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Vol. 20, Issue 12, 2932-2942, June 15, 2009
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Pathology Department and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106
Submitted December 11, 2008;
Revised March 20, 2009;
Accepted April 8, 2009
Monitoring Editor: Karsten Weis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Jaspersen and Winey, 2004; Molk and Bloom, 2006). Nuclear fusion (karyogamy) then leads to encounter of the two parental genomes.
Among proteins that are known to be required for karyogamy are the endoplasmic reticulum (ER) luminal proteins, Kar2p, and Kar8p/Jem1p; the ER transmembrane protein, Kar5p, which localizes near the SPB; Kar7p/Sec71p and Sec63p of the ER translocon; and Prm3p, a protein that, surprisingly, has been localized to the nucleoplasmic face of the inner nuclear membrane (Marsh, 1997; Beilharz et al., 2003). Because karyogamy follows establishment of SPB contact, the mechanism of fusion could be distinct from nuclear fusion in organisms that lack SPBs. An alternate view is that the SPBs ensure spatial proximity and that fusion of the outer membranes, which initiates with contact of their cytoplasmic aspect, involves Snares and the AAA ATPase, Sec18p/NSF, as in topologically equivalent fusion events along the secretory and endocytic paths (Jahn and Scheller, 2006).
For organelles that are enclosed by double membranes, one can envisage distinct models of fusion (see Figure 1A). In the "trans-first" model, the outer membranes (cis, trans) fuse with each other, and the inner membranes fuse with each other. These events could be simultaneous or sequential. If the outer membrane fuses before the inner membrane, fusion of the inner membranes would initiate with contact of their luminal aspect. It is therefore plausible that proteins of the lumen between the outer and inner membranes would be required. Alternatively, the outer and inner membranes of a single organelle could first fuse with each other, and this unit(s) could subsequently fuse with equivalent intermediates generated from the target organelle. This second model is followed during the mitotic cell cycle of higher eukaryotes as part of the events of NE breakdown and reformation (Sheehan et al., 1988; Holaska et al., 2002; Hetzer et al., 2005; Baur et al., 2007; Stewart et al., 2007).
Consistent with the trans-first model for NE fusion, electron micrographs of fertilization in marine algae and sea urchins capture putative intermediates that show selective continuity of outer membranes (Longo and Anderson, 1968; Urban, 1969). A recent report concludes that the outer membrane also fuses seconds before the inner membrane in yeast (Melloy et al., 2007); however, as is explained below, the underlying experimental strategy used in those studies is flawed.
Apart from the importance of microtubules for nuclear congression, there is little information on the cytoskeleton in zygote formation (Hasek et al., 1987). Septins have however been detected at the cell cortex near the waist of zygotes (Ford and Pringle, 1991; Kim et al., 1991).
Little is known regarding the possible independence of parental nuclear genomes during zygote genesis, although studies of fertilization in plants and mice indicate lack of intermixing for at least several generations (Odartchenko and Keneklis, 1973; Rechsteiner and Parsons, 1976; Gleba et al., 1987; Brandriff et al., 1991; Callimassia et al., 1994; Mayer et al., 2005). The relevant genetic and cytological data for diploid yeast are not obviously all in agreement with each other (Kadyk and Hartwell, 1992; Haber and Leung, 1996; Jin et al., 2000; Lorenz et al., 2002).
This study inquires whether fusion of outer and inner nuclear membranes is simultaneous, identifies sequential intermediates in karyogamy, novel conditions that inhibit karyogamy, and examines the extent of genome intermixing which occurs upon karyogamy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein Tagging
Tagging of histone Htb2p and Sik1p was achieved by generating PCR fragments based on pFA6-mRFP-KanMX6 or pFA6-GFP(S65T)-KanMX6 and transforming by standard methods. The viability of the resulting haploids demonstrates the functionality of these integrants.
Two-Step Assay for Zygote Formation
Cells grown to OD600 2–4 were diluted 10 times in growth medium and allowed to grow for 2 more hours before mixing equal numbers of appropriate pairs at OD = 8, adding an equal volume of fresh medium, and reincubating with shaking for 1–2 h. One hundred microliter samples of the cell mixture were then applied to the surface of CSM plates and spread to cover twice the original surface area. After excess liquid had been absorbed (10–15 min), the plates were covered and incubated 3 h at room temperature. The plates included 2 µg/ml nocodazole (Sigma, St. Louis, MO) and 0.5% DMSO, as well as 0.1 M hydroxyurea, to block DNA synthesis. Cells were then rinsed off the plates with complete medium lacking sugar at room temperature, washed two times with medium lacking sugar (including 0.5% DMSO), and reincubated at OD = 1 in appropriate medium with 0.5% DMSO and 0.1 M hydroxyurea at 23 or 37°C, as appropriate. For quantitation of congression and karyogamy, samples were fixed by addition of an equal volume of 4% formaldehyde in PBS, washed, and examined. For examination by Deltavision (Applied Precision Instruments\, Issaquah, WA) they were processed as described in the next paragraph.
Real-Time Deltavision Microscopy
Samples of rapidly growing cells (or cells recovered from nocodazole plates) were mixed and sedimented. One-microliter aliquots of the pellet were applied to 1.5% agarose pads including CSM and additives of interest. After overlaying a coverslip and sealing with petroleum jelly, they were examined at 23°C (unless specified otherwise) using a 100x oil immersion objective without binning (Olympus, Melville, NY; UPlanApo 100x/1.40; 
/0.17/FN26.5). Images were deconvolved using Softworks (Olympus) and processed minimally. A minimum of 20 cells was observed for each condition, and the selected illustrations are representative of the large majority. Brightfield images are in blue.
Photobleaching
Samples on agarose pads were studied with a Zeiss 510 confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY). Typically, squares [0.2–0.5µ]2 were bleached 
80% at 50% laser intensity and then were imaged at 5–15-s intervals with a Plan-Apochromat 100x oil-immersion objective (NA 1.4), 1% laser intensity, using the acquisition software LSM510 (Carl Zeiss MicroImaging).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-factor develop a shmoo, the nucleus elongates and becomes highly polarized, with the SPB at the apex toward the shmoo tip (Figure 1, B and C). Chromatin fills the apical volume, and the nucleolus is at the distal extremity (Stone et al., 2000).
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In crosses in which a tagged nucleolar protein (Gar1p, Sik1p; or histone Htb2p) is contributed by one parent, these proteins gradually relocate to the trans nucleus, with the first trans signal becoming detectable after nuclear contact, but before the nexus is visibly dilated (Figure 1, D and E). Because no such signal is detected in the trans nucleus in crosses of kar1-1 (Figure 1G), which inhibits nuclear congression (Vallen et al., 1992), the trans signal results from intranuclear shuttling of these proteins. Moreover, these observations show that the production of new fluorescent copies of these proteins is negligible over the time period studied. We therefore use the arrival of the tagged nucleolar proteins in the trans nucleus as a temporal landmark.
Mechanism of NE Fusion: HMG1-Green Fluorescent Protein
Investigation of the mechanism and path by which NE membrane proteins such as HMG CoA Reductase 1 (HMG1) access the trans-NE is complicated because known proteins of the outer membrane are also found throughout the ER. On cell–cell fusion, transfer therefore could involve a combination of ER-to-ER fusion and NE-to-NE fusion.
In crosses in which HMG1-green fluorescent protein (GFP; Profant et al., 2000; Wiederkehr et al., 2003) is contributed by one parent, we observe that the labeled nucleus first becomes somewhat pear-shaped (reminiscent of the impact of -factor; Figure 1B). An intriguing focal discontinuity in the HMG1-GFP signal then appears at the apex of the NE that is oriented toward the midline (Figure 2A). Two to 4 min later, visible transfer of HMG1-GFP to the trans-NE is detected. When transfer begins, the signal appears to emerge from the apical discontinuity (Figures 2, A, B, and B' and Supplemental Figure S1). It is striking that little or no signal is seen in the trans cortical ER and that the labeled elements of the cis cortical ER end abruptly just before the midline. Thus, transfer of HMG1-GFP is not primarily via the cortical ER.
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; see below) is to recognize that it must pass through the focal nexus, whose surface area is certainly very small. For HMG1-GFP, as well as GFP-Prm3p and GFP-Esc1p, photobleaching shows that bleaching of one fourth to one third of the perimeter of the NE is followed by recovery with a t1/2 of 3 s (Supplemental Figure S2).
When nuclei establish contact, the parental SPBs do not fuse (Figure 1F). To position the path of transfer of HMG1-GFP relative to the SPBs, we have studied crosses in which each parent expresses a distinct form of Spc42p and one parent expresses HMG1-GFP. Figure 2, B and B', show that the two "sentinel" SPBs remain adjacent to each other during HMG1-GFP transfer and consistently continue to be distinguishable well after karyogamy. Many images suggest that they bracket the site of cis-trans flux of HMG1-GFP. Judging from EM studies, the SPB encounters occur at their "half-bridges" (Byers and Goetsch, 1975; Melloy et al., 2007).
Mechanism of NE Fusion: Transfer of Outer Membrane Proteins, Inner Membrane Proteins, and NPCs Occur Sequentially
To learn whether outer and inner membrane fusion are simultaneous, we have performed a set of crosses in which membrane markers are followed in parallel with a nucleolar marker (Sik1p-mRFP), which indicates the establishment of nucleoplasmic continuity. Crosses between strains that express both HMG1-GFP and Sik1p-mRFP show that HMG1-GFP begins to transfer when the nexus is established, 13.4 ± 5.4 min before transfer of the nucleolar marker is detected (Figure 3A).
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NPCs span the inner membrane and outer membrane of the NE, and previous studies indicate that nucleoporins can transfer upon karyogamy as part of intact NPCs (Belgareh and Doye, 1997; Bucci and Wente, 1997). To establish the timing of their transfer relative to dilation of the nexus, we have crossed strains that express Nup49p-GFP with strains that do not express this tagged protein. Obvious transfer occurs after dilation of the nexus, when the SPB has disengaged from one face of the NE. Interestingly, although several NPCs can remain adjacent to each other, one often finds what appear to be single tagged NPCs deep in trans-NE territory.
Figure 3E summarizes the steps that accomplish karyogamy.
Mechanism of NE Fusion: Interruption in Known Mutants
We have followed pairs of strains what express either Nup49p-GFP or the tagged histone, Htb2p-mRFP, in order to evaluate congression and fusion (Table 1). As an indicator of the extent to which karyogamy is inhibited, we calculate an arrest index (% of cells with nuclei in contact divided by the % of cells with fused nuclei).
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), the first panels illustrate the distribution of Nup49p-GFP and document the establishment and persistence of nuclear contact. The second panels show the transfer of HMG1-GFP. In the prm3 cross, as for wild type, the trans-NE becomes labeled well before the cortical ER. Interestingly, this is somewhat less obvious for kar2-1, perhaps signifying that this mutation does not allow HMG1-GFP transfer via the nexus. The third panels show that the inner membrane does not fuse even well after nuclear contact has been established, as judged by monitoring the distribution of markers of the inner membrane (GFP-Prm3p), nucleolus (Gar1p-GFP), or Nup49p-GFP. For this purpose, these labels are introduced by a single parent.
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; Cao et al., 2003). By contrast, karyogamy is inhibited in sec18-1x sec18-1 crosses, but not in crosses between sec1-1 strains that are temperature sensitive for exocytosis (Carr et al., 1999), indicating that one or more Snares is required. Interestingly, after establishment of contact between the nuclei in sec18-1 crosses, the shape of the nuclei becomes progressively more complex, perhaps because vesicle delivery to the NE/ER remains blocked. Karyogamy is also inhibited upon inclusion of cycloheximide or the reducing agent, dithiothreitol (DTT; Figure 5B and Supplemental Figure S4), which interrupts ER disulfide bond formation and therefore is expected to sequester Kar2p (Bernales et al., 2006).
The impact of DTT is not a consequence of the unfolded protein response (UPR; Bernales et al., 2006), because 1) induction of such a response in the absence of unfolded proteins in the cisternal space of the ER allows karyogamy to occur, e.g., upon expression of an active (spliced) form of the mRNA encoding the HAC1 transcription factor (HAC1i; Cox and Walter, 1996) and 2) DTT is inhibitory in crosses between ire1 strains, which cannot generate a UPR (Sidrauski and Walter, 1997; Table 1). We therefore propose that sequestration of folding equipment within the ER accounts for the impact of DTT. As attempts at overexpression of Kar2p alone do not protect against the effect of DTT (Supplemental Figure S4), it is likely that multiple factors are sequestered, more than one of which is required.
As for kar2-1 and prm3 crosses (Figure 4, A and B), Figure 5C illustrates the arrest of karyogamy in sec18-1 crosses and after treatment with DTT or cycloheximide and shows that the inner membrane does not fuse.
Transfer of ER/NE Content Before Nuclear Contact
In the establishment of ER/NE continuity, different markers are transferred at different times. Thus, content markers (GFP-HDEL, mRFP-HDEL, Figure 6) are actually transferred before nuclear contact (n = 20), whereas HMG1-GFP transfer begins only when the nexus is established. This asynchrony leads us to suspect, as at the bud neck (Luedeke et al., 2005), that the cortical ER does establish cis-trans continuity (allowing flux of tagged HDEL), but allows only a subset of proteins to pass. This is consistent with photobleaching studies that show little cis-trans continuity of HMG1-GFP during zygote formation (Supplemental Figure S4).
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Restriction of genome intermixing could reflect their tethering to SPBs (Loidl, 2003; Kitamura et al., 2007). To learn whether such association is required for restriction, we have performed crosses in which one of the mating partners carries a lacO-tagged ARS plasmid (which lacks a centromere; Velmurugan et al., 2000). In this case tagged loci invade the trans nucleoplasm (Figure 7D). Further evidence that genome tethering is required comes from observations of strains that carry ts mutations in the kinetochore protein, Ndc10p (Goh and Kilmartin, 1993). As shown in Figure 7E, in crosses between an ndc10-1 strain that carries a tetO repeat near CENV (and TetR-GFP) and a ndc10-1 partner that expresses Htb2p-mRFP, transit of the tagged locus can occur. In the protocol used, the cross is initiated at room temperature and the prezygotes are shifted to 37°C once cell fusion has occurred.
Figure 7F provides a diagram of genome organization after karyogamy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ogle et al., 2005; Jaenisch and Young, 2008). During fertilization in certain organisms (e.g., marine algae, sea urchins) nuclei fuse with each other, as opposed to first disassembling their NEs, followed by assembly of a single NE around the composite genome. Unlike yeast, many cell types do not have SPBs; however, the present observations support the idea that the importance of SPBs for nuclear fusion is primarily to bring nuclei together, rather than being functionally central for membrane fusion per se. In Saccharomyces cerevisiae, after nuclear congression, the sequence of events that accomplishes nuclear fusion begins with the focal opening of a gap in the HMG1-GFP signal at the site of imminent fusion. When the HMG1-GFP signal is first detected in the trans-NE, the cortical ER signal is still confined to the cis cell, making it highly likely that transfer occurs only via the nexus. The narrow dimensions of the nexus along with the SPBs that appear to bracket it could account for the slow tempo of transfer. Inner membrane continuity is not established until later, essentially at the same time as for the lamina (Esc1p) and entry of nucleolar proteins. Still later dilation of the waist of the nucleus could result from removal of yet-unidentified structural elements that also define its initial pear-like shape.
A recent study of karyogamy has concluded that the outer membrane of the yeast NE fuses 30 s before the inner membrane (Melloy et al., 2007). These conclusions are based on an EM tomogram and the timing of GFP-HDEL transfer versus nucleoplasmic markers, after correction. Because tagged HDEL transfers before nuclear contact, we consider it not to be a suitable tracer. In our experience, the delay between fusion of the outer membrane and inner membrane is at least 10 min.
The present observations divide karyogamy into five steps: 1) SPB contact, 2) appearance of the apical discontinuity of the NE, 3) transfer of outer membrane proteins, 4) transfer of inner membrane proteins and the lamina, as well as nucleoplasmic continuity, and 5) visible dilation of the nexus, transfer of NPCs and disengagement of the SPB from one face of the NE. There are obvious delays between steps 3 and 4 and between 4 and 5, suggesting that these intervening periods allow appropriate molecular preparations for subsequent events.
As mentioned in the Introduction, the outer membranes and inner membranes of the cis-NE might first fuse with each other (and the outer and inner membranes of the trans-NE do the same). Subsequently, the two "cis-fused" NEs could fuse with each other (Figure 1A). This model has the unattractive property of possibly allowing momentary leakage of nuclear content to the cytoplasm. It nevertheless appears topologically equivalent to mitotic NE breakdown and reformation in higher eukaryotes and in half-open mitosis (e.g., Straube et al., 2005). Moreover, chromosomes can escape from the yeast nucleus during congression in kar1-1 (Dutcher, 1981). Supplemental Figure S5 presents observations inconsistent with this model.
Our investigation of crosses of kar2-1, prm3 and the impact of novel inhibitors of karyogamy (DTT, cycloheximide, inhibition of Sec18p) all show that inner membrane fusion is blocked. The requirement for Sec18p immediately situates karyogamy in the context of cytoplasmic membrane fusion events. Because Sec18p is a Snare disassembly factor, its involvement suggests that the Snare(s) involved in karyogamy (which occurs only once) also participate in other (preceding) ER membrane fusion events. Judging from the lack of involvement of the UPR, we attribute the impact of DTT to sequestration of folding factors of the ER/NE lumen.
The outer and inner membranes fuse sequentially. Two topologically distinct mechanisms are therefore likely to be involved. Fusion initiated by interaction of the cytoplasmic surfaces of the outer membranes could account for the Sec18p requirement, as for vesicle fusion. Subsequent contact between the luminal surfaces of the inner membrane could account for the involvement of proteins that are present in the ER/NE lumen such as Kar2p, Kar5p, and Kar8p. Because the critical feature of DTT treatment appears to be its disruption of ER homeostasis, translocon mutants could have a similar effect, either by compromising the folding of newly synthesized ER proteins or by blocking the removal of those that are not well folded. The role of Prm3p remains obscure, although its accumulation at the SPB (Figure 4A) is consistent with its participation in either step. Perhaps, like other proteins of the inner nuclear membrane, it also gains access to the outer membrane, where it would face the cytoplasm and therefore could be critical for outer membrane fusion.
The kinetics of transfer of tagged HDEL are reminiscent of studies of mitotic cells, in which arrival of this tracer in the cortical ER of the bud occurs before the arrival of membrane proteins (Luedeke et al., 2005). This transfer could be mediated by specialized ER elements or might result from ER–ER fusion being only transient, as in "kiss-and-run" exocytosis (e.g., Sokac and Bement, 2006). In any event, this transfer is exempt from the dramatic restriction of transfer of the membrane protein, HMG1-GFP.
The actin cytoskeleton participates in nuclear orientation during the mitotic cell cycle (Pearson and Bloom, 2004), but its importance for congression is not understood. As in the mitotic cell cycle, it is likely to be required for proper orientation of the nucleus. Consistent with this hypothesis, when latrunculin A is present after cell fusion, nuclei remain for extended periods with their SPBs and microtubule bundles pointing in seemingly arbitrary directions (Supplemental Figure S6).
Several soluble nucleoplasmic proteins and even an ARS plasmid redistribute upon karyogamy, by contrast to chromosomal loci in wild-type strains. Moreover, inactivation of kinetochore function also allows redistribution of chromosomal loci. Mere tethering of the haploid genome to the SPB does not predict persistence of cis-trans genome segregation after karyogamy, a situation which should inhibit recombination between the two contributing genomes during this period. We propose that the lack of coalescence of parental SPBs causes each SPB to continue to coordinate a single genome and thereby imposes spatial restrictions on the distribution of the corresponding chromosomes. So long as the restriction does not preclude random assortment of chromosomes during meiosis, it is not obvious that it would have genetic consequences.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Alan Michael Tartakoff (amt10{at}case.edu)
Abbreviations used: DTT, dithiothreitol; HMG1, HMG-CoA reductase I; NE, nuclear envelope; NPC, nuclear pore complex; SPB, spindle pole body; TBP1, TATA-binding protein; UPR, unfolded protein response; Vac8p, vacuolar membrane protein.
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