It was noted by Theodor Boveri a century ago that “The constant, which we must accept as something given and not at present further analyzable, is the fixed proportion between nuclear volume and protoplasmic volume, namely, the karyoplasmic ratio.” (Wilson, 1925
). This statement was echoed recently by Cavalier-Smith (2005)
, who wrote that “The invariant karyoplasmic ratio is a basic fact of cell biology established for more than a century (but forgotten by two generations of textbooks, which are insufficiently quantitative) and a necessary consequence of the optimization of growth processes by selection for rapid and efficient cell reproduction.”
In this study of the budding yeast nucleus, we have discovered that the karyoplasmic ratio is actively, although not perfectly, maintained in budding yeast. In cell size mutants, nuclear size is altered in rough proportion with cell size. Similarly, as wild-type cells grow, nuclear size increases. The close relationship between cell and nuclear size appeared to overwhelm any direct, immediate nuclear expansions caused by replicating the genome in S-phase, at least at the level of precision afforded by this analysis. A previous analysis of nuclear volume during the S. cerevisiae
cell cycle could not have arrived at this conclusion, as the authors did not monitor cell size (Winey et al., 1997
). This previous study could only conclude that cells in early mitosis had larger nuclei than cells in S-phase, whereas cells in S-phase had larger nuclei than cells in G1-phase. If DNA content were an important and direct factor in setting nuclear volume, a step-like increase in nuclear volume would be expected during S-phase, and we saw no sign of such an increase in either haploid or diploid cells. That nuclear size is more closely correlated with cell size than DNA content is further demonstrated by the small average nuclear size of whi5
Δ cells and cells overexpressing CLN3-1
. FACS analysis demonstrates that asynchronous populations of both strains are enriched for cells with replicated genomes (, C and F).
In one model for how budding yeast measure the critical cell size for cell cycle entry, the highly unstable Cln3 protein increases in concentration in a constant volume nucleus as cells grow and increase their absolute translational capacity (Futcher, 1996
). In this model, a constant G1 nuclear volume acts as the metric against which ribosome content and hence cell volume are measured. But we have demonstrated that nuclear volume increases during G1-phase. This observation renders the model very unlikely, although the fact that the nucleus does not grow quite as quickly as the cytoplasm means the model cannot be completely ruled out. Our observations accommodate the possibility that increased nuclear size is what triggers the Start transition, as previously suggested for S-phase entry in mammalian cells (Yen and Pardee, 1979
). Alternatively, some aspect of DNA quantity and not nuclear volume may be the metric against which cell volume is gauged before Start, because the critical cell size requirement is nearly twice as large in diploid budding yeast (Lorincz and Carter, 1979
Although we estimated cytoplasmic volume by subtracting estimated nuclear volume from estimated cell volume in B, accurately measuring the volume of the cytoplasm is much more complicated. Even before any measurements, it would need to be decided which organelles to include in the definition of cytoplasm. One would almost certainly want to subtract vacuolar volume, which is comparable to the nuclear volume (C). Measuring vacuolar volume would be complicated by the often eccentric shape of this organelle. In addition, the volume of the vacuole may not increase proportionately with cell size. It has been previously noted that cln3
Δ mutant cells, which are large relative to wild-type, have disproportionate increases in total vacuolar volume (Han et al., 2003
). The allometric relationship between vacuole size and cell size remains to be further characterized, as does the possible influence of vacuolar volume on cell size control at Start.
It is generally presumed that the size of the nucleus is set directly by the physical bulk of the chromatin. As the nucleus must house the chromatin, DNA content must directly limit the minimal nuclear size. But even in the smallest yeast, we saw no flattening of the correlation between cell and nuclear size, suggesting that this minimal size has not been reached. Indeed, all of our evidence suggests that DNA content does not directly control nuclear size in growing budding yeast. We therefore anticipate that changes in cell ploidy impact nuclear size indirectly in yeast. By raising the critical cell size threshold at Start (Lorincz and Carter, 1979
), increased DNA content will increase nuclear size due to the unknown mechanisms that maintain the N/C ratio, not because of the greater space occupied by the larger genome. DNA content may also affect nuclear size indirectly in other organisms.
We were unable to obtain any clues as to the mechanism that coordinates nuclear and cell size. Blocking cell growth had little effect in the short-term (<60 min) on nuclear size (). In terms of sheer magnitude, ribosome biogenesis is the dominant process in the yeast cellular economy (Warner, 1999
). Rapamycin treatment rapidly represses ribosome synthesis rates and shrinks the nucleolus (Warner, 1999
; Tsang et al., 2003
), but had no such effects on nuclear size (A). In the inverse experiment, causing ribosomal subunits to accumulate in the nucleus by blocking Crm1-mediated nuclear export also had no effect on nuclear size (). As yeast growing under these conditions double every ~90 min, in 15 min a yeast will synthesize ~
of the total ribosomes synthesized each cell cycle. If nuclear export is completely blocked, then ~
of the total ribosomes synthesized each cell cycle will have accumulated in a space that is
the volume of the cell (), resulting in nuclear ribosomal concentrations twice that of the cytoplasm. These considerations assume no negative feedback to ribosome synthesis rates as well as full blockage of ribosome export during the 15 min. Despite these caveats, it seems reasonable to suppose that these brief LMB treatments strongly increased nuclear protein concentration, and yet there was no effect on nuclear size.
How then might the yeast nucleus expand? Are there dedicated mechanisms that actively maintain the N/C ratio by controlling the density of the chromatin or the structure of the nuclear envelope? Notably, yeast lack the lamin-based meshwork that underlies the nuclear envelope in metazoan cells (Taddei et al., 2004
). Recently, three genes whose loss leads to rampant nuclear membrane expansion have been characterized in budding yeast. All three gene products appear to repress the transcription of enzymes involved in phospholipid biosynthesis (Santos-Rosa et al., 2005
). Remarkably, the nuclear membrane in these cells is no longer round, but displays long extensions that are filled with nucleolar material (Campbell et al., 2006
). It is not clear whether overall nuclear or cell volume is markedly increased in these mutant cells. Mutations that alter nuclear volume without altering cell volume could be very informative.
There are many more interesting questions about nuclear size. Budding yeast do not break down the nuclear envelope during mitosis. But after this closed mitosis, daughter cells clearly have smaller nuclei than mother cells (B and noted by Gasser, 2002
). Is the nuclear volume segregated differentially at anaphase? Or is the nuclear mass segregated equally followed by rapid adjustments in nuclear volume? On a related note, can the nucleus shrink in volume without cell division? It has been demonstrated that parts of the nucleus are engulfed by the vacuole in a process termed piecemeal microautophagy of the nucleus (Roberts et al., 2003
Finally, how is nuclear volume related to cell growth in other organisms? Nuclear volume in metazoan cells can change without alterations in cellular DNA content. A handful of studies with mammalian cells have suggested, but not convincingly demonstrated, that the nucleus may increase in volume before S-phase (Maul et al., 1972
; Yen and Pardee, 1979
; Fidorra et al., 1981
). It is evident in many animal embryos that nuclear volume shrinks during embryonic cleavage cycles as cell volume decreases (Wilson, 1925
; Gerhart, 1980
; Sulston et al., 1983
). Inversely, injecting the nuclei of HeLa cells into much larger frog oocytes can lead to 10-fold or greater increases in nuclear volume, while fusing hen erythrocytes with much larger HeLa cells leads to expansion of the erythrocyte nucleus (Harris, 1967
; Gurdon, 1976
). In the latter case, the expansion was shown to occur independently of DNA replication (Harris, 1967
). Pathologists have used aspects of nuclear morphology, including size, to diagnose and grade cancers for many decades (Zink et al., 2004
). These and many other studies illustrate that nuclear volume is dynamic, but the mechanisms causing nuclear volume alterations remain largely unknown.