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

Abstract

The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. 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. In conclusion, 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 complexmore » composition and stoichiometry.« less

Authors:
 [1];  [2];  [3];  [1]
+ Show Author Affiliations
  1. Pennsylvania State Univ., University Park, PA (United States). Huck Inst. of the Life Sciences, Dept. of Biomedical Engineering
  2. Pennsylvania State Univ., University Park, PA (United States). Dept. of Biomedical Engineering
  3. Pennsylvania State Univ., University Park, PA (United States). Huck Inst. of the Life Sciences, Dept. of Biology
Publication Date:
Research Org.:
Energy Frontier Research Centers (EFRC), Washington, D.C. (United States). Center for Lignocellulose Structure and Formation (CLSF)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)
Contributing Org.:
CLSF partners with Pennsylvania State University (lead); North Carolina State University; University of Rhode Island; Virginia Tech University
OSTI Identifier:
1168144
Grant/Contract Number:  
SC0001090; R01GM100076
Resource Type:
Accepted Manuscript
Journal Name:
Molecular Biology of the Cell
Additional Journal Information:
Journal Volume: 25; Journal Issue: 22; Journal ID: ISSN 1059-1524
Publisher:
American Society for Cell Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 09 BIOMASS FUELS; biofuels (including algae and biomass); bio-inspired; membrane; carbon sequestration; materials and chemistry by design; synthesis (self-assembly)

Citation Formats

Chen, Yalei, Deffenbaugh, Nathan C., Anderson, Charles T., and Hancock, William O. Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex. United States: N. p., 2014. Web. doi:10.1091/mbc.E14-06-1146.
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Chen, Yalei, Deffenbaugh, Nathan C., Anderson, Charles T., & Hancock, William O. Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex. United States. https://doi.org/10.1091/mbc.E14-06-1146
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Chen, Yalei, Deffenbaugh, Nathan C., Anderson, Charles T., and Hancock, William O. Wed . "Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex". United States. https://doi.org/10.1091/mbc.E14-06-1146. https://www.osti.gov/servlets/purl/1168144.
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@article{osti_1168144,
title = {Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex},
author = {Chen, Yalei and Deffenbaugh, Nathan C. and Anderson, Charles T. and Hancock, William O.},
abstractNote = {The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. 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. In conclusion, 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.},
doi = {10.1091/mbc.E14-06-1146},
journal = {Molecular Biology of the Cell},
number = 22,
volume = 25,
place = {United States},
year = {Wed Sep 17 00:00:00 EDT 2014},
month = {Wed Sep 17 00:00:00 EDT 2014}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1091/mbc.E14-06-1146
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Cited by: 44 works
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Figures / Tables:

FIGURE 1 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,more » 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($μ, σ^2$) and Normal($0, δ^2$), 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.« less
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Works referenced in this record:

Bayes Factors
journal, June 1995

Estimating the Dimension of a Model
journal, March 1978

The Cell Biology of Cellulose Synthesis
journal, April 2014

Stoichiometry of the KCNQ1 - KCNE1 ion channel complex
journal, October 2010

  • Nakajo, K.; Ulbrich, M. H.; Kubo, Y.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 44
  • DOI: 10.1073/pnas.1010354107

Interactions among three distinct CesA proteins essential for cellulose synthesis
journal, January 2003

  • Taylor, N. G.; Howells, R. M.; Huttly, A. K.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 3
  • DOI: 10.1073/pnas.0337628100

A Comparison of Step-Detection Methods: How Well Can You Do?
journal, January 2008

Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana
journal, September 2007

  • Desprez, T.; Juraniec, M.; Crowell, E. F.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 39
  • DOI: 10.1073/pnas.0706569104

Stoichiometry and turnover in single, functioning membrane protein complexes
journal, September 2006

  • Leake, Mark C.; Chandler, Jennifer H.; Wadhams, George H.
  • Nature, Vol. 443, Issue 7109
  • DOI: 10.1038/nature05135

Subunit counting in membrane-bound proteins
journal, March 2007

  • Ulbrich, Maximilian H.; Isacoff, Ehud Y.
  • Nature Methods, Vol. 4, Issue 4
  • DOI: 10.1038/nmeth1024

Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma
journal, November 2012

  • Thomas, Lynne H.; Forsyth, V. Trevor; Šturcová, Adriana
  • Plant Physiology, Vol. 161, Issue 1
  • DOI: 10.1104/pp.112.206359

Neck Linker Length Determines the Degree of Processivity in Kinesin-1 and Kinesin-2 Motors
journal, May 2010

Assembly dynamics of microtubules at molecular resolution
journal, June 2006

  • Kerssemakers, Jacob W. J.; Laura Munteanu, E.; Laan, Liedewij
  • Nature, Vol. 442, Issue 7103
  • DOI: 10.1038/nature04928

Tertiary model of a plant cellulose synthase
journal, April 2013

  • Sethaphong, L.; Haigler, C. H.; Kubicki, J. D.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 18
  • DOI: 10.1073/pnas.1301027110

Nanostructure of cellulose microfibrils in spruce wood
journal, November 2011

  • Fernandes, A. N.; Thomas, L. H.; Altaner, C. M.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 47
  • DOI: 10.1073/pnas.1108942108

Kinetic study of de novo chromophore maturation of fluorescent proteins
journal, July 2011

  • Iizuka, Ryo; Yamagishi-Shirasaki, Mai; Funatsu, Takashi
  • Analytical Biochemistry, Vol. 414, Issue 2
  • DOI: 10.1016/j.ab.2011.03.036

Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis
journal, September 2007

  • Persson, S.; Paredez, A.; Carroll, A.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 39
  • DOI: 10.1073/pnas.0706592104

Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro
journal, February 2010

Sucrose Synthase is an Integral Component of the Cellulose Synthesis Machinery
journal, January 2010

  • Fujii, S.; Hayashi, T.; Mizuno, K.
  • Plant and Cell Physiology, Vol. 51, Issue 2
  • DOI: 10.1093/pcp/pcp190

Direct observation of kinesin stepping by optical trapping interferometry
journal, October 1993

  • Svoboda, Karel; Schmidt, Christoph F.; Schnapp, Bruce J.
  • Nature, Vol. 365, Issue 6448
  • DOI: 10.1038/365721a0

Dynamics of Arabidopsis Dynamin-Related Protein 1C and a Clathrin Light Chain at the Plasma Membrane
journal, May 2008

  • Konopka, Catherine A.; Backues, Steven K.; Bednarek, Sebastian Y.
  • The Plant Cell, Vol. 20, Issue 5
  • DOI: 10.1105/tpc.108.059428

Cellulosic Biofuels
journal, June 2009

mixtools : An R Package for Analyzing Finite Mixture Models
journal, January 2009

  • Benaglia, Tatiana; Chauveau, Didier; Hunter, David R.
  • Journal of Statistical Software, Vol. 32, Issue 6
  • DOI: 10.18637/jss.v032.i06

What Do We Really Know about Cellulose Biosynthesis in Higher Plants?
journal, February 2010

An objective, model-independent method for detection of non-uniform steps in noisy signals
journal, November 2008

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    [ × clear filter / sort ]

    Works referencing / citing this record:

    Single Molecule Data Analysis: An Introduction
    book, August 2017

    Nanoscopic Stoichiometry and Single‐Molecule Counting
    journal, May 2019

    • Nino, Daniel; Djayakarsana, Daniel; Milstein, Joshua N.
    • Small Methods, Vol. 3, Issue 10
    • DOI: 10.1002/smtd.201900082

    Kinetics of Nucleotide-Dependent Structural Transitions in the Kinesin-1 Hydrolysis Cycle
    journal, February 2016

    • Mickolajczyk, Keith J.; Deffenbaugh, Nathan C.; Ortega-Arroyo, Jaime
    • Biophysical Journal, Vol. 110, Issue 3
    • DOI: 10.1016/j.bpj.2015.11.1073

    Kinetic analysis methods applied to single motor protein trajectories
    journal, January 2018

    • Nord, A. L.; Pols, A. F.; Depken, M.
    • Physical Chemistry Chemical Physics, Vol. 20, Issue 27
    • DOI: 10.1039/c8cp03056a

    The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State
    journal, July 2016

    • Chen, Geng-Yuan; Mickolajczyk, Keith J.; Hancock, William O.
    • Journal of Biological Chemistry, Vol. 291, Issue 39
    • DOI: 10.1074/jbc.m116.730697

    The S6 gate in regulatory Kv6 subunits restricts heteromeric K+ channel stoichiometry
    journal, October 2018

    • Pisupati, Aditya; Mickolajczyk, Keith J.; Horton, William
    • Journal of General Physiology, Vol. 150, Issue 12
    • DOI: 10.1085/jgp.201812121

    A novel method to accurately locate and count large numbers of steps by photobleaching
    journal, November 2016

    • Tsekouras, Konstantinos; Custer, Thomas C.; Jashnsaz, Hossein
    • Molecular Biology of the Cell, Vol. 27, Issue 22
    • DOI: 10.1091/mbc.e16-06-0404

    Kinetic analysis methods applied to single motor protein trajectories
    journal, January 2018

    • Nord, A. L.; Pols, A. F.; Depken, M.
    • Physical Chemistry Chemical Physics
    • DOI: 10.1101/287748

    The Orphan Kinesin PAKRP2 Achieves Processive Motility Via Noncanonical Stepping
    journal, September 2018

    • Gicking, Allison M.; Wang, Pan; Liu, Chun
    • Biophysical Journal
    • DOI: 10.1101/430736

    Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation
    journal, May 2019

    • Ma, Wenzhe; Chen, Maorong; Kang, Hong
    • Proceedings of the National Academy of Sciences
    • DOI: 10.1101/624882

    Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle
    journal, December 2015

    • Mickolajczyk, Keith J.; Deffenbaugh, Nathan C.; Ortega Arroyo, Jaime
    • Proceedings of the National Academy of Sciences, Vol. 112, Issue 52
    • DOI: 10.1073/pnas.1517638112
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