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Cell division in yeasts
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Experimental Cell Research 123 (1979) 253-259
CELL III.
DIVISION
IN YEASTS
The Biased, Asymmetric Location of the Septum in the Fission Yeast Cell, Schizosaccharomyces pornbe*, ** BYRON F. JOHNSON,’ G. B. CALLEJA,’
ISABELLE
BOISCLAIR’
and BONG Y. YOO*
‘Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KIA OR6, Canada, and 2Department of Biology, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada
SUMMARY Living, dividing, log-phase fission yeast cells (178 pairs) were photographed by fluorescence microscopy of their fluorochromed walls. Analysis of the lengths, volumes, and fission scar distributions of these cells led to the following conclusions: the new septum is sited asymmetrically at division by length parameters, and the asymmetric site is biased toward the newer end (that end generated by the previous cell division) of the dividing cells. The volumes of the resultant sibs, however, are equal. Some rather simple models for siting of the septum are presumed untenable on the basis of the evidence.
For the near-century which has elapsed since the genus was established [l], cell division in Schizosaccharomyces has been considered to be a model of binary fission, with binary in this context meaning division into equal sibs [2]. However, one can see by casual observation many cells dividing to produce unequal sibs. Accordingly, we decided to establish whether these asymmetrically dividing cells were the rule or the exception. In brief, they are the rule, judged by length parameter, though their volumes are equal. METHODS AND MATERIALS The strain of fission yeast used in this study was subisolate 360-2 (ATCC 26192), derived from Schizosaccharomyces aombe NCYC 132, originally provided by Professor J.-M. Mitchison. Cultures were- maintained in continuous log-phase for several weeks by daily subculturing with small inocula. The medium was Malt Extract Broth (Oxoid, 2%). Length measurements of 178pairs of sibs were made 17-791813
with a Vernier calmer on enlareed nhotoeraehs. The photographs were of living, diGding, log-phase cells stained with Calcofluor (ha. 1). a fluorochrome which reacts readily with the primary septum [3] in much the same fashion as does primuline [4]. In earlier work [5] we referred to the primary septum as the annular rudiment of the cell plate, but recently f61 have adopted the more logical -primary and secondary septum- terminoloay of Shannon & Rothman 171. Photomicrographywas done with a Reichert Zetopan microscope equipped for incident light fluorescence, and using an immersion-objective with N.A. 1.25. However, the epi-illuminator was modified rather simply to allow a 30-fold reduction in exposure time so that exposures on Tri-X film required about 2 sec. Fission scars were readily counted on the photomicrographs, with only a small number of sites being questionable (fig. 1). Volume comparisons were made on 29 selected pairs of sibs whose plotted lenaths were found to be nroxima1 to the lineestablished (by least squares analysis) for the entire sample. The breadths (cylindrical diameters) of the selected sibs were measured at their midlengths. The volumes were calculated as V=nr*l, as though the cells were uniform cylinders with flat ends. This should be approximately correct for it overesti* We dedicate this paper to the memory of Professor Masaya Hayashibe, an experimental cell researcher par excellence.
** N.R.C.C. No. 17614. Exp Cell Rrs I23 (1979)
254
Johnson et al.
Fig. I. Fluorescence micrographs of Calcofluorstained, living, log-phase cells of Schizosaccharomyces pombe. Arrows indicate the boundary between
a scar and normal wall laid down by extension. Arrows with questions indicate rare cases where identification of an old scar is equivocal (see text). x3 600.
mated the volumes of the hemispherical ends of the cells, but underestimated the volumes of those scarred regions whose cylindrical diameter exceeds the measurement used [8]. Areas, which are directly convertible to cell surface areas, were established by planimetry on the same photographs used for length and breadth measurement.
is so great, would establish the asymmetry at a high level of significance. The distribution was found (least squares analysis) to fit the line Y=O.260+0.815 X
(with slope significantly different from 1). This means that the longer the progenitor, Length asymmetry the greater the percentage disparity beData from 178 pairs of sibs are presented tween the long and short sib: Conversely, (fig. 2) for a comparison of the length of the the shorter the progenitor, the less the dislong sib vs that of the short sib. Only a parity, with equality obtained at Y, X= 1.41. small number of pairs (nine) were found to No progenitor cell in our sample was so have identical lengths; most pairs differed short. sufficiently from equality that the length of the mean long sib exceeded the length of the Biased length asymmetry mean short sib by about 10%. A simple In view of the pronounced asymmetry dempairing analysis, where the mean disparity onstrated, a basic question is whether one RESULTS
Exp Cell Res 123 (1979)
Sib differences in Schizosaccharomyces
95’
2.5
3.5
pombe
255
4.5
Fig. 2. Abscissa: length (arb. units), long sib; ordinate: length, short sib. Diagram comparing lengths of entire sample of paired sibs of Schizosaccharomyces pombe. One arb. unit equals 2.79 pm. ---, Expected result if pairs of sibs have equal lengths; -, fitted regression line. Sample, 178 pairs.
Fig. 3. Abscissa:
potential sib is apt to be consistently shorter than the other? The answer is easily obtained from the photographs, for it has been demonstrated [9] that at least 90% of the cells grow at only one (primary) end, leaving the other end easily marked by the usual prominence of its most recent fission scar. A simple count of scars per long and per short sib (a scars-by-sib analysis) reveals (fig. 3) that approx. two-thirds (201/308) of all scars were found on short sibs, and indeed, that over one-half (103/178) of the long sibs had no scars derived from preceding cell divisions. By somewhat more complex analysis (sibs-by-scars analysis), the lengths of short and long sibs in pairs were replotted (much as in fig. 2) with the pairs assigned to subsamples according to whether the short sib had more scars (fig. 4a), an equal number of scars (fig. 4b), or fewer scars (fig. 4c) than its longer partner. Comparison (least squares) of the three sub-samples indicated that the slopes did not differ significantly, hence a common slope for the three sub-
samples was derived. However, the intercepts differed significantly, and are tabulated individually (fig. 4). The majority of the pairs (108/178, fig. 4a) were in the subsample characterized by having more scars on the short sib than on the long. Thus, by two analyses, scars-by-sibs and sibs-byscars, the shorter sibs are seen to be more associated with scars than are their longer partners. Only a small number of pairs (27, fig. 46) had scars equally divided between short and long sib. About a quarter (43/178, fig. 4c) were found with more scars on the longer sib. There are obvious differences among the distributions of the three sub-samples (fig. 4a, 6, c), both in terms of apparent coherence of distributions and in terms of significant differences of intercepts. However, the sub-samples are small, and our information about them is insufficient to warrant further comment.
no. of scars/sib (scars derived from earlier cell divisions); ordinate: frequency. Histogram indicating frequency distribution of 308 scars among 178 long and 178 short sibs of Schizosaccharomyces pombe. Short sibs (0) had 201, whereas long sibs (kg) had 107.
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r 2.5
SCARSLONe
t 15 r
25 y=A+OB04X SCARS SHORT = SCA&cm
1.5
c
A=0.215 N=27
///
3.5
25
‘t
1.5,L 1.5
2.5
35
4.5
length, long sib; ordinate: length, short sib (units as in fig. 2). Diagram comparing lengths of sub-samples of paired sibs of Schizosaccharomyces pombe. Solid lines are fitted regression lines (Y=A +0.804 X). In sub-sample a, the short sibs have more fission scars than the long (A =0.287, N= 108); in b, the short and long sibs have equal numbers of fission scars (A=0.215; N=27); in c, the short sibs have fewer fission scars than the long Fig. 4. Abscissa:
(A =0.349; N=43).
Biased length asymmetry at division of recently germinated spores Germinating spores of Schizosaccharomyces pombe swell, extend, and divide.
The septum is asymmetrically located, generating, again, a long and a short sib (fig. 5). The short sib is usually the readily recognizable remnant of the spore, and corresponds with the scarred, short sib-product of log-phase cells. The longer sib is usually the extensile germ-tube, and this is completely comparable to the longer, relatively unscarred, extensile sib-product of the dividing log-phase cell. Exp Cell Res 123 (1979)
Fig. 5. Electron micrograph of longitudinal, median section of germinating spore of Schizosaccharomyces pombe fixed during its first cell division (a, region covered by spore wall; b, region generated by extension).
Bar, 1.0 pm.
Volume symmetry and surface area asymmetry
The failure of dividing fission yeast cells to achieve geometric symmetry by a length parameter leads to the question, do they achieve such symmetry by a volume parameter? (As will be discussed below, the two parameters are not equivalent.) The data
Sib differences in Schizosaccharomyces .
N=29
0
I I
I 2
I 3
I 4
I 5
257
tion. Then one predicts that the obtained data will plot (as the dashed line in fig. 2) as a line of formula:
/
0
pombe
I 6
Fig. 6. Abscissa: vol. long sib (arb. units); ordinate: vol., short sib. Diagram comparing volumes of short and long sibs in 29 pairs of Schizosaccharomyces pombe (see Methods and Materials).
suggest that geometric symmetry by a volume parameter is achieved. In 15 of the 29 pairs (fig. 6), the volume of the long sib slightly exceeded the volume of the short sib; in the remaining 14 pairs, the converse was true. Points representing paired volumes (short sib vs long sib, fig. 6) scatter with few exceptions quite close to a line representing equality. However, the surface areas were not equal-in 25 of the 29 pairs examined, the surface area of the long sib exceeded that of the short sib. In one pair, the volumes were identical, and in three, the short sib had a greater surface area than the long. Once again, the mean disparity was about IO%, and again, a simple pairing analysis would establish asymmetry at a highly significant level. DISCUSSION Symmetry
Before doing the experiment, one can make the simplest of assumptions: that the lengths of the paired sibs are equal, and further, that the dividing cell obtains such equality of length by some geometrizing process (such as triangulation or other selfmeasurement) at the time of septum initia-
y=o+ 1 x (4 with the data points dispersed along the regression line as a function of the length of the progenitor. In fact, the data do not support such assumptions: there is a significant disparity of sib lengths (fig. 2). What basis exists for phrasing the question alternatively-is the septum sited for equal volumes? The cylindrical diameter of the fission yeast cell increases with every cell division, hence the maximum diameter of an individual cell is a function of the number of fission scars it bears [8]. A consequence of this fact is that the short, multiscarred, broader sib just might have the same volume as its longer, lesser-scarred, narrower sib. And indeed, the volumes (fig. 6) are equal. What does the cell measure in establishing its site for a new septum? If the ‘decision’ is concomitant with initiation, then clearly, it is not length; and, just as clearly, not surface area. Because the amounts of cell wall, cytoplasmic membrane and endoplasmic reticulum in this yeast are proportional to surface area, the cell must be ignoring these. In opting for equal volumes of sibs, the cell could be measuring any function of the soluble cytoplasm as a whole-leading to the interesting irony of establishing the site of an elaborate structure on the basis of an unstructured, probably colligative, property of the cytoplasm. Bias
Bias is not an alien phenomenon in fission yeasts: it was established by Mitchison [lo] that the new end of a cell, which was generated during the most recent cell division, is the end which is ordinarily least apt to extend in the sibs’ cell cycle. (We call this
258
Johnson et al.
Mitchison’s Rule [ll].) This new end is copy: [13], fig. 2; [14], fig. Id; [15], fig. 3; biased against extension during its first cell [16], figs 6, 8; fluorescence microscopy: cycle, and thus is easily identified by the [3], fig. 2; [4], fig. 2), let alone to interprominence of its most recent fission scar. ested microscopists, the length differences It is also the end which will have all or between sibs which were quantitated above nearly all of the old fission scars when the should have been apparent. Why then, have cell matures and approaches its own cell notions of identity persisted? Probably bedivision. And, as demonstrated above (figs cause of the tendency to generalize. Gen3, 4), that new septum will usually be laid eralization is appropriate for the early, dedown closer to this end. However, the bias scriptive stages of a science; however, the at division now seems a consequence of more developed, experimental stages of a regional ages of the cell [8], unequal cylin- maturing science require more precision, a drical diameters influencing the septation recognition of the realities which pertain. site in order to yield equal sib volumes, For this reason, it would be better if extrahence, unequal sib lengths. The classical polations to a non-existent ‘idealized cirMitchison’s Rule bias is not so easily ex- cumstance’, such as a recent introductory plained. rhapsody to the balanced growth of S. Consideration of bias leads to another pombe [ 171,were honestly stated to be such comparison. Normal elongation and divi- -extrapolations, idealizations somewhat sion of cells from log-phase cultures of this remote from the realities which remain to strain are unbalanced [12], for no cell at be explored experimentally. division is as long as its progenitor was at General its division. This fact coupled with Mitchison’s Rule and the biased, asymmetric site We define the functional middle of the cell of septum placement, taken together allow as the site of the septum, and we have seen the conclusion that septa are usually associ- that it is displaced by about 5 % (l/2 mean ated with old wall, i.e., wall exceeding one disparity) from the geometric middle of the cell cycle in age, rather than with new wall. cell. The bias is that the functional middle However, one should not conclude that is displaced toward the newer end of the new wall (less than one cycle in age) is a dividing cell. The very fact of bias suggests forbidden site for the septum; unfavored that the asymmetry does not result from a perhaps, but not forbidden. In another vein, basic physiological error in septum placethis combination of unbalanced growth and ment: asymmetry is not accidental! Indeed, biased asymmetric septum location helps it reflects the magnitude of the error inreduce the frequency with which a progen- volved when assuming that the fission yeast itor might be apt to superpose a new septum approximates an isodiametric cylinder. It is worth noting that certain measured exactly upon an old fission scar. Such a topological coincidence should have to oc- distributions of cellular parameters look cur very frequently in an ‘ideal’ cell, hav- skewed when compared with theoretical (ideal) distributions of the same parameter. ing its growth ‘balanced’ by all parameters. Thus, Krasnow [18] directly discussed unPrevious failures equal division leading to skewness of genEven to casual observers of photographed eration time distributions. The asymmetry and bias at division of living fission yeasts (phase contrast microsExp Ceil Res 123 (1979)
Sib differences in Schizosaccharomyces
pombe
259
fission yeasts is quite comparable to the budding yeast process. The division asymmetry in various strains of Saccharomyces, yielding small buds, has been repeatedly emphasized [8, 19-221, while recently Flegel [23] has shown the asymmetry to be true for a large number of budding yeast genera. Bias is of course more readily comprehended in the budding morphogenetic system wherein identification of old and new is obvious, but it is clearly just as real in fission yeasts. In a recent review [24], the relationships between septal location during spore formation and during vegetative growth of the prokaryote Bacillus were discussed. The ‘rules’ for septum location obviously vary with the physiological circumstances in that bacterial system. A precise comparison cannot be made of course, for septa are not laid down in asci of S. pombe during sporulation. But we think that it is instructive to note that in the S. pombe system, the rules of septum location do not seem to vary between ordinary vegetative cell cycles and ‘first’ cell cycles-germination of spores. Model making should be eased by that observation. Finally, we think it useful to observe that an ‘ideal cell cycle model’ system with balanced growth, isodiametric cylinders, and perfectly symmetrical cell division, could be analysed only by perturbing the system. On the other hand, the real cellular system, with small but real differences from ideality, e.g., sib volumes equal but lengths not, invites experimental questions some of which can yet be answered merely by examining the unperturbed system more closely.
Lindner, P, Wochschr brau 10 (1893) 1298. 2. Webster’s third new international dictionary (ed P B G Grove) 17th edn. G & C Meriam, Springfield (1976). 3. Johnson, B F, Yoo, B Y & Calleja, G B, Cell cycle controls (ed G M Padilla, I L Cameron & A Zimmerman) p. 153. Academic Press, New York (1974). 4. Streiblova, E & Beran, K, Folia microbial 8 (1963) 221. 5. Johnson, B F, Yoo, B Y & Calleja, G B, J bacterial 115 (1973) 358. 6. Johnson, B F, Calleja, G B & Yoo, B Y, Eucaryotic microbes as model developmental systems (ed D H O’Day & P A Horgen) p. 212. Dekker, New York (1977). 7. Shannon, J L & Rothman, A H, J bacterial 106 (1971) 1026. 8. Johnson, B F & Lu, C, Exp cell res 95 (1975) 154. 9. Johnson, B F, Exp cell res 39 (1965) 613. 10. Mitchison, J M, Exp cell res 13 (1957) 244. 11. Calleja, G B, Yoo, B Y & Johnson, B F, J cell sci 25 (1977) 139. 12. Johnson, B F, Exp cell res 49 (1968) 59. 13. Robinow, C F & Bakerspigel, A, The fungi (ed G C Ainsworth & A S Sussman) vol. 1, p. 119. Academic Press, New York (1965). 14. Johnson, B F, J bacterial 95 (1968) 1169. 15. Mitchison, J M, Methods in cell physiology (ed D M Prescott) vol. 4, p. 131. Academic Press, New York (1970). 16. McCully, E K & Robinow, C F, J cell sci 9 (1971) 475. 17. Fraser, R S S & Nurse, P, Nature 271 (1978) 726. 18. Krasnow, R A, J theor bio172 (1978) 659. 19. Beran, K, Streiblova, E & Lieblova, J, Proc 2nd symp yeasts, p. 353. Bratislava (1969). 20. Hayashibe, M, Sando, N & Abe, N, J gen appl microbial 19 (1973) 287. 21. Hartwell, L H & Unger, M W, J cell bio175 (1977) 422. 22. Johnson, B F, James, A P, Gridgeman, N T, Lusena, C V & Lee, E-H, Cell cycle regulation (ed J R Jeter, Jr, I L Cameron, G M Padilla & A M Zimmerman) p. 203. Academic Press, New York (1978). 23. Flegel, T W, Can j microbial 24 (1978) 827. 24. Hitchins, A D, Can j microbial 24 (1978) 1103.
We thank Dr N. T. Gridgeman for useful discussions on pairing, J. M. Ridgeway for performing the statistical analyses, R. H. Whitehead for photomicro-
Received January 23, 1979 Revised version received April 19, 1979 Accepted April 24, 1979
scopic advice, H. A. Turner for preparation of the photographic plates, and F. G. Villaume of American Cyanamid Company for a generous gift of the fluorochrome Calcofluor White M2R New. B. Y. Y. was supported by NRC of Canada grant A-3651.
REFERENCES
Exp Cell Res 123 (19791
Experimental Cell Research 123 (1979) 253-259
CELL III.
DIVISION
IN YEASTS
The Biased, Asymmetric Location of the Septum in the Fission Yeast Cell, Schizosaccharomyces pornbe*, ** BYRON F. JOHNSON,’ G. B. CALLEJA,’
ISABELLE
BOISCLAIR’
and BONG Y. YOO*
‘Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KIA OR6, Canada, and 2Department of Biology, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada
SUMMARY Living, dividing, log-phase fission yeast cells (178 pairs) were photographed by fluorescence microscopy of their fluorochromed walls. Analysis of the lengths, volumes, and fission scar distributions of these cells led to the following conclusions: the new septum is sited asymmetrically at division by length parameters, and the asymmetric site is biased toward the newer end (that end generated by the previous cell division) of the dividing cells. The volumes of the resultant sibs, however, are equal. Some rather simple models for siting of the septum are presumed untenable on the basis of the evidence.
For the near-century which has elapsed since the genus was established [l], cell division in Schizosaccharomyces has been considered to be a model of binary fission, with binary in this context meaning division into equal sibs [2]. However, one can see by casual observation many cells dividing to produce unequal sibs. Accordingly, we decided to establish whether these asymmetrically dividing cells were the rule or the exception. In brief, they are the rule, judged by length parameter, though their volumes are equal. METHODS AND MATERIALS The strain of fission yeast used in this study was subisolate 360-2 (ATCC 26192), derived from Schizosaccharomyces aombe NCYC 132, originally provided by Professor J.-M. Mitchison. Cultures were- maintained in continuous log-phase for several weeks by daily subculturing with small inocula. The medium was Malt Extract Broth (Oxoid, 2%). Length measurements of 178pairs of sibs were made 17-791813
with a Vernier calmer on enlareed nhotoeraehs. The photographs were of living, diGding, log-phase cells stained with Calcofluor (ha. 1). a fluorochrome which reacts readily with the primary septum [3] in much the same fashion as does primuline [4]. In earlier work [5] we referred to the primary septum as the annular rudiment of the cell plate, but recently f61 have adopted the more logical -primary and secondary septum- terminoloay of Shannon & Rothman 171. Photomicrographywas done with a Reichert Zetopan microscope equipped for incident light fluorescence, and using an immersion-objective with N.A. 1.25. However, the epi-illuminator was modified rather simply to allow a 30-fold reduction in exposure time so that exposures on Tri-X film required about 2 sec. Fission scars were readily counted on the photomicrographs, with only a small number of sites being questionable (fig. 1). Volume comparisons were made on 29 selected pairs of sibs whose plotted lenaths were found to be nroxima1 to the lineestablished (by least squares analysis) for the entire sample. The breadths (cylindrical diameters) of the selected sibs were measured at their midlengths. The volumes were calculated as V=nr*l, as though the cells were uniform cylinders with flat ends. This should be approximately correct for it overesti* We dedicate this paper to the memory of Professor Masaya Hayashibe, an experimental cell researcher par excellence.
** N.R.C.C. No. 17614. Exp Cell Rrs I23 (1979)
254
Johnson et al.
Fig. I. Fluorescence micrographs of Calcofluorstained, living, log-phase cells of Schizosaccharomyces pombe. Arrows indicate the boundary between
a scar and normal wall laid down by extension. Arrows with questions indicate rare cases where identification of an old scar is equivocal (see text). x3 600.
mated the volumes of the hemispherical ends of the cells, but underestimated the volumes of those scarred regions whose cylindrical diameter exceeds the measurement used [8]. Areas, which are directly convertible to cell surface areas, were established by planimetry on the same photographs used for length and breadth measurement.
is so great, would establish the asymmetry at a high level of significance. The distribution was found (least squares analysis) to fit the line Y=O.260+0.815 X
(with slope significantly different from 1). This means that the longer the progenitor, Length asymmetry the greater the percentage disparity beData from 178 pairs of sibs are presented tween the long and short sib: Conversely, (fig. 2) for a comparison of the length of the the shorter the progenitor, the less the dislong sib vs that of the short sib. Only a parity, with equality obtained at Y, X= 1.41. small number of pairs (nine) were found to No progenitor cell in our sample was so have identical lengths; most pairs differed short. sufficiently from equality that the length of the mean long sib exceeded the length of the Biased length asymmetry mean short sib by about 10%. A simple In view of the pronounced asymmetry dempairing analysis, where the mean disparity onstrated, a basic question is whether one RESULTS
Exp Cell Res 123 (1979)
Sib differences in Schizosaccharomyces
95’
2.5
3.5
pombe
255
4.5
Fig. 2. Abscissa: length (arb. units), long sib; ordinate: length, short sib. Diagram comparing lengths of entire sample of paired sibs of Schizosaccharomyces pombe. One arb. unit equals 2.79 pm. ---, Expected result if pairs of sibs have equal lengths; -, fitted regression line. Sample, 178 pairs.
Fig. 3. Abscissa:
potential sib is apt to be consistently shorter than the other? The answer is easily obtained from the photographs, for it has been demonstrated [9] that at least 90% of the cells grow at only one (primary) end, leaving the other end easily marked by the usual prominence of its most recent fission scar. A simple count of scars per long and per short sib (a scars-by-sib analysis) reveals (fig. 3) that approx. two-thirds (201/308) of all scars were found on short sibs, and indeed, that over one-half (103/178) of the long sibs had no scars derived from preceding cell divisions. By somewhat more complex analysis (sibs-by-scars analysis), the lengths of short and long sibs in pairs were replotted (much as in fig. 2) with the pairs assigned to subsamples according to whether the short sib had more scars (fig. 4a), an equal number of scars (fig. 4b), or fewer scars (fig. 4c) than its longer partner. Comparison (least squares) of the three sub-samples indicated that the slopes did not differ significantly, hence a common slope for the three sub-
samples was derived. However, the intercepts differed significantly, and are tabulated individually (fig. 4). The majority of the pairs (108/178, fig. 4a) were in the subsample characterized by having more scars on the short sib than on the long. Thus, by two analyses, scars-by-sibs and sibs-byscars, the shorter sibs are seen to be more associated with scars than are their longer partners. Only a small number of pairs (27, fig. 46) had scars equally divided between short and long sib. About a quarter (43/178, fig. 4c) were found with more scars on the longer sib. There are obvious differences among the distributions of the three sub-samples (fig. 4a, 6, c), both in terms of apparent coherence of distributions and in terms of significant differences of intercepts. However, the sub-samples are small, and our information about them is insufficient to warrant further comment.
no. of scars/sib (scars derived from earlier cell divisions); ordinate: frequency. Histogram indicating frequency distribution of 308 scars among 178 long and 178 short sibs of Schizosaccharomyces pombe. Short sibs (0) had 201, whereas long sibs (kg) had 107.
Exp Ce// Res g23 (1979)
256
Johnson et al.
r 2.5
SCARSLONe
t 15 r
25 y=A+OB04X SCARS SHORT = SCA&cm
1.5
c
A=0.215 N=27
///
3.5
25
‘t
1.5,L 1.5
2.5
35
4.5
length, long sib; ordinate: length, short sib (units as in fig. 2). Diagram comparing lengths of sub-samples of paired sibs of Schizosaccharomyces pombe. Solid lines are fitted regression lines (Y=A +0.804 X). In sub-sample a, the short sibs have more fission scars than the long (A =0.287, N= 108); in b, the short and long sibs have equal numbers of fission scars (A=0.215; N=27); in c, the short sibs have fewer fission scars than the long Fig. 4. Abscissa:
(A =0.349; N=43).
Biased length asymmetry at division of recently germinated spores Germinating spores of Schizosaccharomyces pombe swell, extend, and divide.
The septum is asymmetrically located, generating, again, a long and a short sib (fig. 5). The short sib is usually the readily recognizable remnant of the spore, and corresponds with the scarred, short sib-product of log-phase cells. The longer sib is usually the extensile germ-tube, and this is completely comparable to the longer, relatively unscarred, extensile sib-product of the dividing log-phase cell. Exp Cell Res 123 (1979)
Fig. 5. Electron micrograph of longitudinal, median section of germinating spore of Schizosaccharomyces pombe fixed during its first cell division (a, region covered by spore wall; b, region generated by extension).
Bar, 1.0 pm.
Volume symmetry and surface area asymmetry
The failure of dividing fission yeast cells to achieve geometric symmetry by a length parameter leads to the question, do they achieve such symmetry by a volume parameter? (As will be discussed below, the two parameters are not equivalent.) The data
Sib differences in Schizosaccharomyces .
N=29
0
I I
I 2
I 3
I 4
I 5
257
tion. Then one predicts that the obtained data will plot (as the dashed line in fig. 2) as a line of formula:
/
0
pombe
I 6
Fig. 6. Abscissa: vol. long sib (arb. units); ordinate: vol., short sib. Diagram comparing volumes of short and long sibs in 29 pairs of Schizosaccharomyces pombe (see Methods and Materials).
suggest that geometric symmetry by a volume parameter is achieved. In 15 of the 29 pairs (fig. 6), the volume of the long sib slightly exceeded the volume of the short sib; in the remaining 14 pairs, the converse was true. Points representing paired volumes (short sib vs long sib, fig. 6) scatter with few exceptions quite close to a line representing equality. However, the surface areas were not equal-in 25 of the 29 pairs examined, the surface area of the long sib exceeded that of the short sib. In one pair, the volumes were identical, and in three, the short sib had a greater surface area than the long. Once again, the mean disparity was about IO%, and again, a simple pairing analysis would establish asymmetry at a highly significant level. DISCUSSION Symmetry
Before doing the experiment, one can make the simplest of assumptions: that the lengths of the paired sibs are equal, and further, that the dividing cell obtains such equality of length by some geometrizing process (such as triangulation or other selfmeasurement) at the time of septum initia-
y=o+ 1 x (4 with the data points dispersed along the regression line as a function of the length of the progenitor. In fact, the data do not support such assumptions: there is a significant disparity of sib lengths (fig. 2). What basis exists for phrasing the question alternatively-is the septum sited for equal volumes? The cylindrical diameter of the fission yeast cell increases with every cell division, hence the maximum diameter of an individual cell is a function of the number of fission scars it bears [8]. A consequence of this fact is that the short, multiscarred, broader sib just might have the same volume as its longer, lesser-scarred, narrower sib. And indeed, the volumes (fig. 6) are equal. What does the cell measure in establishing its site for a new septum? If the ‘decision’ is concomitant with initiation, then clearly, it is not length; and, just as clearly, not surface area. Because the amounts of cell wall, cytoplasmic membrane and endoplasmic reticulum in this yeast are proportional to surface area, the cell must be ignoring these. In opting for equal volumes of sibs, the cell could be measuring any function of the soluble cytoplasm as a whole-leading to the interesting irony of establishing the site of an elaborate structure on the basis of an unstructured, probably colligative, property of the cytoplasm. Bias
Bias is not an alien phenomenon in fission yeasts: it was established by Mitchison [lo] that the new end of a cell, which was generated during the most recent cell division, is the end which is ordinarily least apt to extend in the sibs’ cell cycle. (We call this
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Mitchison’s Rule [ll].) This new end is copy: [13], fig. 2; [14], fig. Id; [15], fig. 3; biased against extension during its first cell [16], figs 6, 8; fluorescence microscopy: cycle, and thus is easily identified by the [3], fig. 2; [4], fig. 2), let alone to interprominence of its most recent fission scar. ested microscopists, the length differences It is also the end which will have all or between sibs which were quantitated above nearly all of the old fission scars when the should have been apparent. Why then, have cell matures and approaches its own cell notions of identity persisted? Probably bedivision. And, as demonstrated above (figs cause of the tendency to generalize. Gen3, 4), that new septum will usually be laid eralization is appropriate for the early, dedown closer to this end. However, the bias scriptive stages of a science; however, the at division now seems a consequence of more developed, experimental stages of a regional ages of the cell [8], unequal cylin- maturing science require more precision, a drical diameters influencing the septation recognition of the realities which pertain. site in order to yield equal sib volumes, For this reason, it would be better if extrahence, unequal sib lengths. The classical polations to a non-existent ‘idealized cirMitchison’s Rule bias is not so easily ex- cumstance’, such as a recent introductory plained. rhapsody to the balanced growth of S. Consideration of bias leads to another pombe [ 171,were honestly stated to be such comparison. Normal elongation and divi- -extrapolations, idealizations somewhat sion of cells from log-phase cultures of this remote from the realities which remain to strain are unbalanced [12], for no cell at be explored experimentally. division is as long as its progenitor was at General its division. This fact coupled with Mitchison’s Rule and the biased, asymmetric site We define the functional middle of the cell of septum placement, taken together allow as the site of the septum, and we have seen the conclusion that septa are usually associ- that it is displaced by about 5 % (l/2 mean ated with old wall, i.e., wall exceeding one disparity) from the geometric middle of the cell cycle in age, rather than with new wall. cell. The bias is that the functional middle However, one should not conclude that is displaced toward the newer end of the new wall (less than one cycle in age) is a dividing cell. The very fact of bias suggests forbidden site for the septum; unfavored that the asymmetry does not result from a perhaps, but not forbidden. In another vein, basic physiological error in septum placethis combination of unbalanced growth and ment: asymmetry is not accidental! Indeed, biased asymmetric septum location helps it reflects the magnitude of the error inreduce the frequency with which a progen- volved when assuming that the fission yeast itor might be apt to superpose a new septum approximates an isodiametric cylinder. It is worth noting that certain measured exactly upon an old fission scar. Such a topological coincidence should have to oc- distributions of cellular parameters look cur very frequently in an ‘ideal’ cell, hav- skewed when compared with theoretical (ideal) distributions of the same parameter. ing its growth ‘balanced’ by all parameters. Thus, Krasnow [18] directly discussed unPrevious failures equal division leading to skewness of genEven to casual observers of photographed eration time distributions. The asymmetry and bias at division of living fission yeasts (phase contrast microsExp Ceil Res 123 (1979)
Sib differences in Schizosaccharomyces
pombe
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fission yeasts is quite comparable to the budding yeast process. The division asymmetry in various strains of Saccharomyces, yielding small buds, has been repeatedly emphasized [8, 19-221, while recently Flegel [23] has shown the asymmetry to be true for a large number of budding yeast genera. Bias is of course more readily comprehended in the budding morphogenetic system wherein identification of old and new is obvious, but it is clearly just as real in fission yeasts. In a recent review [24], the relationships between septal location during spore formation and during vegetative growth of the prokaryote Bacillus were discussed. The ‘rules’ for septum location obviously vary with the physiological circumstances in that bacterial system. A precise comparison cannot be made of course, for septa are not laid down in asci of S. pombe during sporulation. But we think that it is instructive to note that in the S. pombe system, the rules of septum location do not seem to vary between ordinary vegetative cell cycles and ‘first’ cell cycles-germination of spores. Model making should be eased by that observation. Finally, we think it useful to observe that an ‘ideal cell cycle model’ system with balanced growth, isodiametric cylinders, and perfectly symmetrical cell division, could be analysed only by perturbing the system. On the other hand, the real cellular system, with small but real differences from ideality, e.g., sib volumes equal but lengths not, invites experimental questions some of which can yet be answered merely by examining the unperturbed system more closely.
Lindner, P, Wochschr brau 10 (1893) 1298. 2. Webster’s third new international dictionary (ed P B G Grove) 17th edn. G & C Meriam, Springfield (1976). 3. Johnson, B F, Yoo, B Y & Calleja, G B, Cell cycle controls (ed G M Padilla, I L Cameron & A Zimmerman) p. 153. Academic Press, New York (1974). 4. Streiblova, E & Beran, K, Folia microbial 8 (1963) 221. 5. Johnson, B F, Yoo, B Y & Calleja, G B, J bacterial 115 (1973) 358. 6. Johnson, B F, Calleja, G B & Yoo, B Y, Eucaryotic microbes as model developmental systems (ed D H O’Day & P A Horgen) p. 212. Dekker, New York (1977). 7. Shannon, J L & Rothman, A H, J bacterial 106 (1971) 1026. 8. Johnson, B F & Lu, C, Exp cell res 95 (1975) 154. 9. Johnson, B F, Exp cell res 39 (1965) 613. 10. Mitchison, J M, Exp cell res 13 (1957) 244. 11. Calleja, G B, Yoo, B Y & Johnson, B F, J cell sci 25 (1977) 139. 12. Johnson, B F, Exp cell res 49 (1968) 59. 13. Robinow, C F & Bakerspigel, A, The fungi (ed G C Ainsworth & A S Sussman) vol. 1, p. 119. Academic Press, New York (1965). 14. Johnson, B F, J bacterial 95 (1968) 1169. 15. Mitchison, J M, Methods in cell physiology (ed D M Prescott) vol. 4, p. 131. Academic Press, New York (1970). 16. McCully, E K & Robinow, C F, J cell sci 9 (1971) 475. 17. Fraser, R S S & Nurse, P, Nature 271 (1978) 726. 18. Krasnow, R A, J theor bio172 (1978) 659. 19. Beran, K, Streiblova, E & Lieblova, J, Proc 2nd symp yeasts, p. 353. Bratislava (1969). 20. Hayashibe, M, Sando, N & Abe, N, J gen appl microbial 19 (1973) 287. 21. Hartwell, L H & Unger, M W, J cell bio175 (1977) 422. 22. Johnson, B F, James, A P, Gridgeman, N T, Lusena, C V & Lee, E-H, Cell cycle regulation (ed J R Jeter, Jr, I L Cameron, G M Padilla & A M Zimmerman) p. 203. Academic Press, New York (1978). 23. Flegel, T W, Can j microbial 24 (1978) 827. 24. Hitchins, A D, Can j microbial 24 (1978) 1103.
We thank Dr N. T. Gridgeman for useful discussions on pairing, J. M. Ridgeway for performing the statistical analyses, R. H. Whitehead for photomicro-
Received January 23, 1979 Revised version received April 19, 1979 Accepted April 24, 1979
scopic advice, H. A. Turner for preparation of the photographic plates, and F. G. Villaume of American Cyanamid Company for a generous gift of the fluorochrome Calcofluor White M2R New. B. Y. Y. was supported by NRC of Canada grant A-3651.
REFERENCES
Exp Cell Res 123 (19791