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Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

Etheridge TJ, Boulineau RL, Herbert A, Watson AT, Daigaku Y, Tucker J, George S, Jönsson P, Palayret M, Lando D, Laue E, Osborne MA, Klenerman D, Lee SF, Carr AM - Nucleic Acids Res. (2014)

Bottom Line: In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells.We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins.We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.

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Visualization of DNA-bound PCNA in fission yeast. (A) Representative reconstructed images of mEos3.1-PCNA nuclear localization patterns in S-phase and G2 S. pombe cells. (B) Numbers of mEos3.1-PCNA fluorescent localizations per nucleus in elg1+ and elg1Δ cells imaged with an exposure time of 350 ms in either G2 or S-phase cell cycle stages (n = 19, 26, 21 and 20 for elg1+ G2, elg1+ S-phase, elg1Δ G2 and elg1Δ S-phase, respectively). Cells were treated with sodium azide to prevent ATP-dependent PCNA DNA unloading/loading during imaging. Horizontal bar represents median value (elg1+ G2 = 51, elg1+ S-phase = 187, elg1Δ G2 = 60, elg1Δ S-phase = 362). P-value determined by two-tailed students T-test, *P = 8.1 × 10−7, **P = 9.4 × 10−10 ***P = 5.9 × 10−5. (C) Representative reconstructed images of elg1+ and elg1Δ mEos3.1-PCNA nuclear localization patterns in S-phase cells.
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Figure 4: Visualization of DNA-bound PCNA in fission yeast. (A) Representative reconstructed images of mEos3.1-PCNA nuclear localization patterns in S-phase and G2 S. pombe cells. (B) Numbers of mEos3.1-PCNA fluorescent localizations per nucleus in elg1+ and elg1Δ cells imaged with an exposure time of 350 ms in either G2 or S-phase cell cycle stages (n = 19, 26, 21 and 20 for elg1+ G2, elg1+ S-phase, elg1Δ G2 and elg1Δ S-phase, respectively). Cells were treated with sodium azide to prevent ATP-dependent PCNA DNA unloading/loading during imaging. Horizontal bar represents median value (elg1+ G2 = 51, elg1+ S-phase = 187, elg1Δ G2 = 60, elg1Δ S-phase = 362). P-value determined by two-tailed students T-test, *P = 8.1 × 10−7, **P = 9.4 × 10−10 ***P = 5.9 × 10−5. (C) Representative reconstructed images of elg1+ and elg1Δ mEos3.1-PCNA nuclear localization patterns in S-phase cells.

Mentions: For experiments in Figures 1B and 2A, mEos3.1 fluorophores were activated with sequential pulses of the 405-nm laser at power densities ranging from 0.1 to 10 W/cm2, adjusted manually to ensure single-molecule activation. Each activation cycle was followed by imaging of the sample using the 561-nm laser under incident intensities of the order of 1 kW/cm2. Iterative cycling continued until no further in-focus nuclear fluorescence could be detected. Samples for Figures 3 and 4 were imaged using continuous wave illumination of 405 nm (0.1–1 W/cm2) and 561-nm (1 kW/cm2) laser light, in order to reduce experiment duration. Control experiments showed no significant difference in the total number of localization using the pulsed activation method or the continuous wave approach (data not shown). All experiments were carried out at 17.0 ± 0.5°C. The typical number of frames (350 ns): 6000–11 000.


Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

Etheridge TJ, Boulineau RL, Herbert A, Watson AT, Daigaku Y, Tucker J, George S, Jönsson P, Palayret M, Lando D, Laue E, Osborne MA, Klenerman D, Lee SF, Carr AM - Nucleic Acids Res. (2014)

Visualization of DNA-bound PCNA in fission yeast. (A) Representative reconstructed images of mEos3.1-PCNA nuclear localization patterns in S-phase and G2 S. pombe cells. (B) Numbers of mEos3.1-PCNA fluorescent localizations per nucleus in elg1+ and elg1Δ cells imaged with an exposure time of 350 ms in either G2 or S-phase cell cycle stages (n = 19, 26, 21 and 20 for elg1+ G2, elg1+ S-phase, elg1Δ G2 and elg1Δ S-phase, respectively). Cells were treated with sodium azide to prevent ATP-dependent PCNA DNA unloading/loading during imaging. Horizontal bar represents median value (elg1+ G2 = 51, elg1+ S-phase = 187, elg1Δ G2 = 60, elg1Δ S-phase = 362). P-value determined by two-tailed students T-test, *P = 8.1 × 10−7, **P = 9.4 × 10−10 ***P = 5.9 × 10−5. (C) Representative reconstructed images of elg1+ and elg1Δ mEos3.1-PCNA nuclear localization patterns in S-phase cells.
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Related In: Results  -  Collection

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Figure 4: Visualization of DNA-bound PCNA in fission yeast. (A) Representative reconstructed images of mEos3.1-PCNA nuclear localization patterns in S-phase and G2 S. pombe cells. (B) Numbers of mEos3.1-PCNA fluorescent localizations per nucleus in elg1+ and elg1Δ cells imaged with an exposure time of 350 ms in either G2 or S-phase cell cycle stages (n = 19, 26, 21 and 20 for elg1+ G2, elg1+ S-phase, elg1Δ G2 and elg1Δ S-phase, respectively). Cells were treated with sodium azide to prevent ATP-dependent PCNA DNA unloading/loading during imaging. Horizontal bar represents median value (elg1+ G2 = 51, elg1+ S-phase = 187, elg1Δ G2 = 60, elg1Δ S-phase = 362). P-value determined by two-tailed students T-test, *P = 8.1 × 10−7, **P = 9.4 × 10−10 ***P = 5.9 × 10−5. (C) Representative reconstructed images of elg1+ and elg1Δ mEos3.1-PCNA nuclear localization patterns in S-phase cells.
Mentions: For experiments in Figures 1B and 2A, mEos3.1 fluorophores were activated with sequential pulses of the 405-nm laser at power densities ranging from 0.1 to 10 W/cm2, adjusted manually to ensure single-molecule activation. Each activation cycle was followed by imaging of the sample using the 561-nm laser under incident intensities of the order of 1 kW/cm2. Iterative cycling continued until no further in-focus nuclear fluorescence could be detected. Samples for Figures 3 and 4 were imaged using continuous wave illumination of 405 nm (0.1–1 W/cm2) and 561-nm (1 kW/cm2) laser light, in order to reduce experiment duration. Control experiments showed no significant difference in the total number of localization using the pulsed activation method or the continuous wave approach (data not shown). All experiments were carried out at 17.0 ± 0.5°C. The typical number of frames (350 ns): 6000–11 000.

Bottom Line: In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells.We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins.We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.

Show MeSH
Related in: MedlinePlus