Nuclear polarity is maintained throughout interphase
To examine chromosome positioning in yeast, we first identified fixed points of reference within the nucleus from which cell-autonomous measurements could be made. The yeast nucleolus, which represents a single large domain of rDNA transcription and processing, occupies a distinct subnuclear territory, adjacent to the NE ( A, CFP-Nop1). Immunofluorescence (IF) on fixed cells maps the nucleolus to a zone opposite the SPB, an integral NE structure that serves as the microtubule-organizing center in yeast (Yang et al., 1989
; B). Time-lapse imaging has shown that the SPB movement is constrained to very small volumes over 5-min intervals, moving far less than a telomere or centromere (Heun et al., 2001b
). However, it was not clear when the SPB becomes positioned opposite the nucleolus, nor how long this arrangement persists. To visualize this organization in living yeast cells, we have fused a component of the SPB (Spc42) and Nop1, an abundant RNA-binding nucleolar protein, to GFP alone or to CFP in combination with a GFP-Nup49 fusion that labels nuclear pores (Belgareh and Doye, 1997
). Cells were subjected to live time-lapse imaging at 12-s intervals for GFP alone (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200409091/DC1
), or at 3-min intervals for CFP and GFP in combination with capture of the transmission channel using a scanning confocal microscope (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200409091/DC1
). Visual inspection confirmed that normal cell growth was not impaired during or after imaging.
Figure 1. The yeast SPB and nucleolus are aligned with the site of bud emergence throughout interphase. (A) Selected frames from a Zeiss LSM510 confocal time-lapse series of GA-2253 yeast cells as they progress through G2, mitosis and G1, show the NE (Nup49, green) (more ...)
Consistent with IF results, the crescent-shaped nucleolus ( A, CFP-Nop1, red) is found at one end of the nucleus, directly opposite the SPB ( A, 0- and 45-min frames, CFP-SPB in white). Remarkably, the SPB focus is reproducibly positioned on a vector that can be drawn from the nucleolus toward the emerging bud, indicating that nuclear and cellular polarities are linked (). As cells advance to late G2 phase and the nucleus elongates into the daughter cell, the duplicated SPBs separate, and one migrates back toward the nucleolus in the mother cell. These cells traverse mitosis rapidly ( A, 9 min; Video 2), at which point the two SPBs are found at opposite ends of the extended nucleus and the nucleolus spans the length of the spindle. In early telophase, the duplicated nucleolus splits in two, assuming symmetrical positions in mother and daughter nuclei and in G1 phase, the nucleus rotates slightly such that the nucleolus is again localized opposite the SPB ( A, 15–18 min). Thereafter, the nucleolus remains stably positioned opposite both the SPB and the future or actual site of bud emergence throughout G1 and S phase ( B). Statistical support for this observation, comes from scoring SPB position in cells arrested in late G1 phase: in >80% of the cases the SBP falls within 5° of a perpendicular line extending from the nucleolus to the bud neck ( C). We conclude that the nucleolus maintains a position opposite the SPB, which itself maintains a fixed position throughout interphase.
Further evidence that the nucleus does not rotate continuously in G1 phase, is based on GFP-Nup49 FRAP experiments ( D). We irreversibly photobleached the nuclear pore fluorescence within the NE and monitored fluorescence recovery at time intervals relevant to those used to monitor chromatin dynamics of interphase chromatin (i.e., 1.5–10-s intervals over several minutes). If the nucleus were turning rapidly, we would expect to see the bleached zone move from the plane of focus. This does not occur ( D). Instead we observe a slow diffusion of pore fluorescence inwards from the edges of the bleached zone, beginning at ~80 s ( D, arrows). We conclude that the global orientation of the interphase nucleus in yeast is quite stable, not only with respect to the SPB, but also with respect to cytoplasmic structures. Nuclear landmarks such as these can thus be used to monitor relative position of chromosomal tags, and rotation of the nucleus can be ruled out as a source of chromatin mobility.
Juxtaposition of right and left telomeres at the nuclear periphery
Previous studies have shown that yeast telomeres are enriched near the nuclear periphery in G1- and S-phase cells, both when detected individually or through repeat sequences (Gotta et al., 1996
; Hediger et al., 2002
). Nonetheless yeast telomeres are dynamic, shifting irregularly along the NE and occasionally into the nucleoplasm. To explore spatial relationship of pairs of telomeres in vivo we have differentially tagged the two ends of chromosomes 3, 5, 6, and 14, within the most distal unique sequences, such that subtelomeric repeats remain unaltered ( A). We measured distances separating the lacop
insertions, visualized by the binding of CFP- or YFP-fusions to the bacterial repressors, on three-dimensional (3D) confocal stacks of intact cells (). The distributions of 3D measurements (n
= 60–160 for each telomere pair) are plotted in (C and D), and the mean distances between tagged sites are summarized in . At a given moment, the left and right telomeres of Chr 3 and 6 coincide or are immediately adjacent to each other (separation in 3D = 0.2 ± 0.2 μm) in 35–40% of the cells measured. Telomere separation for these two chromosomes is clearly skewed to small distances: >75% of the intra-telomere 3D measurements are under 0.8 μm ( C). This is in contrast to the separation of two peripheral but unlinked telomeres (5L and 14R; or 6L and 14L), which follows a near Gaussian distribution around 1 μm ( D). Indeed, if two telomeres on the same chromosome were to have no bias toward interaction, the distribution of distances should be Gaussian over a range from 0.1 to 2 μm, depending on the compaction ratio of the chromatin and the length of chromosomal arms. Separation distances for right and left telomeres of Chr 5 and Chr 14 are also biased toward values <0.8 μm, but unlike Chr 3 and Chr 6, telomeres are immediately adjacent or superimposed in only ~12% of cells.
Figure 2. 3D position of telomeres relative to each other in intact cells. (A) CFP-lacI and YFP-tetR fusions allow visualization of the inserted lacop and tetop arrays. (B) Image stacks (x-y planes) of 0.2 μm along the z-axis from the Zeiss LSM510 are shown (more ...)
Average 3D telomere–telomere distances
Chromosomes fold back on themselves in interphase
We next analyzed the relationship of telomere pairs to the centromere by combining the double-tagged chromosomes with staining for the SPB (, A–C). Elsewhere we have established that all centromeres cluster within 200–300 nm of the SPB (Bystricky et al., 2004
). By measuring the 3D distance between two telomeric spots and the distance between each telomere and the SPB (, A–C; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200409091/DC1
), we determine the long-range organization of the chromosome and calculate an angle α that subtends telomere separation. Again, right and left telomeres of Chr's 3 and 6 are frequently juxtaposed (>30% at <0.2 μm, 60–70% at <0.6 μm), whereas the telomere (Tel) 5R and Tel5L separation exhibits greater variability ( D). Importantly, we note that right and left telomeres are almost always more closely juxtaposed to each other than either is to the SPB (Fig. S1). This argues for a fold-back structure that is dominant for Chr's 3 and 6, and statistically significant for Chr 5 (see below).
Figure 3. Chr 3 and Chr 6 form whole chromosome loops. Epi- and IF of G1-arrested haploid cells: (A) GA-2195 (SPB, red; 3L::GFP, green; 3R::GFP, green); (B) GA-2201 (SPB, white; 5L::YFP, green; 5R::CFP, red); (C) (more ...)
By triangulation we determined the angle α at between right and left chromosome arms, using the SPB signal as the apex. The distribution of these angles is summarized in E. Mean angle values for each chromosome are 31° ± 32° for Chr 3, 38° ± 26° for Chr 6, and 44° ± 29° for Chr 5. This large variability is inherent to the dynamic nature of telomeres and does not represent different subpopulations (see below). It is noteworthy, however, that among the three chromosomes studied, very few angles are >90° and none are >110°, and ~50% of Chr 3 and Chr 6 arms meet at angles <30°. If telomeres were on 1 μm long arms randomly distributed on the surface of a sphere around a fixed point (the SPB), the subtending angles would have Gaussian distribution around 60°. We can conclude, therefore, that the fold-back organization of Chr's 3 and 6 is statistically significant, reflecting right and left telomere interaction. Chr 5 appears also nonrandomly folded (>70% of the angles are <60°), although Tel5R-5L interactions are less frequent. Finally, the average distance separating Tel14L and 14R () is less than the one separating Tel 5L and 5R, arguing that Chr 14 also assumes a Rabl-like organization.
Right and left telomere interactions are favored by perinuclear constraints
Two parameters may influence telomere–telomere interaction: the length of chromosome arms and their association with the NE. Indeed, the arms of Chr 3 and 6 are both short and of nearly equal lengths (3R/3L = 115 kb/200 kb and 6R/6L = 122 kb/148 kb), which is not true for either Chr 5 or 14. However, short, equal arm length is not alone sufficient to favor interaction of chromosome ends: the chromosomal arms of Tel 5L and Tel 14R are also short and of equal length (152 and 150 kb, respectively), yet these ends are separated on average by ~1 μm (). Thus, chromosome arm length probably only favors telomere–telomere interaction when the arms are physically linked.
We next examined whether the efficiency with which each telomere is found at the GFP-Nup49-tagged NE, correlates with the efficiency of their interaction in trans. We scored telomere position relative to three equal zones of the nucleoplasm, focusing on the peripheral-most zone, which has a width of only 0.184 times the radius (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200409091/DC1
). For all except Tel5R, we monitor a significant enrichment in this zone, with the following hierarchy: Tel14R > 5L ≈ 6R > 14L ≈ 3R ≈ 6L > 3L (). Only Tel5R has a near-random distribution in G1-phase cells. Similarly, nontelomeric loci, such as MATa
, which sits in the middle of Chr 3, or origins of replication located 73 or 437 kb from the nearest telomere (autonomously replicating sequence [ARS] 607 or ARS1, respectively), are either randomly distributed or depleted from the periphery (). Although the well-paired telomeres (those of Chr 3 and 6) tend to be perinuclear, from these measurements one can draw no simple correlation between the efficiency of NE interaction and telomere interaction.
Telomere position and dynamics in G1-phase cells
Two color time-lapse imaging reveals constraints on telomere movement
Every measurement on a fixed cell is, of course, a snapshot of a dynamic chromosomal state, and even telomere–telomere interactions are not static. To monitor directly how stable telomere interactions are, we used live time-lapse imaging to follow the relative movement of differentially tagged telomeres. Up to 250 sequential two-channel (CFP-YFP) confocal images were acquired at 1.5-s intervals without detectable impact on cell-cycle progression. For each strain, we analyze 8–12 independent two-dimensional (2D) time-lapse series (totaling 35–58 min each) of G1-phase nuclei, after the tagged foci by adjusting the focal plane. Representative sequences and videos are shown in and Videos 3–6, available at http://www.jcb.org/cgi/content/full/jcb.200409091/DC1
Figure 4. Live imaging of telomere dynamics. A Zeiss LSM510 confocal time-lapse microscopy (2D) was performed on double-tagged Chr 3, 5, 6, and 14 taking frames every 1.5 s, by adjusting the plane of focus when necessary (see time-lapse series as Videos 3–6). (more ...)
Projection of the paths taken by the individual telomeres onto one plane shows that movements are not only restricted to a fraction of the total nuclear volume, but that the tracks of the Tel 3R-3L and Tel 6R-6L coincide extensively (see examples from typical videos; C). Tel 5R-5L move in close proximity but with little overlap. The juxtaposition does not arise from the methodology used, because movements of unlinked telomeres (Tel 6L-14L) are distinct and uncoordinated, consistent with measurements at fixed time points (). By summing all individual steps over the total time and dividing by the period elapsed, we calculate the average velocity of each individual telomere (). We find that all telomeres except Tel 5L and 5R are significantly less mobile than the tagged centromere-proximal ARS1 locus.
Assuming that chromatin motion resembles a constrained random walk (Marshall et al., 1997
), locus mobility can also be characterized by plotting its mean square displacement (MSD or <Δd2
>) over increasing time intervals. Unconstrained diffusion gives a linear relationship between increasing time intervals and the square of the distance travelled by a particle during that time, where Δd2
; Hediger et al., 2004
). The MSD curve for chromatin with a spatially constrained diffusion process generally reaches a plateau by Δt > 50s. This analysis is highly robust because Δt intervals are pooled from all videos of a given strain.
If we monitor movement as displacement relative to the nuclear center or the nearest point on the NE (d = distance between one fluorescent telomere spot and the center of the nuclear background fluorescence, cf. Heun et al., 2001b
), the resulting MSD curve reflects the dynamics of a given locus relative to the nuclear periphery (radial MSD or radMSD; D). RadMSD curves show that the dynamics of telomeres 5L, 6R and 6L are nearly equally restricted relative to the NE, whereas Tel 5R moves without constraint relative to the NE (). The two telomeres of Chr 3 exhibit NE-constrained movement very similar to Chr 6 (unpublished data). By comparing telomere movements and paths, we conclude that path superposition of right and left telomeres correlates positively with constraint relative to the NE, even though precise distance from the NE may vary. Thus, constrained movement relative to the periphery, whether directly at the NE or not, does correlate with contact between telomeres.
Absolute and relative constraints on telomere dynamics
A more accurate analysis of spatial constraint is based on measurements that reflect the actual distances covered from any one time point to all others (i.e., rather than distances relative to the periphery; A), after an alignment of nuclear centers to eliminate background drift. These d values were then subjected to the similar MSD analysis (here called absolute or absMSD) for both telomeres of Chr 3, 5, and 6. When absolute step sizes are the basis of the curve, the radius of confinement or spatial constraint (rc) determines the plateau of the MSD curve (Ma × MSD). For our geometry, this dependence is Ma × MSD = 4/5 (rc)2 (J. Dorn and Neumann, F., personal communication). Solving for r allows us to calculate the radius of confinement from experimental MSD curves. This analysis shows that Tel 5R and Tel 5L are relatively mobile and do not reach a plateau, yet from the radial analysis we know that Tel 5L tracks along the NE ( E and A). By contrast, movements of Tel 6R, 6L, 3R, and 3L, show clear spatial constraint and rc values ranging from 0.40 to 0.46 μm.
Figure 5. Looping of short chromosomes correlates with reduced telomere mobility. (A) Absolute MSD calculated using the 2D videos as described in for telomeres 5R, 5L, 6R, and 6L using d = actual distance from any one time point to all others (see diagram; (more ...)
The initial slope of the absMSD plot is proportional to the maximal diffusion constant (D = MSD/Δt). These slope values confirm that Tel 5R and 5L are more dynamic than other telomeres, with diffusion rates similar to those of the centromere proximal ARS1
locus (6.9 × 10−11
/s; ). Although Tel 5R and 5L are more mobile than other telomeres, we show here that they move in a paired manner, by scoring the relative separation of the telomere pairs throughout >2,000 frames (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200409091/DC1
). Distances separating telomeres derived from time-lapse series confirm the values determined in 2D and 3D at fixed time points ( and ): Tel 6R-6L and Tel 3R-3L are tightly juxtaposed, with 32–37% of all distances ≤0.2 μm, and >50% ≤0.4 μm. Strikingly, >60% of the separation values for Tel 5R-5L are ≤0.5 μm, whereas the separation of two unrelated telomeres (i.e., Tel 6L-14L) is <0.2 μm in only 5% of all frames. This confirms that Tel 5R-5L are adjacent although rarely interacting.
To quantify the freedom of movement that two telomeres have relative to each other, we plot the change of distances separating the telomeres as a function of t. In this “relative MSD” analysis, d is defined as the distance between two telomeres at any given time point ( B, relative MSD; Berg, 1993
; Marshall et al., 1997
; Vazquez et al., 2001
). These MSD plateaus confirm that all telomere pairs tested undergo obstructed diffusion, yet the values for linked telomere pairs are grouped around <Δd2
> = 0.1–0.14 μm2
. This suggests that two different telomeres move more freely relative to one another than do two identical centromere proximal sites monitored in a diploid cell (for LEU2
> = 0.06 μm2
; Marshall et al., 1997
). It is nonetheless noteworthy that even two unlinked telomeres (Tel6L-14L), which are separated by roughly 1 μm in the nucleus, show a relative radius of constraint of rc
= 0.25 μm. From this one can conclude that, independent of their pairing efficiency, telomeres assume fairly fixed positions in interphase nuclei.
Nuclear order is disrupted in the absence of yKu70 or Sir4
We have recently established that yeast telomeres are bound at the NE through dual pathways. One requires Sir4 and the other yKu (Hediger et al., 2002
; Taddei et al., 2004
). To examine directly whether the observed fold-back organization of chromosomes depends on telomere anchoring, we analyzed the position and dynamics of Tel 6L and 6R after disruption of either YKU
70 or SIR4
. In the absence of the yKu complex, Tel 6R is delocalized from the periphery (Hediger et al., 2002
) becoming randomly distributed in the nucleus, whereas Tel 6L anchoring is only slightly diminished ( A). In contrast, sir4
deletion releases Tel 6L, but not Tel 6R ( A). Confirming the redundancy of the anchoring pathways, we note that all telomeres analyzed to date lose their perinuclear position in double sir4 ku70
mutants (Hediger et al., 2002
; unpublished data). The mobility of Tel 6R and 6L also increases in these mutants, as monitored by live time-lapse imaging and absMSD analysis ( B). Plateau heights correspond to increases in average rc
from 0.38 or 0.43 μm in wild-type cells, to 0.5 μm in the sir4
mutant and >0.6 μm in yku70
Figure 6. Nuclear order is disrupted in the absence of yKu70p or Sir4p. Mobility, telomere–telomere separation, and telomere anchoring of Chr 6 are compared in wild-type, yku70, and sir4 cells. (A) Positions relative to the NE in wt (gray), yku70 (blue), (more ...)
We next asked whether the relative distance between the two telomeres changes significantly in these mutants. The separation between telomere pairs was monitored for mutant and wild-type cells as a function of time ( D). In both yku70 and sir4 mutants, Tel 6L and 6R show significantly greater separation than in wild-type cells (t test P < 0.003, yku70, and P < 0.005 for sir4). In the mutants <23% of the distances measured are ≤0.2 μm, as compared with >30% in wild-type cells. Because the two arms of Chr 6 are short, a 25% increase of the mean distance between the two telomeres corresponds to a large change in the angle between the two chromatids ( E). The average angle α increases from 39° to 48°, which is larger than that observed for Chr 5 in a wild-type strain (44°). Because centromere clustering near the SPB is unaffected by either the yku70 or sir4 deletion (unpublished data), we conclude that the fold-back organization of Chr 6, monitored as telomere–telomere proximity, is severely disturbed when either telomere loses its perinuclear anchoring.
In summary, the loss of yKu or Sir4p should make Chr 6 behave like Chr 5 (i.e., one telomere moves freely and the other is anchored; Fig. 8). Therefore, we plotted the relative MSD between Tel 6L and 6R in the mutant strains ( D), to score their loss of coordination. Indeed, the relative MSD plateau for Tel 6R-6L in the yku mutant is higher, similar to that scored for Tel 5R-5L in a wild-type background and consistent with increased mobility of one end ( B). Nonetheless, the plateau is still quite low, as it is in the sir4 background, suggesting that the ends of a given chromosome preserve a territorial inertia even though they interact less frequently.
Coordinated chromosome dynamics can occur independent of telomere interactions
Do linked telomeres move in a coordinated manner, or simply show constraint relative to each other? To address this we acquired time-lapse videos in 3D (7-image stack of a 300-nm step size) capturing double-tagged telomeres at two wavelengths on the confocal microscope (). Cellular integrity is confirmed by following the imaged cell through the subsequent mitosis. Coordinates of the center of the fluorescent spots were obtained using the IMARIS software, and the nuclear center is interpolated from the YFP-tetR background signal. The nucleus and spot positions for Tel 6L-6R and for Tel 6L-14L were then reconstructed in 3D (, A–C, shown here as projections onto the x, y, and z planes over time). Tel 6L-6R appear frequently, but not always, closely juxtaposed. Even when not juxtaposed, they seem to move in a coordinated fashion, which is not true for 6L and 14L.
Figure 7. 3D and two-color fluorescence time-lapse imaging of telomere dynamics. Time-lapse microscopy in 3D (one 7-plane stack every 3 s) was performed on GA-2201 (A), GA-2805 (B), and GA-2202 (C) as described in Materials and methods. Coordinates of both telomeres (more ...)
The degree to which movement is coordinated can be assessed by a correlation coefficient c (see Materials and methods; no correlation = 0, identical movement = 1). Direction cosines were determined for every vector joining two neighboring points of two separate trajectories, and the mean of Pearson's correlation coefficients (c) in each direction was determined. This was performed both for 2 color 2D and 3D time-lapse series. The movements of Tel 6R-6L have a mean correlation coefficient of 0.39 in 3D (0.26 in 2D), indicative of closely coordinated movement. Confirming our methodology, we found that two tags on the same telomere gave correlation coefficient of 1 (unpublished data). In contrast, Tel 6L-14L movements show no significant coordination (correlation coefficients of 0.03, for a 3D time-lapse series). Thus, the 6R-6L telomeres move with significant coordination over time, whereas unlinked ends do not.
Similar analysis was performed in strains bearing disruptions of YKU70 or SIR4, which compromises both anchoring and telomere–telomere interactions (). Strikingly, however, in yku70 and sir4 mutants the Tel 6R-6L correlation coefficients are ~0.15, which is still half the coordination detected in wild-type cells. In the case of the yku70 mutant, the 3D time-lapse analysis of Tel 6R and 6L trajectories projected onto x, y, and z planes, suggests a low but detectable degree of coordination in the mutants ( B). We predict that this residual coordination in chromosome dynamics can be attributed to their physical contiguity, i.e., that they represent two ends of a single chromosome. The release of one telomere from the NE and the ensuing drop in telomere interaction nonetheless does lead to a significant increase in unlinked movement.