FUSION AND EROSION OF CELL WALLS DURING CONJUGATION … · erosion; suture a,n electron-dense seam...

J. Cell Sci. 25, 139-155 (i977) 139Printed in Great Britain

FUSION AND EROSION OF CELL WALLS

DURING CONJUGATION IN THE FISSION

YEAST (SCHIZOSACCHAROMYCES POMBE)

G. B. CALLEJA, BONG Y. YOO AND BYRON F. JOHNSONDivision of Biological Sciences, National ResearchCouncil, Ottawa, Canada KiA 0R6 and Department ofBiology, University of Neza Brunswick, Fredericton,N.B., Canada E3B 5A3

SUMMARY

Conjugation in Schizosaccharomyces pombe was studied by transmission electron microscopy.Mural and nuclear events were scored from induction, the initial event, to meiosis I, thestart of sporulation. These morphogenic markers were separately identifiable as flocculation,copulation, conjugation-tube formation, cross-wall formation, cross-wall erosion, conjugation-tube expansion, cytoplasmic fusion, de-differentiation of site of union, nuclear migration andkaryogamy. The following were identified as new structural elements: sex hairs, which pre-sumably mediate hydrogen bonding between cells during flocculation; crimp at the site ofunion; dark patch, which presumably serves as a leak-proof seal at the time of cross-wallerosion; suture, an electron-dense seam formed by the union of a copulant pair; and smallelectron-dense particles close to the site of wall erosion. No special structures on the cell wallcould be identified as indicative of specific sites for potential copulatory activity. The dis-continuity of the 2 cell walls at the site of union became so de-differentiated after fusion anderosion that it was no longer possible to pinpoint the site of union.

INTRODUCTION

The fission yeast Schizosaccharomyces pombe is ordinarily cultured as vegetativehaplonts (Fig. 1 A). At the end of the logarithmic growth phase, however, cells may beinduced by aeration to form stable floes (Fig. IB). Some of these flocculated cellspair off and fuse to become transient diplonts, from which ascospores are produced,following 2 meiotic events (Fig. ic). The spores which are subsequently liberated(Fig. 1 D) germinate into vegetative cells (Fig. 1 E) when transferred to fresh medium.

Many interesting morphogenic events occur during this developmental sequence.We have earlier described by electron microscopy morphogenic changes duringcell division (Johnson, Yoo & Calleja, 1973, 1974) and during the period from meiosisI to sporulation (Yoo, Calleja & Johnson, 1973). This report bridges the gap, describingthe ultrastructural changes during the period from flocculation induction to zygoteformation. The emphasis is on the mural events; the behaviour of the nucleus isdiscussed only as it appears temporally related to them. Ultrastructural studies ofconjugation in other species of yeasts have been previously reported (Conti & Brock,1965; Conti & Naylor, i960; Kreger-van Rij & Veenhuis, 1975, 1976; Osumi,Shimoda & Yanagishima, 1974).

140 G. B. Calleja, B. Y. Yoo and B. F. Johnson

Fig. 1. Schematic diagram of the life cycle of Schizosaccharomyces pombe NCYC 132.The division of the diagram into frames reflects our efforts to subdivide the subjectmatter into workable areas of concentration. For emphasis, the dimensions of cells inframes c and D are twice the dimensions of cells in frames B, A and E. free, free cells;floe, flocculation; copu, copulation; conj, conjugation; zygo, zygote formation; me-i,meiosis I; me-2, meiosis II; spor, sporulation; libe, spore liberation; germ, spore germi-nation ; grow, cell division. Steps 3-6 in frame C (bracketed together) are not ordinarilyresolved by light microscopy, hence usually scored together as conjugation.

Conjugation in fission yeast 141

MATERIALS AND METHODS

Culture of organisms

A highly flocculent derivative (360-2) of Schizosaccharomyces pombe NCYC 132 (ATCC26192) was used. Cells were grown in 10 ml of malt extract broth (2 % w/v, Oxoid) for 6-7generations in tightly capped 20-ml bottles. Inoculum per bottle was 1 x io5 cells fromstationary phase cultures. At 24 h after inoculation, each culture was transferred to a 125-mlErlenmeyer flask and shaken at 150 rev/min on a rotary shaker. Incubation temperature was32 °C.

Scoring for flocculation, conjugation and sporulation

Free cells were counted in a haemocytometer after the flocculated cultures were allowed tostand undisturbed in a 15-ml centrifuge tube for 5 min. After they were sampled for free-cellcounts, the cultures were washed with distilled water and deflocculated with pronase (2 figper culture, Calbiochem). The deflocculated cultures were then sampled for total cell counts,conjugants and ascospores. The cell count before deftocculation was substracted from thatafter deftocculation in order to estimate the number of cells in floes.

Electron microscopy

Procedures for electron microscopy have been described in some detail (Yoo et al. 1973).At various times after the start of shaking (Fig. 2), floes were separated from uninduced freecells by gravity sedimentation, briefly washed with distilled water and fixed in a fresh pre-paration of 2 % aqueous KMnO4 for 1 h. After dehydration with acetone, the fixed cells wereembedded in a mixture of Epon and Araldite (Mollenhauer, 1964). Thin sections stained withlead citrate (Reynolds, 1963) were examined with a Philips 200 electron microscope.

RESULTS

The events in the population

Gross flocculation was induced within 1 h of the start of aeration (Fig. 2). Maximumflocculation of 70 % of the cells was attained at 4 h. Copulation (covalently bondedunion of cells, stage 2 of Fig. 1 c) was observed prior to conjugation (stages 3—6) andwas detected as union that was pronase-resistant, but was not yet a heterokaryon.Conjugation (cytoplasmic fusion) was observed at 2 h and reached its maximum at5-6 h (Fig. 2). The copulation curve (not shown in Fig. 2 for the sake of clarity of thefigure) was displaced 30 min to the left of, and essentially parallel to, the conjugationcurve. Sporulation was first observed at 8 h and was maximal as early as 12 h. Duringthis period, the total cell count increased by 15 %. The increase is due to cell divisionof a contaminating free-cell fraction of the cell population that is not immediatelyinducible.

The internal structure of induced cells

The first obvious event after the start of the aeration (induction) procedure is theformation of floes. Elsewhere (Calleja & Johnson, 1977) we have described the kineticsof flocculation which show that the rate-limiting factor is the cellular capacity torespond to the inducing stimulus rather than the formation of floes by already induced

IO CE L 25


cells. Induced cells then may be presumed to have become differentiated, but at theearly stages of flocculation, these cells are not morphologically identifiable: the internalstructure of the induced cell is not very different from that of the uninduced (compareFig. 3 and Johnson et al. 1973, 1974)-

^ 80\O

X 60<yU

40

20

floc

D D -a-th-a-ll-0-

ff

conj

spor

6

D

.6 .

f A

I I I 1 I 110 12 14 16 18

Time (h)

20 22 24 32 48

Fig. 2. Time course of flocculation, conjugation and sporulation of Schizosaccharomycespombe NCYC 132. cell, cell count; floe, cells in floes; conj, conjugant cells (pooled steps3-6 in Fig. 1 c); spor, ascospores. The start of shaking of 24-h still cultures is o h.

The mural events

Predominant among the forces binding cells together are probably hydrogen bonds,plus some degree of hydrophobic interaction, rather than simple electrostatic inter-actions (Calleja, 1974). These bonds appear to be mediated by fibrillar or pseudo-fibrillar structures that are themselves covalently attached to the wall (Fig. 4). However,the linkages flocculated cells have between these structures are not covalent as floesare easily disrupted by heat, urea, guanidinium chloride, or sodium dodecyl sulphate,

Figs. 3-16. A reconstruction of the sequence of ultrastructural changes during conjuga-tion of Schizosaccharomyces pombe NCYC 132. e, electron-dense particle;/, fuscannel;m, mitochondrion; n, nucleus; p, dark patch; s, suture.

Fig. 3. A pair of flocculated cells. Contact is typically pole-to-pole, but typicallynot co-axial. No conjugation tube is apparent yet. These cells are probably not sibsbecause there is no primary septum between them and contact is not co-axial, x 10 720.

Fig. 4. Sex hairs between walls. The poles appear forcibly separated. Note the hairsand their apparent origin in the wall, x 37 250.

Fig. 5. Menage a trois. Copulation between a and b: note the conjugation tube,the expanded area of contact and the asynchronous thinning of the 2-layer cross-wall.Copulation between b and c: very slight deformation is noticeable in cell c. Sex hairsapparently distributed all over the wall (cell b). x 13560.

Fig. 6. Further expansion of the contact area. A pair of copulating cells (2-fuscannelx 2-fuscannel). For the first time, the accumulation of electron-dense particles closeto the site of union becomes noticeable, x 7490.

Fig. 7. A copulant pair (i-fuscannel x 3-fuscannel) farther along toward conjugation.The 2-layer cross-wall has been eroded to such an extent that it has lost its ability towithstand cytoplasmic pressure. Note dark patch at the periphery of the contact area,which is now further expanded, x 6960.

Conjugation in fission yeast

144 G- B- Calleja, B. Y. Yoo and B. F. Johnson

without affecting their ability to reflocculate when these agents are withdrawn (Calleja,1974). Proteinases, such as trypsin, papain and pronase, cause irreversible defloccula-tion; presumably it is these fibrillar structures or their sites of attachment to thewalls that are digested by proteolytic treatment. Soon after flocculation, covalentlinkages are formed. These linkages were scored by light microscopy as unionsresistant to pronase as well as other deflocculating agents. We presume that the originalhydrogen bonds are supplemented by covalent bonds between sugar moieties of theparticipating walls.

Approximately coincident with the onset of resistance to pronase, conjugationtubes were observed (Fig. 5). This event was scored as the deformation of cells incontact.

The next event appears to be the erosion of the walls (Figs. 5-11) where they nowbecome a 2-layer cross-wall. The erosion is outward, i.e. from the inner wall to theouter wall; but it need not be simultaneous nor even bilateral. This erosion of the2-layer cross-wall begins before conjugation-tube formation is finished. It continueslong after the formation of the heterokaryon (fusion of the cytoplasm) and the enlarge-ment of the tube. After a hole appears at the cross-wall (Fig. 9), the tube is enlargedand the suture is finally repaired in such a way that the site of union cannot beidentified.

The nuclear events

Up to now, the noticeable events are mural. After cytoplasmic fusion but beforethe tube is enlarged, the 2 nuclei are mobilized (Figs. 10-12). The nuclei approacheach other so that the most likely region of encounter between them is the conjugationtube. Karyogamy then takes place (Fig. 13), followed by meiosis I, when the nucleiseparate polewards (Fig. 14). The nuclei undergo another division (meiosis II) toform 4 nuclei, which subsequently develop into spores in the heterokaryon cumascus (Figs. 15, 16), as described by Yoo et al. (1973).

Fig. 8. Further erosion of the 2-layer cross-wall. The centre of the cross-wall is thin-nest; this is where the hole in the cross-wall will be found in the next figure. The sutureis now less marked, x 13400.

Fig. 9. The hole in the cross-wall. The erstwhile pair of cells is now a heterokaryon.Note again the electron-dense particles near the site of perforation. The suture is hardlyidentifiable now. The only visible nucleus is still in its original location. The cross-wallmay be mistaken for the septum of a dividing cell but for the absence of the primaryseptum and the eccentric deformity ascribable to conjugation-tube formation, x 12 500.

Fig. 10. Nuclear migration. Nuclei have moved into the patent conjugation tube.The hole in the cross-wall has been expanded; the electron-dense particles are stillat the site of union. There is very little left of the cross-wall, but the conjugation tubeis not yet fully expanded to allow nuclear entry. Arrow points to crimp, x 9100.

Fig. 11. Expanded conjugation tube. The nuclei appear deformed upon entry intothe tube. The crimp at the periphery of the contact area has not been smoothed outand the dark patch is still there, although the suture has already vanished, x 10710.

Fig. 12. The nuclei just prior to fusion. Conjugation tube is fully expanded now.x 11610.


Cytoplasmic events

Electron-dense particles can be seen to concentrate at the site of union (Figs. 6-10).They become apparent only after the initiation of cross-wall erosion and seem mostplentiful at the time of the puncturing of the cross-wall (Fig. 9), only to be scatteredagain by the migrating nuclei. They resemble the electron-dense particles seen atcell division (Johnson et al. 1974) whose origin was ascribed to Golgi bodies.

DISCUSSION

Flocculation structures

The fibrillar structures on the surface of flocculent cells (Figs. 4, 5) are probablyproteinaceous. We have shown that cytoplasmic protein synthesis is required for theinduction of flocculation and that flocculated cells may be readily deflocculated byprotease activity (Calleja, 1973, 1974)- Furthermore, these structures are removed bypronase and are not seen in uninduced cells nor in cells aerated in the presence ofcycloheximide. Hence we conclude that they are the putative protein that mediatesflocculation (Calleja, 1974). Whether it is amorphous as we described it earlier (Yooet al. 1971) or is genuinely fibrillar as seen here cannot be settled. If the appearancehere is due to the forcible separation of a flocculent pair of cells, producing a 'spun-out' pseudofibrillar character, then it is only by chance that the images seem compar-able with the fimbriae shown to occur on the surfaces of many yeasts (Day & Poon,1975; Poon & Day, 1975). Note, however, that fimbriae appear to be associated withnon-sexual flocculation in Saccharomyces cerevisiae (Day, Poon & Stewart, 1975).

As described above, hydrogen-bonded pairs (Fig. 3) may be empirically dis-tinguished from covalently bonded pairs. Distinguishing paired flocculent cells fromincompletely divided sibs is somewhat more complex, but the disposition of fissionscars (or of fuscannels), co-axial or eccentric relationships, the presence of discernibleelectron-transparent primary septum (Shannon & Rothman, 1971; previously calledAR by ourselves, Johnson et al. 1973, 1974) between incompletely divided sibs or

Fig. 13. Azygote. It is difficult to tell whether the nuclei have just fused or the fusednucleus is just about to split into two. Arrow points to crimp, x 10700.

Fig. 14. At the end of meiosis I. Fused nucleus has divided into 2 nuclei, which areen route back to their original locations. Because the crimp at the periphery of the con-tact area has been smoothed out and the black patch and the suture are now gone, itis not possible to tell where the site of union is; it is now de-differentiated. Original cellswere 2-fuscannel (bottom) and i-fuscannel (top). The latter is an example of a conjugat-ing cell with a conjugating tube at the pole proximal to the last divisional event, incontrast to distal as in the upper cell in Fig. 7. x 8720.

Fig. 15. Past meiosis II . The lower 2 nuclei have been derived from a nucleus aftermeiosis I. Only 3 nuclei of a possible 4 are seen in the picture. All of them are nowenclosed by forespore membranes, which also enclose some mitochondria. For details,see Yoo et al. (1973). x 12200.

Fig. 16. A 4-spore ascus. Ascospores are not fully mature. For details, see Yoo et al.(1973). x 14260.


16


the electron-dense flocculent material and the suture between flocculent cells, takentogether, yield unambiguous distinction.

Covalent-linkage formation and cross-wall formation

It seems logical to consider flocculation as a mechanism to hold 2 cells in closecontact while covalent linkages are being forged; the hairs thus function as grapplinghooks. The formation of covalent bonds is what we call copulation, and this stage isoperationally defined as resistance of the union to hydrogen-bond breaking agents aswell as proteinases. Copulation helps stabilize the floes further.

We envisage the formation of covalent bonds between cell wall structural poly-saccharides as the start of cross-wall formation. As the number of covalent linkagesbetween sugar moieties increases, the effective contact area between cells enlarges. Across-wall is scored as soon as the effective contact area attains a diameter of about1 /im. By this time, conjugation tubes are apparent and wall erosion has alreadybegun.

Conjugation-tube formation

Pre-incubating cells of the opposite mating types in a U-tube separated by afilter does not induce conjugation-tube formation nor does it shorten the time ofinduction when the cells are subsequently mixed (Egel, 1971; our unpublished observa-tion). This suggests that no diffusible material brings about induction and thatcontact is necessary for conjugation-tube formation. Thus there can be little doubtthat flocculation is precedent to conjugation-tube formation.

The order of occurrence of covalent-bond formation between walls and conjugation-tube initiation is not easily resolved; perhaps this indicates that there is not an obliga-tory order, but rather, that each occurs more or less independently of the other. Anisolated induced cell which has become recognizably deformed by growing towards itssexual partner may be termed a copulation/conjugation half. It obviously has becomedeformed before generating- enough covalent bonds to make the union resistant toshearing forces. At any rate, there are far fewer of these than there are covalentlylinked pairs in the early hours after induction, suggesting that covalent linkage isoften precedent. Later, at 24 h after induction, one sees a higher frequency of de-formed copulation/conjugation halves, but these may be a consequence of fruitlesspairing.

Erosion of the cross-wall and conjugation-induced lysis

The wall regions in covalent contact become the 2-layer cross-wall upon expansionof the contact area through the increase in the number of covalent linkages. Pre-sumably, the enlargement of the effective contact area is facilitated by softening of thewall region in contact and near the contact area. This softening may be brought aboutcooperatively by conjugation-tube formation and wall erosion. If erosion of thecross-wall occurs to such an extent as to lead to perforation of the cross-wall (andthe cytoplasmic membrane) before a perfect seal is effected at the site of union, lysiswould result. We have observed about 15 % of cells in the process of conjugation to


lyse spontaneously and an even higher percentage may be lysed by sonication orsuspension in distilled water (Calleja, Yoo & Johnson, 1977). We ascribe thisphenomenon, which appears analogous to lethal zygosis in bacteria (Skurray &Reeves, 1973), to premature or hyperactive erosion of the walls. Autolytic activityhas been associated with conjugation in yeast by other authors (Kroning & Egel,1974; Shimoda & Yanagishima, 1972; Lee, Lusena & Johnson, 1975). Wehave suggested that conjugation-induced lysis is apt to be due to faulty fusionor badly controlled lytic activity during cross-wall removal (Calleja et al. 1977).The lytic event indicates that the conjugation process is far from being a perfectsystem. A significant portion of the conjugating population is destined to suicide.

The observation that many of the abortive conjugants are pairs suggests that thelytic events occur after cytoplasmic fusion. However, the fact that lysed copulation/conjugation halves do occur means that lysis can occur before cytoplasmic fusion.As shown here, erosion of the cross-wall may be asynchronous and, in a small per-centage, unilateral. Some non-lysed abortive conjugants are due to failure of onecell to erode its own side of the cross-wall.

The perforation is typically at the centre of the cross-wall (Fig. 9), but occasionallyit may be found at the periphery (see fig. 3 a of Calleja et al. 1977).

Menage a trois and related considerations

Although the number of cells in a floe is very large and therefore wall contactbetween cells is not confined to the poles, copulation, hence conjugation, is usuallybetween 2 poles of 2 cells. Be that as it may, the very tip is not necessarily the mostlikely to be the contact region. Indeed, most of the copulant pairs are not co-axiallyin contact, but rather are eccentric. We occasionally see copulation which may bedesignated as side-to-pole, rather than the normal pole-to-pole. Rarer still is side-to-side copulation. These atypical copulants are less fruitful in that they lead to smallerpercentages of conjugants and asci with 4 spores or with any spores at all. We deducethat there is less cross-wall erosion in these aberrant cases; presumably, the lyticapparatus is more concentrated at the poles. Nevertheless, we detect no struc-tural elements which might indicate a potential site for sexual activity. Certainly theflocculation material is distributed over the entire wall.

Although the random positioning of any cell within a floe should make either poleavailable for copulation, the pole distal to the last fission is favoured, confirming anearlier report by Streiblova & Wolf (1975), and extending Mitchison's rule forvegetative growth (Mitchison, 1957) to sexual growth, i.e. conjugation-tube formation.Consistent with this bias is the possibility that that pole which would be primary(Johnson, 1965) in vegetative growth more readily initiates conjugal activity. Thesecondary pole is also capable of extension (up to 20 % of the cells, Johnson, 1965) and,though less frequently than the primary pole, does participate in conjugal pairing.Indeed, more than pairing can occur, for up to 5 % of copulants may be found asmultiples (most commonly, as a menage a trois, but also as quatre or even cinq; Fig. 5illustrates one cell with 2 partners at one of its ends).


Multiple copulations may be interesting in themselves, but even more interestingis their rarity. For, of the hundreds of thousands of cells that may be found in asingle floe, most must experience multiple contacts, but few of these lead to copulation.Some mechanism must be operative to frustrate supernumerary copulants - arestriction mechanism by which a third participant is excluded. We may call it theprinciple of the excluded third. Thus, when one pole of a cell is already in covalentlinkage with an abutting cell, the left-over pole of either cell is no longer availablefor a third cell. The principle of the excluded third is possibly executed by means ofmural modulation taking place at the pole distal to the pole already activated sexually.Whatever the mechanism, it seems not to lead to significant loss in the hydrogen-bonding activity of the conjugating cells because flocculation is not disturbed butrather strengthened.

Nuclear mobilization and fusion

Nuclear migration toward the conjugation tube begins only after the 2 originalcells have become a heterokaryon but before the conjugation tube is expandedenough to allow nuclear entry. The late part of nuclear mobilization (Fig. 10) and theearly part of meiosis I (Fig. 14) are not readily distinguishable except temporally.However, the former usually occurs even before all the remnants of the cross-wallhave been digested away, before enlargement of the tube and before seam removal iscomplete. Our preparations draw emphasis to these mural markers; KMnO4 fixationdeters us from detailed analysis of nuclear events.

De-differentiation of the site of union

The site of union between the walls of copulating cells is a discontinuity and remainsso until the walls are repaired. There are 3 reliable signs of the site of union when the2-layer cross-wall is gone. These are the electron-dense suture, the electron-densepatch which fills in the gap at the periphery of the contact area, and the crimp. A mostremarkable aspect of the repair system is that once the repair is completed, there isleft no tell-tale sign of the site of union. The wall at this once differentiated site hasbeen morphologically de-differentiated.

The removal of the suture may be the consequence of not only the removal of theelectron-dense material (which might be the protein-rich outer surface of the respect-ive walls) but also further ligating activity between the walls. The removal of thedark patch appears to be the result of further ligating activity; also possibly, theresult of a net deposition of new wall material in the gap that is visualized as a darkpatch. The removal of the crimp does not seem to be just a mechanical unfolding. Thecurvature of the poles is structurally built-in and is not simply ironed out by cyto-plasmic turgor pressure: the typical cell is never a smooth cylinder! We assume amorphogenic activity that is similar to, and an extension of, conjugation-tube forma-tion.


Other considerations

The age of a given cell can be minimally approximated by a count of fission scarsor of fuscannels. Cells having various numbers of scars and fuscannels all seem to beinducible, confirming an earlier report (Streiblova & Wolf, 1975). We have notascertained whether they are inducible with equal probabilities.

Fig. 17. Mural fusion and erosion during conjugation of Schizosaccliaromyces pombeNCYC 132. Black dots are fuscannels. A, ftocculation. Hydrogen-bonded union of2 poles, B, copulation. Covalent union. Initiation of conjugation tube. C, conjugation-tube formation. Expansion of contact area. D, erosion of 2-layer cross-wall. Furtherexpansion of contact area. Conjugation-tube expansion. E, perforation of 2-layer cross-wall. Crimp at site of union smoothed out by dark patch, F, enlargement of hole. Almostall of cross-wall now gone. Further expansion of the conjugation-tube. G, de-differentia-tion of wall complete. Suture, crimp and dark patch now gone. Nuclear migration.H, zygote formation. Nuclear fusion. No further mural activity until spore liberation.

Long before the hole appears, the widening of the contact area - an extension ofcovalent-linkage formation - stops. Hence the enlargement of the tube is not achievedby widening the contact area, although that helps at the start, but presumably bycytoplasmic turgor pressure and by the addition of more wall material, an elaborationof conjugation-tube formation.


The sequence and schedule of mural and nuclear events

A diagrammatic reconstruction of events summarizes how fusion and erosionconvert 2 closed cylindrical walls in contact into 1 continuous wall (Fig. 17). Thesequence of these mural and nuclear events (Table 1) are then mapped in a graphictime table (Fig. 18) to indicate their temporal position in the course of conjugation andsporulation (Fig. 2).

Mur

Pr

Nuc

Time (h)

Fig. 18. Schedule of mural and nuclear events during conjugation of Scliizosaccharo-mycespombe NC YC 132 from induction to meiosis I. Segments represent scorable eventsfrom initiation to completion. Segment numbers correspond to numbers in Table 1.Activity groups are designated by Roman, numerals (i)—(vi). Events in one group aresimilar or associated activities or parts of a continuum. Pop, population; Pr, pair;Mur, mural; Nuc, nuclear. Groups (i) and (ii) represent many individual eventsoccurring in the population. The rest (groups (iii)-(vi)) represent events occurring ina pair of cells. The lengths of the segments are approximated. Groups (ii), (iii) and(iv) may be displaced left or right with respect to one another such that segment 3,for example, may start from either segment 2a or 2b or 2c; or segment 4 may startbefore segment 3. Broken line between segments 5 and 9 indicates discontinuity.By definition, event 8 is a point in time.

The various events may be arranged conveniently into 6 groups. Induction tocompetence, the only event in group (i), is presumably an event that primarilyinvolves protein synthesis: those enzymes needed for the subsequent morphogenicevents and the proteinaceous material of the sex hairs. The early part of inductionmust involve nuclear and cytoplasmic events which we cannot score (instead seeCalleja, 1974). Group (ii) consists of flocculation: the formation of microfiocs,miniflocs and gross floes (for a definition of terms, see Calleja & Johnson, 1977).This group of events is primarily physical. Floes are formed by random collision ofinduced cells which are capable of hydrogen bonding among themselves. Bothgroups (i) and (ii) are considered here as populational events - although only floc-culation is strictly so, induction being an event that properly belongs to the individualcell. The rationale is that in scoring induction we consider not the individual cellbut the population.


Group (iii) events manifest ligating activity between walls. We view cross-wallformation as a consequence and extension of copulation. Further ligating activityoccurs at the time of de-differentiation of the site of union. The events in group (iv)are conjugation-tube formation and conjugation-tube expansion. These morphologicalmarkers are the result of a net glucaneogenic activity. Both synthetic and glucanolyticactivities are obviously involved here, but the end result is a net synthesis of more wallmaterial. On the other hand, group (v), comprising erosion of cross-wall, perforationand perforation expansion, is the consequence of net glucanolytic activity.

Table 1. Sequence of mural and nuclear events during conjugation ofSchizosaccharomyces pombe NCYC 132 from induction to meiosis I

Events Observation

1. Induction to competence

2. Flocculation

3. Copulation or covalent-bondformation

4. Conjugation-tube formation5. Cross-wall formation

6. Erosion of cross-wall

7. Conjugation-tube expansion8. Cytoplasmic fusion or hetero-

karyon formation9. De-differentiation of site of

union10. Nuclear migration

11. Karyogamy or nuclear fusion

12. Meiosis I

Visual: floes of heat-killed cells.EM: sex hairs

Light microscope: (a) microfiocs,(b) miniflocs. Visual: (c) gross floes

Light microscope: pronase-resistantpair

Light microscope: deformed polesLight microscope or EM: expandedcontact area

EM: (a) thin and soft wall, (6)widened perforation

EM: enlarged conjugation tubeEM: hole in the cross-wall (as wellas membrane)

EM: dark patch, crimp and sutureremoved

Light microscope or EM: nuclei inthe conjugation tube

Light microscope or EM: one largenucleus in the conjugation tube

Light microscope or EM: separatednuclei

The nuclear events are placed in group (vi) (Fig. 18). Conjugation-induced lysis ismost likely to occur at 2 h or thereabouts, when the hole in the cross-wall has beenmade but before the nuclear events. Except for group (vi), all the events are mural.When meiosis I has occurred, the mural events associated with conjugation are over.Wall morphogenesis becomes quiescent for a while. As the zygote proceeds totranslate its programme of differentiation toward sporulation, the wall is once morereactivated - this time, for spore liberation, when the activity for the greater partis glucanolytic.

Doubtless there are many nuclear events not scored as well as events that cannotpossibly be scored morphologically. However, the present schedule will hopefullyserve as a basis for temporally mapping out the biochemical activities of differentiation.


We thank Ms Donna Kelly and Mme Isabelle Boisclair-Sarrazin for technical assistance andRobert Whitehead for the preparation of the final plates.

This paper is NRCC contribution No. 15809.

REFERENCESCALLEJA, G. B. (1973). Role of mitochondria in the sex-directed flocculation of a fission yeast.

Archs Biochem. Biophys. 154, 382-386.CALLEJA, G. B. (1974). On the nature of the forces involved in the sex-directed flocculation of a

fission yeast. Can. J. Microbiol. 20, 797-803.CALLEJA, G. B. & JOHNSON, B. F. (1977). A comparison of quantitative methods for measuring

yeast flocculation. Can. J. Microbiol. (in Press).CALLEJA, G. B., YOO, B. Y. & JOHNSON, B. F. (1977). Conjugation-induced lysis of Schizosac-

charomyces pombe. J. Bad. (in Press).CONTI, S. F. & BROCK, T. D. (1965). Electron microscopy of cell fusion in conjugating Hansemila

zoingei. J. Bad. 90, 524-533.CONTI, S. F. & NAYLOR, H. B. (i960). Electron microscopy of ultrathin sections of Schizo-

saccharomyces odosporus. II. Morphological and cytological changes preceding ascosporeformation. J. Bad. 79, 331-340.

DAY, A. W. & POON, N. H. (1975). Fungal fimbriae. II. Their role in conjugation in Ustilagoviolacea. Can. J. Microbiol. 21, 547-557.

DAY, A. W., POON, N. H. & STEWART, G. G. (1975). Fungal fimbriae. III. The effect onflocculation in Saccharomyces. Can. J. Microbiol. 21, 558-564.

EGEL, R. (1971). Physiological aspects of conjugation in fission yeast. Planta 98, 89-96.JOHNSON, B. F. (1965). Autoradiographic analysis of regional cell wall growth of yeasts:

Schizosaccliaromyces pombe. Expl Cell Res. 39, 613-624.JOHNSON, B. F., YOO, B. Y. & CALLEJA, G. B. (1973). Cell division in yeasts: movement of

organelles associated with cell plate growth of Schizosaccliaromyces pombe. J. Bad. 115,358-366.

JOHNSON, B. F., YOO, B. Y. & CALLEJA, G. B. (1974). Cell division in yeasts. II. Templatecontrol of cell plate biogenesis in Schizosaccharomyces pombe. In Cell Cycle Controls (ed.G. M. Padilla, I. L. Cameron & A. Zimmerman), pp. 153-166. New York: AcademicPress.

KREGER-VAN RIJ, N. J. W. & VEENHUIS, M. (1975). Conjugation in the yeast Saccharomycopsiscapsularis. Arch. Mikrobiol. 104, 263-269.

KREGER-VAN RIJ, N. J. W. & VEENHUIS, M. (1976). Conjugation in the yeast Guillienr.ondellaselenospora Nadson et Krasslinikov. Can. J. Microbiol. 22, 960-966.

KRONING, A. & EGEL, R. (1974). Autolytic activities associated with conjugation and sporulationin fission yeast. Arch. Mikrobiol. 97, 241-249.

LEE, E.-H., LUSENA, C. V. & JOHNSON, B. F. (1975). A new method of obtaining zygotes inSaccharomyces cerevisiae. Can. J. Microbiol. 21, 802-806.

MITCHISON, J. M. (1957). The growth of single cells. I. Schizosaccharomyces pombe. Expl CellRes. 13, 244-262.

MOLLENHAUER, H. H. (1964). Plastic embedding mixture for use in electron microscopy.Stain Technol. 39, 111-114.

OSUMI, M., SHIMODA, C. & YANAGISHIMA, N. (1974). Mating reaction in Saccharomycescerevisiae. V. Changes in the fine structure during the mating reaction. Arch. Mikrobiol.97, 27-38.

POON, N. H. & DAY, A. W. (1975). Fungal fimbriae. I. Structure, origin, and synthesis. Can.jf.Microbiol. 21, 537-546.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.

SHANNON, J. L. & ROTHMAN, A. H. (1971). Transverse septum formation in budding cellsof the yeastlike fungus Candida albicans. J. Bad. 106, 1026-1028.

SHIMODA, C. & YANAGISHIMA, N. (1972). Mating reaction in Saccharomyces cerevisiae. III.Changes in autolytic activity. Arch. Mikrobiol. 85, 310-318.


SKURRAY, R. A. & REEVES, P. (1973). Physiology of Escherichia coli K-12 during conjugation:altered recipient cell functions associated with lethal zygosis. J. Bact. 114, 11-17.

STREIBLOVA, E. & WOLF, A. (1975). Role of cell wall topography in conjugation of Schizo-saccharomyces pombe. Can. jf. Microbiol. 21, 1399-1405.

Yoo, B. Y., CALLEJA, G. B. & JOHNSON, B. F. (1971). Electron microscopy of pre-conjugal,flocculating fission yeast cells. Proc. Can. Fedn biol. Soc. 14, 62.

Yoo, B. Y., CALLEJA, G. B. & JOHNSON, B. F. (1973). Ultrastructural changes of the fissionyeast (Schizosaccharomyces pombe) during ascospore formation. Arch. Mikrobiol. 91, 1-10.

(Received 5 November 1976)

FUSION AND EROSION OF CELL WALLS DURING CONJUGATION … · erosion; suture a,n electron-dense seam...

Documents

Transcript of FUSION AND EROSION OF CELL WALLS DURING CONJUGATION … · erosion; suture a,n electron-dense seam...