D ISC U SSIO N

Progress in understanding the molecular mechanisms controlling the cell cycle has emerged from a number of model systems. The early embryonic divisions of invertebrate (clam) or vertebrate (Xenopus) oocytes display an inherent synchrony, as do cells in the early embryos of Drosophila. In the case of yeasts, populations of cells must be synchronized in order to correlate biochemical events of the cell cycle with genetic information. The information so obtained may be added to that from other types of analysis such as the observation of single, living cells. A synchronous culture is particularly useful for the determination of fluctuations of proteins through the cell cycle and to investigate protein modifications and associations. The purpose of this study was to address the question “Does cyclin B oscillate in the fission yeast cell cycle?’. The results presented in this chapter show that synchronous cultures of fission yeast cells are attainable in two ways, either using induction synchrony or by selection synchrony. These synchronization techniques were used to investigate the synthesis and degradation of p63«ki3 in the fission yeast cell cycle.

Perfect synchrony is difficult to achieve due to the inherent variation in cell cycle times in natural cell populations. The combined use of induction and selection synchrony would be particularly useful for the analysis of causal connections between cell cycle events. A combination of the two approaches would be an ideal situation as it would exploit the advantages of each method and possibly eliminate their disadvantages. All methods of synchronisation have inherent dangers. In an attempt to avoid artifacts (Mitchison, 1989), several different methods were used in this study and the results compared.

There are a number of ways in which the synchronised progression of cells through the cell cycle can be monitored. In fission yeast, synchrony is most easily

monitored by counting the percentage of dividing cells to obtain a septation or cell plate index. Live cells containing a septum can be determined microscopically using phase contrast optics. An additional advantage of using 5. pombe is that cells grow only by length extension (Mitchison, 1970; Mitchison

and Nurse, 1985), thus cell length is a direct measure of position in the cell cycle.

In S. pombe ^ centrifugal élutriation is the method of choice as it does not require high g forces or density gradients, in addition cells can be separated in the growth medium used for cell culture. The major disadvantage of this method is that the level of synchrony achieved (between 30-40% in the first cell cycle) is often insufficient to study transient cell cycle events. The number of cells synchronised this way also tends to be low, largely due to the small size of the élutriation chamber but also by the proportion of cells eluted. This limits the number of parameters that can be measured in a given experiment.

With wild type cells synchronized using élutriation in YE medium at 29°C, the first peak of dividing cells occurred 3 hours following inoculation into fresh medium. This is because small wild type cells eluted from the rotor are synchronised at late G1 or early G2 phases of the cell cycle. The peaks of septation observed in this experiment were low. Although the corresponding Western blot shows an oscillation in cyclin levels, it was difficult particularly in the second cell cycle, to determine the point of cyclin accumulation and degradation

In rich growth medium, S. pombe cells grow and divide actively. The G1 phase is short, occupying 10% of the cycle and G2 is much longer, approximately 70% of the cycle (Fantes and Nurse, 1981; Fantes, 1984). Under conditions of nutrient deprevation, G1 is extended as newly divided cells are below the critical size requirement for DNA synthesis to occur. The G2 period is correspondingly

reduced under these conditions. Nutrient limitation can therefore be used to control the entry of cells into stationary phase. Cells entering stationary phase in nitrogen-limiting medium are smaller than those grown in glucose-limiting medium (Costello et al., 1986). Upon return to rich medium however, stationary phase cells re-enter the cell division cycle synchronously. This approach produced a good degree of synchrony which was reflected in the oscillations in p 6 3 « ic i3

levels that were observed (Figure 2.2B).

An alternative method of synchrony is ‘block-release’ or cell cycle arrest and release. In these experiments, cdc mutants are commonly used (King and Hyams, 1982). When a cdc mutant is shifted to the non-permissive temperature it becomes blocked at a cell cycle arrest point unique to that mutant. Cells are normally blocked for two cell cycles to ensure all cells have spent at least one cell cycle at the block point. AVhen shifted back to the permissive temperature, cells should return to the cell cycle from the same point. In fission yeast, cell growth continues during the block period, resulting in larger than usual cells at the time of release. This means that after the first division, cells are already large enough to undergo a second division, thus reducing G2 to a minimum (Fantes and Nurse, 1978). The cell cycles of these mutants initially tend to be shorter than normal (King and Hyams, 1982), but increase to normal cycle time as the cells resume their normal size at division. A possible explanation is that the mitotic size control in oversized cells is cryptic. Alternatively, the fact that there is a requirement for the cell to spend a minimum amount of time traversing G2, is thought to control the cycle to a certain extent (Fantes, 1974; 1984).

wee mutants are accelerated through G2, but their G1 phase is extended as these cells are too small to pass Start when bom and must therefore grow to a sufficient size in G1 (Nurse, 1975). These mutants undergo a normal mitosis generating cells that divide at a reduced size. The change in p63odci3 levels was investigated in

wee mutants at both the permissive and restrictive temperatures. When weel .50 cells were synchronized at 36®C, cell size was small and cyclin levels were clearly periodic. When incubated at 25 ®C, the w eel gene is active and division delayed until cells approach wild type length. The low basal levels of cyclin B in wee 1.50 (Figure 2.3B) might be due to increased degradation of the protein during the extended G1 phase. An alternative explanation could be the small cell size or the inactivation of the wee gene. This possibility could be tested in the cdcl 0.129 mutant which blocks in G l. If p63odci3 levels fall in G1 in c d c l0.129 cells synchronized at 35®C, then it is likely that the low level of p63«ki3 in wee 1.50 cells is due to the size of these cells as cdc 10.129 cells are larger and have a functional weel gene.

Levels of p 6 3 « ic i3 in selection synchronised w eel.50 cells at 36®C have been

determined using an different method of synchronisation (Creanor and Mitchison, 1996). No p63cdci3 was detectable at the start of the cycle followed by a peak just before mitosis. Cyclin levels decreased to almost zero in early G l which is in agreement with the results presented here. A striking difference between wild type and wee cells is that the level of p63odci3 falls to zero in interphase in the wee cells but not in wild type cells. This may be because the length of G l in wild type cells (approximately 0.13 of the cycle) is too short to allow the complete degradation of cyclin. Wee 1.50 cells have a much longer G l which could also explain why p63odci3 levels decrease to such a low level.

Figure 2.5A shows a profile of fission yeast cells synchronized by cell cycle arrest and release. Cdc25.22 cells were arrested at the G2/M transition. Upon release from the temperature block, cells rapidly initiated two synchronous cycles of cell division. Peaks of septation ocurred at 80 and 160 mins. These timings are much earlier than those seen with elutriated wild type cells (Figure 2.1 A) and are due to

the larger size of these mutant cells (see above). The ‘block-release’ methods have the advantages of offering higher levels of synchrony and larger volumes of synchronized cells. Cdc25.22 enters mitosis with 70-80% synchrony ( Booher et a/., 1989; Moreno et aL, 1989) comparable to that originally described for cdc2.33 (King and Hyams, 1982). This superior synchrony can be exploited for the analysis of transient cell cycle events, such as G l and S phases. A disadvantage of using cdc25.22 is that cells already past the G2/M transition at the shift to 36®C, have to complete a cell cycle in order to become blocked at G2/M and levels of cyclin B are high at this point.

Nda3.KM311 cells arrest following spindle pole body duplication but prior to spindle formation (Kanbe et al., 1990). The arrest has been shown to be exceptionally synchronous and can be demonstrated by the highly synchronous separation of chromosomes following return to the permissive temperature (Toda et a i, 1981; Hiraoka et a l, 1984). The marked fluctuation of p63«ki3 seen in Figure 2.6 is a reflection of high level of synchrony possible by the cell cycle arrest and release of nda3.KM3J 1 cells.

In wild type cells, p63cdci3 is not required for S phase to occur and is not present at the point of activation of p34^dc2 kinase in G l. The mechanism responsible for the regulation of p63cdci3 may be due to its constitutive expression coupled with its destruction at mitosis. In some systems there is evidence that B cyclins are degraded rapidly at anaphase; for example in mammalian cells (Girard et al., 1995), and in embryos (Hunt et al., 1992; Luca et al. 1991; Murray, 1995). In other cells, exit from mitosis occurs with no change in B cyclin levels, for example early Drosophila embryos (Gonzales et al., 1994; Edgar et al., 1994), and in Physarum

(Cho and Sauer, 1994). There is a possibility therefore that only a small proportion of p 6 3 c d c i3 i s complexed with p 3 4 c d c 2 and only rapid destruction of this complex allows completion of mitosis.

p34cdc2 is the universal protein kinase responsible for driving cells into M phase (Nurse, 1990). The results presented here show the level of p34ok2 remains constant through the cell cycle and even in cells arrested at G2/M or in M phase (Figures 2.1-2.8). These results agree with previously published data (Simanis and Nurse, 1986; Booher et a/., 1989; Moreno et aL, 1989). In fission yeast and budding yeast, p 34cd c2/C D C 28 kinase activity is required at both transitions (Nurse and Bissett.,1981; Reed and Wittenberg, 1990; Surana et al., 1991). Entry to, and exit from mitosis is governed by the activation and inactivation of p34odc2. The activity of this kinase oscillates during the cell cycle because the activating subunit, cyclin B, is only stable during interphase and is unstable during mitosis (Evans et al., 1993). The rapid proteolysis of cyclin B in mitosis is essential for cell cycle progression. Mitotic cyclins are degraded by the ubiquitin proteolytic pathway and they are targeted to this pathway by an N-terminal consensus sequence called the destruction box (Glotzer et al., 1991). It has been shown that deletion of the cyclin destruction box prevents exit from mitosis by stabilizing cyclin (Murray et al., 1989).

When cell extracts are prepared from S. pombe by breakage with glass beads, some proteins are retained in an insoluble form. p34«k2 and p63cdci3 are present in two forms, a soluble fraction (supernatant) and an insoluble fraction (pellet), (Hayles et al., 1994). When the soluble and insoluble fractions of p34odc2 and