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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.

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Copy number estimation for GFP-AtCESA3 particles. (A) Trace of GFP-AtCESA3 photobleaching (black) with steps fitted by Tdetector2 (blue). (B) BIC values for step detection at increasing number of Gaussians, showing the minimum at k = 6. (C) Estimation of unitary step size (445.4 a.u.) by GMM based on 730 total detected steps. Step size distribution was fitted by six Gaussians, shown in red, green, yellow, pink, and purple. Mean values were 453, 864, 1337, 1799, 2335, and 3082 a.u., relative weights were 0.4953, 0.3325, 0.1252, 0.0367, 0.0074, and 0.0027, and the SD was 160 a.u. Overall fit from GMM is shown in blue. Histogram (black boxes) is also plotted for reference but not used in the GMM fitting. (D) Copy number distribution for GFP-AtCESA3 particles. Two peaks are evident from the histograms, and fitting two Gaussians (red and green curves) gives means of 9.56 and 23.5 and ratio of 0.844 and 0.156, with SD of 4.03.
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Figure 8: Copy number estimation for GFP-AtCESA3 particles. (A) Trace of GFP-AtCESA3 photobleaching (black) with steps fitted by Tdetector2 (blue). (B) BIC values for step detection at increasing number of Gaussians, showing the minimum at k = 6. (C) Estimation of unitary step size (445.4 a.u.) by GMM based on 730 total detected steps. Step size distribution was fitted by six Gaussians, shown in red, green, yellow, pink, and purple. Mean values were 453, 864, 1337, 1799, 2335, and 3082 a.u., relative weights were 0.4953, 0.3325, 0.1252, 0.0367, 0.0074, and 0.0027, and the SD was 160 a.u. Overall fit from GMM is shown in blue. Histogram (black boxes) is also plotted for reference but not used in the GMM fitting. (D) Copy number distribution for GFP-AtCESA3 particles. Two peaks are evident from the histograms, and fitting two Gaussians (red and green curves) gives means of 9.56 and 23.5 and ratio of 0.844 and 0.156, with SD of 4.03.

Mentions: After developing an objective method for estimating subunit copy number for protein complexes tagged with large numbers of fluorophores and assessing its performance on simulated photobleaching data, we applied the technique to a set of photobleaching data for GFP-AtCESA3 particles (Figure 8A). On the basis of the trend of BIC values (Figure 8B), a model consisting of six Gaussians was used to estimate the distribution of predicted step sizes, and the final estimate for a single step was calculated to be 445.4 a.u. (Figure 8C). This step size indicates that the SNR is ∼2–2.5, within the range that our methods can reliably uncover copy number. However, in the final copy number histogram, instead of seeing a single mode as for the simulated data, two modes, one around 10 and the other around 20, are apparent (Figure 8D). This factor of 2 suggests that a subpopulation of the analyzed particles might be composed of two complexes within the focal limited spot, either because there are two populations of CSCs in cells or because pairs of CSCs occasionally exist in close proximity, especially when they are immobile, as was the case for this data set. A fit consisting of two Gaussians identifies peaks at 9.56 and 23.5 copies. Considering that protein misfolding, incomplete maturation of GFP, and bleaching events occurring before data acquisition can all potentially lead to underestimating the true number of GFPs present, we conclude that 10 copies is a lower limit for the estimated number of GFP-AtCESA3 subunits in each CSC particle.


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)

Copy number estimation for GFP-AtCESA3 particles. (A) Trace of GFP-AtCESA3 photobleaching (black) with steps fitted by Tdetector2 (blue). (B) BIC values for step detection at increasing number of Gaussians, showing the minimum at k = 6. (C) Estimation of unitary step size (445.4 a.u.) by GMM based on 730 total detected steps. Step size distribution was fitted by six Gaussians, shown in red, green, yellow, pink, and purple. Mean values were 453, 864, 1337, 1799, 2335, and 3082 a.u., relative weights were 0.4953, 0.3325, 0.1252, 0.0367, 0.0074, and 0.0027, and the SD was 160 a.u. Overall fit from GMM is shown in blue. Histogram (black boxes) is also plotted for reference but not used in the GMM fitting. (D) Copy number distribution for GFP-AtCESA3 particles. Two peaks are evident from the histograms, and fitting two Gaussians (red and green curves) gives means of 9.56 and 23.5 and ratio of 0.844 and 0.156, with SD of 4.03.
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Related In: Results  -  Collection

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Figure 8: Copy number estimation for GFP-AtCESA3 particles. (A) Trace of GFP-AtCESA3 photobleaching (black) with steps fitted by Tdetector2 (blue). (B) BIC values for step detection at increasing number of Gaussians, showing the minimum at k = 6. (C) Estimation of unitary step size (445.4 a.u.) by GMM based on 730 total detected steps. Step size distribution was fitted by six Gaussians, shown in red, green, yellow, pink, and purple. Mean values were 453, 864, 1337, 1799, 2335, and 3082 a.u., relative weights were 0.4953, 0.3325, 0.1252, 0.0367, 0.0074, and 0.0027, and the SD was 160 a.u. Overall fit from GMM is shown in blue. Histogram (black boxes) is also plotted for reference but not used in the GMM fitting. (D) Copy number distribution for GFP-AtCESA3 particles. Two peaks are evident from the histograms, and fitting two Gaussians (red and green curves) gives means of 9.56 and 23.5 and ratio of 0.844 and 0.156, with SD of 4.03.
Mentions: After developing an objective method for estimating subunit copy number for protein complexes tagged with large numbers of fluorophores and assessing its performance on simulated photobleaching data, we applied the technique to a set of photobleaching data for GFP-AtCESA3 particles (Figure 8A). On the basis of the trend of BIC values (Figure 8B), a model consisting of six Gaussians was used to estimate the distribution of predicted step sizes, and the final estimate for a single step was calculated to be 445.4 a.u. (Figure 8C). This step size indicates that the SNR is ∼2–2.5, within the range that our methods can reliably uncover copy number. However, in the final copy number histogram, instead of seeing a single mode as for the simulated data, two modes, one around 10 and the other around 20, are apparent (Figure 8D). This factor of 2 suggests that a subpopulation of the analyzed particles might be composed of two complexes within the focal limited spot, either because there are two populations of CSCs in cells or because pairs of CSCs occasionally exist in close proximity, especially when they are immobile, as was the case for this data set. A fit consisting of two Gaussians identifies peaks at 9.56 and 23.5 copies. Considering that protein misfolding, incomplete maturation of GFP, and bleaching events occurring before data acquisition can all potentially lead to underestimating the true number of GFPs present, we conclude that 10 copies is a lower limit for the estimated number of GFP-AtCESA3 subunits in each CSC particle.

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