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

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

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 cellu lose, 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. 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.

Original languageEnglish (US)
Pages (from-to)3630-3642
Number of pages13
JournalMolecular biology of the cell
Volume25
Issue number22
DOIs
StatePublished - Nov 5 2014

Fingerprint

Photobleaching
Practice Guidelines
Cellulose
Biopolymers
Plant Cells
Arabidopsis
Microscopy
Protein Isoforms
Proteins
Cell Membrane
cellulose synthase

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

@article{94d52734f1d4461a93cbc5ff1d30e7e3,
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 cellu lose, 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. 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.",
author = "Yalei Chen and Deffenbaugh, {Nathan C.} and Anderson, {Charles T.} and Hancock, {William O.}",
year = "2014",
month = "11",
day = "5",
doi = "10.1091/mbc.E14-06-1146",
language = "English (US)",
volume = "25",
pages = "3630--3642",
journal = "Molecular Biology of the Cell",
issn = "1059-1524",
publisher = "American Society for Cell Biology",
number = "22",

}

TY - JOUR

T1 - Molecular counting by photobleaching in protein complexes with many subunits

T2 - Best practices and application to the cellulose synthesis complex

AU - Chen, Yalei

AU - Deffenbaugh, Nathan C.

AU - Anderson, Charles T.

AU - Hancock, William O.

PY - 2014/11/5

Y1 - 2014/11/5

N2 - 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 cellu lose, 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. 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.

AB - 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 cellu lose, 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. 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.

UR - http://www.scopus.com/inward/record.url?scp=84908573135&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84908573135&partnerID=8YFLogxK

U2 - 10.1091/mbc.E14-06-1146

DO - 10.1091/mbc.E14-06-1146

M3 - Article

C2 - 25232006

AN - SCOPUS:84908573135

VL - 25

SP - 3630

EP - 3642

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

SN - 1059-1524

IS - 22

ER -