Superresolution of the rDNA horseshoe loop shows that it is organized as a twisting thread of unequal density

We noticed that the rDNA loop does not appear homogeneously stained with different specific rDNA binding proteins such as Net1 and Cdc14 (Figs 1C and ​and2A).2A). Z-stacking and deconvolution analysis showed segmented beads lengthwise the rDNA loop. We could discern up to nine of these beads or domains, with a median of five per loop (Fig 2B), which we could further confirm by Total Internal Reflection Fluorescence (TIRF) microscopy (Fig 2C). These beads likely represent the previously reported hierarchical organization of functional domains within the rDNA array (Wang et al, 2015; Dauban et al, 2019).

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Figure 2.

Fine characterization of the mid-M ribosomal DNA (rDNA) horseshoe loop.

(A) The rDNA loop comprises domains with distinctly enriched markers. Examples of rDNA loops with two specific markers: Net1 and Cdc14 (both deconvolved z-stack 2D projections). Numbers indicate counted “beads.” (B) Counted beads for the Net1-GFP rDNA loop (n = 50 cells). Central horizontal solid line represents the mean and outer horizontal solid lines represent one SD. (C) The loop and its beads as seen by TIRF microscopy. (D) 120 nm superresolution of the rDNA loop. Inlet “1” features a section with two tight braided threads (“a” and “b”) separated with a constriction. Inlet “2” features a spring-like section. (E) Schematics of two possible and mutually exclusive spatial configurations of the rDNA loop: (a), the loop has two bases, each comprising one of the rDNA flanks; and (b) the loop folds back, having both flanks residing on a single base. The green boxes indicate the tetO arrays inserted at 450 (rDNA proximal flank) and 487 (rDNA distal flank). (F) The rDNA loop in a strain with both 450 and 487 tetOs, the TetR-YFP and the Net1-ECFP (pseudo-coloured in red). All loops presented the configuration (a). BF, bright field. (G) Quantification of rDNA morphologies (Net1-GFP) relative to the main nuclear mass (Hta2-mCherry) as observed from z-stack 2D projections (mean ± SEM; n = 3); a/c, attached/crossing bar; prot bar, protruding bar. Others* mostly include oval and resolved into two rDNA signals. (H) Examples of partial horseshoe rDNA loops as seen by confocal superresolution microscopy (Net1 looks like a protruding bar but Hta2 forms a closed handle); white arrows point to where Net1 and the Hta2 do not overlap. (I) Scale drawing of chromosome XII with rDNA arrays bearing different numbers of its basic repetitive unit. The numbers above indicate Saccharomyces Genome Database (SGD) coordinates. (J) Length of rDNA loops from arrays with ~75 (n = 100 cells) and ~25 (n = 100 cells) copies.

We next approached the fine characterization of the horseshoe loop by adding visualization through confocal superresolution microscopy (CSM). CSM showed that the rDNA loop was ~200 nm thick and comprised twisting of one or two threads, with sections resembling a spring (Fig 2D). We could also distinguish that the two-thread regions were separated by constrictions (“a” and “b” in Fig 2D). The temporal behavior of threads was rather dynamic, although the overall length and shape of the rDNA loop turned out to be quite stable over the course of short time-lapse video-microscopy (Video 1).

The bases of the rDNA horseshoe loop are the flanking sequences of the rDNA array

We reasoned that the tracts of two twisting threads observed in some loops by CSM may comprise either the cohesed sister chromatids, resolved at 120 nm laterally, or domains of topologically packed/knotted rDNA units. In either case, the horseshoe loop would comprise the two sister chromatids travelling all along its length, with the first and the last unit of the repetitive locus at each edge of the loop (Fig 2E; “a” configuration). Alternatively, these two-thread tracts may expose the two halves of a coiled coil rDNA array (Fig 2B; “b”). In support that the latter (“b”) could occur, we often found small gaps between one edge of the Net1 signal and the DAPI (Fig 1A, arrow). To differentiate between these two models, we combined into a single Net1-eCFP strain two bacterial tetOs arrays that we have previously designed to flank the rDNA array; tetO:450 (rDNA proximal flank) and tetO:487 (rDNA distal flank). This strain also carries the tetO-specific binding protein TetR-YFP. If the rDNA loop comprised an extended array that coils back (“b”), we should see both tetOs localizing at the same loop edge. On the contrary, if the rDNA follows the “a” configuration, we should see each tetO at each edge of the loop. We observed the “a” configuration in all cases (Fig 2F). This flanking positioning is in agreement with previous FISH data for the rDNA borders at the chromosome XII right arm (cXIIr) (Fuchs & Loidl, 2004). Aside from the frontal views of horseshoe loops, the “a” configuration for the tetO:450/487 partner was almost always seen for other rDNA loops and bars.

Similar configuration results were observed for horseshoe loops when we used a partner comprising tetO:450 and a tetO:1061, which localizes near the cXIIr telomere. Even though the tetO:1061 showed a looser localization, it was often close to the opposite tetO:450 base (Fig 3A). The distance between the tetO:1061 and the distal rDNA flank ranged from 0.2 to 4 μm (Fig S1A). In short time-lapse movies, the tetO:450 remained immobile and attached to the loop base, whereas the tetO:1061 moved rapidly and extensively (Video 2). Shorter distances to the nearest rDNA flank were measured for the tetO:194, which settles next to the chromosome XII centromere (Fig S1A). However, relative distances (μm/kbp) were the opposite, as the tetO:194 is ~200 Kbs away from the proximal rDNA flank, whereas the tetO:1061 is ~600 Kbs away from the distal rDNA flank (mean relative distances of 0.0025 and 0.0017 μm/kbp, respectively). The resulting mean apparent compaction ratio was 205 for the centromere-proximal flank tract, and 280 for the distal rDNA flank-telomere tract (Fig S1B). Previously, the axial compaction of chromosome arms in mid-M was estimated to be ~140 (Guacci et al, 1994). This implies that both tracts are probably curly or eventually fold back, especially the distal rDNA flank-telomere tract (Fig S1C; see below for further insights).

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Figure 3.

The space between the ribosomal DNA (rDNA) loop and the rest of the nuclear mass is not nuclear.

(A) The heterologous TetR-YFP that freely circulates in the nucleoplasm does not label the SUL and can give the nucleus a doughnut-like appearance. The horseshoe loop comes from a strain with both 450 (proximal rDNA flank) and 1,061 (right arm telomere) tetOs (the two green dots), TetR-YFP and Net1-eCFP (pseudo-coloured in red). The pointing up arrows indicate saturated images. (B, C, D, E, F) The SUL is surrounded by nuclear membranes. (B) The nER (NE+ER) membrane marker Sec61 delimits both the internal and external boundaries of the nuclear space in the doughnut-like nucleus (TetR ring) that contains the rDNA loop (Net1-mCherry). (C) The space within the Hta2 handle that comprises the Net1 loop is surrounded by Sec61. Only the subset of confocal z-planes where the horseshoe loop was in focus were used for the z-projection (see Video 5 for the whole cell). (D) Like in (B) but with the nucleoplasm labelled with an EGFP-NLS construct instead of the TetR-YFP. (E) The rDNA loop (Net1-CFP) is also surrounded by the nuclear pore complex component Nup49. A single confocal z-plane is shown. (F) Transmission electron microscopy images of mid-M arrested cells with a nuclear morphology compatible with the ones shown in (B, C, D, E). The dotted square marks the area of interest, shown in more detail on the right. Electrodense material represents the nucleolus. The SUL is indicated by an asterisk. The arrow and the arrowhead point to the internal (handle-SUL) and the external (handle-cytosol) NE, respectively. (G, H) The SUL can be formed as a result of a highly bent bilobed nucleus. (G) Example of three mid-M cells (their polar axis indicated by a double dotted arrow) with bilobed nuclei, and where lobes touch each other at their poles (the TetR-YFP signal constrictions pointed by the white arrows), making the nucleus look like a doughnut. Each lobe is indicated by a number in the merged image. Note that the rDNA horseshoe loop forms the handle that connects both lobes and that one lobe contains the bulk of the nucleoplasm (1), whereas the rDNA extends through the other one (2). (H) A doughnut-like nucleus that results from both lobes overlapping at their ends (white arrows in the Sec61 image [see also Video 6, lowest cell]). (I) The SUL can contain a NE/ER ladle, which can be seen as a closing Sec61 ring while walking through the z-planes (white arrows) (see also Video 6, upper and mid cells). In micrographs: BF, bright field.

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Figure S1.

Putative configurations of the chromosome XII in the ribosomal DNA (rDNA) loop.

Related to Fig 2E and F. (A) Quantifications of the distance between the tetO:1061 and the distal flank of the rDNA (df-rDNA; identified as the one that did not have the tetO:450 attached) (n = 102 cells), as well as the distance between the tetO:194 (centromere) and its nearest rDNA flank (nf-rDNA; it is not possible to assess whether the nf-rDNA corresponds to the pf-rDNA or the df-rDNA) (n = 120 cells). Boxplot–whiskers represent 5–95 percentile. Mean shown as “+.” Dots represent outliers. (B) Conversion of the data shown in (A) into apparent compaction ratios (acr) for the centromere to pf-rDNA and df-rDNA to right arm telomere tracts. Boxplot: as in (A). (C) Schematics of non-mutually exclusive spatial configurations for the chromosome XII in the horseshoe rDNA loop, as deduced from (B): (a), the rDNA–telomere tract is curlier than the centromere–rDNA tract, giving the impression of an apparent higher compaction ratio; (b) the rDNA–telomere tract folds back, with similar effects on the apparent compaction. The centromere–rDNA tract might adopt a configuration that tends to maximize distance (acr = 204; not far from 140, the reported mitotic compaction ratio [Guacci et al, 1994]). See later figures in this article for further refinement on these configurations. The green boxes indicate the tetO arrays at 194 (centromere), 450 (rDNA proximal flank; pf-rDNA), and 1,061 (right arm telomere). The solid blue lines indicate the distance between the telomere and the df-rDNA. The dotted blue lines indicate the two possible distances measured for the centromere without a reference for either rDNA flank.

Next, we addressed the configuration of the rDNA horseshoe loop relative to the rest of nuclear DNA. To do so, we made use of the histone H2A2 (Hta2) labelled with mCherry to visualize the bulk of the chromatin. A close look at the Net1-GFP Hta2-mCherry strain showed that the Hta2 forms a protruding loop in ~55% of the Nz-blocked cells (Fig 2G). In about four fifth of these horseshoe loops, the rDNA (Net1-GFP) overlapped entirely with the Hta2 loop; however, in one fifth of the cases, the overlapping was partial, suggesting that flanking regions of the rDNA in cXIIr can belong to horseshoe loops (Fig 2H). Single-cell time lapse of these two presentations of the horseshoe loop showed they are interchangeable (Video 3; e.g., nuclei 2, 4, 6, and 13). These, together with the fact that attached/crossed bars (~15% of all Hta2/Net1 morphologies; Fig 2G) can represent top views of horseshoe loops (Fig 1C and Video 3, nuclei 3 and 15; Video 4, nuclei 3–5), imply that horseshoe loops are even more frequent than what can be observed at first glance in 2D projections. Another ~10% of Nz mid-M nuclei presented a protruding bar-like Hta2/Net1 signal, which appear to be anchored to the bulk of the nuclear mass through one base only, with no visible signs of they being perfect side views of horseshoe loops (yet a bunch of cases could be so; see Video 3, nuclei #5).

The size of the rDNA array does not determine the length of the mid-M rDNA loop

Whereas it is undisputed that the rDNA is highly organized in mid-M, previous studies raised concerns about whether the loop is a condensed state of the locus, at least from a longitudinal point of view (Sullivan et al, 2004; Machín et al, 2005). We have repeatedly measured loop lengths in a Net1-GFP strain with ~150 copies of the 9.137-kbp rDNA unit and obtained mean values of ~5 μm (Fig 1F) (Matos-Perdomo & Machín, 2018). This translates into a relative length of ~275 kbp/μm (150 × 9.137/5). Considering that the length of one bp of naked B-form DNA is 0.34 nm, the estimated axial compaction ratio of the rDNA loop results in ~93 (275 × 0.34). In some instances, the loop reached ~9 μm (~152 kbp/μm; compaction ratio ~52) (Matos-Perdomo & Machín, 2018). Hence, the rDNA loop actually appears less compacted than a normal chromosome arm.

We questioned whether the loop length could be shortened by reducing the number of units of the rDNA array. We analyzed the rDNA length in two strains with the rDNA size fixed at around 75 and 25 copies (Figs 2I and S2A and B). This was achieved through the deletion of the FOB1 gene, responsible for the change in size of the array (Kobayashi et al, 1998). Both strains were blocked in mid-M and the length of their rDNA loops was measured. We found no differences in their lengths (Figs 2J and S2C). The length was also equivalent to that of a ~150 copies rDNA (Fig 1E) (Matos-Perdomo & Machín, 2018). This implies that the compaction ratio of the rDNA can be lowered to, at least, ~10 (a loop of 25 copies reached 7.9 μm [0.34 × 25 × 9.137/7.9]). Theoretically, this packing ratio is close to the 10 nm chromatin fiber, assuming that all the array was fully and periodically coated by nucleosomes (Felsenfeld & Groudine, 2003).

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Figure S2.

Confirmation of distinct ribosomal DNA (rDNA) lengths used to check their influence in the size of the rDNA loop.

Related to Fig 2I and J. (A) Unscaled drawing of yeast chromosome XII, with the canonical 150–200 copies rDNA array indicated by a black bar on the right arm. Genetic details of the single repetitive unit are shown below, together with the position and length of the probe used in the Southern blots. 35S and 5S, genes for the corresponding pre-rRNAs; NTS I & II, non-transcribed sequences I & II; ARS, autonomous replication sequence; RFB, replication fork block. (B) Confirmatory pulsed-field gel electrophoresis of two clones with ~75 and ~25 copies rDNA arrays. Running conditions are optimal for maximizing separation by size of chromosome XII (cXII) with rDNA arrays ranging from 2 to >200 copies (Machín et al, 2006). On the left, the ethidium bromide staining of the pulsed-field gel. The band corresponding to chromosome IV (cIV), the largest yeast chromosome with a fixed size (1.53 Mbps), is indicated. The cXII is larger than cIV when the rDNA array is greater than 50 copies. The ×25 lane corresponds to a Δfob1 strain previously constructed with 25 copies (Kobayashi et al, 1998). Accordingly, the size of cXII is smaller than cIV and the cXII band migrates faster than that of cIV. The ×75 lane corresponds to a Δfob1 clone that originates from a ×190 strain that spontaneously suffered from a Fob1-independent shortening of the array. Accordingly, its cXII band migrates slightly slower than cIV. On the right, Southern blot of the PFG with a probe against the rDNA to confirm the cXII bands. (C) Examples of mid-M rDNA loops from both strains. Quantification of loop lengths are in Fig 2J. BF, bright field.

We conclude that (i) the size of the rDNA array does not determine the length of the rDNA loop, at least in a window of 25–150 copies; and (ii), conversely, the loop length is more or less fixed, with the compaction of the rDNA adapting to it, stretching the array if needed. Both the non-homogeneous compaction within the loop (beads) and its spring-like appearance described above may explain why the loop retains a constant length upon reducing the locus size.

The space under the rDNA loop is not nuclear but shapes the entire nucleus

The second remarkable feature of the Nz mid-M arrest is the SUL, the large space between the loop and the main nuclear mass. To our benefit, in the strains that carry the TetR/tetO system not all TetR-YFP molecules bind to the tetOs. A pool of TetR-YFP freely circulates within the nucleus, labelling the nucleoplasm and, hence, marking the shape of the nucleus. When observing the Net1-eCFP horseshoe loops, the free TetR-YFP surrounded the rDNA in all cases. Strikingly, the SUL was void of TetR-YFP, especially evident in frontal views of horseshoe loops (Fig 3A, see saturated TetR-YFP images, and Fig S3A). We hypothesized that this space may comprise other parts of the nucleolus, which could be somehow free of the TetR, perhaps through liquid–liquid phase separation (Feric et al, 2016; Matos-Perdomo & Machín, 2019). However, we found neither rRNA-processing proteins (Fig S3B) nor RNA (Fig S3C). In both cases, the mid-M arrest made these markers adopt a horseshoe-like morphology as well. Alternatively, the SUL could be filled up by part of the nucleus devoid of freely circulating proteins; for instance, heterochromatinized DNA. However, we did not find histone-coated (Hta2-mCherry) DNA, DAPI (after overexposing), or ultrasensitive YOYO-1 DNA stain within that space (Fig S3D and E).

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Figure S3.

The space under the ribosomal DNA (rDNA) loop (SUL) is not nuclear.

Related to Fig 3. (A) The heterologous TetR that freely circulates in the nucleoplasm does not label the SUL and gives the nucleus a doughnut-like appearance. The horseshoe loop comes from a strain with the 194 (cXII centromere) tetOs (green dot), the TetR-YFP and the Net1-eCFP (pseudo-coloured in red). (B) The nucleolar SSU rRNA processome protein Nop1 also forms a horseshoe-like loop. (C) The RNA marker SYTO-RNA, which strongly labels rRNAs, also forms a horseshoe loop. (D) Overexposed DAPI (shown in grey) and fluorescently-tagged histone Hta2 (H2A) co-label the rDNA loop (Net1-GFP), which is seen as a handle in a handbag-like nucleus. The arrow points to the handle, whereas arrowheads point to non-nuclear (mitochondrial) DNA. (E) The ultrasensitive dsDNA stain YOYO-1 also stains a handbag-like nucleus in mid-M. (F) Cross-section intensity profile of a z-stack depth-in-focus 2D projection of the horseshoe loop shown in Fig 3C. On the left, the cross section used for the intensity profile; on the right, the intensity profile. Note that Net1 and Hta2 colours are exchanged relative to Fig 3C. (G, H) The nuclear envelope marker Nup49 weakly stains the SUL in horseshoe rDNA (Net1) loops. In (G), samples were processed for DAPI staining of the nuclear mass and images were captured with WFM (DAPI pseudo-coloured in red). In (H), 0.15-μm-thick z-planes were captured by confocal superresolution microscopy to reconstruct the nucleus in 3D. The figure shows eight spatial perspectives obtained by sequentially rotating 45° clockwise in the y-axis, starting from the frontal (XY) view. The arrow points to a ladle or bucket found in lateral view of the SUL, which is nonetheless poor in Nup49 when compared to the rest of the NE. (I) The same transmission electron microscopy image shown in Fig 3F but with colour lines drawn to label the inner (cyan) and outer (yellow) sheets of NE that protrude out of the main nucleus as a handle. The lines follow the perinuclear space left by inner and the outer membranes of the NE (INM and ONM), which are also indicated by arrows. (J, K) The horseshoe rDNA can be the handle that connects the two lobes of a bilobed nucleus. (J) Another example taken from Fig 3A where a constriction in the TetR-YFP is seen at the bulk of the non-rDNA nucleoplasm (pointed by a white arrow in the enlarged nuclear image underneath). The tetO:1061 is recognized because it is not closely attached to one edge of the Net1 loop, as is the tetO:450. The constriction might indicate the presence of overlapping lobes. On the right, a solid white line has been drawn to indicate a putative spatial configuration of the overlapping lobes. The YFP and CFP channels correspond to a single z plane. In all examples of this kind of horseshoe loop where the Net1-eCFP is seen throughout (i.e., perfect frontal view), the tetO:450 is on focus and the tetO:1061 is out of focus, further supporting the overlapping bi-lobed model. (K) Another Nup49-GFP Net1-CFP example processed as in (H) and in which the Net1 loop connects two terminal lobes that touch each other, forming a constriction in the doughnut-shaped nucleus (white arrowhead). BF, bright field; INM, inner nuclear membrane; ONM, outer nuclear membrane.

In light of these negative labelling, we next questioned whether the SUL was nuclear. For addressing this, we added a third fluorescent marker to the previous set of strains, the ER membrane translocator Sec61, which labels the perinuclear ER, this being a continuum with the outer nuclear membrane of the nuclear envelope (NE). We found that the Net1 loop was surrounded both externally and internally by Sec61 (Figs 3B and C and S3F; see also Video 5 for a full appreciation of the spatial orientation of the second example). A similar pattern was obtained when an EGFP-NLS replaced TetR-YFP as the nucleoplasm reporter (Fig 3D). This was further confirmed by another NE marker, the nuclear pore complex (NPC) protein Nup49. Nup49 is more specific for the NE than Sec61, but its labelling shows a punctate distribution and is more challenging to interpret (Belgareh & Doye, 1997). Nonetheless, we could confirm that Nup49 labels the internal face of the loop as well (Figs 3E and S3G and H). To get further support for the claim that the SUL is not nuclear, we performed transmission electron microscopy (TEM). We found examples where the nucleus appears as a handbag, with a thin handle surrounded by a double membrane, and with the electrodense nucleolus close to or within this handle (Figs 3F and S3I).

The nucleoplasm labelling with both TetR-YFP and EGFP-NLS pinpointed that the nucleus is arranged as a ring-shaped doughnut, with a tendency to asymmetrically distribute the nucleoplasm towards a bulge, where most of the non-rDNA chromatin resides (Fig 3A–D). Frequently, a constriction in the bulge was evident (Fig S3J), which can be deep enough to separate the nucleoplasm signal in two (Fig 3G). 2D projections and 3D reconstructions of the NE (Sec61 and Nup49) showed that in these cases the bulge is made up of two lobes that overlap to various degrees (Figs 3H and S3J and K and Video 6). In addition, in most 3D reconstructions (80%; 16 of 20), the Sec61 signal distributed in depth as a hemisphere in the SUL (Fig 3I and Video 6, top two cells), suggesting that the NE (or the ER) could be ladle-shaped in lateral views, in addition to being doughnut-shaped in frontal views. This ladle was also seen with Nup49, which supports that it is made up of NE; however, Nup49 was much less abundant there, pointing out that NPCs may have restrictions to access the ladle (Fig S3H, arrows).

The space under the rDNA loop is occupied by the vacuole

After realizing that the SUL was not nuclear, we added a cytosolic EGFP to a Sec61-eCFP Net1-mCherry strain to confirm the SUL was cytosolic. To our surprise, the EGFP signal was weaker in the SUL (Fig 4A). A key hint to understand the nature of the SUL came from the visual comparison of the Net1 loop and the Hta2 handle with the entire mid-M cell as seen through the transmission light (bright field). We noticed that most large horseshoe loops appear to either entirely or partially surround the vacuole, which otherwise appears to sit on these loops in partial side views (Fig 4A–C). Hence, we checked whether the vacuole was occupying the SUL. We used several specific vacuolar markers, including the vacuolar lumen vital dyes Blue CMAC and carboxy-DCFDA, the vacuolar membrane (VM) non-vital dye MDY-64, and the VM marker Vph1-GFP. In all cases, at least one vacuole co-localized with the SUL (Figs 4D–I and S4A–D). There were instances where the co-localization of the SUL from a front view horseshoe loop and a sole vacuole was almost perfect, whereas in other cases the vacuole was too large for the SUL, or the SUL was filled with multiple smaller vacuoles (Fig S4A). VM markers such as Vph1-GFP gave the best signal at the equatorial central plane, which in large vacuoles was in a different z plane relative to frontal views of the rDNA horseshoe loop, making them to appear as if they were crossing the vacuole in 2D z-stack projections (Fig 4D). However, 3D reconstructions showed that part of the vacuole was sitting on the SUL (Fig 4G and H). With smaller vacuoles, at least one of them occupies the SUL (Figs 4E and I and S4A–D). Z-stack imaging, fluorescence intensity profiles and orthogonal projections of the vacuolar lumen confirm that the SUL was indeed occupied by these small vacuoles (Figs 4I and S4B–D).

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Figure 4.

The space under the ribosomal DNA (rDNA) loop is occupied by vacuoles.

(A) Cytosolic EGFP weakly stains the SUL. The strain also bears Sec61-eCFP and Net1-mCherry to delimit the SUL (black holes within cell bodies). In the BF, vacuoles (V, pointed by arrows) may appear as balls of different density. Note how SULs and vacuoles colocalize. (B, C, D, E, F, G, H, I) Vacuoles reside in the SUL. (B) The rDNA loop (Net1-GFP and Hta2-mCherry handle) partly surrounds the vacuole (V), pointed with the arrow in the inlet BF image. (C) An example in which the vacuole (V in the BF) sits on a horseshoe loop. (D) A horseshoe loop, too small to engulf the vacuole (labelled with the vacuole membrane reporter Vph1-GFP), appears to cross the organelle in z-stack 2D projections. (E) Vacuole content (labelled with the vacuolar lumen vital dye Blue CMAC) is found in the SUL. (F) For a representative experiment as in (E), quantification of colocalization (for SUL) or juxtaposition (for nuclear flares) of rDNA bars and loops with vacuoles. Nuclear extensions (“flares”) may correspond to either SUL side views or early stages before SUL formation (see below). (G) A walk through z planes of a horseshoe loop with a large vacuole on top. (H) A 3D reconstruction of (G) with serial 45° anticlockwise rotation on the y-axis. Note how the horseshoe loop leans onto the vacuole. (I) Z-plane walk-through of a TetR doughnut-like nucleus where vacuole lumens have been stained with Blue CMAC. In micrographs: BF, bright field; V, vacuole; BC, blue CMAC.

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Figure S4.

The space under the ribosomal DNA (rDNA) loop (SUL) contains one or multiple vacuoles.

Related to Fig 4. (A) Four examples of rDNA loops in which one or multiple vacuoles (stained with MDY-64) are found in the SUL. In the example 1, a group of small vacuoles locate at the SUL. In the example 2, the rDNA appears to cross a large vacuole. This could happen because of either the spatial orientation of the loop (e.g., it is partly seen from the top) or because the SUL only accommodates a portion of the hemisphere of a larger vacuole, so that the vacuole sits on the SUL (see further examples and 3D reconstructions in Fig 4). In examples 3 and 4, the vacuole almost perfectly fits in the SUL (partly lateral view for example 3 and front view for example 4). (B, C, D) The SUL is occupied by vacuolar content. (B) An example of the doughnut-like nucleus that leaves a horseshoe loop with the vacuole lumen occupying the SUL. The strain bears tetO:450 tetO:1061 TetR-YFP, and the vacuole lumen is stained with Blue CMAC (pseudo-coloured in red). (a) Maximum projection of the montage of channels; (b) the TetR-YFP channel (grayscale); (c) the Blue CMAC channel (grayscale). Intensity profiles of a SUL cross section are shown for each channel. (C) Chart of the intensities across the SUL. Note how two peaks of nucleoplasm intensity (in green) lay at the borders of the vacuolar lumen (in red). (D) Orthogonal projections of a second example of a horseshoe loop with the vacuole in the SUL. Note how tetOs lay on different z planes. This is rather common in front views of horseshoe loops.

The rDNA horseshoe loop stems from small rDNA loops and bars that grow and bend around vacuoles

Next, we focused on the origin of the horseshoe rDNA loop with the vacuole in the SUL. To do so, we performed both time-course experiments and time-lapse video-microscopy after Nz addition. We observed that in an asynchronous population, most nuclei were spherical or slightly oval in cells transiting through S/G2/M (counting only the pre-anaphase budded cell subpopulation) (Figs 5A and S5A). Of note, in ~20% of these S/G2/M cells, the nucleus may appear squeezed between the vacuole and the plasma membrane, as if the NE is a malleable body that must seek allocation between two other stiffer bodies, the vacuole and the cell wall (Fig S5A, S/G2/M example). Shortly after Nz addition, budded cells elongate their nucleus (Fig 5A and Video 7), and this occurred with different degrees of symmetry in relation to the amount of nucleoplasm present along the extended nucleus (from pool noodles to finger-like projections; Fig S5B–G) and could carry or not primordial constrictions, which makes nuclei look like cashew nuts (Fig 5B, Video 7 and Video 8, and Fig S5B–F). These nuclear constrictions indicate that bilobulation could be a primordial event in the reshaping of the nucleus in Nz.

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Figure 5.

The ribosomal DNA (rDNA) horseshoe loop stems from small bars and loops of rDNA that protrude out of the main nuclear mass and wrap around the vacuole.

(A) Proportion of the three major nuclear morphologies observed during an Nz time-course experiment (mean ± SEM; n = 3); “extended” comprise a plethora of nuclear morphologies that deviate from the rounded nucleus observed in cycling cells. (B) Three examples of the most common extended morphology seen shortly after Nz addition, the cashew-like nucleus. In this morphology, a seemingly bilobed nucleus is seen, with lobes delimited by a primordial constriction. The rDNA can be located either entirely in one lobe, as a loop (upper cell) or a protruding bar (mid cell), or across both lobes (lower cell). (C) Proportion of the rDNA in one lobe (one-sided) or across both lobes in cashew-like nuclei during an Nz time-course experiment (mean ± SEM; n = 3). (D) Juxtaposition of growing nuclear extensions containing the rDNA and vacuoles during an Nz time-course experiment. “No flare or loop” (in red) indicates that neither extensions (finger-like or lobes) nor SUL were present. (E) An example of a protruding rDNA bar juxtaposed to a large vacuole. (F) An example of a protruding rDNA bar in the context of a protruding Hta2 bar. The arrows point to the denser Hta2 signal ahead of the Net1 tip. (G) Location of selected positions along cXIIr in protruding rDNA bars (from a pooled Nz time-course). “mid+apical” denotes that the corresponding tetOs array locates within the rDNA extension (mean ± SEM; n = 3). (H) Location of the cXIIr telomere in protruding bars during the Nz time-course (mean ± SEM; n = 3). (I) Two examples where the telomeric flank of the rDNA localizes ahead of the protruding rDNA bar, so that most of that chromosome arm (cXIIr) gets away from the nuclear mass. On the left, the protruding cXIIr remains in the mother; on the right, the protruding cXIIr crosses the neck. In micrographs: BF, bright field.

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Figure S5.

The class II horseshoe ribosomal DNA (rDNA) loop is formed when the nuclear flare that contains the protruding rDNA bar folds back and its tip overlaps with the main nucleus.

Related to Fig 3. (A) The reported mid-M arrest morphologies are not present during a normal cell cycle. Representative micrographs taken from an asynchronous log phase culture. Sec61-CFP is shown in grey scale for better visualization. Note that the nucleus and the rDNA are spherical/oval in interphase. An example of an elongated/squeezed nucleus is shown as well. This phenotype is more common in G2/M but does not reach the extensions and nuclear reshaping observed in mid-M arrests. (B) Transmission electron microscopy of a cashew-like extended nucleus. The extension or flare is pointed by an arrow and has the electrodense nucleolus inside. (C) An example of an extended nucleus that resembles a noodle pool rather than a cashew (as deduced from the Sec61 pattern). Here, the rDNA forms a protruding rDNA bar that probably extends to the cXII telomere (as deduced from the position of the tetOs). (D) Nuclear morphologies seen by Sec61 in mid-M arrests. On the top, representative micrographs. Bt, bow-tie nucleus; Ana, dumbbell shaped anaphase nucleus (the cell has escaped from the mid-M arrest). Extended is a mid-M specific category that can take several forms from lobes to finger-like projections (two examples shown). On the bottom, quantification of the observed morphologies in an asynchronous population (DMSO, 3 h) and an Nz mid-M arrest (mean ± SEM, n = 3). (E) An example of a nucleus deformed by localizing between a large vacuole (V) and the cell surface. The nucleus is visualized through the nER marker Sec61-eCFP. Whereas growing nuclear extensions around vacuoles may explain many of the observations reported here, nuclear deformation by squeezing between stiff vacuoles and the cell surface could explain others. It might even aid flare formation and growth. (F) Another example of nuclear deformation by the vacuole as seen by confocal superresolution microscopy. In this case, most of the nucleus, not only the rDNA bar (pointed by an arrowhead), bends around the vacuole. The nuclear DNA mass (Hta2-mCherry) appears unevenly distributed across the bended nucleus, forming constrictions (arrows). (G) Examples of nuclear extensions (finger-like TetR-YFP flares) surrounding the vacuole, whose lumen is stained with blue CMAC (pseudo-coloured in red). These are three separated nuclei, not frames in a time-lapse experiment. The strain also bears the double chromosome labelling tetO:450 tetO:1061. Because one tetOs localizes apically (presumably the tetOs:1061; see Fig 5G and H), it is assumed the flares contain protruding rDNA bars. BF, bright field; V, vacuole.

Nuclear elongation and constriction presentation could occur in two axes relative to the rDNA. The first and more abundant axis entails the spatial separation of the rDNA/nucleolus from the rest of the nuclear material, with the rDNA on one side or lobe (Fig 5B, upper two cells; Fig 5C and Video 7 and Video 8, upper cell). This configuration of having the nucleolus into protruding nuclear fingers/lobes was also confirmed by TEM (Fig S5B), and is in full agreement with the nucleolus-containing NE “flare” previously described by the Cohen-Fix’s laboratory (Campbell et al, 2006; Witkin et al, 2012). In its early presentation, the rDNA often appears packed (oval or any of the small loop morphologies). However, as the nucleus becomes enlarged, this rDNA loop gets larger as well, either blooming into a horseshoe loop (compare the upper cell of Fig 5B with Fig 3C and Video 5; Video 9 for horseshoe bloom) or recoiling entirely one flank of cXII to become a protruding bar (see below for a detailed description of the latter).

The second axis of elongation leads to the formation of an rDNA bar that goes across the extended nucleus (Fig 5B and Video 8, lower cell). More evolved morphologies observed later in Nz suggest that these nuclei continue growing in length while bending, eventually forming a nucleoplasmic bridge that connects the two lobes (also as inferred from Video 7, lower cell). The rDNA is mostly located in that bridge, acquiring the shape of a bent bar (Fig S5C). As the growth and bending continues, the two apical lobes can get closer until they touch each other and even overlap in 2D projections (Figs 3G and S5G and Video 6), resulting in a second class of horseshoe loops.

Strikingly, in cashew-like nuclei, which still lack a proper SUL, a NE ladle was often visible (Fig 5B, two lower cells; Video 8). Even traces of nucleoplasm (EGFP-NLS) were seen in this ladle (Fig 5B, arrowheads in the lowest cell), reasserting that the ladle must be formed by two close sheets of NE instead of ER. The early presence of this ladle points towards a germinal connection between the NE subdomain associated to the rDNA and vacuoles. To determine when the interaction between the rDNA and the vacuole occurs, we again performed time-course experiments and time-lapse video-microscopy (Fig 5D and Video 10). We found that growing rDNA extensions tend to colocalize with vacuoles from the beginning, showing a constant ~7:1 ratio in favor of juxtaposition through the time course (Fig 5D and E). This germinal connection was also seen in time-lapse video-microscopy (Video 10).

A final hint to fully understand the nature and diverse origins of horseshoe loops came from observations that slightly differ from the morphological patterns described above. As stated, the horseshoe rDNA loop is the most remarkable morphology of the rDNA array in mid-M, but protruding rDNA bars are observed as well in 10% of the arrested cells (Figs 2G and ​and5B,5B, mid cell). These protruding bars often contained a brighter Hta2 spot at the tip (Fig 5F, arrow), which must correspond to one of the flanks of cXII, probably up to the corresponding telomere. To precisely determine the arrangement of the chromosome in these protruding bars, we looked at the location of the four tetOs along cXIIr (Fig 5G). We observed that the distal flank of the rDNA (tetO:487) tended to be significantly present in the nuclear projections (~65% of the cases, mostly in an apical position), although in ~18% of the bars we found the proximal flank (tetO:450) in there; and even the centromere in ~10% of bars. The fact that the distal rDNA flank was more frequently found than the proximal flank suggests that distal cXIIr regions are more prone to get into the growing extension. Compared with the rDNA distal flank, we found fewer examples of the cXIIr telomere (tetO:1061) in the projection, strongly pointing out that about half of the rDNA protruding bars are still cXIIr loops (likely side views of the partial rDNA loops we reported in Fig 2G and H). However, as Nz incubation goes by, the cXIIr telomere did move to the tip (Fig 5H and I). Remarkably, these rDNA bars, and their surrounding nucleoplasmic flares, showed extensive bending, which were clearly reminiscent of incomplete states of the horseshoe loop and the overlapping bilobed nucleus described above (Fig 5I. left nucleus; Fig S5C and G); there were also cases where the protruding bar crossed the neck into the bud (Fig 5I, right nucleus).

The horseshoe rDNA loop requires the absence of microtubules

Previous works demonstrated that nuclear extensions and flares are characteristic of cells blocked in mid-M (Witkin et al, 2012). Nz is the most common experimental tool to achieve the mid-M arrest. Nz depolymerizes microtubules, which dismantles the spindle apparatus and activates the spindle assembly checkpoint (SAC) (Jacobs et al, 1988; Hoyt et al, 1991). With an active SAC, the anaphase-promoting complex (APC) activator Cdc20 is tightly bound and inhibited by the SAC components Mad2 and Mad3 (Peters, 2006; Musacchio & Salmon, 2007). Considering the effect of Nz on microtubules and the SAC, we next chose to study the effect of arresting cells in mid-M by other means. We planned two different strategies that preserved the bipolar spindle, yet they differ in the activation state of the SAC (Fig 6A). On the one hand, we depleted Cdc16, an essential component of the APC, by creating a cdc16-aid conditional allele. We used this strategy because thermosensitive alleles require incubation at 37°C, and we and others previously showed that this triggers a mild heat stress response that impinges on the rDNA loop (Shen & Skibbens, 2017; Matos-Perdomo & Machín, 2018). Degradation of Cdc16-aid can be triggered by adding the auxin indole-3 acetic acid (IAA) to the medium (Fig S6A and B) (Nishimura et al, 2009). Under this condition, the APC can be inactivated without interfering with the spindle and/or the SAC, although there is a report about a possible activation of the SAC upon APC inactivation (Lai et al, 2003). On the other hand, we made use of another strain carrying a P_GAL1-MAD2-MAD3 construction, which overexpresses a fusion protein of these two key SAC players (Mad2-Mad3^OE), yielding an active SAC in galactose. Under this condition, the active SAC maintains the mid-M arrest by keeping the APC inactive (Lau & Murray, 2012; Thadani et al, 2018).

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Figure 6.

The ribosomal DNA (rDNA) in mid-M arrests that preserve the microtubules.

(A) Summary of the differences between four mid-M arrests (Nz, depletion of Cdc16, overexpression of the Mad2-Mad3 chimera, and depletion of kinesin motor proteins Cin8 and Kip1). APC, anaphase-promoting complex; SAC, spindle assembly checkpoint; OE, overexpression. The plus sign indicates either active (APC, SAC) or present (spindle); the minus sign indicates the opposite; the question mark indicates that the corresponding activation state is assumed, but there are contradictory data in the literature; bi, bipolar spindle; mo, monopolar spindle. (B) Morphology of the rDNA after the non-Nz mid-M arrests (mean ± SEM; n = 3). (C, D) Representative micrographs of the mid-M arrest observed after depleting Cdc16-aid. (C) An example with labels for the bulk of chromatin (Hta2), the rDNA (Net1), the nucleoplasm (free TetR-YFP), the cXIIr telomere (tetO:1061) and the vacuolar lumen (c-DCFDA), In the green channel, the three latter structures are labelled and differentiated by intensity and morphology; nucleoplasm is the weak signal on the left, the cXIIr telomere is the spot, and the vacuolar lumen is the stronger signal on the right. (D) Like in (C) but with the vacuolar membrane labelled with MDY-64. The strain is the same, but Net1-CFP is completely masked by the much stronger MDY-64 signal. Note in both examples the nuclear disposition across the neck, with the rDNA and the bulk of the DNA mass residing in different cell bodies, and how the rDNA interacts with vacuoles in the receiving body. (E) Length of the rDNA (Net1) in Cdc16-aid plus IAA (>100 cells) versus Cdc16-aid plus Nz (>100 cells). (F, G) Representative micrographs of the neck-crossing rDNA bars observed after overexpressing Mad2-Mad3. (F) Net1 together with DAPI staining. (G) Net1 together with BC staining. (H) Representative micrograph of the mid-M arrest observed after depleting Cin8-aid in a kip1Δ background. The upper cell shows a case where the nucleus and the rDNA remain in the same cell body; note that there is no horseshoe loop. The lower cell shows a neck-crossing rDNA bar, as in other non-Nz mid-M arrests. In micrographs: BF, bright field; V, vacuole; BC, Blue CMAC.

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Figure S6.

Morphology, length and spatial disposition of the ribosomal DNA (rDNA) in non-Nz mid-M arrests.

Related to Fig 6. (A) Spot assays to check Cdc16-aid degradation under auxin. Cdc16 is an essential protein. A strain carrying a cdc16-aid allele plus the auxin-mediated degron system (OsTIR1) was spotted with and without auxin (indol acetic acid; IAA) alongside a strain derivative where OsTIR1 has been eliminated. Note that cdc16-aid OsTIR1 does not grow on YDP plus 5 mM IAA. (B) Western blot (WB) to check Cdc16-aid degradation upon IAA addition. Samples were taken before (0) and 60’ after adding 5 mM IAA. The WB (left picture) is against the myc epitope, which is in both Cdc16-aid (-9myc) and OsTIR1 (-9myc). The latter serves as an internal control. Total protein loading (Ponceau staining) is shown on the right. (C) Comparison of major rDNA morphologies in a cdc16-aid mid-M arrest (top) and an Nz mid-M arrest (bottom). Note the extended bar across the neck in the cdc16-aid, as opposed to the horseshoe loop in Nz. (D) Quantifications of nucleus-rDNA-vacuole arrangements in the cdc16-aid mid-M arrest. From left to right: The first chart represents whether the sum of Hta2 (bulk chromatin mass) and Net1 (rDNA) goes through the bud neck. The second chart quantifies the relative position of the main chromatin mass and the rDNA in those nuclei that cross the neck; “opposite bodies” implies that the bulk of the chromatin locates in one body (either the mother or the bud) and the rDNA in the other, with the bud neck separating them. The third chart quantifies the relative position of the cXIIr telomere in neck crossing nuclei. The location of the telomere in the rDNA extension implies that the rDNA is a protruding bar. The fourth chart shows the juxtaposition of at least one vacuole with the extended rDNA across the neck (the extended Hta2 signals was taken as a reference as MDY-64 was used as vacuolar marker and masked the Net1-CFP). (E) Examples of cells carrying the P_GAL1-MAD2-MAD3 construction after being arrested with Nz for 3 h in glucose. The aim of this experiment was to check that the horseshoe rDNA loop is present in this particular genetic background. (F, G) Length of the rDNA (Net1) in the Mad2-Mad3^OE mid-M arrest. (F) The overexpression of Mad2-Mad3 is achieved by incubation in galactose (left boxplot). In the right boxplot a comparison with the same strain in YPD plus Nz is shown. (G) The effect of galactose versus glucose in rDNA length in the Nz mid-M arrests was determined in the wild type strain. Despite in (F) the rDNA length appears shorter in the Mad2-Mad3^OE mid-M arrest, this is due to the effect of incubating in galactose, as shown in (G). Boxplot–whiskers represent 5–95 percentile. Mean shown as “+.” Dots represent outliers. (H) The nuclear arrangement in the cin8-aid kip1Δ mid-M arrest (mean ± SEM; n = 3). About 80% of the cells arrested in mid-M as mononucleated with or without a bow-tie phenotype. Only these mid-M cells were considered for assessment of the rDNA morphology shown in Fig 6H.

We observed a general pattern that was shared by both non-Nz mid-M strategies, and that greatly differed from Nz-arrested cells. Despite an organized bar-like rDNA (Net1-GFP) was seen in all conditions, the horseshoe loop was largely absent and, instead, either a straight bar or a hook that crossed the bud neck orientated in the chromosome division axis (polar axis) was the major outcome (Figs 6B–F and S6C and D). This shift in the rDNA morphology was not a consequence of the newly introduced alleles, as Nz still leads to horseshoe loops in these strains (Fig S6C and E). Importantly, the crossing bar and the hook appear to bend to interact with and wrap around vacuoles in the second body (either the mother or the bud) (Fig 6C, D, and G). The rDNA length was slightly larger in the cdc16 crossing bars than in the Nz horseshoe loops, pointing out that the rDNA is even more stretched in such configuration (Fig 6E). In the P_GAL1-MAD2-MAD3 strain, the bar was shorter (Fig S6F); however, this was due to the incubation with galactose, as Nz-induced horseshoe loops were also shorter in galactose (Fig S6G).

In addition to these two strains, we included a third approach to arrest cells in mid-M, while keeping polymerized microtubules. This was based in a cin8-aid kip1Δ strain; Cin8 and Kip1 are partly redundant kinesins required for the assembly of the mitotic spindle (Singh et al, 2018). Unlike the previous strains, which are able to form a bipolar spindle, depletion of both kinesins renders cells with a monopolar spindle (Fig 6A). Thermosensitive cin8 kip1 double mutants have been shown to arrest in mid-M at the restrictive condition (Hoyt et al, 1992; Roof et al, 1992). Cin8-aid can be efficiently depleted with IAA (Ayra-Plasencia & Machín, 2019), and we accordingly found that this leads to a similar mid-M arrest (Fig S6H). In terms of the relative position of the nucleus, this arrest was intermediate between what was observed for cdc16 and Mad2-Mad3^OE and what was seen with Nz, that is, ~45% of cells had a nucleus tightly stretched across the neck (bow-tie phenotype), with the rDNA oriented in the polar axis, and ~35% had the nucleus entirely in one cell body (Figs 6H and S6H). Remarkably, horseshoe loops were scarcely present, even in cells where the nucleus locates in a single cell body as in Nz (Fig 6B, last bar; and Fig 6H, upper cell).

We conclude that the absence of microtubules is a prerequisite to acquire the horseshoe rDNA loop. However, the mid-M arrest is sufficient to change the rDNA morphology into an organized bar.

The rDNA protrusion and the nuclear extension depend on active TORC1 and membrane phospholipid synthesis

We have previously shown that conditions that inactivate TORC1 dismantle the rDNA loop (Matos-Perdomo & Machín, 2018). Intriguingly, many reports have demonstrated that TORC1 inactivation activates autophagy, including nucleophagy, thereby promoting the nuclear–vacuolar interaction (Kvam & Goldfarb, 2007; Dawaliby & Mayer, 2010). According to the results shown above, it appears counterintuitive that the horseshoe rDNA loop is absent when the influence of the vacuole on the nucleus should be maximum. To get further insights, we studied the rDNA and nuclear mass morphologies in cells transiting through stationary phase, when TORC1 activity is expected to be low, autophagy high, and most cells appear swollen and with a large vacuole compressing the rest of the cell organelles. In this condition, the rDNA (Net1-GFP) was hyper-compacted, and its structure was barely modified by the vacuole (Fig S7A). Moreover, we could not observe any histone (Hta2-mCherry) handles. The addition of Nz did not change this pattern, demonstrating that Nz only elicits its effects on the rDNA in growing cells, when Nz leads to the mid-M arrest.

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Figure S7.

The ribosomal DNA (rDNA) loop depends on TORC1 and membrane synthesis but is independent of tethering to the nuclear envelope.

Related to Fig 7. (A) The rDNA loop is not present in stationary phase (7 d of continuous growth in a flask) upon Nz. Representative micrographs are shown. Note how the bulk of the chromatin remains rounded, sometimes squeezed between a large vacuole and the cell surface, and the rDNA appears compacted into oval shapes, small bars or small loops. (B) TORC1 is required for rDNA loop formation in the Nz mid-M arrest. Quantification of rDNA morphologies in the presence of Nz alone or co-treatment of Nz plus rapamycin. For the latter, only dumbbell cells (mid-M) were considered in the quantifications. Cell examples are shown in the Fig 7A. (C) Nz does not prompt NE extensions in stationary phase (7 d of continuous growth in a flask). Representative micrographs are shown. Sec61-CFP is shown in grey for better visualization. (D) An independent repetition of part of Fig 7C, which also includes a concurrent Nz rapamycin treatment. The protein electrophoresis was with Phos-tag. (E) Microscopy of the samples taken for the Western blots of Fig 7C (Nem1-HA). Note how the horseshoe loop in Nz gets contracted after TORC1 inactivation (rapamycin addition), as we have shown before (Matos-Perdomo & Machín, 2018). (F) WB to check Nem1-aid degradation upon IAA addition. Samples were taken before (0) and 1 and 2 h after adding 5 mM IAA. The WB is against the myc epitope; OsTIR1 also carries the epitope and acts as an internal reference. (G) Representative micrographs of the nuclear and rDNA morphologies in cells depleted of Nem1, which over-synthetize NE. It can be appreciated that nuclei form extensions that contain the rDNA. Unlike Nz mid-M arrests, the rDNA appears as closed hairpin-like loops, which takes the shape of a protruding bar in lateral views (nucleus on the right). (H) WB to check Fas1-aid degradation upon IAA addition. Samples were taken before (0) and 60’ after adding 5 mM IAA. The upper WB is against the aid epitope; the lower WB is against Pgk1 (reference). The % of protein that remains after IAA is shown as well. (I) Control check that C-tagging of Fas1 with aid does not interfere with loop formation upon Nz. This strain carries the nucleoplasm marker EGFP-NLS, which also contains the auxin degron IAA17 sequence, related to aid. This marker disappears after IAA treatment. Note the bilobed EGFP-NLS signal in the lower cell. (J) Inhibition of fatty acid synthetase with cerulenin also prevents nuclear extension upon Nz treatment. The NE morphology was quantified as in 7I. (K) Example of the NE bubble bridge category, seen as a minor outcome in Fas1-aid upon IAA. This category is closely related to the bow-tie, but with more spherical dumbbell-like appearance. (L) Representative micrographs of the Nz mid-M arrest in a strain without the rDNA tethered to the NE (heh1Δ nur1Δ double mutant). Note how horseshoe loops with SULs and protruding bars in nuclear extensions are still present. (M) The mid-M arrest with Nz still forms horseshoe rDNA loops in mutants for nucleus-vacuole junctions (NVJ). Representative mid-M cells in the ΔNVJ quadruple mutant. The rDNA (Net1) and vacuoles (lumen stained with BC) are shown. BF, bright field; V, vacuole; BC, blue CMAC dye.

Next, we studied the effect of rapamycin addition, a well-known inhibitor of the TORC1 (Heitman et al, 1991; Barbet et al, 1996; Urban et al, 2007). For this, we compared an Nz arrest to both a concomitant Nz plus rapamycin treatment and adding rapamycin to cells previously arrested in Nz. On the two latter, we found that the rDNA morphology was mainly oval without histone handles (Figs 7A and S7B). Alternatively, some mini-loops/handles of Net1/Hta2 (<2 μm) were also visible (Fig 7A, +Nz → rap condition), as we have shown before (Matos-Perdomo & Machín, 2018). We showed above that horseshoe loops and bars are associated with extended nuclei, as seen by rDNA, histone, and NE markers. Thus, we studied nuclear morphology under Nz treatment and TORC1 inhibition by following the NE shape with Sec61-eCFP. In growing cells, most nuclei appear either spherical or oval shaped (Fig S5A and D), except in those cells transiting M phase where the nucleus is stretched along the mother–daughter axis. In such cases, two morphologies are distinguished. The first one is bow-tie shaped, which is shared by cells that are in the late metaphase and early anaphase (Dotiwala et al, 2007). The second one is dumbbell shaped and corresponds to cells in late anaphase. Upon Nz treatment, the NE appears extended, but rarely in the mother–daughter axis (Figs 7B and S5D and E). However, under Nz plus rapamycin the nuclear morphology was mainly spherical/oval again (Fig 7B). Similarly, cells in stationary phase presented a spherical/oval morphology and, once again, the addition of Nz did not change this pattern (Fig S7C).

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Figure 7.

The ribosomal DNA (rDNA) loop requires TORC1, membrane synthesis and functional vacuoles.

(A, B) Rapamycin prevents formation and maintenance of the rDNA loop in Nz-treated cells. (A) Representative cells upon Nz alone, Nz plus rapamycin co-treatment, and 1 h rapamycin after a previous Nz mid-M arrest. The arrowhead points to the Hta2 handle in the Net1 horseshoe loop in Nz alone. (B) Morphology of the NE (Sec61) in Nz and Nz plus rapamycin (mean ± SEM; n = 3; only mid-M cells counted). (C, D, E, F, G) Over-synthesis of NE leads to nuclear and rDNA extensions morphologically distinct to those observed in Nz mid-M arrest. (C) Western blot of Nem1-6HA under Nz alone and Nz followed by 1 h rapamycin. An equivalent rapamycin regime over an asynchronous culture was performed as well for reference. The position of the three expected bands is indicated in the electrophoresis run with Phos-tag (top image). The others were standard SDS–PAGE controls. (D) A representative G2/M cell in asynchronous cultures growing with a depleted Nem1. Note it comprises a seeming protruding bar, which is actually a closed cXIIr loop as deduced from the cXIIr telomere position, into a finger-like flare nuclear projection. Five subcellular structures are labelled: the bulk of chromatin (Hta2), the rDNA (Net1), the nucleoplasm (free TetR-YFP), the cXIIr telomere (tetO:1061), and the vacuolar lumen (c-DCFDA), In the green channel, the three latter structures are labelled and differentiated by intensity and morphology; nucleoplasm is the weak signal on the top that colocalizes with Hta2, the cXIIr telomere is the spot, and the vacuolar lumen is the stronger signal underneath. (E) Quantification of rDNA morphology (loop versus bars) and vacuole juxtaposition in asynchronous cells with depleted Nem1 (mean ± SEM; n = 3). (F) Quantification of rDNA morphologies in a representative experiment where Nz was added to cells depleted of Nem1. Note that the proportion of loops doubled after Nz, yet it does not reach that of Nz alone (from 40% to 70% in other Nz experiments in this work). (G) Length of the rDNA loops and bars in cells depleted of Nem1 before and after Nz addition. Note the additive Nz effect. (H, I) Depletion of the fatty acid synthetase beta subunit Fas1 prevents nuclear extension upon Nz treatment. (H) Representative micrograph with two mid-M cells (whose polar axes are indicated by double dotted arrows in the BF) in which the NE is round and the rDNA are small bent bars (white arrows). (I) Morphology of the NE (Sec61) in Nz (control), IAA → Nz (establishment) and Nz → IAA (maintenance) (mean ± SEM; n = 2; only mid-M cells counted). (J) Quantification of the rDNA morphology in the Nz mid-M arrest in a mutant that does not tether the rDNA to the NE (mean ± SEM; n = 2). (K) Quantification of rDNA morphologies upon Nz in mutants for nucleus-vacuole junctions (mean ± SEM; n = 2). Nucleus-vacuole junction mutants: ΔΔ, mdm1Δ nvj3Δ double mutant; ΔΔΔΔ, mdm1Δ, nvj1Δ nvj2Δ nvj3Δ quadruple mutant. (L) Quantification of the rDNA morphology in the Nz mid-M arrest in mutants that affect vacuole size, inheritance, and function (mean ± SEM; n = 2). In micrographs: BF, bright field; V, vacuole.

In a mid-M block, phospholipid synthesis is unabated, and the nuclear membrane expands around the region that contains the nucleolus (Campbell et al, 2006; Witkin et al, 2012). Several lines of evidence pinpoint the Nem1-Spo7/Pah1 complex as a central player for the control of nuclear membrane expansion. This complex is involved in the balance between membrane phospholipids during growth conditions and lipid droplets during starving conditions; an active Nem1-Spo7/Pah1 complex shifts the balance towards the latter (Siniossoglou et al, 1998; Pascual & Carman, 2013). TORC1 regulates the activity of Nem1-Spo7/Pah1 by keeping Nem1 unphosphorylated and inactive, so that the phospholipid synthesis is favored (Dubots et al, 2014). Accordingly, Nem1-Spo7/Pah1 mutants display nuclear flares and extensions in growing cells (Santos-Rosa et al, 2005; Campbell et al, 2006). For this reason, we studied the phosphorylation status of Nem1 under Nz treatment and TORC1 inhibition. We arrested cells in either Nz alone or Nz followed by rapamycin addition for 1 h, and further compared these conditions with rapamycin treatment in asynchronous cultures (Fig 7C). We observed the same pattern of major phosphorylation shifts that have been described before in asynchronous exponentially growing cells: two bands, P0 and P1, and a third band, P2, after rapamycin addition (Dubots et al, 2014). Strikingly, only the P0 band was seen in Nz. This un(hypo)phosphorylated state suggests a strong Nem1 inhibition in Nz, which was modified only modestly by rapamycin, either after or concomitant to Nz addition (Figs 7C and S7D and E).

Next, we studied the nuclear and rDNA morphologies in cells depleted of Nem1. Instead of using a knockout mutant, we chose a nem1-aid allele (Fig S7F), so that we could control when to elicit NE elongation, and thus avoid carryover effects on the nuclear shape during many generations. We found that an overnight culture (~6 generations) with IAA was sufficient for G2/M nuclei in growing cells to exhibit finger-like nucleolar extensions (flares), in agreement with previous reports (Siniossoglou et al, 1998; Witkin et al, 2012). However, this morphology was more fixed than the plethora of morphologies described above for Nz mid-M arrests. In particular, horseshoe loops were seen in less than 20% of G2/M cells and, instead, protruding bars were the main morphological pattern (Figs 7D–F and S7G). Bars and loops were also shorter than in Nz (Fig 7G; mean length of ~2.5 μm). However, protruding bars turned out to be thicker, and there were examples in which they could be distinguished as hairpin loops (Fig S7G). The position of the cXIIr telomere in the nuclear mass further confirmed that these nem1 protruding bar are in fact rDNA/cXIIr loops. This indicates that nem1 forms rDNA loops that are retrained to blossom into horseshoes. This was partly confirmed as Nz addition doubled the presence of horseshoe loops as well as the overall length of loops and bars (Fig 7F and G). Whether open horseshoe or closed hairpin loops, vacuoles were principally juxtaposed to nem1 flares as well (Fig 7E).

The fact that the rDNA was always found in the nuclear extensions seen in both Nz and nem1, raised the question of whether NE tethering of the rDNA was a prerequisite for this phenotype. Thus, we checked both rDNA morphologies and colocalization with nucleoplasm extensions in the nur1Δ heh1Δ double mutant, in which rDNA-NE tethering is compromised (Mekhail et al, 2008). We still observed loops and bars (Figs 7J and S7L). The overall proportion of the sum of loops and bars was equivalent to that of the wild type strain, although horseshoe loops were less frequent. They both were found within nuclear extension, including finger-like flares, in 62% of all bars and loops. We did not observe extensions without the rDNA; hence, the rDNA-NE tethering is a prerequisite for neither the presence of nuclear extensions nor the rDNA being in these extensions.

In addition to addressing the effects of an excess in phospholipids for membrane synthesis, we decided to investigate the consequences of a defect in these lipids on the phenotypes described here. It has been shown that mRNAs encoding lipogenic enzymes (Acc1, Fas1 and Fas2), all involved in fatty acid synthesis, increased in G2/M (Blank et al, 2017b). Previous studies have also shown that biosynthesis of fatty acids is necessary for the extension of the nuclear membrane (Witkin et al, 2012; Walters et al, 2014, 2019; Male et al, 2020). Thus, we drew our attention to fatty acid synthesis and its relation to nuclear membrane growth in Nz. We made a Fas1-aid chimera and tested both NE (Sec61-eCFP) and rDNA (Net1-mCherry) morphologies when Fas1 was depleted before Nz addition. Degradation of Fas1-aid in IAA was partial (~66% drop in protein levels; Fig S7H and I); however, this was sufficient to prevent NE extensions upon Nz addition (Fig 7H and I). In these round nuclei, neither horseshoe loops nor protruding bars were observed, with the rDNA mostly seen as extremely short bars and loops (Fig 7H, white arrows). When Fas1 depletion was triggered after the Nz mid-M arrest, most mid-M cells maintained an extended nucleus (Fig 7I, subtracting bow-ties and anaphases), implying that lipid biosynthesis is required to attain the NE expansion in mid-M arrests but not for its maintenance. Finally, we corroborated these findings by using cerulenin, a specific inhibitor of fatty acid biosynthesis (Inokoshi et al, 1994). We treated cells with cerulenin, 1 h before the addition of Nz, which also resulted in spherical nuclei (Sec61-eCFP) and compacted Net1-mCherry signals (Fig S7J). Incidentally, we also observed for both Fas1 depletion and cerulenin more spherical NE morphologies within the bow-tie subgroup (Fig 7I and S7J and K, “bubble bridge”), suggesting a stiffer NE when fatty acid biosynthesis is inhibited.

The rDNA loop does not depend on known nuclear–vacuolar interactions

From our previous results, the shape of the malleable NE appears to be highly influenced by the stiffer vacuole. When the NE becomes enlarged in mid-M blocks, the vacuole serves as a template on which the extended NE bends around. In this context, the length of the rDNA in protruding bars and horseshoe loops may depend on how intimate the nuclear-vacuole relationship is. For this reason, we decided to study the role of the nucleus-vacuole junctions (NVJs) in the morphology of the rDNA. The NVJs are formed through the formation of Velcro-like interactions between the vacuolar protein Vac8 and the outer nuclear membrane protein Nvj1, which mediate piecemeal microautophagy of the nucleus (Pan et al, 2000; Roberts et al, 2003). Similarly, NE-vacuole contacts are established as sites for lipid droplet biogenesis, which include the proteins Nvj1, Mdm1, Nvj3, Nvj2, and Vac8 (Henne et al, 2015; Hariri et al, 2018). We tagged the Net1-GFP in wild type, double (mdm1Δ nvj3Δ) and quadruple (mdm1Δ nvj1Δ nvj2Δ nvj3Δ) “Δnvj” mutants, of which, the latter is known to increase the NE–vacuole inter-organelle distance (Hariri et al, 2018). Cells were arrested in Nz for 3 h and the rDNA structure visualized as before. Surprisingly, we still found that most mid-M cells presented an rDNA loop, even in the quadruple Δnvj mutant (Fig 7K). Moreover, when we stained the cells with Blue CMAC in the Δnvj mutant, the loops still wrap the vacuole (Fig S7M). We conclude that NE-vacuole contacts are not a prerequisite for the formation of the horseshoe rDNA loop.

The rDNA loop depends on functional vacuoles

We then checked whether vacuole mutants that impinge on their size, shape, or functionality undermine the ability to form the horseshoe loop. Vacuoles grow and shrink through fusion and fission of vesicles, respectively (Chan & Marshall, 2014). The quick dynamics of such events can be appreciated in the Video 10. Atg18 favors vacuole fission, whereas Apl5, Vps45, and Pep12 are factors involved in delivering vesicles from Golgi to fuse with vacuoles; single knockout mutants for these genes alter vacuolar size and functionality (Becherer et al, 1996; Burd et al, 1997; Cowles et al, 1997; Bryant et al, 1998; Efe et al, 2007). Despite these alterations, we still observed horseshoe loops and all the other bar-like morphologies in proportions equivalent to the wild-type strain (Figs 7L and S8A).

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Figure S8.

The ribosomal DNA structure in mutants that undermine vacuole homeostasis.

Related to Fig 7M. (A) Representative micrographs of mutants that impinge on the fission and fusion dynamics of vacuoles. Note that horseshoe loops are still present, in size and appearance similar to the wild type strain. (B) Representative micrographs of mutants that impinge on the vacuolar maturation and inheritance. Note that horseshoe loops are still present in vac17Δ, whereas in the vac17Δ pep12Δ double mutant, horseshoe loops are less abundant (quantifications in Fig 7M) and more disorganized; that is, the resemblance with a highly bent bar is less evident (either thicker loops, arrow, or Net1 gaps amid the loop, arrowhead). (C) Representative micrograph of the vma1Δ mutant that impinge on the vacuolar acidification and function. Note that loops are absent.

We next checked the vac17Δ mutant, which dampens vacuole maturation and inheritance, and the vac17Δ pep12Δ double mutant, which enhances these phenotypes and even make 40% of cells lack normal vacuoles (Jin & Weisman, 2015). We found a clear drop in the proportion of cells with horseshoe loops in the double mutant (Fig 7L; loops in only ~10% of mid-M cells). In addition, the rDNA array in those loops were less organized than in the wild type (Fig S8B).

Last, we checked the effect of suppressing the vacuolar H+-ATPase (V-ATPase), responsible for vacuolar acidification and thus correct vacuolar function (Stevens & Forgac, 1997). We used the vma1Δ knockout mutant; Vma1 is one V-ATPase subunit. This mutant grew more slowly than wild type cells and only half of the cell population reached a mid-M arrest after 3 h in Nz. However, in these mid-M cells, the horseshoe loop was barely present, and oval and small bars were the most frequent outcome instead (Figs 7L and S8C).

Yeast cell growth and experimental conditions

For experiments, strains were routinely grown overnight in rich YP medium (yeast extract 1% wt/vol plus peptone 2% wt/vol) supplemented with 2% glucose (YPD) at 25°C with moderate orbital shaking (150 rpm; 25 mm orbit). For the standard mid-M arrest, cells were incubated with nocodazole for 180 min. First, nocodazole was added directly to a log phase culture (OD₆₂₀ ~0.8–1.8) at a final concentration of 15 μg/ml, then after 120 min, half the initial concentration was added to the culture media. For aid-based depletion (cdc16-aid, cin8-aid, nem1-aid, fas1-aid, and net1-aid), cells were incubated with indole-3 acetic acid (IAA) at a final concentration of 5 mM (from a 500 mM stock in DMSO). For overexpressing Mad2-Mad3, cells bearing the P_GAL1-MAD2-MAD3 construction were grown in 2% raffinose and then overexpression from the galactose-inducible GAL1 promoter was accomplished by growing cells in 2% raffinose plus 2% galactose for 240 min. For rapamycin experiments, a final concentration of 200 nM was used (from a 2.2 mM stock in DMSO). DMSO was used at a final concentration of 1% vol/vol. Cerulenin was added at a final concentration of 2 μg/ml (from a 5 mg/ml stock in EtOH).

Wide field fluorescence microscopy, staining, and image processing

Two epifluorescence inverted microscopes were used. A Leica DMI6000B with an ultrasensitive DFC350 digital camera was used for single cell visualization with a 63X/1.30 immersion objective as we have reported before (Quevedo et al, 2012; Matos-Perdomo & Machín, 2018). A Zeiss Axio Observer.Z1/7 was also used; this microscope was equipped with an Axiocam 702 sCMOS camera, the Colibri-7 LED excitation system, narrow band filter cubes for co-visualization of CFP, YFP/GFP, and mCherry without emission crosstalk, and a 63X/1.40 immersion objective.

Whenever possible cells were imaged alive. Briefly, 250 μl of cell culture was collected at each time point, centrifuged at 300g for 1 min at room temperature, the supernatant carefully retired, and ~1.5 μl of the pellet was added on the microscope slide. Samples were visualized directly using the appropriate filter cube for each tag/stain. For each field, we first captured either single planes or a series of 10–20 z-focal plane images (0.2–0.6 μm depth between each consecutive image), and then we processed images with the Leica AF6000, Zeiss Zen Blue and ImageJ software. For z-stack 2D projections, we used the maximum intensity method. Deconvolution was performed on z-stacks using Leica AF6000 software (method: blind deconvolution algorithm, 10 total iterations, fast processing). Orthogonal projections were generated using ImageJ. Fluorescence intensity profiles were generated using Leica AF6000 and Zeiss Zen 3.1 lite (blue edition) software. For short time-lapse movies of living cells, Nz-blocked cells were pelleted and spread at a high density onto the slide. Specific conditions are described in the video legends.

Stains: DAPI, YOYO-1, SYTO RNASelect, MDY-64, Blue CMAC, carboxy-DCFDA. Nz-blocked cells were stained as follows:

For DAPI staining, the cell pellet was frozen for at least 24 h at −20°C before thawing at room temperature, and then ~1.5 μl of the pellet was added to ~1 μl of 4 μg/ml of DAPI on the microscope slide.

For YOYO-1, we followed a previously described procedure (Ivanova et al, 2020). Briefly, cells were fixed in 4% formaldehyde for 30 min, washed once in PBS, and re-suspended in 5 mg/ml zymolyase in P solution (1.2 M sorbitol, 0.1 M potassium phosphate buffer, pH: 6.2) for 1 min. Cells were spun down, taken up in P-Solution +0.2% Tween 20 + 100 μg/ml RNAse A and incubated for 1 h at 37°C. After digestion, cells were pelleted and taken up in P-Solution containing 25 μM YOYO-1 and visualized as before.

For SYTO RNASelect, cells were pelleted and stained according to manufacturer’s procedures. Briefly, a solution of RNASelect green fluorescent stain (final concentration 500 nM) in YPD medium was added to the cells and incubated for 30 min. After this, cells were washed twice with fresh YPD, let rest for 5 min and visualized as before.

For carboxy-DCFDA and Blue CMAC, 1 ml of cells were incubated for ~15–30 min in either carboxy-DCFDA (final concentration of 10 μM from a 10 mM DMSO stock solution) or Blue CMAC (final concentration of 100 μM from a 10 mM DMSO stock solution). Then, the cells were pelleted, washed in YPD (PBS if fixed), and visualized accordingly. For MDY-64, cells were incubated for ~3–5 min in of 10 μM from a 10 mM DMSO stock solution.

CSM

Nz-blocked cells were pelleted and imaged in two Zeiss Axio Observer.Z1/7 inverted microscopes equipped for super-resolution confocal microscopy with live cell capabilities (LSM880 with Airyscan and LSM980 with Airyscan 2). The resolution provided in Airyscan mode is lateral (x/y) resolution to 120 nm for 2D and 3D data sets (z-stacks) and 350-nm axial (z) resolution for z-stacks, with an improved resolution up to 1.7X compared with standard confocal (Huff, 2015). The super-resolution images were taken with either a C-Apochromat 63x/NA 1.20 W M27 DICII objective for the LSM880 or a Plan-Apochromat 63x/NA 1.40 Oil M27 DIC objective for the LSM980. The Airyscan detector was used for all single and multiple labellings, with the pinhole automatically set to correct opening according to the selected Airyscan mode. The superresolution mode of the Airyscan detector was used throughout. We used the following laser lines for excitation of fluorescent tags: 405 nm for CFP; 514 nm for YFP; 488 nm for GFP; and 561 nm for mCherry. The bright field (BF) image was acquired with T-PMT detectors (pinhole 1 AU). After imaging, Airyscan processing was conducted. Z-stack 2D projections were generated by either applying the processing “extended depth of focus” using Zeiss Zen Blue 3.2 software or the sum intensity method in ImageJ.

For 3D reconstructions, ~45–50 z planes (0.15 μm thick) were obtained across the entire cell. Both alive and fixed cells were photographed, although fixed cells were generally used in the presented experiments. For cell fixation, cells were incubated in 3.7% wt/vol formaldehyde on a nutating mixer at room temperature for 15–30 min, spun down at 6,800g for 30 s, the pellet washed in 500 μl of filtered PBS 1×, resuspended in another 500 μl of PBS 1×, and stored at 4°C in the dark. Before micrographs were taken, suspensions were sonicated for 8 s in a bath sonicator to separate clumps of cells.

Unless stated otherwise in video captions, long (3 h) time-lapse images were acquired on cells immobilized in Nunc Lab-Tek coverglass eight-wells chambers pretreated with concanavalin A (ConA). ConA pretreatment was undertaken the day of filming by adding 50 μl of ConA (1 mg/ml in PBS) to a well, incubate in the dark at 25°C for 20 min, and washed the well twice with synthetic complete (SC) media. For cell adhesion, a log cell culture was concentrated 2× in SC and 100 μl of the suspension applied to the well and kept at 25°C for 20 min. Non-attached cells were then washed twice with 100 μl of SC media and finally the well was covered with 250 μl of YPD plus Nz at a final concentration of 22.5 μg/ml to start live cell imaging. The higher concentration of Nz used in these experiments is actually equal to the initial plus the reinforcement doses applied in liquid cultures growing in an air incubator. We did this to avoid detachment of the cells by pipetting in the middle of the experiment. Aside from photobleaching and phototoxicity, the horseshoe loop was photosensitive in subtoxic conditions (as determined by comparing end points of exposed versus non-exposed fields). Because of that, laser powers were kept to a minimum and number of z planes reduced. With 405-nm UV irradiation (for CFP), the number of total frames (z planes plus t points) was set to a maximum of 35; without 405 nm, the maximum frame number was 150.

TIRF microscopy

This was adapted from a protocol described before (Barroso-González et al, 2009). Nz-blocked cells were pelleted and imaged with an inverted microscope Zeiss 200 M through a 1.45-numerical aperture objective (α Fluar, 100×/1.45; Zeiss). The objective was coupled to the coverslip using an immersion fluid (n(488) 1.518, Zeiss). The expanded beam of an argon ion laser (Lasos; Lasertechnik GmbH) was band-pass filtered (488/10 nm) and used to selectively excite EGFP-tagged proteins, for evanescent field illumination. The laser beam was focused at an off-axis position in the back focal plane of the objective. Light, after entering the coverslip, underwent total internal reflection as it struck the interface between the glass and the cell at a glancing angle. The images were projected onto a back-illuminated CCD camera (AxioCam MRm; Zeiss) through a dichroic (500 LP) and specific band-pass filter (525/50 nm). Each cell was imaged using Axiovision (version 4.9; Zeiss) with 0.5 s exposition. Image analysis: The raw images were low-pass filtered (3 × 3 pixels) and analyzed with ImageJ.

TEM

The protocol was adapted and modified from Byers and Goetsch (1991). Cells were arrested in nocodazole for 180 min, then pelleted, re-suspended and fixed in phosphate-magnesium buffered (40 mM K₂HPO₄, and 0.5 mM MgCl₂, pH 6.5) 2% glutaraldehyde (EM Grade) + 2% formaldehyde and incubated overnight and stored at 4°C. Then, cells were rinsed twice in 0.1 M phosphate-citrate buffer (170 mM KH₂PO₄ and 30 mM sodium citrate, pH 5.8) and re-suspended in this buffer containing a 1/10 dilution of Lyticase (10 mg/ml 2,000 U stock) + Zymolyase (5 U/μl stock) and incubated at 30°C for 2 h, or until cell walls have been removed. For post fixation, cells were washed twice in 0.1 M sodium acetate (pH 6.1), transferred to a 2% osmium tetroxide fixation solution, and incubate for 4 h in a fume hood. Then, cells were washed with double-distilled water (ddH₂O) and transfer to 1% aqueous uranyl acetate for 60 min of incubation in the dark. After this, cells were washed twice in ddH₂O and dehydrate by transferring them through a series of ethanol concentrations (20, 40, 60, 70 [overnight], 96, and 100). Then, cells were pelleted and resuspended in EMBed 812 resin. Finally, semi-thin and ultra-thin sections were cut on an ultramicrotome (Reichert Ultracut S-Leica) and stained with toluidine blue for semi-thin sections and with uranyl acetate and lead salts (Sato’s Staining Procedure, 5 min) for ultra-thin sections. EM images were captured by a TEM 100 kV JEOL JEM 1010 electron microscope.

Pulsed field gel electrophoresis and Southern blot

Yeast chromosomes extraction was prepared in low-melting point agarose plugs as reported before (Ayra-Plasencia et al, 2021). For each sample, six OD₆₀₀ equivalents were centrifuged and washed twice in ice-cold sterile 1× PBS. Then, cells were re-suspended in Lyticase solution (2,500 U/ml), and embedded into 0.5% (wt/vol) agarose plugs. Finally, full-sized chromosomes were obtained by digesting overnight in RNase A (10 μg/ml) and Proteinase K (1 mg/ml) containing solutions at 37°C. Pulsed field gel electrophoresis, used to assess the chromosome XII size, was performed by using the CHEF DR-III system (Bio-Rad). One third of each plug was placed within the corresponding well of a 1% (wt/vol) agarose gel made in 1× TBE buffer. Then, the wells were filled-up and sealed with additional 1% (wt/vol) agarose. 0.5× TBE was used as the running buffer at 14°C. The electrophoresis was carried out at 3 V/cm for 68 h, including 300 and 900 s of initial and switching time (respectively), and an angle of 120°. The gel was stained with ethidium bromide for 40 min and destained with ddH₂O for 20 min. The chromosomes bands were visualized under UV light using the Gel Doc system (Bio-Rad). To specifically study the chromosome XII, a Southern blot was carried out by a saline downwards transference onto a positively charged nylon membrane (Hybond-N+, Amersham-GE). A DNA probe against the NST1 region within the rDNA was synthesized using the Fluorescein-High Prime kit (Sigma-Aldrich). The fluorescein-labelled probe hybridization was carried out overnight at 68°C. The next day, the membrane was incubated with an anti-fluorescein antibody coupled to alkaline phosphatase (Roche), and the signal was developed using CDP-star (Amersham) as the substrate. The detection was recorded by using the Vilber-Lourmat Fusion Solo S equipment.

Western blotting

Western blotting was carried out as reported before with minor modifications (Matos-Perdomo & Machín, 2018; Ayra-Plasencia & Machín, 2019). Briefly, 5 ml of the yeast liquid culture was collected to extract total protein using the trichloroacetic acid (TCA) method; cell pellets were fixed in 2 ml of 20% TCA. After centrifugation (2,500g for 3 min), cells were resuspended in fresh 100 μl 20% TCA and ~200 mg of glass beads were added. After 3 min of breakage in a homogenizer (P000062-PEVO0-A; Precellys Evolution-Bertin Instruments), extra 200 μl 5% TCA were added to the tubes and ~300 μl of the mix were collected in new 1.5 ml tubes. Samples were then centrifuged (2,500g for 5 min) and pellets were resuspended in 100 μl of PAGE Laemmli Sample Buffer (1610747; Bio-Rad) mixed with 50 μl TE 1X pH 8.0. Finally, tubes were boiled for 3 min at 95°C and pelleted again. Total proteins were quantified with a Qubit 4 Fluorometer (Q33227; Thermo Fisher Scientific). Proteins were resolved in 7.5% SDS–PAGE gels and transferred to PVFD membranes (PVM020C-099; Pall Corporation). For protein phosphorylation states, we used the method for Phos-tag acrylamide gel electrophoresis (Kinoshita et al, 2006). The following antibodies were used for immunoblotting: The HA epitope was recognized with a primary mouse monoclonal anti-HA (1:1,000; Sigma-Aldrich); the Myc epitope was recognized with a primary mouse monoclonal anti-Myc (1:5,000; Sigma-Aldrich); the Pgk1 protein was recognized with a primary mouse monoclonal anti-Pgk1 (1:5,000; Thermo Fisher Scientific) and the aid tag was recognized with a primary mouse monoclonal anti-miniaid (1:500; MBL). A polyclonal goat anti-mouse conjugated to horseradish peroxidase (1:5,000, 1:10,000 or 1:20,000; Promega) was used as secondary antibody. Antibodies were diluted in 5% milk TBST (TBS pH 7.5 plus 0.1% Tween 20). Proteins were detected by using the ECL reagent (RPN2232; GE Healthcare) chemiluminescence method, and visualized in a Vilber-Lourmat Fusion Solo S chamber. The membrane was finally stained with Ponceau S-solution (PanReac AppliChem) for a loading reference.

We kindly thank the Luis Aragón, Frank Uhlmann, Mike Henne, Danesh Moazed, Joris Winderickx and Patricia Kane labs for yeast strains and plasmids. We thank María Moriel-Carretero for sharing unpublished results as well as critical reading of the manuscript. We also thank David Machado from the Pharmacology Unit at Universidad de La Laguna for support on TIRF microscopy and José Manuel Pérez Galván from Servicio Investigación Microscopía Avanzada Confocal y Electrónica–SIMACE–at Universidad de Las Palmas de Gran Canaria for support on confocal and electron microscopy. This work was supported by the Spanish Ministry of Science grant BFU2017-83954-R to F Machín. The Agencia Canaria de Investigación, Innovación y Sociedad de la Información supported S Santana-Sosa and S Medina-Suárez through the predoctoral fellowships TESIS2018010034 and TESIS2020010028, respectively