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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]
  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) (SC-22); 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:
Journal Article: 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.
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. doi:10.1091/mbc.E14-06-1146.
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. doi:10.1091/mbc.E14-06-1146. https://www.osti.gov/servlets/purl/1168144.
@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},
issn = {1059-1524},
number = 22,
volume = 25,
place = {United States},
year = {2014},
month = {9}
}

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Cited by: 16 works
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