J. Cell Sci. 4, 739-749 (1969) 739Printed in Great Britain

ALTERATION OF NUCLEAR DISTRIBUTION IN

5-MUTANT STRAINS OF SCHIZOPHYLLUM

COMMUNE

Y. KOLTIN* AND A. S. FLEXER

Biological Laboratories, Harvard UniversityCambridge, Massachusetts, U.S.A.

SUMMARY

Sexual morphogenesis in Schizophyltum commune, a higher basidiomycete, is controlledby two incompatibility factors, A and B. A key event, the migration of nuclei from each matethroughout the mycelium of the other, is controlled by the B factor and occurs in A = B 4=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 isfound in homokaryons carrying a mutation in the B factor. Because of their developmentalhistory, strains that carry a mutated B factor offer a relatively simple system for the study ofthe events associated with nuclear migration. Growth of mutant-i? germlings occurs in threestages: (I) most cells are binucleate; (II) most cells are uninucleate; (III) cells contain variednumbers of nuclei. The ratio of nuclei: cells remains constant during the transition from stage IIto stage III . Changes in nuclear distribution result from movement of the nuclei from cell tocell, and the movement is associated with the disruption of the dolipore septum. The mutant-fisystem appears to offer an opportunity for the biochemical resolution of the events related tonuclear migration.

INTRODUCTION

Sexual morphogenesis in Schizophyllum is regulated by two incompatibility factors,the A factor and the B factor (Raper, 1966). Each factor regulates part of a morpho-genetic sequence that operates after a mating between two homokaryons and leads tothe formation of a heterokaryon. The morphogenetic sequence consists of the followingevents: (a) hyphal fusion; (b) reciprocal nuclear exchange; (c) migration of nucleifrom each mate through the mycelium of the other; (d) pairing of a resident nucleusand non-resident nucleus in an apical cell; (e) formation of a hook-cell, a lateralprotrusion of an apical cell; (/) conjugate division of the paired nuclei; (g) septationof the hook-cell and the main hypha; (h) fusion of the hook-cell with the subterminalcell to form a clamp connexion.

Only when the two mates have different v4-factor and fi-factor specificities doesthe 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.

740 Y. Koltin and A. S. Flexer

karyon, the dikaryon. With the exception of hyphal fusion, all other events areregulated 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 thesubapical 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 thefunctioning of only part of the morphogenetic sequence and in the formation ofa characteristic heterokaryon (Raper, 1966). Such heterokaryons are mimicked byhomokaryons carrying primary mutations in one of the incompatibility factors(Parag, 1962; Raper, Boyd & Raper, 1965; Koltin & Raper, 1966).

The formation of the dikaryon involves the conversion of mycelia constituted ofuninucleate cells to mycelia constituted of cells containing paired compatible nuclei.This conversion entails the extensive migration of nuclei through an establishedmycelium (Snider & Raper, 1958). The important problem of the regulation ofnuclear migration has acquired a new dimension in the light of recent studies of thefine structure of the complex dolipore septa reported in many higher basidiomycetes(Girbadt, 1966; Moore & McAlear, 1962; Bracker & Butler, 1963; Giesy & Day,1965). As reported by Jersild, Mishkin & Niederpruem (1967), the dolipore septaof Schizophyllum are, in most respects, typical and consist of a septal plate piercedby a dolipore, and a pair of membranous parenthesomes (see below). The septalplate is continuous with, and perpendicular to, the hyphal wall. A central annularswelling in the plate is pierced by a narrow pore, the dolipore. These structures areexternal to the plasma membrane, which is continuous through the dolipore betweenadjacent cells. To either side of the plate and opposed to the dolipore is a perforatedhemisphere of multilayered membrane, the parenthesomes. The dolipore septa ofSchizophyllum appear to differ in two respects from those described in other basidio-mycetes. First, the parenthesomes are not obviously continuous with the endo-plasmic reticulum as is the case in many higher basidiomycetes (Girbardt, 1966).Secondly, the opening of the dolipore into each cell is partially occluded by a smalltorus of electron-opaque material of uncertain function. It is generally held that theseptal apparatus obstructs the intercellular movement of nuclei in homokaryons anddikaryons. The control of nuclear migration, therefore, entails not only the regulationof the direction of movement but also the facilitation of the passage of nuclei throughthe elaborate dolipore septa. The present study supports earlier suggestions thatdisruption of the septal structures permits the migration of nuclei (Giesy & Day,1965; Raper & Raper, 1966).

A system is introduced here that should make possible the study of biochemicalevents implicated in nuclear migration, a study which has previously been hamperedby the constitution of the A = B 4= heterokaryon, a mycelial mosaic consisting ofhomokaryotic as well as heterokaryotic elements. A primary mutation in the B factorremoves the control normally imposed by the B factor. It is therefore possible tosynthesize a fertile dikaryon from two strains carrying the same mutated B factorand different A factors. Basidiospores from such a dikaryon are initially binucleate,but the monosporous mycelia into which they develop are soon transformed intoclose mimics of an A = B 4= heterokaryon. These germlings thus provide a simplified

Nuclear distribution in mutant Schizophyllum 741

model system of what may occur during the initial stages of the interaction of twomated homokaryons and, particularly, of the mechanism of nuclear migration. Theadvantage here is that all cells are genotypically identical with respect to the Bfactor, and their growth is approximately synchronous. By contrast, a mating betweenstrains 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,

1965)-

MATERIALS AND METHODS

The strains employed here carried the mutated B factor originally isolated andcharacterized by Parag (1962).

The distribution of nuclei in mycelia of 5-mutant strains was observed by phase-contrast microscopy in mycelia grown on cellophane membranes that overlaysemi-solid medium as described by Raper (1966). Cultures were grown at 30 °C ona minimal medium (Snider & Raper, 1958) containing 1 % glucose. With the exceptionof the observations of live material, all observations with the light microscope wereof mycelia stained with Mayer's haemalum (McManus & Mowry, i960; Raper &Raper, 1966). The distribution of nuclei was followed from the time spores weresown on the membranes until irregular nuclear distribution was observed.

Materials for electron microscopy were fixed in vapours of glutaraldehyde andpost-fixed in vapours of osmium tetroxide (Hepler & Jackson, 1968), dehydrated inmethyl cellosolve, absolute ethanol and propylene oxide (Feder & O'Brien, 1968),and embedded in a mixture of Epon and Araldite (Mollenhauer, 1964). Sections cutwith a diamond knife on a Porter-Blum Ultramicrotome were collected on uncoatedcopper grids, stained with uranyl acetate and with lead citrate (Reynolds, 1963) andexamined in an RCAEMU-3D electron microscope.

OBSERVATIONS

The developmental history of the spores and germlings of mutant B strains ischaracterized by two striking changes in the distribution of nuclei (Fig. 1 and Table 1).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 ofnuclei 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 describedby Jersild et al. (1967) for germlings of normal homokaryons of S. commune. Thedistribution of nuclei during stage III is typical of the pattern known in A= 54=heterokaryons and in homokaryotic cells of .B-mutant mycelia of S. commune (Raper,1966; Raper & Raper, 1966).

Stage I. The binucleate condition of the spores is maintained in the germlingsfor at least 11 h after inoculation. The transition to stage II involves a sharp changein the proportion of uninucleate and binucleate cells. The proportion of uninucleatecells increases concurrently with the decrease in binucleate cells and reaches amaximum 24 h after inoculation. The inverse relationship between uninucleate and

742 Y. Koltin and A. S. Flexer

u

100 - i

v8 0 -

60 -

40 -

Stage I

I

Ii \

Stage II Stage

l^ f f - *" ,T , t | , 1-10 20 30 40 50

Hours after inoculation

60 70

Fig. i. Nuclear distribution in germlings of B-mutant strains of Schizophyllum. Allhorizontal lines represent the means of statistically homogeneous data (see Table 2).O, anucleate cells; A, uninucleate; V, binucleate; • , multinucleate.

Table 1. Distribution of nuclei in germlings of B-mutant strains

Stage

I

TR*

II

TR*

III

Time(h)

0

46-5

1 1

172O-S

2 42 8

30

31

4552

5355

586469

No.of cellsscored

109

115

i ° 5110

133114

113118

56109

103

108

1 0 2

105

108

99114

OA

No.

0

0

0

0

1

42

2

4456

316

3732

3 i

0//o

0

0

0

0

I

42

2

7456

315

3432

27

Cells with

1A

No.

17

91

4

5178

i°5106

4494879578764 0

32

45

• TR =

specified no.

/o

16

81

3

3868

939 0

78868487767 2

37324 0

No.

9 i

i ° 51 0 2

1 0 6

793°

61 0

69

1 0

6

15

918

232 2

transition.

of nuclei

2A

S

%

8391

9797

5926

51 0

11

81 0

6

15

917

23

19

>

No.

1

1

2

0

2

2

0

0

2

2

1

1

64

131 2

16

Pooled

2

/o

I

I

2

O

2

2

O

O

42

1

1

64

1 2

13

14

value

Total noof

nuclei

2 0 2

2 2 1

211

2 1 6

215144

117

126

62

1 2 0

113110

133106

127

123

148

(205-69

NucleiCells

i -8

1-92 0

2 0

•6• 3

• 0

• 1

•1

• i

•1

•0

•3•0

• 2

[ • 2

i -3h) I I S

Nuclear distribution in mutant Schizophyllum 743

binucleate cells (Fig. 1) suggests the operation of a mechanism for the coordinatedcontrol of cellular and nuclear division that leads to a 1:1 ratio of nuclei to cells.

Stage II. The uninucleate condition is fully established by 24 h after inoculationand is maintained for 28 h. More than 80% of the cells in stage II germlings areuninucleate. A constant but low frequency of anucleate, binucleate and multinucleatecells is observed in stage II germlings. These are interpreted as the result of errorsin the coordinate control of cellular division and nuclear distribution. The transitionfrom stage II to stage III is characterized by a sharp and rapid decrease in thefrequency of uninucleate cells and a concurrent increase in the porportion of anucleate,binucleate and multinucleate cells. This alteration of the distribution of nuclei iscompleted within 6 h.

Stage III. The proportion of anucleate, uninucleate, binucleate and multinucleatecells is stabilized 58 h after inoculation and is then maintained indefinitely.

Table 2. x2 contingency tests for the homogeneity of certain data from Table 1

Anucleate cells

t = 11X?» = o-oo

o-o%

t = 17Transitional

t = 20-5-53A'fj) = 6-14

3-6%

' =.55Transitional

t = 58-69A'?3) = 1-14

3 " %

Uninucleate cells

t = 0-20-5Transitional

t = 24-52*?.> = 8-74

87-S%

t = 53 and 55Transitional

t = 58-69Xr.) = 1-19

36-4%

Binucleate cells

t = 0-205Transitional

t = 24-52XM = 3-°5

7-7%

t = 53 and 55Transitional

t = 58-69tfj) = I-42

19-6%

Multinucleate cells

t - 0-52A'fn) = 9-iS

I- I %

t = 53 and 55Transitional

t = 58-69Xfn ~ °'25

12-7%

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 nucleardistribution from stage II to stage III might be the result of (a) the unobstructedmovement of nuclei from cell to cell, or (b) an uncoupling of the coordinated divisionof nuclei and cells. If the former interpretation were correct, the mean ratio ofnuclei 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 duringstage III would differ from the ratio in stage II as a result of the unequal rates ofnuclear and cellular division. The data (Tables 1, 2 and Fig. 2) show clearly thatthe ratio of nuclei to cells remains constant during stages II and III and during the

744 Y- Koltin and A. S. Flexer

transition between them. Moreover, this same ratio was also observed in mutant-5mycelia 120 h after inoculation. These data are consistent with the interpretationthat there is no uncoupling of nuclear and cellular division and that the irregulardistribution of nuclei in fact results from the movement of nuclei from cell to cell,initiated during the transitional period and continued during stage III.

2 0 -

40 60

Hours after inoculation80

Fig. 2. Nuclei/cell during development of fi-mutant germlings. All data beyond 17 h(Table 1) are statistically homogeneous on a ,Y* contingency test Q'(?,, = 5-676,P = 0-8-0-9). The pooled value of 1 15 is significantly greater than i-oo(xf1} = 12096,P < o-ooi), which suggests that, beyond 17 h, cellular division consistently lagssomewhat behind nuclear division.

The movement of nuclei during stage III was confirmed by phase-contrast micro-scopy of living germlings in stages II and III. During stage II only intracellularnuclear movement was observed, whereas during stage III nuclei were observed topass through the intercellular septa.

The intercellular movement of nuclei was thought to involve the disruption of thedolipore septum, which is generally considered to prevent such nuclear movement.The fine structure of the septa of germlings in stages II (24 and 48 h after inoculation)and III (64 h) was accordingly compared. Only normal dolipore septa were foundin stage II germlings (Fig. 4). By contrast, many of the septa in stage III germlingswere disrupted (Figs. 5-7). These and many other micrographs show that disruptionmay occur in either of two ways: (a) disruption may occur at or near the junctionof the hyphal wall and the septal plate with the plate, rather than the dolipore orparenthesomes, being the structure primarily affected; or (b) disruption may occur

Nuclear distribution in mutant Schizophyllum 745

in the central region of the septal apparatus with the disintegration of the parenthe-somes and dolipore. Incomplete septa comparable to those observed in stage IIIgermlings have been reported in A = 5 #= heterokaryons of Coprinus by Giesy &Day (1965) and in Schizophyllum by Jersild et al. (1967). Here, however, the dis-ruption of the dolipore septa can more confidently be correlated with the intercellularmovement of nuclei than in either previous report.

Comparisons among numerous germlings at various stages revealed a high degreeof uniformity throughout the course of development. The uniformity andjrelativelysynchronous development of 5-mutant germlings make this system an attractive onefor the study of the biochemical events associated with nuclear migration and withthe disruption of dolipore septa.

0-50 -1

0-40 -

3 0-30 -

0 2(H

0-10 -

30 °C, 1 % glucose ;O . 23 °C, 1 % glucose/ A

Disruption ofnuclear

distributionat 30 °C

si--*

'Disruption ofnucleardistribution

— 30 °C, 005% glucose

24—I 1 164 84 104Hours after inoculation

124 144

Fig. 3. Dependence of mycelial growth on environmental conditions. Shake culturesinitiated from 8-6 x io6 spores in liquid minimal medium were established for eachtreatment.

For this system to be useful in a search for the enzymes involved in the disruptionof dolipore septa, it would be necessary to manipulate the environmental conditionsso as to maximize the synthesis of the proteins that are directly involved and to mini-mize the synthesis of other species of proteins. To determine the feasibility ofachieving a temporal separation of the phenomena of generalized synthesis of proteinsand alterations of nuclear distribution, increases in dry weight and in the distributionof nuclei were followed under various conditions (Fig. 3).

In cultures of germlings grown at 23 °C in liquid medium containing 1 % glucose,alteration of nuclear distribution occurred 120 h after inoculation and coincidedwith the major increase in dry weight—an unfortunate coincidence. When cultureswere grown at 30 °C in liquid minimal medium containing either 1 % or 0-05 %

47 Cell Sci. 4

746 Y. Koltin and A. S. Flexer

glucose, however, the alteration of nuclear distribution preceded the major increasein dry weight. It was also observed that a further decrease in the concentration ofglucose below 0-05% enhanced somewhat the alteration of nuclear distribution.It should thus be feasible, by manipulation of environmental conditions, such astemperature and the concentration of glucose, to attain conditions in which thesynthesis of protein species involved in the disruption of dolipore septa will berelatively amplified.

DISCUSSION

Recent reports of the fine structure of A = B 4= heterokaryons of Schizophyllumand Coprinus have shown that the dolipore septa in such mycelia are frequentlydisrupted (Giesy & Day, 1965; Jersild et al. 1967). These observations are inconclusive,however, because it was impossible to determine whether the cells observed were infact located in heterokaryotic hyphae. Furthermore, occasional abnormal septa wereinterpreted in earlier reports as stages in the disruption of these structures, but thesecould equally well be considered stages in the synthesis of intercalary septa (Raper &Raper, 1966). The .B-mutant system described here avoids these ambiguities: thecells are genotypically uniform and have the phenotype of the A = B 4= heterokaryon.It is thus possible to correlate the intercellular movement of nuclei directly withultrastructural changes in the dolipore septa. The alteration in the distribution ofnuclei in B-mutant germlings, from predominantly one per cell to an irregulardistribution, corresponds in time with initiation of intercellular movements of nucleiobserved in living material and with the disruption of septal fine structure. It cantherefore be concluded that the disruption of the dolipore septa permits facile inter-cellular movement of nuclei, and this, in turn, results in an irregular distribution ofnuclei in older (more than 60 h) germlings. As long as the integrity of the doliporeseptum is maintained, each cell of a germling contains a single nucleus.

The main objective in future biochemical studies is the elucidation of the enzymicprocesses involved in the disruption of dolipore septa. Studies with A= B 4=heterokaryons are complicated by the need to eliminate the biochemical constituentscontributed by the individual strains, because the goal is the detection of the productsproduced following the interaction between the two homokaryons and not theindividual products of the interacting strains. This difficulty does not exist in theB-mutant system, because of the genotypic uniformity of the germlings. The B-mutantsystem may also be utilized to seek correlations between the activities of specificenzymes and ultrastructural changes during the various shifts in the distributionof nuclei in developing germlings. The structure of the dolipore septum suggeststhe presence of at least two major components, a membranous component anda polysaccharide component. Current biochemical studies are designed to detect,during the transition between stages II and III, elevated activities of enzymes thatmay play a role in the degradation of the major components of the septum. Currentfine-structural studies are designed to clarify the course of the disruption of doliporesepta during nuclear migration in matings of certain morphological mutants.

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 ofHealth Nos. AI06124 and GM06637.

REFERENCESBRACKER, C. E. & BUTLER, E. E. (1963). The ultrastructure and development of septa in

hyphae of Rliizoctonia solani. Mycologia 55, 35-58.FEDER, N. & O'BRIEN, T. P. (1968). Plant microtechnique: some principles and new techniques.

Am.J. Bot. 55, 123-142.GIESY, R. M. & DAY, P. R. (1965). The septal pores of Coprinus lagopus (Fr.) sensu Buller

in relation to nuclear migration. Am. J. Bot. 52, 287—294.GIRBARDT, M. (1966). Probleme der Struktur, Dynamik und Genese cytoplasmatischer

Membranen. Biol. Rdsch. 4, 1-25.HEPLER, P. K. & JACKSON, W. T. (1968). Microtubules and the early stages of cell-plate

formation in Hemantkiu catlierinae Baker. J. Cell Biol. 38, 437-446.JERSILD, R., MISHKIN, S. & NIEDERPRUEM, D. J. (1967). Origin and ultrastructure of complex

septa in Sciiizophyllum commune development. Arch. Mikrobiol. 51, 20-32.KOLTIN, Y. & RAPER, J. R. (1966). Schizophyllum commune: New mutations in the B incom-

patibility factor. Science, N.Y. 154, 510-511.MCMANUS, J. F. A. & MOWRY, R. W. (i960). Staining Methods, Histological and Histochemical.

New York: Hoeber.MOLLENHAUER, H. H. (1964). Plastic mixtures for electron microscopy. Stain Technol. 39,

111-114.

MOORE, R. T- & MCALEAR, J. H. (1962). Fine structure of Mycota. 7. Observations on septaof Ascomycetes and Basidiomycetes. Am. J. Bot. 49, 86—94.

PARAG, Y. (1962). Mutations in the B incompatibility factor in Schizophyllum commune.Proc. natn. Acad. Sci. U.S.A. 48, 743-750.

RAPER, C. A. & RAPER, J. R. (1966). Mutations modifying sexual morphogenesis in Schizo-phyllum. Genetics, Princeton 54, 1151-1168.

RAPER, J. R. (1966). Genetics and Sexuality in Higher Fungi. New York: Ronald Press.RAPER, J. R., BOYD, D. H. & RAPER, C. A. (1965). Primary and secondary mutations at the

incompatibility loci in Schizophyllum. Proc. natn. Acad. Sci. U.S.A. 53, 1324-1332.REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in

electron microscopy. J. Cell Biol. 17, 208-212.SNIDER, P. J. & RAPER, J. R. (1958). Nuclear migration in the Basidiomycete Schizophyllum

commune. Am. J. Bot. 45, 538-546.SNIDER, P. J. & RAPER, J. R. (1965). Nuclear ratios and genetic complementation in common-^4

heterokaryons of Schizophyllum commune. Am. J. Bot. 52, 547-552.

{Received 29 April 1968—Revised 30 November 1968)

47-2

748 Y. Koltin and A. S. Flexer

Fig. 4. Septal apparatus of a typical stage I germling. The laminate nature of thehyphal wall, septal plate and parenthesome membranes is evident. A torus of electron-opaque material partially occludes the opening of the dolipore, but note the cytoplasmiccontinuity, x 28000.

Fig. 5. A disrupted septum from a stage II germling. The plane of the section isdisplaced slightly from the axis of the dolipore. The parenthesomes appear to beunaffected, x 31000.

Figs. 6, 7. Disrupted septa from a stage III germling. Profiles of migrating nuclei areevident. Fig. 6, x 30000; Fig. 7, x 32030.

Nuclear distribution in mutant Schizophyllum