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Fusion of haploid cells of S. cerevisiae generates zygotes. We observe that the zygote midzone includes a septin annulus and differentially affects redistribution of supramolecular complexes and organelles. Redistribution across the midzone of supramolecular complexes (polysomes and Sup35p-GFP [PSI+]) is unexpectedly delayed relative to soluble proteins; however, in [psi−] × [PSI+] crosses all buds eventually receive Sup35p-GFP [PSI+]. Encounter between parental mitochondria is further delayed until septins relocate to the bud site, where they are required for repolarization of the actin cytoskeleton. This delay allows rationalization of the longstanding observation that terminal zygotic buds preferentially inherit a single mitochondrial genotype. The rate of redistribution of complexes and organelles determines whether their inheritance will be uniform.
Asymmetric cell division can occur if the two ends of the spindle reside in compositionally distinct regions of the cytoplasm (Barral and Liakopoulos, 2009; Knoblich, 2008; Pereira and Yamashita, 2011; Rando, 2006; Rujano et al., 2006). The present study is concerned with the causes of such inhomogeneity, using as a model the elongated zygotes of S. cerevisiae that result from fusion of haploid cells. When buds form at the termini of zygotes, mitochondrial genomes contributed by a single parent are preferentially inherited by buds that emerge at the corresponding end (Birky, 1975; Dujon, 1981; Lukins et al., 1973; Nunnari et al., 1997; Okamoto and Shaw, 2005; Strausberg and Perlman, 1978). A partial explanation of this asymmetry is provided by the observation that, although nuclei congress and fuse soon after cell-cell fusion, e.g. (Melloy et al., 2007; Molk and Bloom, 2006; Tartakoff and Jaiswal, 2009), parental mitochondria encounter and fuse with each other in the midzone of the zygote significantly later (Hoppins et al., 2007; Nunnari et al., 1997; Okamoto et al., 1998). Mechanisms underlying this genetic and cell biological puzzle have not been investigated.
These considerations also provide a point of reference for understanding the distribution and mitotic inheritance of supramolecular complexes, including the prion form of Sup35p. When in the [psi−] conformation, Sup35p acts as a translation termination factor. In an alternate (“aggregated”) conformer prion form(s), [PSI+] self-associates and allows read-through of termination codons. [PSI+] can template the conversion of [psi−] to [PSI+], and in [PSI+] cells monomeric-oligomeric forms of Sup35p interconvert, e.g. (Cox, 1965; Liebman and Chernoff, 2012; Paushkin et al., 1996; Serio and Lindquist, 1999; Wickner et al., 2007). Several prions, both in fungi and in mammalian cells, are toxic (Halfmann et al., 2011; Mathur et al., 2012; McGlinchey et al., 2011; Vishveshwara et al., 2009; Wickner et al., 2010). Moreover, some can form macroscopic aggregates which reduce mitotic inheritance, as do certain oxidized proteins (Aguilaniu et al., 2003; Bagriantsev et al., 2008; Derdowski et al., 2010; Erjavec et al., 2007; Tyedmers et al., 2010; Uptain et al., 2001). By visualizing Sup35p-GFP contributed by one parent during zygote formation, one can investigate the timing of its aggregation and its transmission. Barriers within the zygote could restrict these events.
In mitotic yeast, a cortical patch or ring of five septins (Cdc3, Cdc10, Cdc11, Cdc12, Shs1) accumulates along with the myosin, Myo1p, at the site of the incipient bud. This unit then forms an hourglass-like structure that encircles the bud neck. Prior to cytokinesis, the hourglass is replaced by two rings, a further ring of actin forms between the rings, and contraction of this unit promotes cytokinesis (Bi et al., 1998; Caudron and Barral, 2009; Lippincott et al., 2001; Longtine and Bi, 2003; Spiliotis and Gladfelter, 2012). A “fence” function of septins has been described at the bud neck where they limit diffusion of cortical proteins (Caudron and Barral, 2009; Longtine and Bi, 2003). The present study considerably extends the significance of septins by showing that a septin-containing partition subcompartmentalizes non-cortical portions of the zygote cytoplasm. Moreover, relocation of septins to the site of bud formation promotes polarization of the actin cytoskeleton.
The non-uniform distribution of components of mammalian cells could be limited by barriers equivalent to those that we characterize in this study. Moreover, the yeast bud neck resembles the cytokinetic bridge of animal cells. Both structures include septins and actin (Balasubramanian et al., 2004; Estey et al., 2010; Hurley and Hanson, 2010; Liu et al., 2012; Seshan and Amon, 2004; Steigemann et al., 2009).
A further point of interest in studying zygotes pertains to transgenerational inheritance. In cells that result from fusion of distinct precursors, if mitosis occurs before thorough mixing of parental complexes and organelles, distinct characteristics can be passed to subsets of progeny, i.e. from an initial generation to the third generation.
When haploid yeast of opposite mating type are mixed at room temperature, zygotes form after 1.5–2.0 hr. To establish the relative timing of cis-to-trans redistribution of parental proteins and organelles, we used time-lapse microscopy and visualized fluorescent marker proteins. Population-based estimates of relative timing agree with time-lapse observations, but the temporal dispersion of these events makes it more informative to use time-lapse, which also can illustrate the suddenness of redistribution. The selected images and time-lapse series illustrated below are in all cases representative of examination of at least twenty cells.
We initially observed that redistribution of distinct organelles and supramolecular complexes is by no means synchronous. We therefore have inquired whether cytoskeletal barriers partition the cytoplasm, beginning with septins.
After treatment of MAT a haploid cells with mating factor for 2–3 hr, the tagged septin, GFP-Cdc3p, forms a collar at the cell cortex distal to the tip of the mating projection, as previously described (Ford and Pringle, 1991; Kim et al., 1991; Longtine et al., 1998) (Figure 1A). This collar has a composite organization in which lobes are joined at their apical ends and become increasingly splayed as they extend distally. A pool of diffuse cytoplasmic fluorescence is also evident.
When a mating pair meets, the two plasma membranes form a flattened “zone of contact” (Byers and Goetsch, 1975). In crosses in which one partner expresses GFP-Cdc3p, contact is followed by entry of a diffuse signal into the acceptor cell. Within 5 minutes, the collar in the acceptor cell then becomes symmetrically labeled (Figure 1B, 4–6 min time points). Such behavior is expected if a soluble pool of GFP-Cdc3p permeates cis to trans, and if assembly of the collar is dynamic.
Just prior to nuclear contact, a GFP-Cdc3p-positive structure appears at the interface between the two parental domains (e.g. Figure 1B–C). It can also be detected in cells expressing other tagged septins (GFP-Cdc10p, GFP-Cdc11p or GFP-Cdc12p) (not shown). In face view, it appears as an annulus (Figure 1C – insert). Its diameter is somewhat greater than the septin hourglass at the bud neck, which is brighter and wider. In typical experiments in which cells expressing GFP-Cdc3p are examined 2 hr after mixing, this structure is evident in >90% of zygotes for which plasma membrane fusion has occurred. Parental nuclei congress and fuse with each other when it is already in place (Figure 1B–C). The concentration of GFP-Cdc3 at the midzone is obvious for ~15 min. A patch of GFP-Cdc3p at the site of future bud emergence then appears (Figure 1C).
GFP-Myo1p is not detected at the cortex of the mating projection; however, it also forms an annulus in the midzone (Figure 1D). Judging from the distribution of GFP-tagged Act1p, and the actin-binding protein, Abp140/Trm140, and from staining fixed preparations with rhodamine-phalloidin, actin patches and cables are widespread but are not characteristically concentrated or oriented in the midzone at this time (see below).
Photobleaching (FRAP) of GFP-Cdc3 in zygotes shows that the cortical collar, annulus, patch, and bud neck filaments are all dynamic. Since the estimates of half-time for recovery (13.5+/−3.4 sec – 27.1+/−8.7 sec, n=5–21) and mobile fraction (28+/−2.8% – 38+/−8.5%, n=5–21, S.D.) are relatively uniform among these structures, the successive redistribution of septins is likely to result from the progressive elimination and/or appearance of binding sites. Additional evidence of the dynamic nature of the annulus comes from FLIP experiments: repeated photobleaching of the diffuse pool of GFP-Cdc3p in the zygote cytoplasm (avoiding the annulus itself) progressively weakens the signal in the annulus (Figure S1).
We therefore suggest the following sequence of septin morphogenetic intermediates (Figure 1E): 0: the collar of the mating projection, 1: the symmetric collar of early zygotes, 2: the annulus, 3: the patch at the site of bud formation, 4: the bud neck itself. As is explained below, these events are concurrent with changes in the actin cytoskeleton.
Since the medial concentration of septins becomes most obvious when parental nuclei establish contact, we explored the possibility that the annulus has a continuing association with the nucleus. In support of this possibility, we observe that 1) a narrowed segment of the nuclear envelope spans the midpoint of the zygote even after karyogamy and displacement of the chromatin mass to one side (Figure 1F), 2) nuclear pores are generally not detected at this point (Figure 1F), and 3) the annulus encircles the narrowed segment (Figure 1G).
Shortly after cell-cell contact is established, soluble DsRed (~120kDa) – like GFP-Cdc3 - suddenly redistributes from the donor to acceptor cell. Surprisingly, at this point polysomes (including Rps3p-GFP or Rpl25p-mCherry) remain in their initial parental domain (Figure 2A). In fact, they begin to redistribute only when nuclei are about to establish contact, well after rupture of the plasma membrane, as judged by the apical elimination of the plasma membrane protein, Mid2p-GFP (Figure 2B–D) that is present at the zone of contact prior to rupture. The equilibration of polysomes then requires ~10 min. The slow pace of these events suggests the presence of a barrier, given the apparent full disruption of the plasma membrane, the significant mobility of polysomes (t1/2 for recovery after photobleaching ~16 sec (Figure S2)), and electron micrographs that show a 0.5–1 micron gap between the nuclear envelope and the cortex of the zygote, e.g. (Byers and Goetsch, 1975).
As illustrated above, while fluorescent polysomes gradually shift from cis to trans, there is a sharp discontinuity in their signal intensity. To learn whether the nucleus is responsible for this discontinuity and impedes transit through the midzone, we studied kar1 × wt crosses, in which nuclei do not congress, e.g. (Molk and Bloom, 2006) (Table 1). The presence of the nucleus at the midzone does not appear to contribute, since a) a sharp discontinuity in polysome distribution is evident in such crosses (Figure S3), and b) there is no significant acceleration of the rate of polysome redistribution in kar1 × wt crosses by comparison to wt × wt crosses. Moreover, the redistribution of polysomes still precedes redistribution of the mitochondrial signal – as for wt × wt crosses (Figure S4).
Since septins concentrate at the zygote midzone, we also asked whether they contribute to the slow pace of flux. Indeed, temperature increase from 23°C to 37°C causes a modest increase in the rate of flux in crosses between temperature-sensitive (ts) conditional septin mutants (cdc12-6), while no comparable increase is seen when wt control crosses are studied at the same temperatures (Table 1).
To learn whether Sup35p [PSI+] readily traverses the midzone, we followed Sup35p-GFP in crosses in which one mating partner expresses a functional integrated copy of Sup35p-GFP from the MFA1 promoter, that is turned off upon cell fusion. The same partner also expresses soluble DsRed or Rpl25-mCherry. In [psi−] cells, cytoplasmic Sup35p-GFP is smoothly distributed and can diffuse freely, while in [PSI+] cells the signal is generally inhomogeneous (Greene et al., 2009; Kawai-Noma et al., 2006; Satpute-Krishnan and Serio, 2005). In the “strong” [PSI+] strains that we use, Sup35-GFP has a mottled/irregular appearance at steady-state. The designation “strong” signifies that translation termination defects are suppressed more efficiently than by “weak” forms.
Redistribution of aggregated Sup35p-GFP in crosses between [PSI+] cells is restricted at the midzone, judging from examination of intermediate time points (Figure 3A). This is reminiscent of previous studies of the [HET-s] prion (Mathur et al., 2012). The restriction is not simply due to the position of the nucleus, since an equivalent discontinuity is also evident in wt × kar1 crosses in which the lack of karyogamy is ensured by following a marker of the ER/nuclear envelope (mRFP-HDEL) (Figure 3B). Moreover, in such crosses, redistribution of Sup35 still occurs before redistribution of mitochondria (not shown).
Further experiments help characterize the medial barrier and the in vivo hydrodynamic properties of Sup35p. First, Sup35p-GFP (91kDa) [psi−] and soluble DsRed (~120kDa) have similar hydrodynamic properties in vivo: both transfer over the same period of time in crosses between [psi−] cells (Figure 3C). Transfer of Sup35p-GFP is essentially complete before initiation of polysome equilibration (Figure 3D).
Moreover, progressive coalescence of diffuse Sup35p can be detected, possibly as a reflection of conformational maturation. Thus, when Sup35p-GFP from a [psi−] “donor” enters a [PSI+] “acceptor” environment, tiny just-discernable foci appear in the acceptor cell domain during the initial 30 minutes. These foci provide a first suggestion - at this resolution - of conformational change and possible oligomer/aggregate formation. The donor cell domain, as in studies of heteroallelic conversion of the HET-s and HET-S proteins (Mathur et al., 2012), could receive non-fluorescent seeds of [PSI+] Sup35 in such experiments. We do not, however, see progressive changes in the donor compartment over the same period of time, perhaps because the size of any seeds precludes their rapid exchange (Figure 3Ea). More visible foci and extensive “mottling” do appear in both domains, but this occurs only gradually over 1–2 hours, e.g. (Figure 3Eb/c). Curiously, an earlier study has described more rapid Sup35-GFP conversion during zygote formation (Satpute-Krishnan and Serio, 2005).
Furthermore, the overall polydispersity of Sup35p in a [PSI+] environment appears comparable to that of polysomes, i.e. in [PSI+] × [PSI+] crosses in which both Sup35p-GFP and tagged polysomes are expressed by one parent, the transfer of Sup35p-GFP aggregates extends over at least as long a period as for polysomes (Figure 3A).
After polysome and prion flux and completion of nuclear fusion, tagged mitochondria extend precisely up to the midzone, as though abutting on an invisible barrier (Figure 4A/B). During this period their position is however not further restricted - as in haploid cells, they can move extensively. Only after a 15–30 min delay do matrix markers contributed by one parent quickly access much of the mitochondrial labyrinth of the trans domain of the zygote (Figure 4A/B), presumably as a result of sudden fusion between the parental mitochondria (Hermann et al., 1998; Nunnari et al., 1997; Okamoto et al., 1998) – see also Figure S5. In Figure 4B, note that, prior to redistribution of the mitochondrial marker, a patch of GFP-Cdc3 appears at the site of bud formation (asterisk) – as in Figure 1C. The consistency of this order is evident in experiments in which a parent that expresses GFP-Cdc3p was crossed with a parent that expresses the matrix marker, Cox4-DsRed: At a time point when redistribution of the matrix marker had occurred in half of the zygotes, a GFP-Cdc3-positive cortical patch or bud neck was evident in all zygotes (83/83).
The timing of redistribution of DsRed-Cox4 and the sequential morphogenesis of septin-containing structures suggests a “sequestration” hypothesis: that the zygote midzone initially impedes encounter of parental mitochondria, that the site of incipient budding then recruits components from the midzone, including septins, and that the integrity of the midzone becomes so impaired that cis-trans encounter of parental mitochondria can occur. Relocation of selected proteins from the midzone to the bud neck could also cause secondary changes that promote redistribution – as is further discussed below.
To learn whether septin integrity affects encounter of parental mitochondria, we first evaluated redistribution of Cox4-DsRed in crosses between cdc12-6 strains. These “two-step crosses” were initiated at 23°C and then reincubated for up to 40 min at 37°C vs 23°C. As shown in Figure 4C, redistribution is little affected at 37°C vs 23°C for the cdc12-6 cross, and the rate is nearly identical at both temperatures for wt cells. In these experiments, multiple pools of septins are perturbed, i.e. any medial barrier could be weakened and any role for septins at the site of bud emergence could also be compromised.
We therefore studied redistribution of Cox4-DsRed in crosses of mutants that inhibit bud emergence (Figure 4C). In each case, one of the parents also expressed GFP-Cdc3p. Relevant mutants are a) an exocytosis ts mutant that stops budding, sec1-1, e.g. (Togneri et al., 2006), and b) the ts cyclin-dependent kinase mutant, cdc28-13, that stops both budding and deposition of septins at the site of bud formation in haploid cells (Cid et al., 2001). As expected, no zygotic buds formed in either cross at 37°C.
In sec1-1 × sec1-1 two-step crosses, a cortical patch of GFP-Cdc3p appeared in ~2/3 of zygotes within 40 min during the reincubation at 37°C and the annulus became less evident with time (Figure 4D - left). Moreover, redistribution occurred at essentially the same rate at both 37°C and at 23°C (Figure 4Cd). In cdc28-13 × cdc28-13 crosses, the annulus is readily detected, but there was no cortical patch of GFP-Cdc3p after incubation at 37°C (Figure 4D - right). Moreover, the cdc28-13 crosses consistently showed slower redistribution at 37°C than at 23°C (Figure 4Cc).
Parallel two-step crosses show that actin polymerization is required in order for redistribution of Cox4-DsRed: addition of latrunculin A during the second incubation halts redistribution (Table 2). This treatment does not cause an obvious change in the distribution of GFP-Cdc3 (not shown).
Thus, encounter and fusion of parental mitochondria are delayed when the septin annulus is conspicuous and bud neck filaments have not formed. In this sense, by checking on the progress of bud formation, the timing of encounters between parental mitochondria is adjusted.
To learn whether the presence of the nucleus at the midzone delays the encounter of parental mitochondria, we evaluated redistribution of Cox4-DsRed in kar1 × wt crosses, by comparison to wt × wt crosses, and observed that redistribution occurs earlier in the kar1 x wt crosses (Figure 4Ce). Both septins and the nucleus thus contribute to the delay of mitochondrial encounter and fusion.
Some forms of Sup35p are not efficiently inherited during mitotic growth. Nevertheless, time-lapse observations of individual [PSI+] × [PSI+] and [PSI+] × [psi−] zygotes in which aggregated Sup35p-GFP is introduced from a [PSI+] parent show that all buds – including the smallest that are encircled by septins - receive aggregated Sup35p-GFP (e.g. Figure 3Eb/c), a process that is favored by fragmentation of prion units (Liebman and Chernoff, 2012; Paushkin et al., 1996). This is also true in [psi−] × [PSI+] crosses in which Sup35p-GFP is contributed by the [psi−] parent. Indeed, there is no visible distinction between buds originating at the two distinct ends in [psi−] × [PSI+] crosses. Thus, although these aggregates of Sup35pstrong-GFP are delayed at the midzone, and although there can be quantitative differences in the relative abundance of aggregates among cells, they are transmitted to all progeny. It will be of interest to investigate the extent to which the [RNQ+]/[PIN+] status of cells and other forms of Sup35 (e.g. weak vs strong) may influence transit between parental domains and entry into buds.
To learn whether delayed fusion of parental mitochondria causes terminal buds to be enriched in a single parental mitochondrial genome, we conducted crosses in which one parent expressed tagged proteins of mitochondrial nucleoids - Abf2p-GFP or Mgm101p-GFP (Kucej et al., 2008; Meeusen et al., 1999; Okamoto et al., 1998) - and the other expressed Cox4-DsRed. Indeed, tagged nucleoid(s) consistently associate with adjacent nascent terminal buds well before cis-trans fusion of mitochondria, e.g. (Figures 5A and S6).
The biased inheritance of mitochondrial genomes could signify that there is only a brief time window for association of mitochondria with terminal buds. Alternatively, mitochondria could retain continuity into the bud for an extended period of time, but nearby (cis) nucleoids that enter early might outnumber nucleoids derived from the distant parent, or associate with a finite number of binding sites. It is therefore important to learn for how long mitochondria remain continuous across the bud neck. Published images show continuity when buds are present, e.g. (Boldogh et al., 2005; Garcia-Rodriguez et al., 2009; Weisman, 2006).
Since buds form before entry of the nucleus, we have used two protocols to take this analysis a step further, showing that continuity of Cox4-GFP into buds continues when the nucleus spans the bud neck: 1) When the cell cycle is arrested by inactivating the mitotic exit network kinase, Dbf2p – (Figure 5B), and 2) After depletion of the activator of the anaphase promoting complex (APC), Cdc20p (Komarnitsky et al., 1998) – (Figure 5C). In the latter case we have used FRAP to assess functional continuity. When the bud is bleached, the signal can a) diminish in the bud but not change in the zygote, b) immediately diminish in both bud and in the zygote, or c) recover in the bud only after a delay. Each outcome is observed with comparable frequency. Thus, although mother-bud continuity can be intermittent, it persists until after entry of the nucleus.
Both parental types of mitochondria are present in most diploid cells that originate from medial buds, although one type is lost (at random) within a few generations (Birky, 1978; Dujon et al., 1974; Okamoto et al., 1998; Thomas and Wilkie, 1968). As there has been no indication of whether parental mitochondria fuse with each other before entry into buds, we have studied early stages of medial bud emergence using parents that express Cox4-DsRed vs Cox4-GFP. One readily finds examples in which both types of mitochondria extend to the bud neck but have not fused, suggesting that the two types of parental mitochondria generally fuse with each when they enter the bud – Figure 5D. Thus, as for non-medial events, fusion occurs when bud formation is already underway. Interestingly, fusion of parental vacuoles also does not occur before entry into medial buds (Weisman, 2006).
Why do parental mitochondria not fuse with each other long before bud formation ? Is a medial barrier strongly restrictive or do bud formation, the arrival of septins at the neck, and actin polarization also have a positive effect ? To explore this issue, we first localized mitochondria along with actin cables by following Cox4-DsRed and a GFP-tagged copy of the actin filament-binding protein, Abp140 (Yang and Pon, 2002) (Figure 5E). Prior to cell-cell fusion, actin cables and mitochondria orient toward the zone of contact. Upon fusion, actin orientation becomes less obvious, the midzone often appears depleted of filaments, and – as detailed above – mitochondria extend only to the midpoint of the zygote. When buds become visible, actin has reorganized to generate cables that extend from the bud neck and extend either a) in roughly symmetric fashion toward each parental domain (when budding is medial), or b) along the long axis of the zygote (when budding is non-medial). In each case, mitochondria appear to align with cables.
In mitotic cells, septins and the formin, Bnr1p, localize to the bud neck and are required for nucleating linear actin filaments in the mother cell. A second formin, Bni1p, localizes to the bud tip and plays a similar role for organization of actin in the bud (Buttery et al., 2007; Pruyne et al., 2004). We therefore investigated the interdependence of septin localization, Bnr1p and actin polarization in zygotes.
In crosses between wildtype cells, we observe that GFP-tagged Bnr1p becomes visible only when zygotic buds emerge, i.e. approximately when actin becomes repolarized and parental mitochondria fuse. At this time it colocalizes with septins at the bud neck (Figure 5F).
To learn whether septins at the bud neck are required for colocalization of Bnr1p and for actin polarization throughout the zygote, we allowed bud formation to begin and then inactivated one septin at the bud neck. In these experiments, we formed cdc11-6 zygotes at 23°C, using a pair of temperature-sensitive strains that express mCherry-tagged Cdc3p and GFP-tagged Bnr1p. When the zygotes were then shifted to 36–37°C, both tagged Cdc3p and tagged Bnr1p disappeared from the bud neck (Figure 5G, panel 1). Moreover, in equivalent protocols in which one of the haploid parents expressed tagged Abp140, actin cables became dramatically concentrated in the elongated buds and remarkably absent from the rest of the zygote (Figure 5G, panels 2–4). No such changes occurred in parallel experiments with wildtype zygotes. Since bnr1Δ strains do not form conventional zygotes with good efficiency, it has not been possible to inquire whether Bnr1p itself is needed (unpublished observations).
There thus is a close connection between arrival of septins at the bud neck and the polarization of actin in the body of the zygote. We propose that this repolarization of actin facilitates fusion of parental mitochondria in zygotes.
The ultimate redistribution of parental constituents during zygote formation occurs asynchronously (Figure 6A). Why is this ? During the mitotic cell cycle, septin filaments that encircle the bud neck contribute to the restricted diffusion of cortical proteins between mother and bud. The present observations indicate that septin-containing structures serve a broader “gatekeeper” function, in that they can also control the flux of entities that are not primarily associated with the cortex: polysomes and Sup35-GFP [PSI+], as well as mitochondria, are delayed at the level of the zygote midzone, that includes a septin annulus. Subcompartmentalization of the cytoplasm in the absence of membrane barriers is in fact characteristic of many cell types, e.g. (Caudron and Barral, 2009; Galiano et al., 2012; Kissel et al., 2005; Lin et al., 2009; Merisko et al., 1986; Mollenhauer and Morre, 1978; Song et al., 2009; Wolosewick and Porter, 1979).
Interestingly, the septin annulus continues to encircle the nucleus well after karyogamy and one report concludes that septins at the bud neck restrict transit of nuclear pores into buds (Shcheprova et al., 2008). The efficacy of the midzone barrier therefore could be promoted by its association with both the nuclear envelope and the plasma membrane, perhaps due to binding of septins to phosphoinositides (Casamayor and Snyder, 2003). Nevertheless, constriction around the nucleus does not account for all barrier function of the midzone: significant delay in redistribution persists even when nuclei do not congress. The functional significance of this barrier could normally be to buffer motion and thereby promote the surely intricate multi-step process by which nuclei fuse (Melloy et al., 2007; Tartakoff and Jaiswal, 2009). Further interesting possibilities are that the populations of RNPs on either side of the midzone are distinct and that, given the restricted transit of Sup35 [PSI+], the fidelity of translation termination is different within different parts of the cytoplasm.
Well after the flux of polysomes and Sup35p-GFP, parental mitochondria contact each other and fuse. What causes this delay ? Fusion is linked to relocalization of septins to the bud neck and polarization of the actin cytoskeleton. Most strikingly, in septin mutant zygotes at the restrictive temperature, actin cables vanish from the body of the zygote and become dramatically concentrated in emerging buds. The present observations thus strongly reinforce the concept that septins coordinate the organization of other components of the cytoskeleton, e.g. (Kusch et al., 2002; Pruyne et al., 2004; Spiliotis and Gladfelter, 2012). Moreover, the rearrangement of actin filaments that occurs in septin mutants is likely to contribute to their inability to accomplish cytokinesis (Hartwell, 1971).
We suggest that slight translocation of both types of parental mitochondria across the midzone normally is required for their encounter, and that this step requires reorganization of the actin cytoskeleton. Concurrent remodeling of the midzone – including removal of septins - may facilitate these encounters.
Prior to cell fusion, actin cables in parental cells run anti-parallel relative to each other, e.g. (Slaughter et al., 2009) (Figure 6B). After cell fusion, cables in the body of the zygote have a less defined orientation until they extend either a) from the neck of medial buds into each parental domain, or b) from the neck of lateral or terminal buds along the full length of the zygote. In both situations, the actin filament nucleator, Bnr1p, localizes to the bud neck. Judging from our observations, mitochondrial fusion occurs in the middle of the zygote in both situations. This could signify that parallel alignment of linear actin filaments is required for in vivo mitochondrial fusion to occur.
We therefore suggest that several parameters determine which mitochondrial genomes are inherited by terminal buds. These include the timing of alleviation of the medial impasse and the timing of repolarization of the actin cytoskeleton. Since the progressive sequestration of septins away from the midzone to the bud neck couples these events, encounter and fusion of parental mitochondria is expected to be delayed but efficient. Further considerations also oppose entry of cis nucleoids into trans buds: 1) nucleoids are tethered to transmembrane proteins (Boldogh and Pon, 2007), and 2) nucleoids would likely encounter significant difficulty were they – after cis-trans fusion - to enter the trans mitochondrial reticulum of tubules and then need to pass beyond nucleoids of the other parent to gain access to the bud.
The uniparental inheritance of mitochondria that is characteristic of many organisms suggests that there is an advantage to protecting at least some copies of the mitochondrial genome from recombination. Alternatively, mitochondrial-nuclear interactions could become deleterious in the presence of more than one mitochondrial genome (Birky, 2001; Lewontin, 1971), or consequences of possible genetic incompatibilities could become manifest, e.g. (Saupe, 2011). Interestingly, several conditions affect inheritance of mitochondria in man, e.g. (Dimauro and Davidzon, 2005) and the segregation of distinct mitochondrial genomes when they coexist (Jokinen and Battersby, 2012). Moreover, septin integrity is critical for proper differentiation of spermatozoa and compartmentalization of their mitochondria, e.g. (Ihara et al., 2005; Kissel et al., 2005; Lin et al., 2009), and cell polarity defects and formin mutations have been linked to several diseases (DeWard et al., 2010; Stein et al., 2002).
Spatially separate cytoplasmic characteristics of cells can be differentially passed to daughter cells if the ends of the mitotic spindle extend into these distinct regions. For such a mechanism to function, the underlying characteristics could either be locally tethered (Spokoini et al., 2012) or restricted by barriers.
Yeast strains were primarily derivatives of W303 or S288C (Table S1, Supplemental Experimental Procedures). Table S2 listing yeast plasmids is also in Supplemental Experimental Procedures. Cells were grown in complete synthetic medium or appropriate drop-out media at room temperature, supplemented with adenine sulfate. As needed, cells were precultured overnight, or induced for 1–2 hr, in medium supplemented with 1% galactose and 1% raffinose, as indicated in figure legends. All chemicals were from Sigma-Aldrich Chem. Co. except for latrunculin that was from Millipore. Drug stocks (100×) were prepared in DMSO.
Strains (MAT a, MAT α) grown to OD600 ~2 were diluted 20× in medium and allowed to regrow with shaking for 2–3 hrs before mixing equal numbers of appropriate pairs at OD ~ 4 in fresh medium. 50–100 µl samples were then applied to the surface of CSM-glucose plates at 23°C. When the samples had dried (~15 min), the plates were covered and incubated for 1.5–2.0 hr to generate early, unbudded, zygotes. To quantitate redistribution of mitochondria (Cox4-DsRed), samples washed off plates was either fixed at once or first reincubated at OD600 = 1 in appropriate medium at 23°C or 37°C. For fixation, an equal volume of 4% formaldehyde in PBS was added on ice. After 5 min, the samples were washed with water and examined by epifluorescence.
Zygotes that were sufficiently mature to have a smooth concave contour at the middle and lacking any demarcation of the midpoint were scored in blinded fashion according to whether the mitochondrial marker was confined to a single parental domain. In selected experiments (see text) one partner expressed cytoplasmic GFP so that attention could be restricted to those zygotes for which the GFP was certain to have equilibrated. In fact, equilibration was seen in >85% of zygotes, as identified by strictly anatomic criteria.
For time-lapse microscopy of zygotes, samples of mating mixtures were rinsed off plates with complete medium at the indicated times and sedimented. 1µl samples of the pellet were applied to 1.5% agarose pads including medium and additives of interest. After overlaying a coverslip and sealing with Vaseline, they were examined by DeltaVision microscopy at 23°C (unless specified otherwise).
For DeltaVision microscopy, we used a 100× oil immersion objective without binning (Olympus UPlanApo 100×/1.40; ∞/0.17/FN26.5). z-stacks were deconvolved using Softworx and processed minimally. At least 20 cells were observed for each condition and the selected illustrations are representative of the large majority (> 80%). Brightfield images are in blue. Successive z-planes were generally collected at 0.2–0.4 µ intervals and complete through-focal series were examined in all cases. Images were collected at 15 second to 30 minute intervals, as appropriate. For most experiments, single planes are illustrated to optimize resolution. Other relevant information was not evident in alternative planes or in projections. Any use of entire z-stack projections is specified in the legends.
To visualize actin, cells were fixed 10 min in ice-cold 70% ethanol, washed in PBS, stained for 2 min at room temperature with 0.14 µM rhodamine-phalloidin (Sigma 77418), washed repeatedly with PBS and examined.
Photobleaching was performed with a Zeiss 510 confocal microscope using a Plan-Apochromat 100× oil-immersion objective (NA 1.4). After bleaching ~80% of the signal at 50% laser intensity, cells were imaged at 1% laser intensity using the acquisition software LSM510 (Carl Zeiss MicroImaging, Inc.). When transformants that express Cox4-DsRed (ATY3129) were entirely photobleached and then followed over the next hour at room temperature, only minimal recovery of signal was seen (not shown). Therefore the contribution of new synthesis of Cox4-DsRed is equally minor over this period of time.
All quantitative experiments are expressed as mean +/− standard deviation. P values were calculated using Student’s t-tests. The illustrated time-lapse series are representative of at least 20 independent cells or zygotes.
We thank Dr. D. McDonald for use of microscopic facilities and A. Camacho, V. Cheruvu, A. Feczko, C. Hollegien, M. Khan, M. Lam, M. Mattera, F. Najm, C-L. Ni, R. Patel, S. Rinonos, S. Roy, J. Toska and Y. Zhang for help with experiments. Thanks to the following for materials: Y. Barral, E. Bi, R. Davis, V. Doye, S. Emr, P-E. Gleizes, E. Grote, W. Huh, A. Johnson, R. Jensen, M. Kucej, S. Liebman, P. Novick, J. Nunnari, E. O’Shea, D. Pellman, E. Pon, M. Rose, R. Rothstein, K. Runge and T. Serio. Thanks to D. Goldfarb, K. Weis and to the anonymous reviewers for comments on the manuscript. This work was supported by NIH Grants P30 CA43703-12 and R01GM089872.
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