ALTERATION OF NUCLEAR DISTRIBUTION IN 5-MUTANT STRAINS OF SCHIZOPHYLLUM COMMUNE

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1 J. Cell Sci., (99) 739 Printed in Great Britain ALTERATION OF NUCLEAR DISTRIBUTION IN 5-MUTANT STRAINS OF SCHIZOPHYLLUM COMMUNE Y. KOLTIN* AND A. S. FLEXER Biological Laboratories, Harvard University Cambridge, Massachusetts, U.S.A. SUMMARY Sexual morphogenesis in Schizophyltum commune, a higher basidiomycete, is controlled by two incompatibility factors, A and B. A key event, the migration of nuclei from each mate throughout the mycelium of the other, is controlled by the B factor and occurs in A = B = matings. The distribution of nuclei in the resulting heterokaryon is irregular, and anucleate, uninucleate, binucleate and multinucleate cells are found. A similar distribution of nuclei is found in homokaryons carrying a mutation in the B factor. Because of their developmental history, strains that carry a mutated B factor offer a relatively simple system for the study of the events associated with nuclear migration. Growth of mutant-i? germlings occurs in three stages: (I) most cells are binucleate; (II) most cells are uninucleate; (III) cells contain varied numbers of nuclei. The ratio of nuclei: cells remains constant during the transition from stage II to stage III. Changes in nuclear distribution result from movement of the nuclei from cell to cell, and the movement is associated with the disruption of the dolipore septum. The mutant-fi system appears to offer an opportunity for the biochemical resolution of the events related to nuclear migration. INTRODUCTION Sexual morphogenesis in Schizophyllum is regulated by two incompatibility factors, the A factor and the B factor (Raper, 9). Each factor regulates part of a morphogenetic sequence that operates after a mating between two homokaryons and leads to the formation of a heterokaryon. The morphogenetic sequence consists of the following events: (a) hyphal fusion; (b) reciprocal nuclear exchange; (c) migration of nuclei from each mate through the mycelium of the other; (d) pairing of a resident nucleus and non-resident nucleus in an apical cell; (e) formation of a hook-cell, a lateral protrusion of an apical cell; (/) conjugate division of the paired nuclei; (g) septation of the hook-cell and the main hypha; (h) fusion of the hook-cell with the subterminal cell to form a clamp connexion. Only when the two mates have different v-factor and fi-factor specificities does the entire sequence function and ultimately lead to the formation of a fertile hetero- Present address: Department of Food and Biotechnology, Israel Institute of Technology, Haifa, Israel.

2 7 Y. Koltin and A. S. Flexer karyon, the dikaryon. With the exception of hyphal fusion, all other events are regulated by the incompatibility factors. The B factor controls nuclear migration; the A factor controls all other events except the fusion of the hook-cell with the subapical cell, a process that is jointly controlled by the A and B factors. Therefore, a mating between two homokaryons that differ at only one factor will result in the functioning of only part of the morphogenetic sequence and in the formation of a characteristic heterokaryon (Raper, 9). Such heterokaryons are mimicked by homokaryons carrying primary mutations in one of the incompatibility factors (Parag, 9; Raper, Boyd & Raper, 95; Koltin & Raper, 9). The formation of the dikaryon involves the conversion of mycelia constituted of uninucleate cells to mycelia constituted of cells containing paired compatible nuclei. This conversion entails the extensive migration of nuclei through an established mycelium (Snider & Raper, 958). The important problem of the regulation of nuclear migration has acquired a new dimension in the light of recent studies of the fine structure of the complex dolipore septa reported in many higher basidiomycetes (Girbadt, 9; Moore & McAlear, 9; Bracker & Butler, 93; Giesy & Day, 95). As reported by Jersild, Mishkin & Niederpruem (97), the dolipore septa of Schizophyllum are, in most respects, typical and consist of a septal plate pierced by a dolipore, and a pair of membranous parenthesomes (see below). The septal plate is continuous with, and perpendicular to, the hyphal wall. A central annular swelling in the plate is pierced by a narrow pore, the dolipore. These structures are external to the plasma membrane, which is continuous through the dolipore between adjacent cells. To either side of the plate and opposed to the dolipore is a perforated hemisphere of multilayered membrane, the parenthesomes. The dolipore septa of Schizophyllum appear to differ in two respects from those described in other basidiomycetes. First, the parenthesomes are not obviously continuous with the endoplasmic reticulum as is the case in many higher basidiomycetes (Girbardt, 9). Secondly, the opening of the dolipore into each cell is partially occluded by a small torus of electron-opaque material of uncertain function. It is generally held that the septal apparatus obstructs the intercellular movement of nuclei in homokaryons and dikaryons. The control of nuclear migration, therefore, entails not only the regulation of the direction of movement but also the facilitation of the passage of nuclei through the elaborate dolipore septa. The present study supports earlier suggestions that disruption of the septal structures permits the migration of nuclei (Giesy & Day, 95; Raper & Raper, 9). A system is introduced here that should make possible the study of biochemical events implicated in nuclear migration, a study which has previously been hampered by the constitution of the A = B = heterokaryon, a mycelial mosaic consisting of homokaryotic as well as heterokaryotic elements. A primary mutation in the B factor removes the control normally imposed by the B factor. It is therefore possible to synthesize a fertile dikaryon from two strains carrying the same mutated B factor and different A factors. Basidiospores from such a dikaryon are initially binucleate, but the monosporous mycelia into which they develop are soon transformed into close mimics of an A = B = heterokaryon. These germlings thus provide a simplified

Nuclear distribution in mutant Schizophyllum 7 model system of what may occur during the initial stages of the interaction of two mated homokaryons and, particularly, of the mechanism of nuclear

3 Nuclear distribution in mutant Schizophyllum 7 model system of what may occur during the initial stages of the interaction of two mated homokaryons and, particularly, of the mechanism of nuclear migration. The advantage here is that all cells are genotypically identical with respect to the B factor, and their growth is approximately synchronous. By contrast, a mating between strains that differ only in the B factor results in the formation of an A= + heterokaryon, of which only a portion of the cells are heterokaryotic (Snider & Raper, 95)- MATERIALS AND METHODS The strains employed here carried the mutated B factor originally isolated and characterized by Parag (9). The distribution of nuclei in mycelia of 5-mutant strains was observed by phasecontrast microscopy in mycelia grown on cellophane membranes that overlay semi-solid medium as described by Raper (9). Cultures were grown at 3 C on a minimal medium (Snider & Raper, 958) containing % glucose. With the exception of the observations of live material, all observations with the light microscope were of mycelia stained with Mayer's haemalum (McManus & Mowry, i9; Raper & Raper, 9). The distribution of nuclei was followed from the time spores were sown on the membranes until irregular nuclear distribution was observed. Materials for electron microscopy were fixed in vapours of glutaraldehyde and post-fixed in vapours of osmium tetroxide (Hepler & Jackson, 98), dehydrated in methyl cellosolve, absolute ethanol and propylene oxide (Feder & O'Brien, 98), and embedded in a mixture of Epon and Araldite (Mollenhauer, 9). Sections cut with a diamond knife on a Porter-Blum Ultramicrotome were collected on uncoated copper grids, stained with uranyl acetate and with lead citrate (Reynolds, 93) and examined in an RCAEMU-3D electron microscope. OBSERVATIONS The developmental history of the spores and germlings of mutant B strains is characterized by two striking changes in the distribution of nuclei (Fig. and Table ). Three successive stages thus occur: stage I, in which most cells are binucleate; stage II, in which most cells are uninucleate; and stage III, in which the distribution of nuclei is irregular, and cells may be anucleate, uninucleate, binucleate or multinucleate. The pattern of nuclear distribution during stages I and II is similar to that described by Jersild et al. (97) for germlings of normal homokaryons of S. commune. The distribution of nuclei during stage III is typical of the pattern known in A= 5= heterokaryons and in homokaryotic cells of.b-mutant mycelia of S. commune (Raper, 9; Raper & Raper, 9). Stage I. The binucleate condition of the spores is maintained in the germlings for at least h after inoculation. The transition to stage II involves a sharp change in the proportion of uninucleate and binucleate cells. The proportion of uninucleate cells increases concurrently with the decrease in binucleate cells and reaches a maximum h after inoculation. The inverse relationship between uninucleate and

4 7 Y. Koltin and A. S. Flexer -i v 8- I Stage I Stage II Stage - - I i \ u l^ff- *",T, t, Hours after inoculation 7 Fig. i. Nuclear distribution in germlings of B-mutant strains of Schizophyllum. All horizontal lines represent the means of statistically homogeneous data (see Table ). O, anucleate cells; A, uninucleate; V, binucleate;, multinucleate. Table. Distribution of nuclei in germlings of B-mutant strains Cells with specified no. of nuclei Stage Time (h) No. of cells scored No. O A /o No. A /o No. A % S No. > /o Total no of nuclei Nuclei Cells I TR* II TR* III -5 7 O-S i i I i i i TR = transition Pooled I I O O O 3 value (5-9 i i 3 [ i-3 h) IIS

5 Nuclear distribution in mutant Schizophyllum 73 binucleate cells (Fig. ) suggests the operation of a mechanism for the coordinated control of cellular and nuclear division that leads to a : ratio of nuclei to cells. Stage II. The uninucleate condition is fully established by h after inoculation and is maintained for 8 h. More than 8% of the cells in stage II germlings are uninucleate. A constant but low frequency of anucleate, binucleate and multinucleate cells is observed in stage II germlings. These are interpreted as the result of errors in the coordinate control of cellular division and nuclear distribution. The transition from stage II to stage III is characterized by a sharp and rapid decrease in the frequency of uninucleate cells and a concurrent increase in the porportion of anucleate, binucleate and multinucleate cells. This alteration of the distribution of nuclei is completed within h. Stage III. The proportion of anucleate, uninucleate, binucleate and multinucleate cells is stabilized 58 h after inoculation and is then maintained indefinitely. Table. x contingency tests for the homogeneity of certain data from Table Anucleate cells Uninucleate cells Binucleate cells Multinucleate cells t = X?» = o-oo o-o% t = 7 t = A'fj) = - 3-% t = --5 t = -5 *?.> = S% t = -5 t = -5 XM = % t - -5 A'fn) = 9-iS I-I % ' =.55 t = 53 and 55 t = 53 and 55 t = 53 and 55 t = 58-9 A'? 3 ) = - 3" % t = 58-9 Xr.) = -9 3-% t = 58-9 tfj) = I- 9-% t = 58-9 Xfn ~ ' 5-7% t = time (h) after inoculation. None of the values for,\'* is significant at the 5 % level. Numbers in bold type give pooled values for the mean proportion of cells with the specified number of nuclei. These observations are open to at least two interpretations. The change in nuclear distribution from stage II to stage III might be the result of (a) the unobstructed movement of nuclei from cell to cell, or (b) an uncoupling of the coordinated division of nuclei and cells. If the former interpretation were correct, the mean ratio of nuclei to cells would be expected to remain constant during stages II and III. If, however, the latter interpretation were correct, the mean ratio of nuclei:cells during stage III would differ from the ratio in stage II as a result of the unequal rates of nuclear and cellular division. The data (Tables, and Fig. ) show clearly that the ratio of nuclei to cells remains constant during stages II and III and during the

6 7 Y - Koltin and A. S. Flexer transition between them. Moreover, this same ratio was also observed in mutant-5 mycelia h after inoculation. These data are consistent with the interpretation that there is no uncoupling of nuclear and cellular division and that the irregular distribution of nuclei in fact results from the movement of nuclei from cell to cell, initiated during the transitional period and continued during stage III. - Hours after inoculation Fig.. Nuclei/cell during development of fi-mutant germlings. All data beyond 7 h (Table ) are statistically homogeneous on a,y* contingency test Q' (?,, = 5-7, P = -8--9). The pooled value of 5 is significantly greater than i-oo(xf } = 9, P < o-ooi), which suggests that, beyond 7 h, cellular division consistently lags somewhat behind nuclear division. The movement of nuclei during stage III was confirmed by phase-contrast microscopy of living germlings in stages II and III. During stage II only intracellular nuclear movement was observed, whereas during stage III nuclei were observed to pass through the intercellular septa. The intercellular movement of nuclei was thought to involve the disruption of the dolipore septum, which is generally considered to prevent such nuclear movement. The fine structure of the septa of germlings in stages II ( and 8 h after inoculation) and III ( h) was accordingly compared. Only normal dolipore septa were found in stage II germlings (Fig. ). By contrast, many of the septa in stage III germlings were disrupted (Figs. 5-7). These and many other micrographs show that disruption may occur in either of two ways: (a) disruption may occur at or near the junction of the hyphal wall and the septal plate with the plate, rather than the dolipore or parenthesomes, being the structure primarily affected; or (b) disruption may occur 8

7 Nuclear distribution in mutant Schizophyllum 75 in the central region of the septal apparatus with the disintegration of the parenthesomes and dolipore. Incomplete septa comparable to those observed in stage III germlings have been reported in A = 5 #= heterokaryons of Coprinus by Giesy & Day (95) and in Schizophyllum by Jersild et al. (97). Here, however, the disruption of the dolipore septa can more confidently be correlated with the intercellular movement of nuclei than in either previous report. Comparisons among numerous germlings at various stages revealed a high degree of uniformity throughout the course of development. The uniformity andjrelatively synchronous development of 5-mutant germlings make this system an attractive one for the study of the biochemical events associated with nuclear migration and with the disruption of dolipore septa C, % glucose ;O. 3 C, % glucose / A - - 'Disruption of nuclear distribution (H - - Disruption of nuclear distribution at 3 C si--* 3 C, 5% glucose I 8 Hours after inoculation Fig. 3. Dependence of mycelial growth on environmental conditions. Shake cultures initiated from 8- x io spores in liquid minimal medium were established for each treatment. For this system to be useful in a search for the enzymes involved in the disruption of dolipore septa, it would be necessary to manipulate the environmental conditions so as to maximize the synthesis of the proteins that are directly involved and to minimize the synthesis of other species of proteins. To determine the feasibility of achieving a temporal separation of the phenomena of generalized synthesis of proteins and alterations of nuclear distribution, increases in dry weight and in the distribution of nuclei were followed under various conditions (Fig. 3). In cultures of germlings grown at 3 C in liquid medium containing % glucose, alteration of nuclear distribution occurred h after inoculation and coincided with the major increase in dry weight an unfortunate coincidence. When cultures were grown at 3 C in liquid minimal medium containing either % or -5 % 7 Cell Sci.

8 7 Y. Koltin and A. S. Flexer glucose, however, the alteration of nuclear distribution preceded the major increase in dry weight. It was also observed that a further decrease in the concentration of glucose below -5% enhanced somewhat the alteration of nuclear distribution. It should thus be feasible, by manipulation of environmental conditions, such as temperature and the concentration of glucose, to attain conditions in which the synthesis of protein species involved in the disruption of dolipore septa will be relatively amplified. DISCUSSION Recent reports of the fine structure of A = B = heterokaryons of Schizophyllum and Coprinus have shown that the dolipore septa in such mycelia are frequently disrupted (Giesy & Day, 95; Jersild et al. 97). These observations are inconclusive, however, because it was impossible to determine whether the cells observed were in fact located in heterokaryotic hyphae. Furthermore, occasional abnormal septa were interpreted in earlier reports as stages in the disruption of these structures, but these could equally well be considered stages in the synthesis of intercalary septa (Raper & Raper, 9). The.B-mutant system described here avoids these ambiguities: the cells are genotypically uniform and have the phenotype of the A = B = heterokaryon. It is thus possible to correlate the intercellular movement of nuclei directly with ultrastructural changes in the dolipore septa. The alteration in the distribution of nuclei in B-mutant germlings, from predominantly one per cell to an irregular distribution, corresponds in time with initiation of intercellular movements of nuclei observed in living material and with the disruption of septal fine structure. It can therefore be concluded that the disruption of the dolipore septa permits facile intercellular movement of nuclei, and this, in turn, results in an irregular distribution of nuclei in older (more than h) germlings. As long as the integrity of the dolipore septum is maintained, each cell of a germling contains a single nucleus. The main objective in future biochemical studies is the elucidation of the enzymic processes involved in the disruption of dolipore septa. Studies with A= B = heterokaryons are complicated by the need to eliminate the biochemical constituents contributed by the individual strains, because the goal is the detection of the products produced following the interaction between the two homokaryons and not the individual products of the interacting strains. This difficulty does not exist in the B-mutant system, because of the genotypic uniformity of the germlings. The B-mutant system may also be utilized to seek correlations between the activities of specific enzymes and ultrastructural changes during the various shifts in the distribution of nuclei in developing germlings. The structure of the dolipore septum suggests the presence of at least two major components, a membranous component and a polysaccharide component. Current biochemical studies are designed to detect, during the transition between stages II and III, elevated activities of enzymes that may play a role in the degradation of the major components of the septum. Current fine-structural studies are designed to clarify the course of the disruption of dolipore septa during nuclear migration in matings of certain morphological mutants.

9 Nuclear distribution in mutant Schizophyllum The technical assistance of Mrs Lucille P. Gatchell is gratefully acknowledged. The research reported in this paper was supported by grants provided by the Atomic Energy Commission of the United States, No. AT (3o-i)-3875, and the National Institutes of Health Nos. AI and GM37. REFERENCES BRACKER, C. E. & BUTLER, E. E. (93). The ultrastructure and development of septa in hyphae of Rliizoctonia solani. Mycologia 55, FEDER, N. & O'BRIEN, T. P. (98). Plant microtechnique: some principles and new techniques. Am.J. Bot. 55, 3-. GIESY, R. M. & DAY, P. R. (95). The septal pores of Coprinus lagopus (Fr.) sensu Buller in relation to nuclear migration. Am. J. Bot. 5, GIRBARDT, M. (9). Probleme der Struktur, Dynamik und Genese cytoplasmatischer Membranen. Biol. Rdsch., -5. HEPLER, P. K. & JACKSON, W. T. (98). Microtubules and the early stages of cell-plate formation in Hemantkiu catlierinae Baker. J. Cell Biol. 38, 37-. JERSILD, R., MISHKIN, S. & NIEDERPRUEM, D. J. (97). Origin and ultrastructure of complex septa in Sciiizophyllum commune development. Arch. Mikrobiol. 5, -3. KOLTIN, Y. & RAPER, J. R. (9). Schizophyllum commune: New mutations in the B incompatibility factor. Science, N.Y. 5, 5-5. MCMANUS, J. F. A. & MOWRY, R. W. (i9). Staining Methods, Histological and Histochemical. New York: Hoeber. MOLLENHAUER, H. H. (9). Plastic mixtures for electron microscopy. Stain Technol. 39, -. MOORE, R. T- & MCALEAR, J. H. (9). Fine structure of Mycota. 7. Observations on septa of Ascomycetes and Basidiomycetes. Am. J. Bot. 9, 8 9. PARAG, Y. (9). Mutations in the B incompatibility factor in Schizophyllum commune. Proc. natn. Acad. Sci. U.S.A. 8, RAPER, C. A. & RAPER, J. R. (9). Mutations modifying sexual morphogenesis in Schizophyllum. Genetics, Princeton 5, 5-8. RAPER, J. R. (9). Genetics and Sexuality in Higher Fungi. New York: Ronald Press. RAPER, J. R., BOYD, D. H. & RAPER, C. A. (95). Primary and secondary mutations at the incompatibility loci in Schizophyllum. Proc. natn. Acad. Sci. U.S.A. 53, REYNOLDS, E. S. (93). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 7, 8-. SNIDER, P. J. & RAPER, J. R. (958). Nuclear migration in the Basidiomycete Schizophyllum commune. Am. J. Bot. 5, SNIDER, P. J. & RAPER, J. R. (95). Nuclear ratios and genetic complementation in common-^ heterokaryons of Schizophyllum commune. Am. J. Bot. 5, {Received 9 April 98 Revised 3 November 98) 7-

10 78 Y. Koltin and A. S. Flexer Fig.. Septal apparatus of a typical stage I germling. The laminate nature of the hyphal wall, septal plate and parenthesome membranes is evident. A torus of electronopaque material partially occludes the opening of the dolipore, but note the cytoplasmic continuity, x 8. Fig. 5. A disrupted septum from a stage II germling. The plane of the section is displaced slightly from the axis of the dolipore. The parenthesomes appear to be unaffected, x 3. Figs., 7. Disrupted septa from a stage III germling. Profiles of migrating nuclei are evident. Fig., x 3; Fig. 7, x 33.

11 Nuclear distribution in mutant Schizophyllum

Studies on Basidiospore Development in Schizophyllum commune

Journal of General Microbiology (1976), 96,49-41 3 Printed in Great Britain 49 Studies on Basidiospore Development in Schizophyllum commune By SUSAN K. BROMBERG" AND MARVIN N. SCHWALB Department of Microbiology,