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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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Estimating copy number for kinesin-4xGFP. (A) Trace of kinesin-4xGFP bleaching (black) with steps fitted by Tdetector2 (red). (B) The BIC search leads to a best fit of k = 4 Gaussians for fitting the step size distribution. (C) Estimating the unitary step size (60.8 a.u.) from the step size distribution (455 total detected steps). The mean values of the four modes were 63.9, 109.9, 165.8, and 258.1 a.u., relative weights were 0.622, 0.289, 0.062, and 0.027, and the SD was 19.6 a.u. (D) Copy number distribution. There were two peaks, centered at 3.28 and 6.65. These peaks are consistent with the binomial nature leading to a slight shift from four toward lower copy number and with a double-aggregate population at roughly twice the copy number of the first peak. Histograms (black boxes) are also plotted in C and D for reference but not used in the GMM fitting.
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Figure 7: Estimating copy number for kinesin-4xGFP. (A) Trace of kinesin-4xGFP bleaching (black) with steps fitted by Tdetector2 (red). (B) The BIC search leads to a best fit of k = 4 Gaussians for fitting the step size distribution. (C) Estimating the unitary step size (60.8 a.u.) from the step size distribution (455 total detected steps). The mean values of the four modes were 63.9, 109.9, 165.8, and 258.1 a.u., relative weights were 0.622, 0.289, 0.062, and 0.027, and the SD was 19.6 a.u. (D) Copy number distribution. There were two peaks, centered at 3.28 and 6.65. These peaks are consistent with the binomial nature leading to a slight shift from four toward lower copy number and with a double-aggregate population at roughly twice the copy number of the first peak. Histograms (black boxes) are also plotted in C and D for reference but not used in the GMM fitting.

Mentions: To validate the ability of the developed methods to estimate copy numbers from a protein with a known number of GFP subunits, we engineered a kinesin construct containing four GFPs (see Materials and Methods). Proteins were attached to the coverslip surface through nonspecific interactions and imaged using TIRF microscopy (Shastry and Hancock, 2010). Steps were fitted to the 71 acquired photobleaching traces using the Tdetector2 algorithm (Figure 7A), resulting in 455 detected steps. The step size distribution was fitted using the Gaussian mixture model, and on the basis of the calculated BIC values, the optimal number of modes was determined to be four (Figure 7B). When the step size distribution was fitted using four modes, the corresponding unitary step size was determined to be 60.8 a.u. (Figure 7C). On the basis of this step size and the SD of noise in the traces, the SNR was calculated to be 1.1 for these measurements.


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)

Estimating copy number for kinesin-4xGFP. (A) Trace of kinesin-4xGFP bleaching (black) with steps fitted by Tdetector2 (red). (B) The BIC search leads to a best fit of k = 4 Gaussians for fitting the step size distribution. (C) Estimating the unitary step size (60.8 a.u.) from the step size distribution (455 total detected steps). The mean values of the four modes were 63.9, 109.9, 165.8, and 258.1 a.u., relative weights were 0.622, 0.289, 0.062, and 0.027, and the SD was 19.6 a.u. (D) Copy number distribution. There were two peaks, centered at 3.28 and 6.65. These peaks are consistent with the binomial nature leading to a slight shift from four toward lower copy number and with a double-aggregate population at roughly twice the copy number of the first peak. Histograms (black boxes) are also plotted in C and D for reference but not used in the GMM fitting.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Estimating copy number for kinesin-4xGFP. (A) Trace of kinesin-4xGFP bleaching (black) with steps fitted by Tdetector2 (red). (B) The BIC search leads to a best fit of k = 4 Gaussians for fitting the step size distribution. (C) Estimating the unitary step size (60.8 a.u.) from the step size distribution (455 total detected steps). The mean values of the four modes were 63.9, 109.9, 165.8, and 258.1 a.u., relative weights were 0.622, 0.289, 0.062, and 0.027, and the SD was 19.6 a.u. (D) Copy number distribution. There were two peaks, centered at 3.28 and 6.65. These peaks are consistent with the binomial nature leading to a slight shift from four toward lower copy number and with a double-aggregate population at roughly twice the copy number of the first peak. Histograms (black boxes) are also plotted in C and D for reference but not used in the GMM fitting.
Mentions: To validate the ability of the developed methods to estimate copy numbers from a protein with a known number of GFP subunits, we engineered a kinesin construct containing four GFPs (see Materials and Methods). Proteins were attached to the coverslip surface through nonspecific interactions and imaged using TIRF microscopy (Shastry and Hancock, 2010). Steps were fitted to the 71 acquired photobleaching traces using the Tdetector2 algorithm (Figure 7A), resulting in 455 detected steps. The step size distribution was fitted using the Gaussian mixture model, and on the basis of the calculated BIC values, the optimal number of modes was determined to be four (Figure 7B). When the step size distribution was fitted using four modes, the corresponding unitary step size was determined to be 60.8 a.u. (Figure 7C). On the basis of this step size and the SD of noise in the traces, the SNR was calculated to be 1.1 for these measurements.

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