J. Cell Sci. 25, 139-155 (i977)
Printed in Great Britain
139
FUSION AND EROSION OF CELL WALLS
DURING CONJUGATION IN THE FISSION
YEAST (SCHIZOSACCHAROMYCES POMBE)
G. B. CALLEJA, BONG Y. YOO AND BYRON F. JOHNSON
Division of Biological Sciences, National Research
Council, Ottawa, Canada KiA 0R6 and Department of
Biology, 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, the
start of sporulation. These morphogenic markers were separately identifiable as flocculation,
copulation, conjugation-tube formation, cross-wall formation, cross-wall erosion, conjugationtube expansion, cytoplasmic fusion, de-differentiation of site of union, nuclear migration and
karyogamy. The following were identified as new structural elements: sex hairs, which presumably mediate hydrogen bonding between cells during flocculation; crimp at the site of
union; dark patch, which presumably serves as a leak-proof seal at the time of cross-wall
erosion; suture, an electron-dense seam formed by the union of a copulant pair; and small
electron-dense particles close to the site of wall erosion. No special structures on the cell wall
could be identified as indicative of specific sites for potential copulatory activity. The discontinuity of the 2 cell walls at the site of union became so de-differentiated after fusion and
erosion that it was no longer possible to pinpoint the site of union.
INTRODUCTION
The fission yeast Schizosaccharomyces pombe is ordinarily cultured as vegetative
haplonts (Fig. 1 A). At the end of the logarithmic growth phase, however, cells may be
induced by aeration to form stable floes (Fig. IB). Some of these flocculated cells
pair 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 during
cell division (Johnson, Yoo & Calleja, 1973, 1974) and during the period from meiosis
I to sporulation (Yoo, Calleja & Johnson, 1973). This report bridges the gap, describing
the ultrastructural changes during the period from flocculation induction to zygote
formation. The emphasis is on the mural events; the behaviour of the nucleus is
discussed only as it appears temporally related to them. Ultrastructural studies of
conjugation 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).
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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 subject
matter into workable areas of concentration. For emphasis, the dimensions of cells in
frames 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 germination ; grow, cell division. Steps 3-6 in frame C (bracketed together) are not ordinarily
resolved 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 (ATCC
26192) was used. Cells were grown in 10 ml of malt extract broth (2 % w/v, Oxoid) for 6-7
generations in tightly capped 20-ml bottles. Inoculum per bottle was 1 x io5 cells from
stationary phase cultures. At 24 h after inoculation, each culture was transferred to a 125-ml
Erlenmeyer flask and shaken at 150 rev/min on a rotary shaker. Incubation temperature was
32 °C.
Scoring for flocculation, conjugation and sporulation
Free cells were counted in a haemocytometer after the flocculated cultures were allowed to
stand undisturbed in a 15-ml centrifuge tube for 5 min. After they were sampled for free-cell
counts, the cultures were washed with distilled water and deflocculated with pronase (2 fig
per culture, Calbiochem). The deflocculated cultures were then sampled for total cell counts,
conjugants and ascospores. The cell count before deftocculation was substracted from that
after 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 free
cells by gravity sedimentation, briefly washed with distilled water and fixed in a fresh preparation of 2 % aqueous KMnO4 for 1 h. After dehydration with acetone, the fixed cells were
embedded in a mixture of Epon and Araldite (Mollenhauer, 1964). Thin sections stained with
lead citrate (Reynolds, 1963) were examined with a Philips 200 electron microscope.
RESULTS
The events in the population
Grossflocculationwas induced within 1 h of the start of aeration (Fig. 2). Maximum
flocculation of 70 % of the cells was attained at 4 h. Copulation (covalently bonded
union of cells, stage 2 of Fig. 1 c) was observed prior to conjugation (stages 3—6) and
was 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 at
5-6 h (Fig. 2). The copulation curve (not shown in Fig. 2 for the sake of clarity of the
figure) was displaced 30 min to the left of, and essentially parallel to, the conjugation
curve. Sporulation was first observed at 8 h and was maximal as early as 12 h. During
this period, the total cell count increased by 15 %. The increase is due to cell division
of a contaminating free-cell fraction of the cell population that is not immediately
inducible.
The internal structure of induced cells
The first obvious event after the start of the aeration (induction) procedure is the
formation of floes. Elsewhere (Calleja & Johnson, 1977) we have described the kinetics
of flocculation which show that the rate-limiting factor is the cellular capacity to
respond to the inducing stimulus rather than the formation of floes by already induced
IO
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G. B. Calleja, B. Y. Yoo and B. F. Johnson
cells. Induced cells then may be presumed to have become differentiated, but at the
early stages offlocculation,these cells are not morphologically identifiable: the internal
structure of the induced cell is not very different from that of the uninduced (compare
Fig. 3 and Johnson et al. 1973, 1974)-
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22
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48
Time (h)
Fig. 2. Time course offlocculation,conjugation and sporulation of Schizosaccharomyces
pombe NCYC 132. cell, cell count;floe,cells infloes;conj, conjugant cells (pooled steps
3-6 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 interactions (Calleja, 1974). These bonds appear to be mediated by fibrillar or pseudofibrillar structures that are themselves covalently attached to the wall (Fig. 4). However,
the linkages flocculated cells have between these structures are not covalent as floes
are easily disrupted by heat, urea, guanidinium chloride, or sodium dodecyl sulphate,
Figs. 3-16. A reconstruction of the sequence of ultrastructural changes during conjugation 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 typically
not co-axial. No conjugation tube is apparent yet. These cells are probably not sibs
because 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 hairs
and 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 hairs
apparently distributed all over the wall (cell b). x 13560.
Fig. 6. Further expansion of the contact area. A pair of copulating cells (2-fuscannel
x 2-fuscannel). For the first time, the accumulation of electron-dense particles close
to 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 to
withstand cytoplasmic pressure. Note dark patch at the periphery of the contact area,
which is now further expanded, x 6960.
Conjugation in fission yeast
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- - 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 deflocculation; presumably it is these fibrillar structures or their sites of attachment to the
walls that are digested by proteolytic treatment. Soon after flocculation, covalent
linkages are formed. These linkages were scored by light microscopy as unions
resistant to pronase as well as other deflocculating agents. We presume that the original
hydrogen bonds are supplemented by covalent bonds between sugar moieties of the
participating walls.
Approximately coincident with the onset of resistance to pronase, conjugation
tubes were observed (Fig. 5). This event was scored as the deformation of cells in
contact.
The next event appears to be the erosion of the walls (Figs. 5-11) where they now
become a 2-layer cross-wall. The erosion is outward, i.e. from the inner wall to the
outer wall; but it need not be simultaneous nor even bilateral. This erosion of the
2-layer cross-wall begins before conjugation-tube formation is finished. It continues
long after the formation of the heterokaryon (fusion of the cytoplasm) and the enlargement of the tube. After a hole appears at the cross-wall (Fig. 9), the tube is enlarged
and the suture is finally repaired in such a way that the site of union cannot be
identified.
The nuclear events
Up to now, the noticeable events are mural. After cytoplasmic fusion but before
the tube is enlarged, the 2 nuclei are mobilized (Figs. 10-12). The nuclei approach
each other so that the most likely region of encounter between them is the conjugation
tube. Karyogamy then takes place (Fig. 13), followed by meiosis I, when the nuclei
separate polewards (Fig. 14). The nuclei undergo another division (meiosis II) to
form 4 nuclei, which subsequently develop into spores in the heterokaryon cum
ascus (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 thinnest; this is where the hole in the cross-wall will be found in the next figure. The suture
is 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 hardly
identifiable now. The only visible nucleus is still in its original location. The cross-wall
may be mistaken for the septum of a dividing cell but for the absence of the primary
septum 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 still
at the site of union. There is very little left of the cross-wall, but the conjugation tube
is 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 into
the tube. The crimp at the periphery of the contact area has not been smoothed out
and 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.
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G. B. Calleja, B. Y. Yoo and B. F. Johnson
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 most
plentiful at the time of the puncturing of the cross-wall (Fig. 9), only to be scattered
again by the migrating nuclei. They resemble the electron-dense particles seen at
cell division (Johnson et al. 1974) whose origin was ascribed to Golgi bodies.
DISCUSSION
Flocculation structures
Thefibrillarstructures on the surface of flocculent cells (Figs. 4, 5) are probably
proteinaceous. We have shown that cytoplasmic protein synthesis is required for the
induction of flocculation and that flocculated cells may be readily deflocculated by
protease activity (Calleja, 1973, 1974)- Furthermore, these structures are removed by
pronase and are not seen in uninduced cells nor in cells aerated in the presence of
cycloheximide. Hence we conclude that they are the putative protein that mediates
flocculation (Calleja, 1974). Whether it is amorphous as we described it earlier (Yoo
et al. 1971) or is genuinely fibrillar as seen here cannot be settled. If the appearance
here is due to the forcible separation of a flocculent pair of cells, producing a 'spunout' pseudofibrillar character, then it is only by chance that the images seem comparable 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 with
non-sexual flocculation in Saccharomyces cerevisiae (Day, Poon & Stewart, 1975).
As described above, hydrogen-bonded pairs (Fig. 3) may be empirically distinguished from covalently bonded pairs. Distinguishing paired flocculent cells from
incompletely divided sibs is somewhat more complex, but the disposition of fission
scars (or of fuscannels), co-axial or eccentric relationships, the presence of discernible
electron-transparent primary septum (Shannon & Rothman, 1971; previously called
AR 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 fused
nucleus 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 are
en route back to their original locations. Because the crimp at the periphery of the contact area has been smoothed out and the black patch and the suture are now gone, it
is not possible to tell where the site of union is; it is now de-differentiated. Original cells
were 2-fuscannel (bottom) and i-fuscannel (top). The latter is an example of a conjugating cell with a conjugating tube at the pole proximal to the last divisional event, in
contrast 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 after
meiosis I. Only 3 nuclei of a possible 4 are seen in the picture. All of them are now
enclosed 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.
Conjugation in fission yeast
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G. B. Calleja, B. Y. Yoo and B. F. Johnson
the electron-dense flocculent material and the suture between flocculent cells, taken
together, yield unambiguous distinction.
Covalent-linkage formation and cross-wall formation
It seems logical to consider flocculation as a mechanism to hold 2 cells in close
contact while covalent linkages are being forged; the hairs thus function as grappling
hooks. The formation of covalent bonds is what we call copulation, and this stage is
operationally defined as resistance of the union to hydrogen-bond breaking agents as
well as proteinases. Copulation helps stabilize the floes further.
We envisage the formation of covalent bonds between cell wall structural polysaccharides as the start of cross-wall formation. As the number of covalent linkages
between sugar moieties increases, the effective contact area between cells enlarges. A
cross-wall is scored as soon as the effective contact area attains a diameter of about
1 /im. By this time, conjugation tubes are apparent and wall erosion has already
begun.
Conjugation-tube formation
Pre-incubating cells of the opposite mating types in a U-tube separated by a
filter does not induce conjugation-tube formation nor does it shorten the time of
induction when the cells are subsequently mixed (Egel, 1971; our unpublished observation). This suggests that no diffusible material brings about induction and that
contact is necessary for conjugation-tube formation. Thus there can be little doubt
that flocculation is precedent to conjugation-tube formation.
The order of occurrence of covalent-bond formation between walls and conjugationtube initiation is not easily resolved; perhaps this indicates that there is not an obligatory order, but rather, that each occurs more or less independently of the other. An
isolated induced cell which has become recognizably deformed by growing towards its
sexual partner may be termed a copulation/conjugation half. It obviously has become
deformed before generating- enough covalent bonds to make the union resistant to
shearing forces. At any rate, there are far fewer of these than there are covalently
linked pairs in the early hours after induction, suggesting that covalent linkage is
often precedent. Later, at 24 h after induction, one sees a higher frequency of deformed copulation/conjugation halves, but these may be a consequence of fruitless
pairing.
Erosion of the cross-wall and conjugation-induced lysis
The wall regions in covalent contact become the 2-layer cross-wall upon expansion
of the contact area through the increase in the number of covalent linkages. Presumably, the enlargement of the effective contact area is facilitated by softening of the
wall region in contact and near the contact area. This softening may be brought about
cooperatively by conjugation-tube formation and wall erosion. If erosion of the
cross-wall occurs to such an extent as to lead to perforation of the cross-wall (and
the cytoplasmic membrane) before a perfect seal is effected at the site of union, lysis
would result. We have observed about 15 % of cells in the process of conjugation to
Conjugation infissionyeast
149
lyse spontaneously and an even higher percentage may be lysed by sonication or
suspension in distilled water (Calleja, Yoo & Johnson, 1977). We ascribe this
phenomenon, which appears analogous to lethal zygosis in bacteria (Skurray &
Reeves, 1973), to premature or hyperactive erosion of the walls. Autolytic activity
has been associated with conjugation in yeast by other authors (Kroning & Egel,
1974; Shimoda & Yanagishima, 1972; Lee, Lusena & Johnson, 1975). We
have suggested that conjugation-induced lysis is apt to be due to faulty fusion
or 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 perfect
system. A significant portion of the conjugating population is destined to suicide.
The observation that many of the abortive conjugants are pairs suggests that the
lytic 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 percentage, unilateral. Some non-lysed abortive conjugants are due to failure of one
cell to erode its own side of the cross-wall.
The perforation is typically at the centre of the cross-wall (Fig. 9), but occasionally
it 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 contact
between cells is not confined to the poles, copulation, hence conjugation, is usually
between 2 poles of 2 cells. Be that as it may, the very tip is not necessarily the most
likely to be the contact region. Indeed, most of the copulant pairs are not co-axially
in contact, but rather are eccentric. We occasionally see copulation which may be
designated as side-to-pole, rather than the normal pole-to-pole. Rarer still is side-toside copulation. These atypical copulants are less fruitful in that they lead to smaller
percentages of conjugants and asci with 4 spores or with any spores at all. We deduce
that there is less cross-wall erosion in these aberrant cases; presumably, the lytic
apparatus is more concentrated at the poles. Nevertheless, we detect no structural elements which might indicate a potential site for sexual activity. Certainly the
flocculation material is distributed over the entire wall.
Although the random positioning of any cell within a floe should make either pole
available for copulation, the pole distal to the last fission is favoured, confirming an
earlier report by Streiblova & Wolf (1975), and extending Mitchison's rule for
vegetative 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. The
secondary 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 as
multiples (most commonly, as a menage a trois, but also as quatre or even cinq; Fig. 5
illustrates one cell with 2 partners at one of its ends).
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G. B. Calleja, B. Y. Yoo and B. F. Johnson
Multiple copulations may be interesting in themselves, but even more interesting
is their rarity. For, of the hundreds of thousands of cells that may be found in a
single floe, most must experience multiple contacts, but few of these lead to copulation.
Some mechanism must be operative to frustrate supernumerary copulants - a
restriction mechanism by which a third participant is excluded. We may call it the
principle of the excluded third. Thus, when one pole of a cell is already in covalent
linkage with an abutting cell, the left-over pole of either cell is no longer available
for a third cell. The principle of the excluded third is possibly executed by means of
mural 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 hydrogenbonding activity of the conjugating cells because flocculation is not disturbed but
rather strengthened.
Nuclear mobilization and fusion
Nuclear migration toward the conjugation tube begins only after the 2 original
cells have become a heterokaryon but before the conjugation tube is expanded
enough to allow nuclear entry. The late part of nuclear mobilization (Fig. 10) and the
early 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-wall
have been digested away, before enlargement of the tube and before seam removal is
complete. Our preparations draw emphasis to these mural markers; KMnO4 fixation
deters 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 remains
so until the walls are repaired. There are 3 reliable signs of the site of union when the
2-layer cross-wall is gone. These are the electron-dense suture, the electron-dense
patch which fills in the gap at the periphery of the contact area, and the crimp. A most
remarkable aspect of the repair system is that once the repair is completed, there is
left no tell-tale sign of the site of union. The wall at this once differentiated site has
been morphologically de-differentiated.
The removal of the suture may be the consequence of not only the removal of the
electron-dense material (which might be the protein-rich outer surface of the respective walls) but also further ligating activity between the walls. The removal of the
dark patch appears to be the result of further ligating activity; also possibly, the
result of a net deposition of new wall material in the gap that is visualized as a dark
patch. The removal of the crimp does not seem to be just a mechanical unfolding. The
curvature of the poles is structurally built-in and is not simply ironed out by cytoplasmic turgor pressure: the typical cell is never a smooth cylinder! We assume a
morphogenic activity that is similar to, and an extension of, conjugation-tube formation.
Conjugation in fission yeast
151
Other considerations
The age of a given cell can be minimally approximated by a count of fission scars
or of fuscannels. Cells having various numbers of scars and fuscannels all seem to be
inducible, confirming an earlier report (Streiblova & Wolf, 1975). We have not
ascertained whether they are inducible with equal probabilities.
Fig. 17. Mural fusion and erosion during conjugation of Schizosaccliaromyces pombe
NCYC 132. Black dots are fuscannels. A, ftocculation. Hydrogen-bonded union of
2 poles, B, copulation. Covalent union. Initiation of conjugation tube. C, conjugationtube formation. Expansion of contact area. D, erosion of 2-layer cross-wall. Further
expansion of contact area. Conjugation-tube expansion. E, perforation of 2-layer crosswall. Crimp at site of union smoothed out by dark patch, F, enlargement of hole. Almost
all of cross-wall now gone. Further expansion of the conjugation-tube. G, de-differentiation 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 of
covalent-linkage formation - stops. Hence the enlargement of the tube is not achieved
by widening the contact area, although that helps at the start, but presumably by
cytoplasmic turgor pressure and by the addition of more wall material, an elaboration
of conjugation-tube formation.
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G. B. Calleja, B. Y. Yoo and B. F. Johnson
The sequence and schedule of mural and nuclear events
A diagrammatic reconstruction of events summarizes how fusion and erosion
convert 2 closed cylindrical walls in contact into 1 continuous wall (Fig. 17). The
sequence of these mural and nuclear events (Table 1) are then mapped in a graphic
time table (Fig. 18) to indicate their temporal position in the course of conjugation and
sporulation (Fig. 2).
Mur
Pr
Nuc
Time (h)
Fig. 18. Schedule of mural and nuclear events during conjugation of Scliizosaccharomycespombe NC YC 132 from induction to meiosis I. Segments represent scorable events
from initiation to completion. Segment numbers correspond to numbers in Table 1.
Activity groups are designated by Roman, numerals (i)—(vi). Events in one group are
similar or associated activities or parts of a continuum. Pop, population; Pr, pair;
Mur, mural; Nuc, nuclear. Groups (i) and (ii) represent many individual events
occurring in the population. The rest (groups (iii)-(vi)) represent events occurring in
a 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 start
before 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 to
competence, the only event in group (i), is presumably an event that primarily
involves protein synthesis: those enzymes needed for the subsequent morphogenic
events and the proteinaceous material of the sex hairs. The early part of induction
must involve nuclear and cytoplasmic events which we cannot score (instead see
Calleja, 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 of
induced cells which are capable of hydrogen bonding among themselves. Both
groups (i) and (ii) are considered here as populational events - although only flocculation is strictly so, induction being an event that properly belongs to the individual
cell. The rationale is that in scoring induction we consider not the individual cell
but the population.
Conjugation infissionyeast
153
Group (iii) events manifest ligating activity between walls. We view cross-wall
formation as a consequence and extension of copulation. Further ligating activity
occurs 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 morphological
markers are the result of a net glucaneogenic activity. Both synthetic and glucanolytic
activities are obviously involved here, but the end result is a net synthesis of more wall
material. On the other hand, group (v), comprising erosion of cross-wall, perforation
and perforation expansion, is the consequence of net glucanolytic activity.
Table 1. Sequence of mural and nuclear events during conjugation of
Schizosaccharomyces pombe NCYC 132 from induction to meiosis I
Events
1. Induction to competence
2. Flocculation
3. Copulation or covalent-bond
formation
4. Conjugation-tube formation
5. Cross-wall formation
6. Erosion of cross-wall
7. Conjugation-tube expansion
8. Cytoplasmic fusion or heterokaryon formation
9. De-differentiation of site of
union
10. Nuclear migration
11. Karyogamy or nuclear fusion
12. Meiosis I
Observation
Visual: floes of heat-killed cells.
EM: sex hairs
Light microscope: (a) microfiocs,
(b) miniflocs. Visual: (c) gross floes
Light microscope: pronase-resistant
pair
Light microscope: deformed poles
Light microscope or EM: expanded
contact area
EM: (a) thin and soft wall, (6)
widened perforation
EM: enlarged conjugation tube
EM: hole in the cross-wall (as well
as membrane)
EM: dark patch, crimp and suture
removed
Light microscope or EM: nuclei in
the conjugation tube
Light microscope or EM: one large
nucleus in the conjugation tube
Light microscope or EM: separated
nuclei
The nuclear events are placed in group (vi) (Fig. 18). Conjugation-induced lysis is
most likely to occur at 2 h or thereabouts, when the hole in the cross-wall has been
made 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 to
translate its programme of differentiation toward sporulation, the wall is once more
reactivated - this time, for spore liberation, when the activity for the greater part
is glucanolytic.
Doubtless there are many nuclear events not scored as well as events that cannot
possibly be scored morphologically. However, the present schedule will hopefully
serve as a basis for temporally mapping out the biochemical activities of differentiation.
154
G. B. Calleja, B. Y. Yoo and B. F. Johnson
We thank Ms Donna Kelly and Mme Isabelle Boisclair-Sarrazin for technical assistance and
Robert Whitehead for the preparation of the final plates.
This paper is NRCC contribution No. 15809.
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(Received 5 November 1976)