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. 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.
authors.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.
authors.forename: Yalei
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biomedical Engineering, Huck Institutes of the Life Sciences, University Park, PA 16802.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Deffenbaugh
authors.affiliation: Interdisciplinary Graduate Degree Program in Cell and Developmental Biology, Huck Institutes of the Life Sciences, University Park, PA 16802 Department of Biology, Pennsylvania State University, University Park, PA 16802.
authors.forename: Charles T
authors.initials: CT
authors.lastname: Anderson
authors.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 wohbio@engr.psu.edu.
authors.forename: William O
authors.initials: WO
authors.lastname: Hancock
mesh_terms: D000465:Algorithms; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D038761:Photobleaching; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; C516179:At5g05170 protein, Arabidopsis; D005964:Glucosyltransferases