Introduction

Cellular aging results in conserved phenotypes, including chromosome mis-segregation, fragmentation of mitochondria, accumulation of damaged or misfolded proteins, and alteration of nuclear morphology^1–3. Importantly, the nuclear pore complex (NPC) is remodelled in aging across evolutionary lineages, as shown in fungi, worms, and mammals ^4–6. As the primary gateway between the nucleus and cytoplasm, the NPC controls mRNA export and protein distribution between these two cellular compartments. The most prominent change in the NPCs of old cells is the loss of their nuclear basket, which lies on the nucleoplasmic side of the NPC. Nuclear basket loss is observed both in dividing cells and upon expression of the progeric variant of Lamin A, progerin^8–9. The protein TPR (Mlp1 and Mlp2 in S. cerevisiae) is the main structural component of the nuclear basket in mammals. The nuclear basket serves as a critical processing hub for mRNA export and nuclear retention of pre-mRNA, protein quality control and gene regulation^10–15. Altering the structure or function of the NPC or its nuclear basket could have diverse and wide-ranging effects on cellular function given the NPC’s broad range of functions and central position in eukaryotic cellular information flow. Despite this and the fact that the reorganization of NPC architecture with age is prominent in a wide range of organisms, little is known about whether and how NPC remodelling contributes to aging.

The unicellular fungus Saccharomyces cerevisiae, budding yeast, is a remarkably useful system for studying the role of NPCs in cellular aging^1,3. Budding yeast undergoes replicative aging, during which the mother cell produces a finite number of daughter cells before dying¹⁶. After the mother cell enters senescence, the rate of cell division slows. Importantly, several studies have emphasized that the yeast NPC is remodelled during replicative aging^4–6, but the exact consequences of its reorganization are not fully understood.

Extrachromosomal DNA circles (also called eccDNAs) accumulate in the mother cell nucleus and are one of the key drivers of aging in the budding yeast^7–9. These DNA circles form during the cell’s lifetime through excision of genomic segments, generally by recombination between repeated sequences¹⁰. The vast majority of these circles (>99%) stems from the highly repetitive rDNA locus and are referred to as extrachromosomal rDNA circles (ERCs)⁷. As the cell age, these circles accumulate exponentially because they are replicated in S-phase and retained in the mother cell at mitosis^7,11. By the end of their life, yeast cells contain about 1000 ERCs, which represents roughly the same amount of DNA as the rest of the genome¹¹. Importantly, their retention in the mother cell relies on their anchorage to NPCs via the acetyltransferase complex SAGA^5,12. These NPC-ERC units are then confined to the mother part of the dividing nucleus by a lateral diffusion barrier forming in the nuclear envelope in the future plane of cleavage^12–14. Yeast cells undergo a closed mitosis, meaning that they keep their nuclear envelope intact throughout mitosis. Remarkably, anchorage of ERCs to NPCs results in the dissociation of their nuclear basket, which is a direct result of SAGA-driven acetylation of nucleoporins on the nucleoplasmic side of the NPC, including Nup60¹². Consequently, most of the NPCs of old yeast cells lack a nuclear basket⁵. However, it is unknown whether nuclear basket displacement has consequences for the physiology and survival of the cell or contributes to the aging process.

One way to approach the question of whether nuclear basket displacement from the NPCs contributes to the aging process is to determine whether other aging phenotypes depend on basket displacement and if so, how. In this study, we focused on chromosome instability in old yeast cells as a case study of an aging phenotype. In many metazoans, including humans, aging results in increasing frequency of chromosome mis-segregation and aneuploidy, correlating with increasing risk of cancer^15,16. Our understanding of the molecular mechanisms of chromosome segregation errors in old cells remains rudimentary. Here, we report evidence that NPC remodelling during yeast aging leads to chromosome loss from old mother cells and characterize the underlying mechanisms.

Results

Aging yeast cells lose chromosomes

To examine whether aging affects the karyotype of yeast cells, we visualized and followed the fate of individual chromosomes in replicatively aging mother cells, using either TetO-labelled chromosome II or chromosome IV as reporters. Chromosome IV is the second longest chromosome, while chromosome II is of average size. Both chromosomes were labelled with an array of 256 TetO repeats in the vicinity of their centromere^17,18. These chromosomes were visualized by expressing fluorescently tagged versions of the TetR protein, which binds to TetO repeats (TetR-GFP or TetR-mCherry; Figures 1A-B). We then monitored budding yeast mother cells throughout their entire replicative lifespan using live-imaging microscopy combined with a microfluidics platform^5,19. The replicative age of each mother cell was measured by counting the number of daughters that it budded off over time, defining how many budding events they have already completed at a given time point (completed budding events, CBE). Bud size and the number and position of the spindle pole bodies (SPBs, labelled with mCherry) were used to identify the cell cycle stages at which the cells were imaged (Figure 1A).

Figure 1

Analysis of these images showed that all young mother cells (∼0-3 CBE; t=0h) and their daughters contained a fluorescent dot, documenting the presence of the labelled chromosomes in all of them. However, when reaching the age of 18 to 22 CBE (t=24h), 10-15% of these now old mother cells had lost their dot (Figures 1B-C). This was observed irrespective of which chromosome was labelled, and which fluorescent tag was used. Fluorescence of the labelled SPB was not affected, indicating that dot loss was not caused by fluorophore instability in old cells. A chromosome dot could be lost from aging mother cells in three possible ways: 1) Chromosome mis-segregation during anaphase 2) Migration of the entire nucleus into the daughter cell 3) Through the loss of a TetO array. The SPB is located in the nuclear envelope, so the presence of the SPB but the absence of the chromosome points to chromosome loss during anaphase, rather than the mis-segregation of the entire nucleus into the daughter cell (Figure 1A). Nuclear mis-segregation into the daughter cells was previously reported during replicative aging and is marked by the segregation of both SPBs to the daughter compartment (Sup Figure 1A-B)²⁰. While we do observe whole nuclei mis-segregation in ∼3% of the cells, this is a distinct event from chromosome loss, which occurs in ∼10-15% of final replicative divisions.

To test if the loss of fluorescent chromosomal foci was due to the loss of the TetO array specifically or of the entire chromosome, we included a second label (an array of 256 repeats of the LacO sequence) in the middle of the long arm of chromosome IV (Figure 1B) and asked whether the cells that lost the TetO array lost the LacO repeats as well. Analysis of these images showed that 85% of the cells lacking the TetO array had indeed lost the LacO array (50/59). Thus, these cells had probably lost the entire chromosome. Supporting this notion, loss of the TetO array was fatal to the cells. More than 99% (145/146 chromosome II loss events) of the dot-less cells failed to divide further, as expected for a haploid cell losing an entire chromosome. Thus, chromosome loss took place specifically in the last division of old mother cells and likely caused their death.

Finally, we examined whether the loss of a reporter chromosome points to general chromosome instability in old cells. To assess this possibility, we quantified how often aging cells simultaneously lost both chromosome II and chromosome IV (Figure 1B-C). As expected, the co-loss rate (∼5%) was lower than the individual loss rates (∼10-15%), but notably higher than the ∼1% expected by chance alone. Therefore, the loss of a single chromosome does not necessarily accompany the loss of another, but the high rate of simultaneous mis-segregation points to general chromosome instability in old cells. Considering that the budding yeast haploid genome has 16 chromosomes, the high loss frequency of a single chromosome and the fact that individual chromosomes are lost in not fully independent manners we estimate that 35-70 % of aging mother cells likely lose several chromosomes in their final division, whereas the other cells die with an intact karyotype. We concluded that much like many other organisms, yeast undergoes a burst of chromosome instability in old age (Figures 1A, C).

In contrast to chromosomes, extra-chromosomal DNA circles accumulate in the yeast mother cell with age rather than being lost^7,12. Similarly, deleting the centromere from a chromosome causes the retention of both sister chromatids in the mother cell¹⁸. Thus, we wondered whether centromeres drive chromosome loss in old mother cells. DNA circles containing a centromeric sequence (CEN) were lost from aged mother cells at a similar rate to full-size chromosomes (∼20% of old cells; Figures 1B-C). Removing the centromere of the exact same circle was shown in previous work to cause its retention in the mother cell rather than its loss^5,12,13. Therefore, centromeres are necessary and sufficient for chromosomes and mini chromosomes to be lost from aging mother cells. To exclude the possibility that the TetO array used to label them is the cause of chromosome and mini-chromosome loss in aging, we also characterized the stability of a circular mini-chromosome expressing short-lived GFP as a marker instead of TetO repeats. Scoring of the GFP signal demonstrated that old mother cells lost these mini-chromosomes at essentially the same frequency as they lose the TetO labelled one (Figures 1B-C). We concluded that centromeres drive chromosome loss in old cells.

Aged cells asymmetrically partition both sister chromatids to the bud

Given the prominent role of the centromere in chromosome loss, we rationalized that the loss of chromosomes in old mother cells could be due to mitotic mis-segregation events. To determine whether it was the case, we next focused our analysis on cells imaged during mitosis in time-lapse recordings of aging cells (Figure 2A). Analysis of anaphases in old mother cells revealed a striking increase in the frequency of chromosome mis-segregation, using labelled chromosome II, IV and minichromosome as reporters (Figure 2B). In 600 total anaphase events captured in our movies we found that 66 (11%) of them co-segregated the labelled sister chromatids to a single pole with, 54/66 (82%) of the mis-segregation events resulting in asymmetric partition of the chromatids to the bud of these old mother cells (Figure 2C). This was virtually never observed in young cells (Figure 2A-B). Remarkably, the frequency of chromosome loss in the old mother cells and of sister chromatids segregating asymmetrically to the bud were in the same size order, irrespective of whether chromosome II or chromosome IV was labelled. Therefore, chromosome loss from old mother cells seems fully explained by a rise in sister chromatid non-disjunction with age, resulting in co-segregation of the sister chromatids asymmetrically to the bud. Next, we examined the mechanism behind this peculiar mode of directed chromosome mis-segregation.

Figure 2

In budding yeast, the pre-existing (old) SPB, which the cell inherited from the previous mitosis, segregates in a highly biased manner to the bud (∼95% of mitoses)^21–23. Consequently, labelling of stable SPB components, such as the core SPB protein Spc42, with mCherry causes the pre-existing SPB to shine bright and segregate in 95% of mitoses to the bud (Figure 2A, red arrowhead), while the new one is dim and remains in according proportions in the mother cell. This useful effect is a result of the folding kinetics of mCherry. In short, mCherry matures slowly (∼45 minutes half-time) relative to the duration of yeast mitosis (∼50 minutes from spindle assembly to completion of cytokinesis), and therefore the new mCherry-labelled SPB is dimmer than the old one. Given the mother-daughter bias of SPB inheritance and sister chromatid mis-segregation with age, we wondered whether the non-disjoining sister chromatids of old cells attach to and co-segregate with the old SPB. Thus, we examined the partition of the labelled chromosomes relative to the pre-existing and new SPBs in old mother cells (t=24h, Figures 2D, E). In about 10% of old cells, SPB inheritance is inverted, meaning that the old SPB goes to the mother cell instead of the daughter^21,22. In a strong majority of these cases (80%), the non-disjoining sister chromatids followed the old SPB to the mother cell, instead of going to the bud (Figures 2D, E). We conclude that non-disjoining sister chromatids of old mother cells remained attached to the pre-existing SPB and partitioned with it to the bud, causing them to lose these chromosomes. In other words, SPB identity mattered more than mother-daughter identity in determining which cell inherited the mis-segregated chromosomes.

In budding yeast, sister chromatids both initially attach to the pre-existing SPB, which is available first. These syntelic attachments are then dissolved by the aurora B kinase, allowing sister chromatids to reorient, reach bi-orientation and partition symmetrically in mitosis^24,25. Failure to correct these initial attachments errors causes sister chromatids to co-segregate with the old SPB to the bud²⁶. The similarity between this phenotype and the mechanism of chromosome loss in old mother cells suggested, therefore, that the error correction pathway might be impaired in old yeast cells. To test this possibility, we analysed the effects of dampening the function of aurora B (called Ipl1 in budding yeast) during aging. Young cells carrying the temperature sensitive ipl1-321 mutant allele and grown at a permissive temperature (27°C) showed no detectable chromosome loss (Figure 2F). However, they started losing chromosomes early in age (Figure 2F), resulting in a shortened lifespan (Sup Fig 1C). Strikingly, the chromosome loss frequency of the old ipl1-321 mutant cells (t=24h) was indistinguishable from that of the wild type cells, indicating that the effects of age and reducing Ipl1 activity were not additive (Figure 2F). This suggests that age and the ipl1-321 mutation affect the same pathway. We concluded that old cells lose chromosomes due to defects in correcting chromosome attachment errors.

ERC accumulation drives chromosome loss in aging

To identify mechanisms behind the deterioration of mitotic error correction with age, we investigated whether chromosome loss is causally linked to other aging events, particularly to the displacement of the basket from NPCs⁵. Given that basket displacement is triggered by accumulating ERCs and their SAGA-dependent anchorage to NPCs^5,12,13, we first investigated whether ERC accumulation promotes chromosome loss. Mutant cells lacking the deacetylase Sir2, which inhibits recombination in the rDNA locus, form ERCs at an increased rate, accumulate them quicker, and their replicative lifespan is shorter than that of wild type cells^7,27. Supporting the notion that ERC accumulation might promote chromosome loss, these mutant cells also showed a strong and premature loss of the reporter chromosome (Figure 3A). To further test the effect of ERC accumulation, we asked whether reducing their formation rate delays chromosome loss. Recombination in the rDNA locus is stimulated by the presence of a replication fork barrier between rDNA units²⁸. While these barriers prevent the transcription machinery to collide with replication forks coming from neighbouring rDNA units, the stalling forks are fragile, causing high rates of double strand breaks. Therefore, removing these fork barriers, for example by inactivating the fork barrier protein Fob1, drastically decreases the rate at which ERCs form and accumulate, stabilizes the nuclear baskets of NPCs and prolongs the life span of the cells^5,28. Strikingly, the fob1Δ mutant cells trapped in our microfluidics platform lost chromosome II at a substantially reduced frequency compared to wild type cells, even after dividing 20 times or more (Figure 3A). Introducing an artificial replicative DNA circle with a different sequence than an ERC into wild type cells also promoted chromosome loss, supporting the idea that DNA circles promote chromosome loss with age independently of their sequence (see below). Taken together, the chromosome loss rates seen in cells with artificial DNA circles, along with those in sir2Δ and fob1Δ cells, show that chromosome loss correlates well with the accumulation of extrachromosomal DNA circles. Since the primary known effect of DNA circle accumulation is to cause the displacement of the nuclear basket from NPCs, we wondered next whether this event was causally linked to chromosome loss. Supporting this notion, removal of the SAGA subunit Sgf73, which mediates ERC attachment to NPCs and the subsequent displacement of their nuclear basket, phenocopied the effect of the fob1Δ mutation¹²: the sgf73Δ mutant cells failed to show any significant increase in chromosome loss frequency at any age (Figure 3A). Thus, our data suggested that ERC formation and anchorage to NPCs triggered chromosome loss in old yeast cells.

Figure 3

Nuclear basket remodelling drives chromosome loss

To investigate whether basket displacement promotes chromosome loss in some manner, we next perturbed the nuclear basket and measured whether this accelerated chromosome loss and aging. To do this, we disrupted the MLP1 gene, encoding the major isoform of the structural basket protein TPR in yeast. In stark contrast to the sgf73Δ and fob1Δ single mutants and the wild type cells, the mlp1Δ single mutant cells exhibited a rapid increase in chromosome loss frequency as they aged, as determined by using our reporter chromosome II, along with a shortened replicative lifespan (Figures 3A-B). Thus, our data suggest that displacement of the basket from NPCs as the cells age promotes chromosome loss through some yet unknown mechanism.

Introns in genes of the aurora B pathway drive asymmetric chromosome partitioning in aging

Together our data suggested that the displacement of the nuclear basket from NPCs upon ERC anchorage weakened the molecular pathway that corrected chromosome attachment errors (Figure 3A). This impaired ability to correct erroneous chromosome attachments in turn caused old mother cells to lose chromosomes to their buds. To test this hypothesis, we next investigated how basket displacement could lead to asymmetric chromosome partitioning. The basket of the nuclear pore has been involved in various processes, such as mRNA export²⁹, protein quality control³⁰, docking of environmentally regulated genes to the nuclear periphery upon their induction³¹, the retention of faulty mRNAs in the nucleus, such as unspliced pre-mRNAs^32,33, as well as docking Mad1 and Mad2, two key components of the mitotic spindle assembly checkpoint (SAC), to the NPC^34,35.

The SAC components Mad1 and Mad2 bind Mlp1, Mlp2 and Nup60 at the basket of NPCs³⁵. However, basket dissociation, which delocalizes Mad1/2 from the nuclear periphery, does not appear to interfere with their function³⁵. Nevertheless, we investigated whether SAC dysfunction could account for aging phenotypes caused by basket dissociation and for the premature aging phenotype seen in mlp1Δ mutant cells. Arguing against this hypothesis, the mad1Δ and mad2Δ single mutant cells exhibited no change in replicative lifespan compared to wild type cells and did not phenocopy mlp1Δ mutant cells (Sup Fig 2A). Therefore, we searched for other mechanisms through which basket displacement could trigger chromosome loss.

While perusing the list of genes involved in chromosome segregation (Figure 4A) we noticed that only three of them contain an intron, NBL1, MCM21 and GLC7. Surprisingly, these three genes all function with Ipl1/aurora B in preventing syntelic chromosome attachment. Nbl1/borealin is a component of the chromosome passenger complex of which aurora B/Ipl1 is the catalytic subunit. Mcm21/CENP-O is a docking receptor for Ipl1/aurora B at kinetochores. Glc7/PP1A is the protein phosphatase 1, which counteracts aurora B by dephosphorylating its targets at the outer-kinetochore^36–38. Intrigued by this remarkable convergence, we wondered whether asymmetric chromosome partitioning in aging could stem from the presence of these three introns in error correction genes. We rationalized that displacement of the basket from NPCs could potentially affect the regulation, export and hence expression of intron-containing transcripts, specifically, thereby affecting the function of the NBL1, MCM21 and GLC7 genes.

Figure 4

To investigate the possibility that they contribute to the high rate of chromosome loss in old cells, we removed the introns of GLC7, MCM21, NBL1 (-Δi alleles) and imaged these mutant cells throughout their replicative lifespan, monitoring chromosome II segregation. Removing each intron individually or all three simultaneously (3xΔi strain) had no detectable effects on chromosome segregation in young cells (Figure 4B), cell growth and mitotic timing (Sup. Figure 3A-C), Interestingly, whereas the removal of GLC7’s intron decreases the expression of the gene by about 50% and affects cell proliferation ^39–41, we note that the GLC7-Δi allele is duplicated in our strain, probably owing to recombination between transposon LTRs surrounding it (Sup Figure 3E-F). We suspect that this duplication explains why our GLC7-Δi mutant strain grew as well as wild type cells. Simultaneously removing all three introns nearly completely suppressed chromosome loss in old cells (Figure 4B-C). Removing them individually or in pair did so as well, albeit the effects were milder. Importantly, the simultaneous removal of NBL1, MCM21 and GLC7 introns did not affect the duration of the last budding event (Sup Figure 3D), indicating that it suppressed chromosome loss without alteration cell cycle progression. Removing the introns of the two α-tubulin genes TUB1 and TUB3 simultaneously had no effect, indicating that the observed effects were specifically related to the introns of NBL1, MCM21 and GLC7 and not to introns in general (Figure 4B). The effects observed were similar for chromosome II and chromosome IV, indicating that the role of these introns was not chromosome specific (Sup Figure 2B). Furthermore, removing these three introns also suppressed the occurrence of asymmetric anaphases in old cells (Figure 4C), supporting the view that asymmetric chromosome segregation and chromosome loss go hand-in-hand and are under the control of these three introns. Strengthening this conclusion, removal of these introns also suppressed chromosome loss in the ipl1-321 mutant cells (Sup Figure 1D).

Remarkably, simultaneous removal of MCM21, NBL1 and GLC7 introns had also a substantial effect on the longevity of the cells, extending their replicative lifespan up to ∼20% (Figure 4D). A similar effect was observed in the ipl1-321 mutant cells (Sup Fig1C). Thus, chromosome loss may indeed be the first direct cause of death for many yeast cells upon aging, although other mechanisms eventually take over when chromosome loss is abrogated.

Basket destabilization causes intron-dependent chromosome loss in young cells

Our data were consistent with the hypothesis that basket dissociation from NPCs promotes chromosome loss through an intron-dependent dampening of the error correction pathway. To test this hypothesis in greater depth we asked whether mutations affecting the nuclear basket also promote chromosome loss outside of the aging context. Thus, we characterized chromosome segregation in young mlp1Δ cells (Figure 5A-C). Analysis of chromosome II segregation indicated that the mlp1Δ mutant cells not only exhibited a four-fold increase in their chromosome mis-segregation frequency compared to the young wild type cells, but ∼80% of these mis-segregation events followed the old SPB, as in old cells (Figure 5C). Furthermore, this chromosome loss was suppressed upon removal of the introns of MCM21, NBL1 and GLC7 (Figure 5B). Therefore, although to a lower extent than in old cells, defects in the nuclear basket result in asymmetric chromosome partitioning in an intron-dependent manner in young cells as well. Interestingly, removal of these three introns also rescued the age dependent chromosome loss and partially restored the longevity of the mlp1Δ mutant cells (Figure 5D-E). We concluded that intron-dependent dampening of the error correction pathway is one, even if not the only progeric effects of the mlp1Δ mutation. Possibly, other introns promote additional aging phenotypes. Thus, we conclude that basket displacement promotes chromosome loss in young mutant cells and old wild type mother cells.

Figure 5

Intron deletion suppresses the effect of DNA circle accumulation on chromosome loss

As seen above, chromosome loss correlates well with conditions that promote ERC accumulation and their anchorage to NPCs. This idea gained in traction as we progressed, because ERC attachment, and the attachment of any DNA circle, to NPCs is known to mediate nuclear basket displacement⁵.Thus, we further investigated the relationships between DNA circle accumulation and chromosome loss by investigating how the removal of NBL1, MCM21 and GLC7 introns affected chromosome loss observed upon DNA circle accumulation. For this, we followed two parallel approaches. First, we tested whether introducing a replicative DNA circle into the cells is sufficient to drive chromosome loss during aging, and if so, whether this event required the presence of the introns of NBL1, MCM21 and GLC7. Thus, we transformed the wild type and the 3xΔi mutant strains carrying the labelled chromosome II as reporter with the yeast replicative plasmid YRp17⁴². We then monitored the frequency at which these transformed cells lost the reporter chromosome as they underwent aging. YRp17 contains a replication origin (ARS1) but no centromere, such that it accumulates in the mother cells with each replicative cycle (Figure 6A). As expected⁷, the cells carrying YRp17 showed a much shorter lifespan (10 CBE median RLS, Figure 6B). Remarkably however, the effect of YRp17 on longevity was substantially suppressed by the 3xΔi mutations (Figure 6B). Analysis of chromosome loss indicated that introducing this DNA circle promoted chromosome loss, starting early in life and increasing rapidly with age (Figure 6C). Furthermore, removal of the introns of NBL1, MCM21 and GLC7 (3xΔi cells) largely suppressed the effect of YRp17 on chromosome loss. However, note that the suppressive effect of the 3xΔi mutations on longevity and chromosome loss was only partial, indicating that YRp17 promotes chromosome loss through both a mechanism that involves these three introns, and at least one additional mechanism independent of these introns.

Figure 6

As a second approach, we investigated whether removing the introns of NBL1, MCM21 and GLC7 mitigates the chromosome loss phenotypes of the sir2Δ mutant cells. Supporting this hypothesis, the 3xΔi triple mutation significantly, though modestly, improved the longevity phenotype of these cells (Figure 6D). As in cells carrying YRp17, the triple intron removal indeed ameliorated the premature chromosome loss phenotype of the sir2Δ mutant cells (Figure 6E). As for YRp17, the effect of the 3xΔi was substantial and significant, but partial. Thus, we concluded that the accumulation of DNA circles, such as ERCs and others, which promote the dissociation of the basket from NPCs and thereby drive chromosome loss at least in part through the introns of NBL1, MCM21 and GLC7. While this effect appeared to be the dominant one in physiologically aged cells, additional effects seem to further promote chromosome loss in the sir2Δ mutant cells and cells carrying an exogenous DNA circle, such as YRp17.

Old cells leak pre-mRNA to the cytoplasm

Putting our different observations together, our data suggested that removal of the nuclear basket from NPCs in response to the accumulation of DNA circles with age causes old yeast mother cells to lose chromosomes to their last bud. Furthermore, the effect of nuclear basket removal from NPCs was primarily mediated by three introns. This suggested that the nuclear basket of NPCs enforces proper chromosome segregation through its function in pre-mRNA processing or quality control. To test this possibility, we next investigated whether old mother cells show defects in pre-mRNA quality control. Since, basket mutant cells fail to retain unspliced pre-mRNAs in the nucleus ^32,33, we reasoned that old yeast mother cells also might leak pre-mRNAs to the cytoplasm.

As a first proof of principle, we characterized the localization of intronic RNA sequences in young and old cells, using single molecule RNA fluorescence in situ hybridization (smRNA FISH). Most yeast introns are short (50-150 nucleotides) and well below the minimal size detectable by smRNA FISH (>500 nts, ideally >1 kb). In the yeast genome, only 20 introns are longer than 500 nucleotides. Thus, we created probes for the longest introns, namely those of DBP2 (766 nts) and YRA1 (1002 nts), and of GLC7 because of its role in chromosome segregation (525 nts). The introns of NBL1 (66 nts) and MCM21 (88 nts) are too short for smRNA FISH. Cohorts of old cells were collected using the mother enrichment program (MEP)⁴³ followed by FACS sorting to reach high homogeneity (see methods). By inducing cell death in the daughter cells specifically, the MEP ensures that aging mother cells are diluted linearly instead of exponentially in the population, greatly facilitating the isolation of very old cells. FACS sorting ensured that the cohorts were free of young cells and young cell debris. With each of these three series of smRNA FISH probes, we detected one or two nuclear foci in young cells (Figure 7A). Only very few cells ((<3%) showed a focus in the cytoplasm. No foci were detected upon deleting the corresponding introns (Δi), demonstrating probe specificity (Sup Figure 4A-B). In contrast, for each of the three introns tested, smRNA FISH detected intronic RNA sequences in the cytoplasm of most old cells. This was not due to an increase in overall smRNA FISH foci number during aging (See Sup Figure 4C), but due to the change in foci distribution between nucleus and the cytoplasm. For example, in the case of GLC7, ∼70% of smRNA FISH foci were found in the cytoplasm of old mother cells, compared to ∼3% in young cells (Figure 7A-B). Thus, 3 out of 3 tested introns showed a strong tendency to leak to the cytoplasm in old mother cells. However, this first test did not clarify whether the full pre-mRNA or only the intron escaped from the nucleus.

Figure 7

To directly address this question, we turned to a functional assay and monitored the translation of an unspliced pre-mRNA reporter in the cytoplasm. This fluorescent reporter contains the coding sequence for mCherry in the intron and expresses GFP upon proper splicing of the nascent transcript. Therefore, mCherry is only translated when the unspliced pre-mRNA leaks out of the nucleus (Figure 7C)⁴⁴. Cells carrying this construct integrated at the TRP1 locus were trapped and imaged in our microfluidics platform. As cells aged, we measured the number of completed budding events and the fluorescence levels of the two reporter fluorophores. Fluorescence signal analysis established that the mCherry/GFP ratio increased in old compared to young cells, demonstrating elevated pre-mRNA translation in the cytoplasm of a large majority of aged cells (Figures 7D-E).

Consistent with ERC-dependent leakage and translation of this reporter, the mCherry/GFP ratio increased prematurely and to a higher level in the sir2Δ mutant, compared to the wild type cells (Figures 7D-F). The fact that signal was already observable in young sir2Δ mutant cells suggests that the pleiotropic functions of Sir2 cause a fraction of these effects. Conversely, the fob1Δ mutant cells showed a lowered mCherry/GFP ratio, which did not increase much during their lifespan (Figures 7D-F). Thus, we conclude that ERC formation and accumulation promotes pre-mRNA leakage during yeast aging, as expected from their effect on the nuclear basket.

Pre-mRNA leakage causes intron-dependent chromosome loss in young cells

Given the results above, we wondered whether chromosome loss in old cells is linked to their failure to retain pre-mRNAs in the nucleus. Thus, we asked whether inducing pre-mRNA leakage in young cells would trigger chromosome loss. The two yeast SR-protein Gbp2 and Hrb1 have been implicated in the retention of pre-mRNAs in the nucleus by binding intron-containing transcripts and delaying their export until they are properly spliced or degraded^45,46. Accordingly, the hrb1Δ gbp2Δ double mutant cells leak pre-mRNA to the cytoplasm at a higher rate than wild type cells^45,47. Thus, we asked whether these double mutant cells lost the labelled chromosome II at an increased frequency. As a control, we asked whether the snu66Δ mutation, which affects the spliceosome itself directly^48,49, lead to similar phenotypes.

The young spliceosome-defective snu66Δ mutant cells did exhibit a five-fold elevated level of chromosome mis-segregation compared to young wild type cells. However, this mis-segregation was not biased towards any specific SPB (Figures 8A-B), suggesting that it involved a distinct mechanism than that observed in old cells and in young cells lacking the basket protein Mlp1. In contrast to the snu66Δ mutant, the hrb1Δ gbp2Δ double mutant cells, showed an even further increased frequency of chromosome mis-segregation, which was more than ten-fold above that of young wild type cells (Figure 8A). Furthermore, sister chromatid mis-segregation was strongly biased towards the old SPB (Figure 8B). Removing the introns of GLC7, MCM21 and NBL1 suppressed this chromosome segregation defect (Figure 8A). Therefore, pre-mRNA leakage is sufficient to induce asymmetric chromosome partitioning and is contingent on the presence of introns in the error correction genes NBL1, MCM21 and GLC7. The fact that mutations affecting the spliceosome did not promote asymmetric chromosome segregation to any specific SPB with age indicates that the chromosome loss observed in old cells is not due to splicing defects.

Figure 8

We conclude that accumulation of extrachromosomal DNA causes the displacement of the NPC basket and subsequent leakage of pre-mRNAs. Leakage of intron-containing transcripts into the cytoplasm impairs correction of syntelic attachments by the Ipl1/aurora B pathway, likely because three genes involved in this pathway contain introns. This results in asymmetric partitioning of sister chromatids, and age-associated chromosome loss. In summary, basket displacement and pre-mRNA leakage impair error correction via an as-yet unknown mechanism involving the introns of NBL1, MCM21 and GLC7.

Discussion

Aging manifests itself through a broad diversity of conserved cellular phenotypes, but in most cases, we know little about how these are causally linked to each other^1–3. In this study we investigated whether NPC remodelling in old yeast cells contributed to the emergence of other aging phenotypes, using chromosome loss as a case study⁵. We showed that NPC remodelling is both necessary and sufficient to drive the age-associated increase in chromosome loss and we identified the mechanisms involved. Specifically, interventions that promoted displacement of the NPC basket, or basket defects more broadly, enhanced non-disjunction and mis-segregation of sister chromatids to the bud, thereby promoting chromosome loss in old yeast cells. These interventions included mutations that stimulated ERC formation and removed the main yeast TPR isoform, Mlp1. Conversely, interventions that prevented NPC remodelling, such as delaying ERC accumulation or preventing their anchorage and the recruitment of SAGA to NPCs, restored proper chromosome segregation in old cells and supressed chromosome loss. Thus, NPC remodelling appeared to be the pivotal point for triggering chromosome loss in old cells.

Two potential caveats should be considered. First, the sir2Δ and fob1Δ mutations each have effects beyond influencing ERC formation rates. The sir2Δ mutant cells lose silencing of the hidden mating type loci, genetically behaving as diploids. They fail to silence subtelomeric regions and act through some as-yet unknown mechanism on the maintenance of proteostasis^50–53. Likewise, the fob1Δ mutation renders the rDNA locus unstable, which might interfere with rRNA transcription and ribosome biosynthesis⁵⁴. However, their only clear common denominator is that they both affect ERC formation, and hence NPC remodelling even if in opposite manners^27,28. Moreover, the sgf73Δ mutation phenocopies the effects of the fob1Δ mutation. Here again the only known functional overlap between Fob1 and Sgf73 is that they promote ERC accumulation in the mother cell indirectly (Fob1) or directly (Sgf73), ERC anchorage to NPCs and the consequent displacement of NPC basket^5,12. Finally, introducing another DNA circle in the form of a non-centromeric replicative plasmid, a process that also causes basket displacement⁵, promoted chromosome loss as well. Thus, our data establish that DNA circle accumulation and attachment to NPCs trigger chromosome loss. Destabilizing the basket independently of DNA circle accumulation also promotes the premature onset of chromosome loss, indicating that it is the basket displacement from NPCs that triggers chromosome loss upon DNA circle accumulation.

A second possible caveat is that chromosome loss could be linked to aging in some other manner. To address this possibility, we investigated the molecular mechanisms that link basket displacement and chromosome loss. Our data reveal that chromosome loss is due to the non-correction of syntelic chromosome attachments in old cells. In young cells, the kinase aurora B and its accessory factors enforce such corrections²⁴. Our data also show that the inhibition of error correction in old cells requires the presence of three introns, those of the genes NBL1, MCM21 and GLC7. These three genes are the only chromosome segregation machinery genes that contain an intron. Strikingly, the proteins Nbl1, Mcm21 and Glc7 are all involved in the error correction pathway, together with aurora B/Ipl1^36–38. Removing their introns restored proper attachment correction and chromosome segregation in old cells and supressed the premature chromosome loss phenotype of the ipl1-321 mutant cells grown at semi-permissive temperature. Intron removal delays cell death but does not abrogate aging. Furthermore, removing these introns not only supresses chromosome mis-segregation in old wild type cells, but also in young mlp1Δ mutant cells. Thus, intron removal separates aging and chromosome loss, indicating that these introns are part of the proximal cause of chromosome loss and act independently of age. Therefore, our data indicate that there is a direct, intron-dependent link between basket displacement from NPCs and chromosome loss. Our data indicate that this link involves the leakage of unspliced pre-mRNAs out of the nucleus upon basket removal and affects the basket’s function in mRNA quality control. The fact that another, independent set of mutations that also interfere with pre-mRNA retention in the nucleus, such as the gbp2Δ hrb1Δ double mutant cells, also increase the frequency of chromosome loss through the same three introns strengthens this conclusion. Thus, together our data establish that displacement of the nuclear basket from NPCs in old mother cell directly triggers chromosome non-disjunction and loss through causing the leakage of the pre-mRNAs of the intron containing genes NBL1, MCM21 and GLC7.

This conclusion has several consequences and opens new questions. The key question stems from our observation that releasing pre-mRNA from the nucleus relaxes the control that cells impose on chromosome attachment. The question is, how does this work? Does it involve the translation of the pre-mRNAs in the cytoplasm? Does it come about through the correlated loss of properly mature mRNAs and hence, the reduced expression of their products? Two arguments speak against these interpretations. First, if any of these two options were to account for the effect of pre-mRNA leakage, mutations affecting splicing efficiency would lead to the same effects. However, tempering with the spliceosome does not affect the correction of chromosome attachment errors to any extent and specificity close to the effects of pre-mRNA leakage. Second, since Mcm21 and Glc7 have opposite effects on chromosome correction it is unclear how impairing the expression of both would lead to the synergistic effects that we observe between them. Finally, analysis of RNA-seq data from replicatively aged cells indicate that if aging indeed impairs the splicing of some transcripts, such as those encoding ribosomal proteins, it stimulates that of others⁵⁵. This suggests that aging has a non-linear effect on intron-containing transcripts. It will be important to determine whether basket displacement contributes to these effects, how it does so and whether this could explain the effect it has on the error correction pathway.

Our observation that basket displacement has such profound and highly specific impacts on processes functionally very distant from that of the NPC, such as chromosome segregation, has several consequences as well. First, it means that NPC remodelling could affect many more cellular functions than only chromosome segregation. Interestingly, beyond mRNA quality control the basket of the NPC is also involved in protein quality control and the activation of environmentally regulated genes, two processes that are impaired in old cells^1,2. Thus, basket displacement might contribute to these phenotypes in aging cells. It is also very likely that the role of the basket in mRNA quality control could affect more than only chromosome segregation. Curiously, while budding yeasts have kept only a subset of the introns of their ancestors, these introns are not distributed randomly and seem to be enriched in very specific processes, such as genes coding for ubiquitin-conjugating enzymes and proteasome subunits, components of the mitochondrial respiratory chain and ribosomal proteins⁵⁶. Interestingly, decay of the ubiquitin-mediated protein degradation, mitochondrial membrane potential and of ribosome assembly are classical and ubiquitous hallmarks of aging^1–3. Thus, it is possible that basket displacement in old cells contributes to the emergence of these aging phenotypes as well.

A second type of consequences emerging from our observations stems from the fact that displacement of the NPCs’ basket is also observed in stressed cells and not solely in the context of aging. For example, heat shock also causes basket displacement in yeast⁵⁷. Therefore, it is possible that the downstream effects we observe in old cells also take place during stress response. In this respect, it is interesting to note that heat shock has been associated with a strong rise in aneuploidy in yeast^58–60. Furthermore, aneuploidy is a common response to environmental stress and a primary mechanism for the emergence of stress resistant clones in fungi⁶¹. Therefore, it is tempting to speculate that the chromosome mis-segregation resulting from basket displacement might be a selected response to stress. It might be selectively advantageous for cells to actively promote karyotype variations when their survival is in question. It is therefore worth considering that a similar process, perhaps using similar mechanisms, underlies the aneuploidy of cancer cells.

Third, the role of basket displacement in the emergence of aging phenotypes is likely not a peculiarity of yeast. Using proteomics, basket displacement has been inferred to take place in aging cells such as the hepatocytes of old mice and humans^62,63. Little is known about whether the nuclear basket of metazoans plays a similar role in pre-mRNA retention in the nucleus as that of yeast, but it is striking that intron-retention is one of the hallmarks of aging on the metazoan transcriptome, and that pre-mRNA processing genes are important for mitotic stability^64–67. Intron retention would be expected if some transcripts escaped the nucleus prematurely. More strikingly, perhaps the most ubiquitous effect of the expression of the progeric variant of lamin A, progerin, across cell types is the displacement of the nuclear basket protein TPR from NPCs⁶⁸. We know still very little about the impact of basket displacement in the aetiology of the Gilford-Hutchinson Progeria Syndrome, but our study suggest that it might be relevant. If so, investigating the potential effects of progerin on pre-mRNA leakage might become an essential step towards understanding how progerin expression causes premature aging.

In a broader sense, our findings might bring fundamental insights towards our understanding of aging. For decades, the role of ERCs in aging has been perceived as a yeast peculiarity. However, the effect it has on NPCs may close the gap. On one hand, it seems to illuminate what might be the commonality between ERC-driven aging in yeast and other aging processes in other eukaryotes. Indeed, the basket-centric view of aging that we propose here would explain how ERCs actually cause aging in yeast and indicate that while ERC accumulation might be a yeast idiosyncrasy, its downstream effects are not. On the other hand, this idea open two corollary questions. 1) What is the biological significance of eccDNAs promoting basket displacement in yeast? 2) What causes basket displacement in other aging systems? We are intrigued by the idea that ERCs and any replicating eccDNA for that matter are the closest mimics that laboratory yeast strain may encounter of pathogenic DNA of exogenous origin. In that perspective, basket displacement and the ensuing effects might have first appeared as a response to infections. In other words, yeast aging might reflect the activity of a death pathway that normally functions in pathogen control. It might represent a fungal version the inflammatory or auto-immune response in metazoans. Therefore, it will be interesting to investigate whether exogenous or non-chromosomal DNA, such as that of viruses, also triggers the displacement of the nuclear basket from NPCs in yeast and other systems, and whether this contributes to either or both of anti-viral defence and aging. If so, aging might have emerged as an anti-viral defence.

Materials and methods

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Yves Barral (yves.barral@bc.biol.ethz.ch).

Materials availability

This study generated ∼50 novel yeast strains, derived either by crossing or transformation. All strains, primers and RNA FISH probes are listed in Supplementary table 1. All resources are available to the public upon request. All the raw data used in this manuscript is available in Supplementary file 1.

Experimental Model Details

Strains

All yeast strains and plasmids used in this study are listed in the Supplementary table 1. Strains are derived from S288C background. For every strain derived from a cross, an appropriate control strain was always selected from the same tetrad. Protein C-terminal yeGFP-tag and knock-out strains were generated using methods described in Janke et al 2004 ⁶⁹. All cultures were grown using standard conditions at 160 rpm, in YPD or synthetic drop-out medium supplemented with 0.1% BSA for aging chips (SD-medium; ForMedium, Norfolk, UK) at 25°C or 30°C.

Intron-less strains

Intron deletions were conducted in two independent colonies of the strain JPY10I, as outlined previously in Parenteau et al 2008 ⁴⁰. Briefly, each intron was individually deleted using a modified two-step process (pop-in / pop-out), ensuring the absence of any additional sequence or markers post-deletion. The deletions were done in diploid strains, confirmed via PCR, and three distinct haploid strains bearing the confirmed deletions were chosen for subsequent analyses. Combinations of different Δ-intron strains have been derived by crossing and validated by PCR for the absence of the intron.

Chromosome and Plasmid reporter strains

Multiple chromosomes and minichromosome visualization strains have been used in this study: 1) Chromosome II reporter strain, with the centromere proximal TetO array (POA1 locus) strain was kindly provided by Elisa Dultz from the Weis Lab, ETH, institute of Biochemistry (internal reference KWY3759). 2) Chromosome IV TetO (Centromere proximal) and LacO (centromere distal) were acquired from strains used in Neurohr et al 2011 ¹⁷, originally derived from Vas AC et al 2006 ⁷⁰ 3) Ubi-GFP minichromosome (PYB 2665) was derived by cloning UBIYdkGFP* from Houser et al 2012 (PNC 1136) ⁷¹ into a PYB 2640 backbone by homologous recombination in yeast. 4) A minichromosome reporter containing the TetO array was previously described in Denoth-Lippuner et al 2014 ¹².

Plasmids

For the assessment of centromeric plasmid loss using the TeO-TetR-GFP system, we used the plasmid already published in Dennoth-Lipptuner et al 2014¹². For construction of centromeric plasmid encoding GFP as a reporter, we used a short-lived GFP (UBIYΔkGFP*–SpHis5–TIM9 in pUC118) available from Addgene(pNC1136). Ubi-GFP was amplified from PNC1136 with primers MM 311/312 (listed in Supplementary table1). The resulting PCR product contained 50 bp homologous overhangs to another plasmid containing a centromere, pADH1 promoter and a pUG23 backbone (PYB2640). The template plasmid (PYB2640) was digested with MscI and Sac II and co-transformed with amplified UBIYΔkGFP* PCR product to yeast to assemble the plasmid with CEN and pADH1-UBIYΔkGFP* via homologous recombination. Successful recombinants were then selected on -LEU media after transformation. Recombinant plasmid was extracted from yeast and transformed into bacteria. The correct plasmid assembly was validated by sequencing. A full sequence of the assembled plasmid is available in supplementary data. For transformation of yeast with a non-centromeric plasmid, we have utilized Yeast Replicative Plasmid 17(YRp17) from Mann et al 1983. The full sequence of YRp17 is available online and in the supplementary data.

Materials and methods

Microscopy

For confocal fluorescent microscopy, yeast cells were precultured in YPD and then washed in synthetic drop-out medium. One ml of cells from exponential growing cultures with OD <1 was concentrated by centrifugation at 1500 rcf, washed in SD complete, and resuspended in ∼10 µl of low fluorescent SD-medium, spotted on a round coverslip and immobilized with a SD/agar patch. The cells were imaged in >15 z-stacks slices with 0.3 μm spacing, with a 100×/1.4 NA objective on a DeltaVision microscope (Applied Precision) equipped with a CCD HQ2 camera (Roper), 250 W Xenon lamps, Softworx software (Applied Precision) and a temperature chamber set to 30°C.

Aging microfluidic platform

Chromosome segregation and protein intensity during aging were assessed using the high-throughput yeast aging analysis (HYAA) microfluidics dissection platform ¹⁹. The PDMS (polydimethylsiloxane) microchannel is made by soft-lithography and bonded on the 30 mm micro-well cover glass in the 55 mm glass bottom dish (Cellvis, CA, USA). For lifespan analyses, a chip with a new cell trapping design was used ⁵, to ensure excellent retention of old cells.

To start the aging experiment, yeast cells were pre-cultured for 24 hr in SD-full media supplemented with 0.1% Albumin Bovine Serum (protease free BSA; Acros Organics, Geel, Belgium). Young cells from an exponentially growing culture were captured in the traps of the microfluidic chip; the chip was continuously flushed with fresh medium at a constant flow of 10 μl/min, using a Harvard PHD Ultra syringe pump (Harvard Apparatus, Holiston, MA, USA) with 60 ml syringe per lane, with inner diameter 26.7 mm (Becton Dickinson, Franklin Lakes, NJ, USA). Bright field images in a single z focal plane were recorded every 15 min throughout the duration of the entire experiment to measure replicative age of cells. To record fluorescent signals, images with a florescent lamp and at least 13x z-stacks (0.5 μm step) were acquired in 12h intervals. For imaging we used an epi-fluorescent microscope (TiE, Nikon Instruments, Tokyo, Japan) controlled by Micro-Manager 1.4.23 software ⁷², with a Plan Apo 60 × 1.4 NA objective. For fluorescence illumination of the GFP and mCherry labelled proteins, a Lumencor Spectra-X LED Light Engine was used. Z stacks of >13 slices with 0.5 μm spacing were recorded for fluorescent imaging every 12 hours to cover the entire volume of the nucleus during the aging process. Transmitted light imaging was done in a single Z plane to minimize the light exposure of the cells. The age of the cell was defined by the number of daughter cells that emerged during the budding cycles (CBE).

Chromosome loss assessment

For the assessment of chromosome and mini-chromosome loss and mis-segregation in aging, trapped cells in the chip that had the labeled chromosomes at the initial time point of 0h (∼virtually all cells) were followed in transmitted light channel and fluorescent images were acquired every 12 hours. Only cells where the entire volume of the nucleus was in focus and imaged, and where SPB was clearly visible were considered in the analysis. The background fluorescence was subtracted in FIJI using a subtract background function. The aged nuclei were examined by scrolling through the z stack (>13 stacks, 0,5um step) in both channels, to validate the presence of the SPBs and TetR foci in the mother or the daughter cell. “Chromosome loss” refers to two quantified events: 1) The absence of the chromosome from a mother or a daughter cell compartment during anaphase 2) A G1 mother cell that just underwent its final division and is clearly viable, with a present SPB dot, but no chromosome dot. When “Chromosome loss” is listed, it pertains to the sum of these two events. When “chromosome loss in anaphase” is listed, it only pertains to the loss frequency observed during anaphase. Cells which lost chromosomes due to abnormal mitotic events in the last replicative division, such as SPB overamplification or full nuclei migration into the bud (Combined ∼10% of all cells in the final replicative division) were discarded from the quantifications. All cells were examined in the DIC channel for signs of disrupted morphology indicating cell death and followed up in DIC to confirm cell death after chromosome loss. For the assessment of minichromosome loss in aging (TetO array or ubi-GFP minichromosome) the yeast cultures were grown in SD-LEU or SD-URA selective media enabling minichromosome maintenance in all starting young cells. When loaded onto an aging chip cell were transferred to SD complete media, allowing them to lose the minichromosome during aging without inducing cell death. As the cells were grown in selective media, the minichromosome loss rate at 0h is N/A. In general, aging chips are not suitable for measurement of chromosome loss frequency in young cells, as cells within the young population that are missegregating chromosomes are usually stressed and enlarged, and thus cannot be loaded into the microfluidic traps intended for normal young cells. For chromosome mis-segregation frequency in young cells, we have used classical confocal imaging on a patch of SD agar, as described in the Microscopy section.

Measurements of chromosome loss per CBE

To measure chromosome loss probability per Completed Budding Event (CBE), we utilized at least 150 cells per genotype and quantified the number of cells that lost the reporter chromosome during a specific replicative division. The number of losses at a specific CBE was then divided by total number of cells which passed through mitosis at that CBE number to give the probability of chromosome loss per CBE. Categories of 5x CBE and its mean value of chromosome loss were plotted along with its SEM.

Mother Enrichment Program (MEP) with FACS and RNA FISH

Mother Enrichment Program was performed according to the standard procedure, using the strains acquired from the Gotchling Lab ⁴³. Cells were labeled with Biotin and aged for 28h, after which they were fixed in 4% PFA for 30 minutes. The cells were then stained with streptavidin conjugated with a FACS compatible fluorophore (647nm) and sorted using FACSAria III at the Flow Cytometry Core Facility at ETH Zurich to enrich for old mother cells. After FACS sorting, single molecule RNA FISH with Stellaris® probes designed for introns of GLC7, YRA1, DBP2 was performed as described in Rahman et al 2013 ⁷³.

Quantification and Statistical analysis

Statistical analyses were performed using GraphPad Prism 10 software

Resources table

Supplementary Figure Legends

Figure S1

Figure S2

Figure S3

Figure S4

Acknowledgements

We acknowledge Dr.Dan Jarosz, Dr. Gabriel Neurohr, Dr. Madhav Jagannathan, Dr. Patrick Meraldi, Dr Anna Marzelliusardottir for critical reading and suggestions on the Manuscript. We acknowledge the ScopeM facility at the Institute of Biochemistry, ETH for their technical assistance. We acknowledge Elisa Dultz for providing the Chromosome II strain with the POA1::TetO array.

Additional information

Data and materials availability

All the raw data generated in the manuscript will be deposited in the Mendelay online repository.

Author Contributions

Conceptualization: MM, ACM, YB Investigation: MM, JM, ACM Formal Analysis: MM,JM, ACM,CD Writing-original draft: YB,MM Writing – review & editing: MM,JM,JP,SAE,YB Resources: JP,SSL Supervision: YB Funding acquisition: YB

Funding

YB: grant 31003A-105904 from the Swiss National Science Foundation (SNSF)

MM: ETH postdoctoral fellowship 19-1 FEL-10

JP: CIHR (Canadian institute of health research) 201809PJT-407549-BMB-CFDA-53473.

SSL: Scope M facility of ETH Zurich, Institute of Biochemistry

SAE: CIHR (Canadian institute of health research) 201809PJT-407549-BMB-CFDA-53473.

Funding

Swiss National Science Foundation (31003A-105904)

Canadian Institutes of Health Research (201809PJT-407549-BMB-CFDA-53473)