Nuclear pore formation and the cell cycle in Saccharomycescerevisiae E. G. JORDAN, N. J. SEVERS* and D. H. WILLIAMSON,’ Biology Department, Queen Elizabeth College, Universiw of London, Campden Hill, London W8 7AH and ‘National Institute for Medical Research, Mill Hill, London NW7 IAA, UK Summary. The total number of nuclear pore complexes/nucleus was determined at intervals through the first cycle of synchronous growth in the yeast, Saccharomyces cerevisiae, using electron micrographs of freeze-fracture replicas of nuclei. Nuclear pore number increased in early GO phase, attaining a plateau by late GO which was maintained throughout the S phase. This was followed by a second increase at the time of nuclear division. The significance of these changes in relation to other events of the cell cycle is discussed.
The existence of the nuclear pore has been known for a quarter of a century [l] but its function is still far from being fully understood. Many reviews have appeared which weigh the mounting evidence in favour of a role in the transport of macromolecules, especially ribonucleoproteins [2-91, although there have also been some indications that the nuclear pore might be involved in DNA replication [lo] and chromosome organization [ 1l-1 31. One approach clarifying the function of nuclear pores is that of attempting to correlate changes in their number with macromolecular synthetic events. Conclusions from this type of experiment must be drawn with caution because pore number is not necessarily directly related to the rate of transport of macromolecules across the nuclear envelope. Indeed, there is evidence that changes in cell physiology may be accompanied by changes in the functional capacity of the pores themselves [14-B]. Nevertheless, certain significant changes in * Present address: Cell Pathology Unit, The School of Pathology, The Middlesex Hospital Medical School, London WlP 7LD, UK. Exp Cell Res 104 (1977)
nuclear pore number during the cell cycle do at least offer clues towards understanding their function. Notably it has been found that pore number may increase rapidly in the new post-mitotic daughter nuclei [ 10, 191indicating a role in the resumption of growth following cell division. Another pattern of pore number increase, at the time of DNA synthesis, has also been reported [lo]. Additionally, there is evidence that the number of pores is related to general cell activity [20-231. In this paper we report the results of a study into nuclear pore number in relation to the cell cycle in a lower eukaryote-the yeast Saccharomyces cerevisiae. This organism is of particular interest because it keeps an intact nuclear envelope through nuclear division, and also a number of the cell cycle parameters are known [24, 251. A preliminary report of some of this work has appeared elsewhere .
Materials and Methods Saccharomyces cerevisiae was grown and synchronized by a feed-starve regime followed by selection according to the method of Williamson & Scopes . Samples of 100 cm3 were removed from the synchronous cultures at 0, 30, 60, 90, 115, 145, 160 and 195 min after inoculation. Cells were harvested by vacuum filtration, washed in fresh growth medium and rapidly pelleted, the whole sampling operation taking less than 5 min. Samples of the concentrated cell suspension were mounted in specimen holders and rapidly frozen in melting Freon 12. It should be emphasised that no fixation or glycerol treatment were carried out before freezing. Freeze-fracture replicas were prepared by the method of Bullivant  and electron micrographs were taken with an AEI EM6B electron microscope operated at 60 kV. Exposures were made of nuclei showing face-fractured membranes at a standard magnification of x 15000. Images of these negatives were projected at a final magnification of x60000 using an enlarger, and areas of nuclear envelope were traced onto paper. Regions which were steeply inclined, as judged by density of shadow and the appearance of nuclear pores were excluded. The number of pores in each outline was counted and the areas of the outlines determined using a planimeter. The mean nuclear pore frequency (i.e., nuclear pores pm-*) for each time interval of the cell cycle was calculated. In selecting
Preliminary notes areas of nuclear envelope for pore frequency detcrminations, very small and therefore possibly unrepresentative areas were excluded. Resting cell nuclei (0 mitt) have a large indented area devoid of nuclear pores  which was estimated to occupy at least 3.2 pm2 of the nuclear surface area. Account was therefore taken of the fact that fractures through this pore-free area may not have been recognised as nuclear envelope. For nuclear surface area estimates, cross-fractured nuclei were utilized. Tracings were made from images of negatives at a final magnification of X40000. Measurement of nuclear profiles was performed using a map measurer and an estimate of nuclear surface area obtained by treating each profile as the circumference of a sphere. Most of the profiles used represented near-median cross sections of the nuclei because of the tendency of face fractures, but not cross-fractures, to occur towards the ends of the nuclei. The method is therefore judged to give a reliable comparative guide to changes in nuclear surface area. In the 145-and 160min samples, the surface area value was corrected to take account of the proportion of the cells in mitosis by doubling that part of the figure which represented the proportion of cells with dividing dumb-bell shaped nuclei. The total number of pores/nucleus was calculated from the pore frequency and nuclear surface area measurements for each cell cycle stage investigated. The morphological markers used to relate this information to the exact cell cycle position were (i) percentage of cells budding; (ii) cell division. Budding cells were counted at intervals using samples viewed by phase contrast. Cell number was assayed after sonication of samples using a Coulter counter.
Fig. 1. Abscissa;
time after inoculation (min); ordinnte: (lefr) no. of nuclear pores/nucleus (0); nuclear pore frequency (A); (right) nuclear surface area @m*) (0). Changes in nuclear pore frequency, nuclear surface area and number of pores per nucleus during the cell cycle of Saccharomyces cerevisiue. The sigmoid curves show the time of appearance of young buds (A) and divided cells (0) as percentages of the population. The S and M periods are positioned in relation to budding and cell division in accordance with earlier studies . The rise in pores occurring during the time of mitosis has been corrected for the proportion of the sample that has completed nuclear division. This is necessary because most cross-fractures obtained at this time represent only half of a dumb-cell shaped nucleus.
unchanged until the time of nuclear division. During nuclear division and up until cell division (which is coincident with the start of the next S phase) there was another rapid increase in both nuclear surface area Results and nuclear pore number bringing both valThe number of nuclear pores showed sig- ues close to the levels at which they stabinificant fluctuations through the cell cycle lized in the first round of budding and DNA (fii. 1) which can be related to nuclear synthesis. During the periods of nuclear envelope swelling, DNA synthesis and nuclear division. The cells spend the first hour recover- and nuclear pore increase, both the early ing from the feed-starve cycling and syn- GO and the later rise around the time of nuchronisation procedure in a ‘GO’ phase clear division show a similar relationship. which ends at the time of bud emergence Nuclear envelope surface area shows a and DNA synthesis; these events follow higher rate of increase than does pore numcell division without any lag in the next ber in the earlier half of the rise but a higher cycle. During the first growth phase (GO), rate of increase in pore number than in enthe nuclear surface area enlarged rapidly velope area occurs in the later half of the from 17 to 23 pm2. Over this same period rise. The synthesis of both pores and memthe number of nuclear pores increased from 250 to 350 per cell. With the onset of DNA brane must be particularly rapid during the synthesis and budding there was a stabiliza- time of mitosis because the number of pores tion of both nuclear surface area and pore and surface area of the nuclei have reached number which then remained more or less nearly two-thirds of their maximum interExp Cell Res 104 (1977)
phase levels by the time the nuclei are formed. Discussion The effect of changes in RNA synthesis on nuclear pore number cannot be judged easily from the normal cell cycle of Saccharomyces cerevisiae because RNA/cell increases at a constant rate, not even apparently showing a drop during nuclear division [24, 25, 30, 311. However, in the yeast synchronized by the method we have used, it is possible to follow pore number through changes in cellular RNA synthesis. The first rise in nuclear pore number occurred during a time of very little RNA synthesis in ‘GO’, and the second during a period of extensive RNA synthesis  (which presumably represents the resumption of RNA synthesis in the growing cells). There is no obvious or direct relationship between either total cell RNA, or RNA synthesis and nuclear pore number. These facts do not exclude a function for nuclear pores in RNA translocation and may be interpreted in terms of variable flow rates of RNA through the pores [14-181. The RNA synthesized in the ‘GO’ phase may be different from that synthesized later. Hartwell  has likened the events which follow release from starvation, i.e. the GO phase, to a differentiation. It could be argued that during this change, new mRNAs must leave the nucleus and many chromosomal proteins must enter, and that these processes may be related to the changes in pore number. In higher eukaryotes, a biphasic increase in pores occurs during the cell cycle, the first is immediately post-mitotic starting in telophase and the second coincides with DNA synthesis [lo, 191.In yeast, considering the cell cycle after the ‘GO’ phase, we found the former rise in pores but the latter Exp CeNRes 104 (1977)
was absent. Although there was a rise at the time of mitosis, this can be considered as part of the post-mitotic repopulation of the nuclear envelope with pores. Since the nuclear envelope does not suffer disintegration at mitosis, events that would necessarily be delayed until its arrival in other eukaryotes can start earlier in yeast. This may also be seen from the fact that both budding and DNA synthesis occur without any delay following cell division. However, there is a point which occurs close to cell division at which the cycle may be arrested in conditions of starvation  and it is interesting that the cells at the start of the experiment have a pore number equivalent to that to be expected in early post-mitotic nuclei, indicating that the arrest by starvation may stop the G 1 rise in nuclear pore number. This explanation would also imply that pore number becomes uncoupled from other events because the arrested cells proceed as far as the next S phase in other respects. The events of ‘GO’ may thus be considered as the completion of processes delayed by the block besides involving the necessary reprogramming steps. There is no indication in yeast that pore number has any relation to DNA synthesis, either in its initiation or continuance. Neither is there any sign that pores must change in number during the time of chromosome separation, a process which precedes nuclear division in yeast . These facts make it unlikely that nuclear pores have a function that is directly related to DNA synthesis or chromatin organization as suggested from some work on higher eukaryotes [l&13]. Finally, the relationship between the increase in nuclear surface area and total pore number is also interesting. Notwithstanding the general trend for nuclear surface area and pore number to increase together, a
N. J. Severs was in receipt of an award from the S.R.C. at the time this work was carried out.
28. Bullivant, S, Advanced techniques in biological electron microscopy (ed J K Koehler) p. 67. Springer-Verlag, Heidelberg (1973). 29. Severs, N J, Jordan, E G & Williamson, D H, J ultrastruct res 54 (1976) 374. 30. Williamson, D H, Synchrony in cell division and growth (ed E Zeuthen) p. 351. Wiley & Sons, New York (1964). 31. Sogin, S J, Carter, B L A & Halvorson, H 0, Exp cell res 89 (1974) 127. 32. Severs, N J & Jordan, E G, Experientia 31 (1975) 1276.
Received October 14, 1976 Accepted October 20, 1976
feature also noted in some higher organisms [8, 19, 321, there is a tendency for the increase in nuclear membrane to precede that in pores, indicating that control of the pore number may be effected somehow via nuclear membrane synthesis.
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