2 Institute of Applied Mathematics, University of Lausanne, 1015 Lausanne, Switzerland
Correspondence to Susan M. Gasser:susan.gasser{at}fmi.ch
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Thierry Laroche and Susan M. Gasser's present address is Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.
Abbreviations used in this paper: 2D, two-dimensional; 3D, three-dimensional; ARS, autonomously replicating sequence; Chr, chromosome; IF, immunofluorescence; MSD, mean square displacement, NE, nuclear envelope; rc, radius of confinement or spatial constraint; SPB, spindle pole body; Tel, telomere.
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Introduction |
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One means for the positioning of chromosomes may be the anchorage of specific chromosomal elements. It is well established in unicellular organisms like yeasts, Plasmodia and Trypanosoma, that telomeres are grouped in clusters at the nuclear envelope (NE; Funabiki et al., 1993; Scherf et al., 2001). This organization is well-characterized in Saccharomyces cerevisiae, where there are five to eight discrete perinuclear foci, each containing five to seven telomeres (Gotta et al., 1996). Moreover, yeast centromeres cluster near the membrane-embedded spindle pole body (SPB; Guacci et al., 1997; Jin et al., 1998; Heun et al., 2001a; Bystricky et al., 2004). Theoretically, these clustering events are compatible with a Rabl-like arrangement for yeast interphase chromosomes (Ostashevsky, 2002), in which centromeres and telomeres would be found at opposite poles. However, no imaging study to date has specifically tagged both right and left telomeres of a given yeast chromosome to formally demonstrate a Rabl configuration. A recent cross-linking study suggests that subtelomeric regions of the budding yeast chromosome (Chr) 3 interact preferentially in living cells (Dekker et al., 2002). Because Chr 3 is a small yeast chromosome which bears unusual GC-rich isochores (Bradnam et al., 1999) and active and silent mating type loci, it was unclear whether other yeast chromosomes would yield similar cross-linking results.
Here, we directly examine the organization of chromosomes in vegetatively growing yeast cells, exploring the relationship of this organization to mechanisms that anchor telomeres at the NE. Exploiting two different bacterial repressor proteins with high affinity for integrated operator site arrays (lacop or tetop arrays), we analyze chromatin structure in vivo at high resolution with live fluorescence microscopy (Belmont, 2001). Past studies have used such tools to examine the dynamics of individually tagged chromosomal loci, revealing rapid and constant, yet spatially constrained movements. Typical loci shift position frequently (0.10.5 µm/s) within restricted subnuclear volumes (Marshall et al., 1997; Heun et al., 2001b), characteristics that seem to be conserved from yeast to man. Yeast centromeres and telomeres, on the other hand, move within more tightly restricted zones and remain near the nuclear periphery (Heun et al., 2001b; Hediger et al., 2002). By using GFP derivatives fused to different bacterial repressors (i.e., lacI and tetR), we are able to use similar techniques to study the global folding of chromosomes in vivo, avoiding artifactual trans-interactions between arrays of like repressor molecules (Aragon-Alcaide and Strunnikov, 2000). We further examine the positions of differentially tagged telomeres relative to subnuclear landmarks such as the SPB, the nucleolus and NE.
The right and left telomeres of several budding yeast chromosomes interact frequently, but not stably. The interaction is most pronounced for two small chromosomes, Chr 3 and Chr 6, which have relatively short chromosomal arms of roughly equal length, yet the resulting Rabl-like organization is demonstrated for two other larger chromosomes. Telomeretelomere interactions are compromised in cells lacking the proteins involved in perinuclear anchoring, namely yKu and Sir4p. Nonetheless, even in strains lacking these tethers, the movement of telomeres on opposite chromosome arms is coordinated, which is not observed for telomeres of unlinked chromosomes. We suggest that centromere anchorage and telomeretelomere interactions, together with the general compaction of chromatin into a 30-nm fiber (Bystricky et al., 2004), determine chromosome position in the yeast interphase nucleus.
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Results |
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Further evidence that the nucleus does not rotate continuously in G1 phase, is based on GFP-Nup49 FRAP experiments (Fig. 1 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., 1.510-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 (Fig. 1 D). Instead we observe a slow diffusion of pore fluorescence inwards from the edges of the bleached zone, beginning at 80 s (Fig. 1 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 (Fig. 2 A). We measured distances separating the lacop and tetop insertions, visualized by the binding of CFP- or YFP-fusions to the bacterial repressors, on three-dimensional (3D) confocal stacks of intact cells (Fig. 2, A and B). The distributions of 3D measurements (n = 60160 for each telomere pair) are plotted in Fig. 2 (C and D), and the mean distances between tagged sites are summarized in Table I. 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 3540% 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 (Fig. 2 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 (Fig. 2 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.
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Right and left telomere interactions are favored by perinuclear constraints
Two parameters may influence telomeretelomere 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 (Table I). Thus, chromosome arm length probably only favors telomeretelomere 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 (Table II). 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 (Table II). 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.
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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 <d2>) 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
d2 = (d(t)d(t+
t))2 (Berg, 1993; Hediger et al., 2004). The MSD curve for chromatin with a spatially constrained diffusion process generally reaches a plateau by
t > 50s. This analysis is highly robust because
t 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; Fig. 4 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 (Fig. 4, D and E). 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; Fig. 5 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 x MSD). For our geometry, this dependence is Ma x 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 (Fig. 4 E and Fig. 5 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.
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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 (Fig. 5 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 <d2> = 0.10.14 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/Cen3, <
d2> = 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 YKU70 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 (Fig. 6 A). In contrast, sir4 deletion releases Tel 6L, but not Tel 6R (Fig. 6 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 (Fig. 6 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 cells.
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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 (Fig. 6 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 (Fig. 5 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 (Fig. 7). 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 (Fig. 7, AC, 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.
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Similar analysis was performed in strains bearing disruptions of YKU70 or SIR4, which compromises both anchoring and telomeretelomere interactions (Fig. 6). 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 (Fig. 7 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.
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Discussion |
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Multiple elements constrain chromatin mobility to help define chromosome position
We provide novel evidence that long-range interactions between telomeres can be altered in vegetatively growing yeast by interfering with the telomere-associated proteins yKu and Sir4p. These same factors are directly involved in the anchorage of yeast telomeres to the NE. Indeed, silencing-incompetent forms of each protein are sufficient to relocate an otherwise internal locus to the nuclear periphery (Hediger et al., 2002; Taddei et al., 2004). Importantly, the disruption of anchorage at just one end of Chr 6 significantly reduces telomeretelomere interaction (Fig. 7). Correlation analysis of movement in 3D argues that despite their separation, Tel 6R and 6L continue to move in a partially coordinated manner in these yku70 or sir4 mutant cells. Because unlinked telomeres do not behave in a similar fashion, we conclude that not only direct interaction, but the contiguity of the chromosomal fiber influences chromatin movement, even though telomeres are separated by several hundred kilobases and a kinetochore.
It has been questioned whether the notion of chromosome "territories" is appropriate for yeast due to the relatively large rc monitored for individual loci (rc = 0.50.65 µm) and the small size of the yeast nucleus (nuclear radius = 1 µm). The movement we document here indicates that two linked telomeres move in a partially coordinated manner, thus providing a quantifiable parameter for a "chromosomal territory". In contrast to this, a 16-kb ring of chromatin released from its chromosomal context by an inducible recombinase, traverses the nucleoplasm freely and randomly, moving in all directions (rc 0.8 µm for the ring vs. 0.6 µm for the chromosomal locus; Gartenberg et al., 2004). The unconstrained movement of this ring further stresses the impact of chromatid contiguity both on the relative positioning of linked telomeres and on general chromosome positioning in interphase nuclei. In conclusion, we propose that chromosome position is defined by three types of constraint: the contiguity and compaction of the chromosomal fiber, sites of anchorage to less mobile nuclear landmarks (centromeres to the SPB and telomeres to the NE) and finally, reversible interactions between right and left chromosome ends.
Our data strongly support the looped Chr 3 model proposed from an assay that scores the efficiency of cross-linking in vivo (Dekker et al., 2002). Chr 3 is unique among yeast chromosomes in that it carries three homologous mating type loci that participate in a gene conversion event required for mating type switching. Chr 3 also has unique, strongly pronounced GC-rich "isochores" of 3050 kb (Bradnam et al., 1999), which are not found on the other chromosomes analyzed here. It is conceivable that the folded structure of Chr 3 reflects its propensity for recombination between MAT (on the right arm) and HML (on the left arm) in MATa cells. However, because Chr 6 forms a whole chromosome loop as efficiently as Chr 3, these Chr 3-specific features are unlikely to be critical for its folding pattern in vivo. We note that the interactions of telomeres on Chr 3 and 6 may well be aided by the fact that these two small chromosomes have similar arm lengths and compaction ratios (Bystricky et al., 2004). Conversely, one might assume that grossly different chromosome arm lengths limit pairing. Finally, we note that chromatid arm length is not a sufficient criterion to determine stable pairing events, because the telomeres of 5L and 14R do not interact despite the equal length of these chromosome arms.
Extended sequence homology is not critical for telomere pairing
Does sequence homology contribute to selective telomeretelomere interactions? It was suggested that transient contact between homologues, or chromosome "kissing" events, would facilitate homology searches in meiotic prophase (Kleckner and Weiner, 1993; Pryde and Louis, 1999). One might imagine that once in contact, sequence homology could in turn promote more stable interactions in trans through ligand binding. Such trans-interactions have been proposed to facilitate silencing, but also are thought to help coordinate the timing of replication of right and left telomeres in budding yeast (Raghuraman et al., 2001). Our study, however, demonstrates that the selective interactions of the Tel 3R and 3L and Tel 6R and 6L does not result simply from sequence homology. Neither pair of telomeres shares any homology other than the universal TG-rich and STR/core X element repeats. Moreover, a pair of telomeres that shares >90% homology over 16 kb (Tel 6L and 14L), almost never interact despite the presence of highly conserved Y' elements. Consistently, entire chromosomal homology also has little impact on pairing: in a yeast strain that serendipitously contains a duplication of the double-tagged Chr 3 (bearing Tel 3R-tetop and 3L-lacop sequences) the two homologous chromosomes are far apart within the nucleus and each forms a separate fold-back structure (unpublished data).
Heterochromatin factors anchor telomeres and contribute to trans-interaction
In budding yeast, a strong candidate for contributing to telomeretelomere interactions could be silent chromatin itself. Silencing efficiency, like the availability of Sir proteins and telomeretelomere pairing, varies from end to end (Pryde and Louis, 1999). Sir4p, a 174-kD protein bears a COOH-terminal coiled-coil domain that is necessary for homo-dimerization as well as interaction with Sir3, Rap1, and yKu (for review see Gasser and Cockell, 2001). The Sir4 COOH-terminal domain has often been compared with nuclear lamins in higher eukaryotes, although Sir4 requires another perinuclear protein, Esc1p, or yKu to ensure its anchoring (Taddei et al., 2004). To test rigorously whether or not subtelomeric heterochromatin directly influences pairing, it will be necessary to compare the efficiency of native telomere repression and the efficiency of native telomere interaction systematically in a single strain background. Whereas yku70 mutations compromise telomeric silencing, they do not impair mating type silencing, suggesting that reduced interactions do not significantly disrupt the repressive state.
In analogy to Sir4p, HP1 has been proposed to mediate interactions between repressed domains in higher eukaryotes in trans (Ryan et al., 1999). However, delocalization of HP1 can occur in mammalian cells without the disruption of the chromocenter (Peters et al., 2001). The elimination of Sir4 and yKu oblate telomere associated silencing, much like spTaz1, which is required in fission yeast both for telomere-associated silencing (Kanoh and Ishikawa, 2001) and meiotic clustering. Surprisingly, however, loss of mitotic clustering is governed by the fission yeast RNAi machinery, not Taz1, and mutation of this ironically does not derepress subtelomeric silencing or perinuclear anchorage (Hall et al., 2003). Therefore, although subsets of heterochromatin components may contribute to long-range chromosomal contacts, the loss of interactions in trans, is not necessarily correlated with changes in repression status. Rather, critical components for telomeretelomere interactions, among which may figure cohesin molecules, may simply associate with heterochromatin, participating to different degrees in both repression and trans-interactions (Partridge et al., 2002).
Limited chromosomal mobility can define a territory
Besides specific patterns of chromosome folding and centromere/telomere positioning, we provide evidence that the coordinated movement of the two distal regions of a yeast chromosome is compromised by mutation. We envision this coordinated movement as a sort of "chromosomal inertia," which reflects the tendency of a chromosome to move as one body, even when specific interactions are compromised. Given the mass of a mammalian chromosome, if similar coordination occurs, then this alone could account for the infrequency with which human chromosomes change their territorial distribution (for review see Spector, 2003). Reproducible positioning and limited mobility of chromosomal domains has been documented as well for Drosophila cells (Marshall et al., 1996, 1997; Vazquez et al., 2001). We propose that the general inertia of whole chromosome territories in higher eukaryotic cells, may be linked to a phenomenon we quantify herethat of chromosome-wide coordination of constrained movement.
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Materials and methods |
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Quantitative analyses of distance, position and dynamics
Distances were measured using the Zeiss LSM510 Confocal software version 2.5. Y/CFP and IF signals were scored on 3D stacks using 40160 nuclei per point, monitoring nuclear integrity through nucleolar shape and nuclear diameter. Tagged telomere position percentages in zone 1 were compared with a random distribution by t test (Hediger et al., 2002), with a 95% confidence interval. 2D time-lapse series of GFP or YFP-CFP spots were analyzed with MetaMorph Offline v. 4.6r6 (Universal Imaging). For each strain 812 videos from two to three independent cultures were combined and averaged. MSD analysis was performed as described previously (Heun et al., 2001b; Vazquez et al., 2001) with modifications as detailed in Results.
The IMARIS software (Bitplane) was used to determine coordinates of the center of the fluorescent spots imaged in 3D over time. Reliable coordinates in 3D could be obtained from 4 out of 12 videos taken for each strain. Videos in 2D were also analyzed. Representations of the trajectories projected onto the three imaged planes were obtained using Mathematica. Direction cosines were determined for every vector, which joins two neighboring points of the trajectories of two separate fluorescent spots (frames analyzed for 3D videos were as follows: n =162 for wt, n = 178 for yku70, n = 70 for sir4). Similar analysis was performed on 2D videos for which the numbers of frames were as follows: n = 1099 for wt, n = 1053 for yku70, n = 829 for sir4, n = 971 for Tel6L-14L. The mean of correlations (Pearson's correlation coefficient, c) in each (x, y, z) direction (or x, y in the 2D videos) was determined. This value (c) expresses the degree of linear relationship between two variables and is equal to the average cross product of the variables in standardized form. Pearson's c values can range between 1.00 and +1.00, with the latter signifying a perfect positive relationship, whereas 1.00 shows a perfect negative relationship. The smallest correlation is zero.
Online supplemental material
Fig. S1 shows chromosomes 3, 5, and 6 loop back on themselves. Fig. S2 shows the position of telomeres relative to the NE. Fig. S3 shows 2D distances between the telomeres during time-lapse imaging. Videos 16. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200409091/DC1.
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Acknowledgments |
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Our research is supported by the Swiss National Science Foundation and "Frontiers in Genetics" NCCR program.
Submitted: 20 September 2004
Accepted: 16 December 2004
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References |
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