An important question in cell biology is what controls organelle shape. Throughout most of the cell cycle, the yeast nucleus is round, but the underlying structure responsible for this shape is unknown. Here, we analyzed the phenotype of a budding yeast mutant that has a nonround nucleus, displaying a single nuclear flarelike extension. Our results show that the spo7
Δ nuclear flare colocalizes with the nucleolus (). In a wild-type cell, the membrane adjacent to the nucleolus seems indistinguishable from the membrane that surrounds the rest of the nucleus, although it was previously noted that some nuclear periphery proteins, such as Mlp1p and Esc1p, are excluded from the nucleolus (Andrulis et al., 2002
; Galy et al., 2004
; Taddei et al., 2004
). Our analysis of the spo7
Δ mutant phenotype suggests that the nuclear membrane associated with the nucleolus has certain properties that distinguish it from the membrane surrounding the rest of the nucleus, because in the absence of Spo7p function only the nucleolus-associated membrane becomes extended (). The involvement of Spo7p in inhibiting phospholipid biosynthesis (Santos-Rosa et al., 2005
) strongly suggests that the flare is a result of membrane proliferation due to elevated levels of phospholipids, rather than an expansion of the nucleolus itself. Indeed, we found that when the nucleolus was no longer intimately associated with the nuclear envelope, such as in a strain in which the only rDNA copies were on plasmids and expressed by Pol II (Oakes et al., 1998
), Spo7p inactivation did not alter nucleolar shape, but it still led to the appearance of flares (). Thus, the nuclear membrane associated with the nucleolus differs from the nuclear membrane associated with the bulk of the DNA in its susceptibility to expansion when phospholipid levels are elevated.
Our findings raise an interesting question as to how the crescent-shaped nucleolus is normally formed. This structure could, in principle, be an inherent property of the nucleolus. However, we observed that when the nuclear membrane expands, such as in a spo7
Δ mutant, the nucleolus loses its crescent shape. Moreover, under these conditions, the rDNA, which resides within the nucleolus, spreads throughout the entire flare (). Because, as mentioned above, flare formation is independent of nucleolar expansion, we favor the idea that as the flare forms, nucleolar components spread into this extended space. This suggests that the nucleolus does not have an inherent crescent-shaped structure but that it is confined by the nuclear membrane. In wild-type cells, at the end of anaphase, the nuclear membrane must be remodeled by a yet unknown mechanism to absorb or remove the extra membrane that connects the two DNA masses (, wild type). Because the nucleolus divides only at the end of anaphase (Fuchs and Loidl, 2004
; Pereira and Schiebel, 2004
), trailing behind the rest of the DNA, it is possible that as the nucleus regains its round shape, the nucleolus is compacted by the nuclear membrane into a crescent shape (). In the absence of Spo7p function, this compaction may fail (, spo7
Δ), resulting in an extended nucleolus. Thus, the nuclear membrane itself may contribute to the shape of the budding yeast nucleolus, and by limiting the amount of nuclear membrane synthesized, Spo7p may play a role maintaining proper nucleolar shape. The question of what determined nucleolar shape could be further extended to diploid cells, where under wild-type conditions there is only one nucleolus, despite having two copies of chromosome XII, each carrying an rDNA array. How the single nucleolus forms in diploid cells is unknown, but our results show that the nuclear membrane plays an important role in maintaining nucleolar structure, because nuclei of spo7
Δ mutant cells exhibit a high frequency of two nucleoli (). Hence, the yeast nucleus and its nucleolus provide an interesting paradigm for elucidating the role of intracellular membranes in ensuring organelle shape.
Figure 8. Schematic model to explain the mechanism of flare formation. (A) The structural changes experienced by the nucleus and nucleolus of dividing cells. Cell body, gray; the bulk of the DNA mass, blue; nucleolus, green; and spindle pole body, purple. Note (more ...)
Our finding that the spo7
Δ flares are associated with the nucleolus, whereas the bulk of the DNA remains tightly associated with the nuclear membrane, led us to examine how another ER-associated membrane, the peripheral ER, responds to the absence of Spo7p. We found that in the absence of Spo7p, the peripheral ER is also extended, no longer exhibiting the typical tubular structure (). Because the nuclear membrane is contiguous with the ER, our data suggest that all ER membranes, with the exception of the nuclear membrane that is associated with the bulk of the DNA, exhibit membrane expansion in the absence of Spo7p. How does the nuclear membrane associated with the bulk of the DNA resist membrane expansion? It is tempting to speculate that this membrane domain is tethered to the DNA, either directly or indirectly, by a chromosome associated factor(s) that is absent from the region occupied by the nucleolus (). This kind of tethering would be analogous to the role played by lamins in shaping nuclei of higher eukaryotes, although a yeast laminlike protein has yet to be identified. When phospholipid levels increase, this mechanism would restrict membrane expansion around the bulk of the DNA. In budding yeast, several proteins are known to tether DNA, and specifically telomeric regions, to the nuclear periphery. These include Sir4p, the Ku70p/Ku80p complex, and Esc1p (reviewed in Taddei et al., 2005
). In an attempt to determine whether these proteins also act to prevent membrane expansion in the spo7
Δ mutant, we deleted these genes individually and in combination (i.e., sir4
Δ or esc1
Δ) and examined what effect these combined mutations have on nuclear structure. In none of the mutant combinations could we detect an exacerbation of the spo7
Δ phenotypes, and there was no noticeable membrane detachment from the bulk of the DNA (our unpublished data). This suggests that these proteins are not necessary to resist membrane expansion, although we cannot rule out the possibility that they play a nonessential role in this process. In this regard, a study from the Tartakoff laboratory showed that overexpression of Esc1p leads to dramatic elaborations of the nuclear envelope that include nuclear pores but contain a limited amount of nucleoplasm (Hattier, Andrulis, and Tartakoff, personal communication). The mechanism by which this occurs remains to be determined. We also examined whether the simultaneous deletion of the Mlp1p and Mlp2p proteins, whose localization pattern made them tempting candidates for playing a role in membrane tethering, might affect the spo7
Δ phenotype, but found no effect (our unpublished data). It was previously found that the spo7
Δ and nup84
Δ mutations are synthetically lethal, namely, that cells can survive with either, but not both, mutations (Siniossoglou et al., 1998
). The Nup84 protein is part of the nuclear pore, and in its absence nuclear pores cluster into discrete domains rather than distribute throughout the nuclear envelope. The synthetic lethal interaction between spo7
Δ and nup84
Δ is suggestive of a possible role for nuclear pores in tethering the nuclear envelope of the bulk of the DNA, although additional components must be involved because nuclear pores are present in the membrane associated with the nucleolus. It will be possible to examine the involvement of Nup84p in membrane tethering once an appropriate system for effectively activating and inactivating Spo7p and/or Nup84p is developed.
If there is membrane tethering mechanism, why and how it is excluded from the nucleolus are not known. It is possible that it is advantageous to the cell to keep the nucleolus-associated membrane amenable to expansion. This raises interesting questions as to how the yeast nucleus grows in size as cells undergo division: can membrane components be incorporated at any place on the nuclear membrane, or is there a specific “entry site”? Because the flare can form at various points of the cell cycle, does this mean that the nucleolus remains associated with the same “susceptible” membrane domain? An insight into how proteins partition between the nucleolus and the rest of the nucleus was gained from our experiment examining the localization of Mlp1p in the rdn1ΔΔ/pGAL7 rDNA spo7Δ strain. Because Mlp1p remained associated with the bulk of the DNA and did not spread into the flare despite the existence of a much smaller nucleolus (), it seems likely that the intranuclear distribution of Mlp1p is determined not by nucleolar exclusion but by an affinity to one or more components that reside in the nonnucleolar compartment of the nucleolus. Further experiments are needed to determine what these components are.
Why do flares always form in the vicinity of the nucleolus? One possibility is that the expansion in the vicinity of the nucleolus is influenced by the nucleolus itself. Alternatively, the colocalization of the nucleolus and the flare could be a coincidence created by the mechanics of nuclear division. The nucleolus is the last region of DNA to segregate, placing it at the same nuclear membrane region that must be remodeled during nuclear division (). A nuclear landmark that is in a fixed relationship to the nucleolus is the spindle pole body (SPB), which is embedded in the nuclear membrane and serves to nucleate both cytoplasmic and spindle microtubules. The SPB, which is in the leading edge of the dividing nucleus, and the nucleolus, which is at the trailing end of the nucleus, are located on opposite sides of the nucleus and this organization is maintained throughout most of the cell cycle (Yang et al., 1989
; Bystricky et al., 2005
). Interestingly, we found that in the rdn1
strain, as in the wild-type and spo7Δ strains, the nucleolus was typically found opposite the spindle pole, thus precluding the determination of whether flare formation is dependent on a particular nuclear membrane domain (i.e., opposite the spindle pole) or whether it depends on the proximity to the nucleolus. However, because the nucleoli in this strain are significantly smaller than nucleoli of wild-type cells, and consequently occupy only a very small fraction of the spo7
Δ flare (), we favor the possibility that the site of flare formation is independent of the nucleolus. We speculate that the membrane opposite the spindle pole, which has to remodel after the completion of anaphase, has properties that make it distinct from the nuclear membrane that surrounds the DNA. Our results show that the spo7
Δ flare may form at the end of anaphase due to incomplete remodeling of the nuclear membrane located between the segregated DNA masses (Figures and ). We also find that a flare may form later in the cell cycle, independent of nuclear division, but again coincident with the nucleolus (Figures and ). If indeed nuclear division results in a distinct membrane domain that is sensitive to expansion, one possibility is that its colocalization with the nucleolus contributes to its maintenance in this distinct state. This raises an interesting question: does the nucleolus remain associated with a particular membrane region throughout the cell cycle, and if so, what is the functional importance of this association?
Together, our results suggest that the nuclear membrane has discrete domains that respond differently to changes in phospholipid levels. We propose that this is due to an asymmetric distribution of membrane tethering factors that are present throughout the nuclear envelope except in the newly reorganized region. What these factors are and whether the nucleolus plays a role in preventing membrane tethering awaits further analysis.