title: Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells.
abstract: Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h.
authors.affiliation: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, China.
authors.forename: Xiaohua
authors.identifier: http://orcid.org/0000-0003-2320-4543
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory for Genetics and Breeding of Forest Trees and Ornamental Plants, Ministry of Education, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Deng
authors.affiliation: Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Unité Mixte de Recherche (UMR) 5004 Centre National de la Recherche Scientifique (CNRS) UMR 0386 Institut National de la Recherche Agronomique/Montpellier SupAgro/Université Montpellier 2, Montpellier, France.
authors.forename: Doan-Trung
authors.initials: DT
authors.lastname: Luu
authors.affiliation: Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Unité Mixte de Recherche (UMR) 5004 Centre National de la Recherche Scientifique (CNRS) UMR 0386 Institut National de la Recherche Agronomique/Montpellier SupAgro/Université Montpellier 2, Montpellier, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Maurel
authors.affiliation: Key Laboratory for Genetics and Breeding of Forest Trees and Ornamental Plants, Ministry of Education, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China.
authors.forename: Jinxing
authors.initials: J
authors.lastname: Lin
mesh_terms: D017360:Arabidopsis / Q000166:cytology*; D029681:Arabidopsis Proteins / Q000032:analysis*; D005453:Fluorescence; D008565:Membrane Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000379:methods; D061848:Optical Imaging / Q000379:methods; D059828:Plant Cells / Q000737:chemistry* / Q000648:ultrastructure; D055503:Protein Multimerization; D021122:Protein Subunits / Q000032:analysis
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D021122:Protein Subunits