Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres
- Janelia Farm Research Campus, Howard Hughes Medical Institute, United States
- National Cancer Institute, National Institutes of Health, United States
Figures
Figure 1 with 2 supplements
Internal tagging of Cse4 confers exclusive centromeric localization and preserves wild type phenotype.
(A) Alternative tag locations at Leu81 (internal XbaI site) or at the C-terminus of Cse4 are indicated by green triangles. Unstructured N-terminal tail (aa1-135) is depicted in grey while region …
Figure 1—figure supplement 1
Fluorescence of centromeric clusters containing C-terminally tagged Cse4 is slightly elevated but does not double in anaphase.
(A) Representative examples of metaphase and telophase cells (outlined) shown at the same brightness scale. Clusters in surrounding cells may be out of focus. (B) Intensity of individual centromeric …
Figure 1—figure supplement 2
Internally tagged Cse4 accumulates at levels comparable to wild-type protein.
(A) Whole-cell lysates of cells containing wild-type or tagged Cse4 were probed with affinity-purified anti-Cse4 antibody. Arrows indicate bands corresponding to wild-type Cse4 (27 kDa) and Cse4 …
Figure 2
Pre-existing Cse4 is removed and exchanged for new Cse4 molecules at G1/S transition.
(A) Relevant fluorescence states of a tdEos-tagged protein molecule are depicted schematically after its synthesis and folding, fluorophore maturation and irreversible photoconversion. Excitation …
Figure 3
Cell cycle-dependent increase in centromere cluster intensity is a result of fluorophore maturation.
(A) Relative intensity of centromeric clusters in asynchronously growing cells as the function of cell cycle stage and approximate time since entry into S phase. Values were corrected per 16 …
Figure 4
Removal of pre-existing Cse4 is associated with DNA replication.
(A) Experimental scheme to assess role of DNA replication on the removal of pre-existing Cse4. α-factor synchronized cells were released into control medium or one with 0.2 M hydroxyurea (HU). …
Figure 5
There is no additional Cse4 deposition in anaphase.
(A) Scheme of the experimental test for Cse4 deposition in anaphase. All operations were carried on a selected metaphase cell in the specified order. 3D diffraction-limited spot, generated with a …
Figure 6 with 1 supplement
Centromeric clusters become more compact during anaphase.
(A) An example of MFM-PALM image with nine simultaneously acquired Z-planes. Gold Nanorods are indicated (blue circles) and distance above the glass surface is listed for other tiles. Anaphase cell …
Figure 6—figure supplement 1
tdEos fluorophore undergoes transitions between multiple fluorescent and dark states.
Different states of tdEos fluorophore (and mEos variants, including mIRIS [Wiedenmann et al., 2011]) are shown schematically. Newly synthesized non-fluorescent tdEos undergoes fast folding followed …
Figure 7 with 1 supplement
Two Cse4 molecules are present on each centromere.
(A) Comparison of Cse4-GFP centromere clusters with TetR-GFP bound to arrays of 7, 14 or 21 tetO, displayed within the same brightness range. Representative telophase cells are outlined. Clusters in …
Figure 7—figure supplement 1
Wavelet filtering allows precise separation of cluster signal from nuclear and cellular background.
(A) Images of the representative telophase cells (outlined) are shown for Cse4-GFPinternal strain or strains containing integrated tetO arrays (7, 14, 21 or 112 repeats) and TetR-GFP under …
Figure 8 with 2 supplements
Scm3 dynamically interacts with centromeres at levels equivalent to Cse4.
(A) Scm3-tdEos fluorescence recovery after targeted photobleaching. The experiment was performed essentially as shown in Figure 5A, except that recovery was monitored by repetitive imaging, without …
Figure 8—figure supplement 1
Scm3-tdEos is present on centromeres and in the nucleus at every stage of the cell cycle.
(A) Distribution of Scm3-tdEos in live cells. Cell cycle stages are indicated in DIC panels and red fluorescence, after photoconversion, is shown as in Figure 1. (B) Majority of Scm3 is dispersed …
Figure 8—figure supplement 2
Similar amounts of Cse4 and Scm3 molecules reside at centromeres.
(A) Average intensity (photons/s, ±standard deviation) of centromeric clusters containing Cse4-GFPinternal or Scm3-GFP (n = 8), measured during continuous excitation. (B) Sample images of cells …
Figure 9
Model of Cse4 replacement and re-establishment of point centromere during cell cycle (see text for details).
https://doi.org/10.7554/eLife.02203.021Videos
Video 1
3D representation of tdEos fluorophore distribution in anaphase cell from Figure 6C.
Each event is depicted as a dot 50 nm across instead of the actual average localization precision (20 nm lateral/50 nm axial). The original color coding of axial distance from Figure 6C is …
Video 2
3D representation of Cse4-tdEos distribution in G1 centromere cluster from Figure 6D.
Each event is depicted as a dot 20 nm across instead of the actual average localization precision (20 nm lateral/50 nm axial). The box encloses 1 μm3 volume and two artificial sizing marks (red and …
Video 3
3D representation of Cse4-tdEos distribution in late anaphase centromere cluster from Figure 6E.
Each event is depicted as a dot 20 nm across instead of the actual average localization precision (20 nm lateral/50 nm axial). The box encloses 1 μm3 volume and two artificial sizing marks (red and …
Tables
Table 1
Recovery time of centromeric Scm3-tdEos after targeted photobleaching
| Cell cycle stage | Mean recovery time (min) | Standard deviation | Sample size |
|---|---|---|---|
| G1 | 5.2 | 2.5 | 9 |
| S | 4.5 | 1.6 | 4 |
| metaphase | 4.9 | 2.3 | 7 |
| anaphase | 4.7 | 2.0 | 13 |
| telophase | 5.1 | 2.7 | 9 |
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Note: After targeted photobleaching of photoconverted Scm3-tdEos centromere clusters with 551 nm dye-laser, cells were imaged with stepwise focus changes within -1 μm to +1 μm Z range (7 steps, 333nm apart, 5 sec. exposure per step). G2 clusters were excluded due to their extended size.
Table 2
Saccharomyces cerevisiae strains
| Strain | Genotype |
|---|---|
| MBY507* | MATa ade2 CSE4-GFP-CSE4 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 RAD5 |
| JBY119† | MATa ADE2 dynLC::hphMX4 cse4::natMX4 can1-100 his3-11,15 leu2-3,112::LEU2-CSE4-tdEOS-CSE4 trp1-1 ura3-1 RAD5 |
| JBY111‡ | MATa ADE2 dynLC::hphMX4 SCM3-tdEOS-kanMX4 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 RAD5 |
| JBY251§ | MATa ADE2 can1-100 his3-11,15 leu2-3,112::LEU2-Δ80ura3p-TetR-GFP-TAP-ADHt trp1-1 ura3-1::pRS406-7xtetO RAD5 |
| JBY252§ | MATa ADE2 can1-100 his3-11,15 leu2-3,112::LEU2-Δ80ura3p-TetR-GFP-TAP-ADHt trp1-1 ura3-1::pRS406-14xtetO RAD5 |
| JBY253§ | MATa ADE2 can1-100 his3-11,15 leu2-3,112::LEU2-Δ80ura3p-TetR-GFP-TAP-ADHt trp1-1 ura3-1::pRS406-21xtetO RAD5 |
| JBY254§ | MATa ADE2 can1-100 his3-11,15 leu2-3,112::LEU2-Δ80ura3p-TetR-GFP-TAP-ADHt trp1-1 ura3-1::pRS306-112xtetO RAD5 |
| MSY173# | MATa his3-1 leu2-0 ura3-0 Cse4-GFP::SpHIS5 |
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Notes: *Cse4-GFPinternal, Xiao et al., 2011.
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†
Cse4-tdEosinternal, this paper.
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‡
Scm3-tdEosC-terminal, this paper.
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§
7, 14, 21 or 112 tetO repeats, respectively, and TetR-GFP, this paper.
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#
Cse4-GFPC-terminal, Shivaraju et al., 2012.
Table 3
Light sources and filters used for wide field fluorescence imaging
| Fluorophore | Light source & channel | Filter cube | ||
|---|---|---|---|---|
| Excitation | Beamsplitter | Emission | ||
| GFP | Colibri/LED470* Spectra-6/C§ | FF01-475/28† LL01-488/1† | T495LP‡ | FF01-525/50† |
| tdEos (red emission) | Colibri/LED555* Spectra-6/GY§ | BP550/25* FF01-543/3† | FT570* FF568-Di01† | BP605/70* FF01-593/46† |
| tdEos (photoconversion) | Colibri/LED405* Spectra-6/V§ | FF01-405/10† | 59004BS‡ | 59004M‡ |
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Source: *Zeiss.
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†
Semrock.
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‡
Chroma.
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§
Lumencor.
Additional files
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Supplementary file 1
Batch FITS converter macro for ImageJ. This macro converts 16-bit TIFF files from a selected folder into FITS files (re-assigned into 32-bit floating-point space) and saves them in a destination folder. The content of the file should be saved as ‘Batch FITS Converter.txt’ into Macro folder of ImageJ.
- https://doi.org/10.7554/eLife.02203.024
