Limits...
Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex.

Chen Y, Deffenbaugh NC, Anderson CT, Hancock WO - Mol. Biol. Cell (2014)

Bottom Line: The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data.The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle.These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Huck Institutes of the Life Sciences, University Park, PA 16802 Interdisciplinary Graduate Degree Program in Cell and Developmental Biology, Huck Institutes of the Life Sciences, University Park, PA 16802.

Show MeSH

Related in: MedlinePlus

In vivo photobleaching of GFP-AtCESA3. (A) Photobleaching trace of a single GFP-AtCESA3 particle in hypocotyl cells of Arabidopsis seedling. Video is recorded at 5 fps, and total time is 100 s to allow most GFP to be photobleached. Representative Movie S1 is included in the Supplementary Data. Inset, ensemble average of 77 photobleaching traces with exponential fit to the data. (B) Quantitative model describing photobleaching. The fluorescence signal is assumed to fall over time with constant step sizes, matching the quantal fluorescence of a single GFP. The GFP fluorescence and the background signal are treated as Gaussian distributions, Normal(μ, σ2) and Normal(0, δ2), respectively. The time before fluorophore bleaching, T, is assumed to be exponentially distributed with mean τ = 1/λ, where λ is the photobleaching rate constant. The SNR is defined as the step size divided by the SD. (C) Simulated photobleaching trace from 12 fluorophores with μ = 500 a.u. and σ = δ = 250 a.u. (D) Simulated stepping data such as a kinesin walking along a microtubule in an optical trap experiment, with μ = 1, σ = 1, and 10% backward steps.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230622&req=5

Figure 1: In vivo photobleaching of GFP-AtCESA3. (A) Photobleaching trace of a single GFP-AtCESA3 particle in hypocotyl cells of Arabidopsis seedling. Video is recorded at 5 fps, and total time is 100 s to allow most GFP to be photobleached. Representative Movie S1 is included in the Supplementary Data. Inset, ensemble average of 77 photobleaching traces with exponential fit to the data. (B) Quantitative model describing photobleaching. The fluorescence signal is assumed to fall over time with constant step sizes, matching the quantal fluorescence of a single GFP. The GFP fluorescence and the background signal are treated as Gaussian distributions, Normal(μ, σ2) and Normal(0, δ2), respectively. The time before fluorophore bleaching, T, is assumed to be exponentially distributed with mean τ = 1/λ, where λ is the photobleaching rate constant. The SNR is defined as the step size divided by the SD. (C) Simulated photobleaching trace from 12 fluorophores with μ = 500 a.u. and σ = δ = 250 a.u. (D) Simulated stepping data such as a kinesin walking along a microtubule in an optical trap experiment, with μ = 1, σ = 1, and 10% backward steps.

Mentions: To estimate the copy number of GFP-AtCESA3 in membrane-localized particles in living cells of A. thaliana, we mounted 5- to 6-d-old light-grown seedlings expressing GFP-AtCESA3 (Desprez et al., 2007) in an imaging chamber and carried out recordings of GFP bleaching in hypocotyl cells containing low densities of GFP-AtCESA3 particles (Supplemental Movie S1). Imaging was performed using variable-angle epifluorescence microscopy (Konopka and Bednarek, 2008), which, like TIRF microscopy, reduces background fluorescence but allows for the imaging of proteins farther from the coverslip, such as those in the plasma membrane of plant cells that are separated from the coverslip by the cell wall (Konopka et al., 2008; Konopka and Bednarek, 2008). To quantify photobleaching rates, time lapse recordings were collected (Supplemental Movie S1), and fluorescence intensity traces for individual GFP-containing particles were measured using ImageJ (see Materials and Methods). Instead of exhibiting discrete steps, the intensity changes during photobleaching for many traces appeared to be relatively smooth (Figure 1A and Supplemental Movie S1), suggesting that the number of fluorophores per particle is relatively high.


Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex.

Chen Y, Deffenbaugh NC, Anderson CT, Hancock WO - Mol. Biol. Cell (2014)

In vivo photobleaching of GFP-AtCESA3. (A) Photobleaching trace of a single GFP-AtCESA3 particle in hypocotyl cells of Arabidopsis seedling. Video is recorded at 5 fps, and total time is 100 s to allow most GFP to be photobleached. Representative Movie S1 is included in the Supplementary Data. Inset, ensemble average of 77 photobleaching traces with exponential fit to the data. (B) Quantitative model describing photobleaching. The fluorescence signal is assumed to fall over time with constant step sizes, matching the quantal fluorescence of a single GFP. The GFP fluorescence and the background signal are treated as Gaussian distributions, Normal(μ, σ2) and Normal(0, δ2), respectively. The time before fluorophore bleaching, T, is assumed to be exponentially distributed with mean τ = 1/λ, where λ is the photobleaching rate constant. The SNR is defined as the step size divided by the SD. (C) Simulated photobleaching trace from 12 fluorophores with μ = 500 a.u. and σ = δ = 250 a.u. (D) Simulated stepping data such as a kinesin walking along a microtubule in an optical trap experiment, with μ = 1, σ = 1, and 10% backward steps.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230622&req=5

Figure 1: In vivo photobleaching of GFP-AtCESA3. (A) Photobleaching trace of a single GFP-AtCESA3 particle in hypocotyl cells of Arabidopsis seedling. Video is recorded at 5 fps, and total time is 100 s to allow most GFP to be photobleached. Representative Movie S1 is included in the Supplementary Data. Inset, ensemble average of 77 photobleaching traces with exponential fit to the data. (B) Quantitative model describing photobleaching. The fluorescence signal is assumed to fall over time with constant step sizes, matching the quantal fluorescence of a single GFP. The GFP fluorescence and the background signal are treated as Gaussian distributions, Normal(μ, σ2) and Normal(0, δ2), respectively. The time before fluorophore bleaching, T, is assumed to be exponentially distributed with mean τ = 1/λ, where λ is the photobleaching rate constant. The SNR is defined as the step size divided by the SD. (C) Simulated photobleaching trace from 12 fluorophores with μ = 500 a.u. and σ = δ = 250 a.u. (D) Simulated stepping data such as a kinesin walking along a microtubule in an optical trap experiment, with μ = 1, σ = 1, and 10% backward steps.
Mentions: To estimate the copy number of GFP-AtCESA3 in membrane-localized particles in living cells of A. thaliana, we mounted 5- to 6-d-old light-grown seedlings expressing GFP-AtCESA3 (Desprez et al., 2007) in an imaging chamber and carried out recordings of GFP bleaching in hypocotyl cells containing low densities of GFP-AtCESA3 particles (Supplemental Movie S1). Imaging was performed using variable-angle epifluorescence microscopy (Konopka and Bednarek, 2008), which, like TIRF microscopy, reduces background fluorescence but allows for the imaging of proteins farther from the coverslip, such as those in the plasma membrane of plant cells that are separated from the coverslip by the cell wall (Konopka et al., 2008; Konopka and Bednarek, 2008). To quantify photobleaching rates, time lapse recordings were collected (Supplemental Movie S1), and fluorescence intensity traces for individual GFP-containing particles were measured using ImageJ (see Materials and Methods). Instead of exhibiting discrete steps, the intensity changes during photobleaching for many traces appeared to be relatively smooth (Figure 1A and Supplemental Movie S1), suggesting that the number of fluorophores per particle is relatively high.

Bottom Line: The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data.The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle.These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Huck Institutes of the Life Sciences, University Park, PA 16802 Interdisciplinary Graduate Degree Program in Cell and Developmental Biology, Huck Institutes of the Life Sciences, University Park, PA 16802.

Show MeSH
Related in: MedlinePlus