtoc, was directly attached to the outer membrane the of cell nucleus without any intervening microtubules. To analyze the fine details of the centrosome perimeter, and especially the space between the cell nucleus and mtoc, we used 0.1 % Triton X-100 and different concentrations of NaCl to remove ob structive cytoplasm around the centrosome and the cell nucleus. The centrosome complex was shown to be composed of 2 centrioles, mtoc, link, ppks, extension, crown and lattice. With 100 mM NaCl, the connection between the centrosome complex and the cell nucleus remained intact and was visible as a cell-nuclear appendage even by light microscope. This linkage was maintained even when 2 mM Ca2+ ion was added to the samples to depolymerize and eliminate the effects of microtubules. This shows that the structure which connects the centrosome complex to the cell nucleus is mtoc, not microtubules. Electron micrography using whole mounts and thin sections of the treated amoeba cells demonstrated that the cell nucleus retained traces of its outer and inner membranes, and that the mtoc of the centrosome complex was directly connected to the outer membrane of the cell nucleus.

Acknowledgements

This work was supported by a Grant-in Aid (T. K.) for Original and Creative Research Project on Biotechnology on the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.

References

Aldrich, H. C. 1968. The development of flagella in swarm cells of the myxomycete Physarum flavicomum. J. gen. Microbiol. 50: 217-222. Goodman, E. M. 1980. Physarum polycephalum: A review of a model system using a structure function ap proach. Int. Rev. Cytol. 63: 1-58. Kawano, S., Anderson, R. W., Nanba, T. and Kuroiwa, T. 1987. Polymorphism and uniparental inheritance of mitochondrial DNA in Physarum polycephalum. J. Gen. Microbiol. 133: 3175-3182. Kilmartin, J. V., Wright B. and Milstein, C. 1982. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J. Cell Biol. 93: 576-582. McIntosh, J. R. 1983. The centrosome as an organizer of the cytoskeleton. Mod. Cell Biol. 2: 115-142. Meindl, U. 1983. Cytoskeletal control of nuclear migration and anchoring in developing cells of Micrasterias denticulata and the change caused by the anti-microtubular herbicide amiprophosmethyl (APM). Protoplasma 118: 75-90. Miller, O. L., Hamkalo, B. A. and Thomas Jr., C. A. 1970. Visualization of bacterial genes in action. Science (Wash. D. C.) 169: 392-395. Ohta, T., Kawano, S. and Kuroiwa, T. 1991. Migration of the cell nucleus during the amoebo-flagellate trans formation of Physarum polycephalum is mediated by an actin-generated force that acts on the centro some. Protoplasma 163, 114-124. Raff, J. W. and Glover, D. M. 1989. Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos. Cell 57: 611-619. Schuster, F. L. 1965. Ultrastructure and morphogenesis of solitary stages of true slime molds. Protistologica 1:49-62. Uyeda, T. Q. P. and Furuya, M. 1985. Cytoskeletal changes visualized by fluorescence microscopy during amoeba-to-flagellate and flagellate-to-amoeba transformations in Physarum polycephalum. Pro toplasma 126: 221-232. Wheatley, D. N. 1982. The Centriole: A central enigma of . Elsevier Biomedical Press. Am sterdam, North Holland. Wright, M., Moisand, A. and Mir, L. 1979. The structure of the flagellar apparatus of the swarm cells of Phy sarum polycephalum. Protoplasma 100: 231-250. -, Mir, L. and Moisand, A. 1980a. The structure of the pro-flagellar apparatus of the amoebae of Physarum polycephalum. Protoplasma 103: 69-81. 1992 Centrosome Is Connected to Cell Nucleus 523

- , Moisand, A. and Mir, L. 1980b. Centriole maturation in the amoebae of Physarum polycephalum. Pro toplasma 105: 149-160 .-, Mir, L. and Moisand, A. 1985. Spatial relationships between the anterior centriole and the mitotic center during interphase in the amoebae of the myxomycete Physarum polycephalum. Developmental Genetics 5: 227-238. Zolakar, M. and Erk, I. 1976. Division and migration of nuclei during early embryogenesis of . J. Microbiol. Cell. 25: 97-106.