Lysosomal compartmentation: ultra- structural aspects of the origin, development, and func- tion of vacuoles in root cells of Lepidium sativum. Ann. Bot. 36:73-81.
Vol. 116, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1973, p. 981-988 Copyright 0 1973 American Society for Microbiology
Ultrastructural Aspects of a Mutant of Schizophyllum commune with Continuous Nuclear Migration M. RAUDASKOSKI AND Y. KOLTIN Department of Botany, University of Turku, Turku, Finland, and Genetics Unit, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel Received for publication 24 July 1973
Study of a mutant of the basidomycete Schizophyllum commune with continuous migration of nuclei revealed that the mutant is characterized by vacuolization, bundles of fibrillar-like material, and microtubules. The bundles of fibrillar-like material and microtubules extend through degraded septa to adjacent cells and are found in proximity to nuclei.
Extensive intercellular migration of nuclei is heterokaryon formed by two mates with the known to occur during the sexual development same A but different B factors (15). This of some of the higher fungi (21). The cellular mutant is characterized by the breakdown of organization during the migration of nuclei in intercellular septa and continuous movement of these fungi may shed some light on the mecha- nuclei from cell to cell (9, 14, 15). Because nuclear migration is otherwise normally a trannism responsible for this phenomenon. In the basidiomycete Schizophyllum com- sient phase, the latter characteristic, together mune the morphogenetic sequence leading to with the genetic homogeneity of the mutant, the formation of the sexually fertile heterokar- makes it useful for studies of nuclear moveyon, the dikaryon, is initiated by fusions be- ment. In addition, a high occurrence of nuclear tween monokaryotic cells of two homokaryotic migration can be obtained by using monospormates and terminates in the establishment of ous cultures of a certain age (9). a mycelium with binucleate cells. The entire sequence is regulated by two incompatibility facMATERIALS AND METHODS tors, A and B, and is fully operative when the mates differ in both factor specificities (17). The B mutant of S. commune used in the study is When the mates differ in the specificity of one the one found and described by Parag (15). Synchronous development of the cells was obtained of the two factors, only part of the morphogeby the method described by Koltin and Flexer (9). A netic sequence becomes operative. After hyphal fusion and nuclear exchange dikaryon was formed between two strains carrying the mutation in the B factor and different A factors. have occurred between two homokaryons, nu- same from the fruiting bodies of the dikaryon were clei received from one of the homokaryons begin Spores spread on cellophane membrane overlying complete in other from to cell the homoto migrate cell medium. Well-separated monosporous colonies were karyon and vice versa if the two mates carry sampled after 64 h of growth at 30 C, by which time different B factor specificities. The movement the migration of nuclei had been occurring for several of the nuclei is directed toward the apical cells hours (9). The same procedure was followed with a of the homokaryons. If the mates also differ in dikaryon from two wild-type strains to permit comtheir A factor specificities, pairing between parison with normal homokaryotic mycelia. Howresident and nonresident nuclei takes place, ever, because the results did not reveal any additional beyond those reported earlier for wild-type and the paired nuclei start to divide synchro- details homokaryotic and dikaryotic mycelia (18, 19), only nously. After each division, the four nuclei are photographs of the mutant mycelium are presented in so distributed in the subsequently developing the current report. apical and subapical cells that each cell conPreparations for electron microscopy followed the tains two nuclei with different A and B factors. procedure reported by Raudaskoski (18). All the Homokaryotic strains are known which, be- specimens were viewed and photographed with an cause of a mutation in the B factor, mimic the AEI EM6B electron microscope. 981
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RESULTS Three basic features distinguish the mutant hyphae with continuous migration from the wild-type hyphae from which intercellular migration of nuclei is absent: (i) intense vacuolization, (ii) the present of bundles of fibrillar-like material extending through two or more cells, and (iii) cytoplasmic microtubules extending through a number of cells. Intense vacuolization is found especially in subapical cells of the mutant (Fig. 2, 3, 5) and is sometimes associated with poorly delimited longitudinal vacuoles (Fig. 2). Isolated pieces of cytoplasm occasionally occur in the vacuoles, which suggests that phagocytosis takes place within them (Fig. 3, 6). In the vacuolated cells, a space is noticeable between the plasma membrane and the septa; likewise, the nucleus in these cells is often separated from the cytoplasm by what appear to be vacuoles (Fig. 2). Unlike the situation in the monokaryotic and dikaryotic cells (18, 19), in the mutant the endoplasmic reticulum is not apparent, either in apical cells (Fig. 1) or in older cells (Fig. 2, 3, 5). However, some configurations (Fig. 4) suggest that the endoplasmic reticulum may be involved in the development of the vacuoles. Lomasome-like structures (11) are regularly observed in association with the cell walls (Fig. 3, 5), but the complex membranous structures recently described in the germinating hyphae of the basidiomycete Volvariella volvacea (3), and which are also commonly found in the apical cells of wild-type hyphae of S. commune (18, 19), are only seldom observed in the cells of the mutant. The bundles of fibrillar material are most conspicuous in the vacuolated cells, sometimes extending into several adjacent cells (Fig. 3). One such bundle could be traced for at least 40 ,um. The fibrillar bundles are not surrounded by a membrane, and very few ribosomes are found within the bundle, although many are found around it and between the vacuoles. The fibrillar bundles appear to be of uniform diameter, approximately 300 nm for long stretches. At high magnification the fibrils within the bundle become visible and prove to have a diameter of approximately 10 nm (Fig. 7). The longest fibril traced in the same plane was 270 nm in length. The fibrils are not always organized in bundles. Sometimes they are diffuse and less conspicuous (Fig. 5, 8, 9). Electron-dense material, which is membrane bound and connected with vesicles, often occurs in association with the diffuse fibrils (Fig. 5, 9). No clear connection between the fibrils and the nucleus is noticeable (Fig. 5).
J. BACTERIOL.
Microtubules are commonly found in the mycelium of the mutant and are frequently in proximity to nuclei. The organization of microtubules around a nucleus is shown in Fig. 10-12, which represent 3 of a series of 11 sections taken through a nucleus occupying a cell. The septa near both ends of the nucleus are degraded. One septum lacks the septal complex (Fig. 10, 11); the other is perforated at the point of connection between the septum and the lateral wall of the cell (Fig. 12), and the septal complex remains intact (Fig. 11). The nucleus might be undergoing mitotic division in view of the constriction at its center (Fig. 10), the condensation of the chromatin (Fig. 10, 11), and the appearance of the kinetochore equivalent (7) (Fig. 11, 14). However, the absence of intranuclear microtubules and cytoplasmic microtubules originating fromi the kinetochore equivalent makes this questionable. The contours of the microtubules at both ends of the nucleus indicate that they develop toward the perforation in the cross walls and continue into the adjacent cells (Fig. 10-16). The shape of the nucleus in some sections in relation to the microtubules (Fig. 13) suggests a connection between the microtubules and the nuclear envelope at some site other than that of the kinetochore equivalent.
DISCUSSION The correlation between the occurrence of intercellular migration of nuclei and specific cytological features indicates some causal relationship. Many types of vesicles and vacuoles have been described in fungi (3, 6, 8), although little is known concerning their function. In the present case, the abundance of vacuoles in the mutant hyphae and their absence in the control wild-type hyphae might reflect the disturbance of the completion of the morphogenetic sequence in the mutant. The events which lead to degradation of the complex septum and allow the migration of nuclei are normally transient. In the mutant, only part of the morphogenetic sequence is operative; and this part, which involves septal dissolution and nuclear migration, functions continuously and involves hydrolytic activity (22). The appearance of some of the vacuoles in the mutant hyphae suggests that they might contain hydrolytic enzymes (2). Excessive accumulation of these enzymes also is suggested by the gross morphology of the mutant hyphae, in which lesions in the cell wall are prominent (16, 17). Cytological observations have suggested that microfibrils are implicated in cellular move-
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FIG. 3. A bundle of fibrillar material among small vacuoles (v). Note the isolated cytoplasm in some ot the vacuoles (bars); cw, cross wall of a dissolved septum; lo, lomasome. FIG. 4. A vacuole (v) connected with endoplasmic reticulum (er). FIG. 5. A complex of fibrils, electron-dense material, and vacuoles-starting close to a nucleus (n). FIG. 6. Pieces of cytoplasm surrounded by vacuoles. 984
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NUCLEAR MIGRATION IN SCHIZOPHYLLUM
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7. The bundle of fibrillar material at a higher magnification. 8. Diffuse filaments (bars) close to the cell wall; cw, cross wall of a dissolved septum. 9. Fibrils (bars), electron-dense material (em), and vacuoles (v) from the same complex as in Fig. 5, but different section.
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FIG. 10-12. Three sections of a cell with a nucleus (n). The opening in the middle of the upper septum is evident in Fig. 10 and 11, and that of the lower septum is evident in Fig. 12. The septal swelling (ss) and the pore cap (pc) are intact in the lower septum, and the opening occurs in the cross wall (cw) of the septum. Microtubules surround the nucleus in all sections; ch, chromatin; kce, kinetochore equivalent. For a more detailed explanation, see text. 986
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FIG. 13. Another section Of the nucleus seen in Fig. 10-12 showing the end of the nucleus (n) surrounded by microtubules close to the opening in the upper septum; cw, cross wall of the dissolved septum. FIG. 14. Enlargement of the upper part of the nucleus (n) in Fig. 11; kce, kinetochore equivalent. FIG. 15. Microtubules around the same nucleus as in Fig. 10-14. Note the electron-dense material (em) at the end of some of the microtubules. FIG. 16. A bundle of microtubules passing through the opening in the lower septum. Enlargement of the lower part of Fig. 12. 987
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RAUDASKOSKI AND KOLTIN
J. BACTERIOL.
hyphae of the imperfect fungus Drechslera sorokiniment and movement of organelles (20, 23). ana. J. Ultrastruct. Res. 41:563-571. Furthermore, microfibrils have been shown to 5. Girbardt, M. 1968. Ultrastructure and dynamics of the contain actin-like or actomyosin-like proteins moving nucleus, p. 249-259. In P. L. Miller (ed.), Aspects of cell motility. Symp. Soc. Exp. Biol., 22nd. (1, 12, 13), which also indicate their involveUniversity Press, Cambridge, England. ment in the movement of cells and organelles. 6. Girbardt, M. 1969. Die Ultrastruktur der Apikalregion The bundles of fibrillar material and the diffuse von Pilzhyphen. Protoplasma 67:413-441. microfibrils in the mutant resemble the strands 7. Girbardt, M. 1971. Ultrastructure of the fungal nucleus. II. The kinetochore equivalent (KCE). J. Cell Sci. (10) or microfibrils (4) described recently in the 2:453-473. hyphae of the fungi Erysiphe graminis hordei 8. Grove, S. N., and C. E. Bracker. 1970. Protoplasmic and Drechslera sorokiniana in association with organization of hyphal tips among fungi: vesicles and nuclear movement. In S. commune, microfibrils spitzenkorper. J. Bacteriol. 104:989-1009. so far have been observed in the apical cells of 9. Koltin, Y., and A. S. Flexer. 1969. Alteration of nuclear distribution in B-mutant strains of Schizophyllum the dikaryon (18) and in the mutant mycelium commune. J. Cell Sci. 4:739-749. of the current study. Nuclear migration occurs 10. McKeen, W. E. 1972. Nuclear movement in Ervsiphe in both cases, following the nuclear division in graminis hordei. Can. J. Microbiol. 18:1333-1336. the apical cell of the dikaryon, and is continuous 11. Moore, R. T., and J. H. McAlear. 1961. Fine structure of mycota. V. Lomasomes-previously uncharacterized in the mutant. In the mutant mycelium, mihyphal structures. Mycologia 53:194-200. crofibrils are always detected in cells with 12. Nachmias, V. T., H. E. Huxley, and D. Kessler. 1970. dissolved septa, which also suggests an interreElectron microscope observations on actomyosin and actin preparations from Physarum polycephalum, and lation between the migration of nuclei and the on their interaction with heavy meromyosin subfragmicrofibrils. ment I from muscle myosin. J. Mol. Biol. 50:83-90. A correlation between the occurrence of mi- 13. Nachmias, V. T., and W. C. Ingram. 1970. Actomyosin crotubules and intracellular movements of nufrom Physarum polycephalum: electron microscopy of myosin-enriched preparations. Science 170:743-745. clei has been observed by Girbardt (5) in the D. J. 1971. Kinetic studies of septum basidiomycete Polystictus versicolor. The pre- 14. Niederpruem, synthesis, erosion, and nuclear migration in a growing valence of microtubules in the mutant hyphae B-mutant of Schizophyllum commune. Arch. Microof S. commune, their pattern of association with biol. 75:189-196. the nuclei, and their penetration into adjacent 15. Parag, Y. 1962. Mutations in the B incompatibility factor of Schizophyllum commune. Proc. Nat. Acad. Sci. cells indicate that they are also involved in the U.S.A. 48:743-750. intercellular migration of nuclei. If the nucleus 16. Raper, C. A., and J. R. Raper. 1966. Mutations modifying (Fig. 10-12) is dividing and the movements of sexual morphogenesis in Schizophyllum. Genetics 54:1151-1168. daughter nuclei are directed by the cytoplasmic microtubules, it is possible to envisage how the 17. Raper, J. R. 1966. Genetics of sexuality in higher fungi. Ronald Press, New York. openings in the septa and the orientation of the 18. Raudaskoski, M. 1970. Occurrence of microtubules and microtubules will enable these nuclei to find microfilaments, and origin of septa in dikaryotic hyphae of Schizophyllum commune. Protoplasma their way into adjacent cells.
ACKNOWLEDGMENT We thank Y. Ben-Shaul for the opportunity to start this study at the Laboratory of Electron Microscopy, Tel-Aviv University. LITERATURE CITED 1. Adelman, M. R., and E. W. Taylor. 1969. Further purification and characterization of slime mold myosin and slime mold actin. Biochemistry 8:4976-4988. 2. Berjak, P. 1972. Lysosomal compartmentation: ultrastructural aspects of the origin, development, and function of vacuoles in root cells of Lepidium sativum. Ann. Bot. 36:73-81. 3. Chang, S. T., and K. Tanaka. 1971. An electron microscope study of complex membranous structures in the basidiomycete, Volvariella volvacea. Cytologia 36:639-651. 4. Cole, G. T. 1972. Microfibrils in the cytoplasm of fertile
70:415-422. 19. Raudaskoski, M. 1973. Light and electron microscope study of unilateral mating between a secondary mutant and a wild-type strain of Schizophyllum commune. Protoplasma 76:35-48. 20. Rebhun, L. I. 1972. Polarized intracellular particle transport: saltatory movements and cytoplasmic streaming. Int. Rev. Cytol. 32:93-137. 21. Snider, P. J. 1968. Nuclear movements in Schizophyllum, p. 261-283. In P. L. Miller (ed.), Aspects of cell motility. Symp. Soc. Exp. Biol., 22nd. University Press, Cambridge, England. 22. Wessels, J. G. H. 1969. Biochemistry of sexual morphogenesis in Schizophyllum commune: effect of mutations affecting the incompatibility system of cell-wall metabolism. J. Bacteriol. 98:697-704. 23. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. Luduena, E. L. Taylor, J. T. Wrenn, and K. M. Yamada. 1971. Microfilaments in cellular and developmental processes. Science 171:135-143.
