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Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli.

Hur KH, Mueller JD - PLoS ONE (2015)

Bottom Line: Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes.We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements.The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

View Article: PubMed Central - PubMed

Affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
The brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

No MeSH data available.


Related in: MedlinePlus

MSQ curves.(A) MSQ-curve (triangles) for EGFP in U2OS cell and fit (solid line) to MSQ model with Q = 0.0193 and a diffusion time of 0.8 ms. (B) MSQ-curve (triangles) of EGFP in yeast cell. Fit (blue line) of data with TS < 1.6 s to Eq 8 yielded Q = 0.0237 and τD = 1.2 ms. Fit (red line) of data with TS > 1.6 s to Eq 12 determined Q = 0.0238 and kD = 4.46 × 10−3 s-1. (C) MSQ-curve (triangles) of EGFP in E.coli cell and fit (solid line) to Eq 14 with Q = 0.028, τD = 2.7 ms and kD = 2.7 × 10−2 s-1. (D) MSQ-curve (triangles) of NTF2-EGFP in E.coli cell and fit (solid line) to Eq 14 with n = 2.1, τD = 10 ms and kD = 5.3 × 10−2 s-1. The dashed line in each panel represents the reference Q-value of EGFP in solution, which was measured at the same power as the corresponding MSQ data.
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pone.0130063.g003: MSQ curves.(A) MSQ-curve (triangles) for EGFP in U2OS cell and fit (solid line) to MSQ model with Q = 0.0193 and a diffusion time of 0.8 ms. (B) MSQ-curve (triangles) of EGFP in yeast cell. Fit (blue line) of data with TS < 1.6 s to Eq 8 yielded Q = 0.0237 and τD = 1.2 ms. Fit (red line) of data with TS > 1.6 s to Eq 12 determined Q = 0.0238 and kD = 4.46 × 10−3 s-1. (C) MSQ-curve (triangles) of EGFP in E.coli cell and fit (solid line) to Eq 14 with Q = 0.028, τD = 2.7 ms and kD = 2.7 × 10−2 s-1. (D) MSQ-curve (triangles) of NTF2-EGFP in E.coli cell and fit (solid line) to Eq 14 with n = 2.1, τD = 10 ms and kD = 5.3 × 10−2 s-1. The dashed line in each panel represents the reference Q-value of EGFP in solution, which was measured at the same power as the corresponding MSQ data.

Mentions: We calculated the MSQ-curve for FFS data of EGFP measured in U2OS, yeast and E. coli (Fig 3A, 3B and 3C) and observed a clear dependence of MSQ on TS. Similarly, repeating the process on data from E. coli expressing NTF2-EGFP resulted in a pronounced dependence of MSQ on TS (Fig 3D). The MSQ-curve from the U2OS cell expressing EGFP (Fig 3A) comes closest to the ideal behavior. The MSQ-factor stays essentially constant for TS > 1s and only appreciably drops for TS less than ~0.4s. Performing the same experiment in yeast cells resulted in a MSQ-curve (Fig 3B) with a similar decline at short segment times as seen with the U2OS cells. However, unlike the U2OS cells, the MSQ-curve rises at long segment times, indicating an apparent increase in brightness. We previously demonstrated that photobleaching, which leads to a depletion of the fluorophores within the small volume of the yeast cell, introduces artificially inflated brightness values [8]. Because the volume of a U2OS cell vastly exceeds that of yeast, the same photobleaching process results in an entirely negligible depletion of the fluorophore population in the larger cell [8]. The MSQ-curves for E. coli (Fig 3C and 3D) are graphed with a logarithmic y-axis and display the same general behavior as observed for yeast, only more pronounced.


Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli.

Hur KH, Mueller JD - PLoS ONE (2015)

MSQ curves.(A) MSQ-curve (triangles) for EGFP in U2OS cell and fit (solid line) to MSQ model with Q = 0.0193 and a diffusion time of 0.8 ms. (B) MSQ-curve (triangles) of EGFP in yeast cell. Fit (blue line) of data with TS < 1.6 s to Eq 8 yielded Q = 0.0237 and τD = 1.2 ms. Fit (red line) of data with TS > 1.6 s to Eq 12 determined Q = 0.0238 and kD = 4.46 × 10−3 s-1. (C) MSQ-curve (triangles) of EGFP in E.coli cell and fit (solid line) to Eq 14 with Q = 0.028, τD = 2.7 ms and kD = 2.7 × 10−2 s-1. (D) MSQ-curve (triangles) of NTF2-EGFP in E.coli cell and fit (solid line) to Eq 14 with n = 2.1, τD = 10 ms and kD = 5.3 × 10−2 s-1. The dashed line in each panel represents the reference Q-value of EGFP in solution, which was measured at the same power as the corresponding MSQ data.
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Related In: Results  -  Collection

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pone.0130063.g003: MSQ curves.(A) MSQ-curve (triangles) for EGFP in U2OS cell and fit (solid line) to MSQ model with Q = 0.0193 and a diffusion time of 0.8 ms. (B) MSQ-curve (triangles) of EGFP in yeast cell. Fit (blue line) of data with TS < 1.6 s to Eq 8 yielded Q = 0.0237 and τD = 1.2 ms. Fit (red line) of data with TS > 1.6 s to Eq 12 determined Q = 0.0238 and kD = 4.46 × 10−3 s-1. (C) MSQ-curve (triangles) of EGFP in E.coli cell and fit (solid line) to Eq 14 with Q = 0.028, τD = 2.7 ms and kD = 2.7 × 10−2 s-1. (D) MSQ-curve (triangles) of NTF2-EGFP in E.coli cell and fit (solid line) to Eq 14 with n = 2.1, τD = 10 ms and kD = 5.3 × 10−2 s-1. The dashed line in each panel represents the reference Q-value of EGFP in solution, which was measured at the same power as the corresponding MSQ data.
Mentions: We calculated the MSQ-curve for FFS data of EGFP measured in U2OS, yeast and E. coli (Fig 3A, 3B and 3C) and observed a clear dependence of MSQ on TS. Similarly, repeating the process on data from E. coli expressing NTF2-EGFP resulted in a pronounced dependence of MSQ on TS (Fig 3D). The MSQ-curve from the U2OS cell expressing EGFP (Fig 3A) comes closest to the ideal behavior. The MSQ-factor stays essentially constant for TS > 1s and only appreciably drops for TS less than ~0.4s. Performing the same experiment in yeast cells resulted in a MSQ-curve (Fig 3B) with a similar decline at short segment times as seen with the U2OS cells. However, unlike the U2OS cells, the MSQ-curve rises at long segment times, indicating an apparent increase in brightness. We previously demonstrated that photobleaching, which leads to a depletion of the fluorophores within the small volume of the yeast cell, introduces artificially inflated brightness values [8]. Because the volume of a U2OS cell vastly exceeds that of yeast, the same photobleaching process results in an entirely negligible depletion of the fluorophore population in the larger cell [8]. The MSQ-curves for E. coli (Fig 3C and 3D) are graphed with a logarithmic y-axis and display the same general behavior as observed for yeast, only more pronounced.

Bottom Line: Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes.We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements.The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

View Article: PubMed Central - PubMed

Affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
The brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

No MeSH data available.


Related in: MedlinePlus