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

Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; Hancock, William O.

doi:10.1091/mbc.E14-06-1146

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

Journal Article · Wed Sep 17 00:00:00 EDT 2014 · Molecular Biology of the Cell

DOI:https://doi.org/10.1091/mbc.E14-06-1146· OSTI ID:1168144

Chen, Yalei ^[1]; Deffenbaugh, Nathan C. ^[2]; Anderson, Charles T. ^[3]; Hancock, William O. ^[1]

Pennsylvania State Univ., University Park, PA (United States). Huck Inst. of the Life Sciences, Dept. of Biomedical Engineering
Pennsylvania State Univ., University Park, PA (United States). Dept. of Biomedical Engineering
Pennsylvania State Univ., University Park, PA (United States). Huck Inst. of the Life Sciences, Dept. of Biology

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.

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Research Organization:: Energy Frontier Research Centers (EFRC), Washington, D.C. (United States). Center for Lignocellulose Structure and Formation (CLSF)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)

Contributing Organization:: CLSF partners with Pennsylvania State University (lead); North Carolina State University; University of Rhode Island; Virginia Tech University

Grant/Contract Number:: SC0001090; R01GM100076

OSTI ID:: 1168144

Journal Information:: Molecular Biology of the Cell, Vol. 25, Issue 22; ISSN 1059-1524

Publisher:: American Society for Cell BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 44 works

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Kinetics of Nucleotide-Dependent Structural Transitions in the Kinesin-1 Hydrolysis Cycle Mickolajczyk, Keith J.; Deffenbaugh, Nathan C.; Ortega-Arroyo, Jaime Biophysical Journal, Vol. 110, Issue 3 https://doi.org/10.1016/j.bpj.2015.11.1073	journal	February 2016
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