Yeast techniques and live fluorescence microscopy
For live imaging, cultures were grown in synthetic medium to a concentration of less than 1 × 105 cells/ml. Microscopy was performed at 30°C on cells spread on agarose patches containing synthetic complete medium with 4% glucose. Images for zoning measurements were captured on a Metamorph driven Olympus IX70 widefield microscope equipped with a Coolsnap HQ camera from Roper Scientific Photometrics. Cells expressing GFP-NUP49 and lacI-GFP bound to lacO were imaged by acquiring stacks with a step size of 0.2m using a wave length of 475nm for excitation. For cells bearing the nup133 N-terminal deletion, CFP-Nup49 was used to identify pore clusters and lacO/GFP-lacI was used to monitor the targeted locus. Stacks of 21 images with a step size of 0.2 μm were acquired, alternating once per stack the wavelength between 437 nm (CFP) and 517 nm (YFP). A 100X / 1.4 Oil Plan-Apochromat objective from Zeiss was used.
Deconvolution was performed using the Huygens software (calculated PSF) and Imaris® (Biplane) Software for analysis of colocalization. The Fisher's Exact Test for Count Data was performed used the R-project software package (www.r-project.org). Plasmids and yeast strains used in this study are described in Supplementary materials. Deletion and modification of genes were achieved by plasmid-borne or PCR based methods (Longtine et al, 1998) and were verified by PCR and phenotypic analysis. Telomeric positioning data was analyzed as described (Hediger et al., 2006).
97
Mating assay
MATa and MAT cells were grown overnight in synthetic complete (SC) medium to a concentration of less than 1 × 105 cells/ml and mixed in equal amounts. The cells were applied to a filter by vacuum elution of the media. The filter coated with cells was transferred to solid SC medium and incubated for 90 min. Next the cells were washed off the filter using SC medium, transferred to a Ludin Chamber and mounted on a Metamorph driven Till5 wide field microscope. Zygotes were imaged every 3 min at minimal excitation and with 400nm Z-step size. Only zygotes that displayed normal progression through the cell cycle were used for analysis.
Abbreviations
CC Cajal Body
NE Nuclear Envelope NP Nuclear Periphery NPC Nuclear Pore Complex GFP Green Fluorescent Protein
98
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Figure legends
Figure 1
yKu80 does not recruit chromatin to the nuclear pore complex. (A) Yeast strain GA-4584 was used which bears nup133N’, CFP-Nup49, lacI-GFP and lexA-lacO integrated at LYS2. In this strain pores cluster at one site of the NE as visualized by CFP-Nup49. Either lexA, lexA-yku80-4 or lexA-Nup84 fusion proteins were expressed and the position of the integrated array of lacO-lexA-sites visualized as a lacI-GFP focus, was scored by its position relative to the CFP-Nup49 cluster. (B) Fusion proteins were analyzed for their ability to recruit an internal chromosomal locus (LYS2) to the pore cluster in S-phase cells.
Examples of what was scored as colocalized or not colocalized are shown. G1- and S-phase cells were scored by the presence or absence of a bud, as illustrated.
Figure 2
yKu-mediated NE anchoring in S phase requires its interaction with Tlc1. (A) Scheme of different yku80 proteins and their interaction partners relevant for this study (B) An assay for targeted anchoring of an internal locus close to ARS607 tagged with lacO repeats and LexA-binding sites in strain GA-1461 was performed as described in Taddei et al. (2004). (C) GA-1461 + lexA-yku80-4 (G1 n=147, S n=113); GA-1461 + lexA-yku80-135i (G1 n= 215, S n=169); GA-1461 + lexA-yku80-135i-4 (G1 n=195, S n=129); (D) GA-1461 + lexA alone (G1 n=158,
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S n=87), GA-1461 + 4 (G1 n=147, S n=113); GA-2841 + lexA-yku80-4 (48tlc1) (G1 n=255, S n=170).
Figure 3
Est2 can relocate a randomly distributed locus to the NE. The targeted anchoring assay was performed as described in Figure 2B. (A) GA-1461 + lexA alone (G1 n=158, S n=87), GA-1461 + Est2 (G1 n=190, S n=167); GA-1950 + lexA-Est2 (esc1) (G1 n=269, S n=120) (B) Est2 does not recruit chromatin to the nuclear pore complex. (A) Yeast strain GA-4584 as in Figure 1. Fusion protein of lexA-Est2 was analyzed.
Figure 4
(A) Scheme of in vivo analysis of native telomere 6R positioning in strain GA-1459 (as described in Hediger et al., 2002b). Insets on the right show fluorescence imaging of several typical samples; (B) GA-1459 (Tel6R WT) G1 n=87, S phase n=61 conferred to GA-1459 (Tel6R) + 2 plasmid encoding mps3-N’-tetRmCherry = mps3-N’, G1 phase n= 61, S phase n=56; (C) GA-1867 (Tel6R sir4) G1 phase n=62, S phase n=57 and GA-1867 (Tel6R sir4) + mps3-N’, G1 phase n= 51, S phase n=53; (D) GA-1459 (Tel6R WT) G1 n= 87 S phase n=61 cf. to GA-1867 (Tel6R sir4) G1 n=62, S phase n=57 cf to est2 in GA-1867 (Tel6R sir4) G1 n=52, S phase n=36
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Figure 5
(A) Targeted anchoring of internally localized ARS514 in strain 3690; (B) GA-3690 + lexA, G1 phase n=251, S phase n=180; GA-GA-3690 + lexA-Est2, G1 phase n=271, S phase n=142; GA-3690 + lexA-Est2 + mps3-N’, G1 phase n=282, S phase n=170; (C) GA-3690 + lexA, G1 phase n=239, S phase n=221; GA-3690 + lexA-yku80-4 G1 phase, n=228, S phase n=158; GA-3690 + lexA-yku80-4 + mps3-N’, G1 phase n=317, S phase n=150.
Figure 6
Synthetic interaction between tel1 and mps3-N’ overexpression. (A) Brightfield images of WT yeast strain GA-426 (Tel5R-ADE2-TG), GA-1370 (tel1 in GA-426) and GA-911 (yku70 in GA-426) transformed with either empty 2 (left panels) or with +mps3-N’ (right panels). Red fluorescence shown is mps3-N’-tetRmCherry; Scale bar = 5m (B) Comparison of unbudded, budded, large budded dumbbell shaped cells in WT (GA-426), tel1 cells (GA-1370) and yku70 (GA-911) cells expressing either empty 2 or with 2+mps3-N’. In the order of appearance n= 138; 270, 338; 159; 178; 198. Examples of each category are shown on the bottom. (C) Southern blot of XhoI digested genomic DNA probed with TG repeats as described (Craven and Petes, 1999).
Figure 7
Visualizing elongating telomeres. (A) The mating assay designed for detection of telomerase activity was performed as in (Forstemann et al., 2000; Teixeira et al.,
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2004). Telomerase positive or negative donors (GA-3362 and GA-3363) were mated to a telomerase negative recipient that has short telomeres (GA-3361).
One of the telomeres (Tel5R-ADE2-TG1-3) in the recipient was fluorescently tagged with lacO/lacI-GFP. Inset on the right shows an example of the fluorescence of a zygote (B) Zygotes were analysed microscopically as shown in this montage. Image stacks are acquired every 3min. A z-projection is shown.
Zygotes were then categorized as S phase cells according to presence or absence of a bud, and as entering mitosis when the nucleus invades the daughter cell. Note that zygotic nuclei appear elongated in the first cell cycle, whereas they are round in the following cell cycles. (C) Sketch of critical zygotic states.
Figure 8
(A) Examples of fluorescent images of zygotes bearing a tagged telomere and GFP-Nup49 at 6 min or 3 min before mitosis, and at the onset of mitosis. Images where deconvolved using the Huygens® software and the projection is shown.
(B) The position of Tel5R relative to the GFP-Nup49-tagged NE was scored in the 1st cell cycle and the results from positive and telomerase-negative zygotes is shown. Average distances of the telomere from the NE are plotted, and all measurements are synchronized to the cell’s entry into mitosis.
Each point is the average of more than 50 values. (C) same as (B), 1st cell cycle is compared to 2nd cell cycle of telomerase positive zygotes.
107
lexA G1
lexA-bs
3 zones of equal surface
random 20
lexA G1
Mps3
lexA-yku80-4
Δyku70 WT
URA3 2μ
A
B
C
Δtel1
mps3-N’
0%
20%
40%
60%
80%
100%
WT WT Δtel1
dumbbell and large budded budded
unbudded
Δtel1 Δyku70
Δyku80 Δtel1
WT
mps3-N’ - + - + + - - +
Δyku70
8 7 6 5 4 3 2 1
mps3-N’
2μ mps3-N’
2μ mps3-N’
2μ
113
4
late S phase: The short, fluoresntly labeled telomere is elongated:
WHERE?
1
cell cycle arrest in G1, shmoo
3
Zygote enters S-phase 2
nuclear fusion, EST1 loads on short recipient telomeres
2nd cell cycle: nuclei appear round
avarage distance from the NE 200 250 300 350 400 450 500
-18 -15 -12 -9 -6 -3
est1, short telos
min before mitosis 1st vs. 2nd cell cycle
200 250 300 350 400 450 500
-18 -15 -12 -9 -6 -3
min before mitosis
B C
A
EST1, short telos
2nd cc EST1+
1st cc EST1+
-3 min
-6 min onset of Mitosis
short telomere NE short telomere NE
115