title: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy.
abstract: Probing the oligomeric state of abundant molecules, such as membrane proteins in intact cells, is essential, but has not been straightforward. We address this challenge with a simple counting strategy that is capable of reporting the oligomeric state of dense, membrane-bound protein complexes. It is based on single-molecule localization microscopy to super-resolve protein structures in intact cells and basic quantitative evaluation. We validate our method with membrane-bound monomeric CD86 and dimeric cytotoxic T-lymphocyte-associated protein as model proteins and confirm their oligomeric states. We further detect oligomerization of CD80 and vesicular stomatitis virus glycoprotein and propose coexistence of monomers and dimers for CD80 and trimeric assembly of the viral protein at the cell membrane. This approach should prove valuable for researchers striving for reliable molecular counting in cells.
authors.affiliation: Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Fricke
authors.affiliation: Department for Bioinformatics and Functional Genomics, Bioquant and Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, and Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 267, 69120 Heidelberg, Germany.
authors.forename: Joel
authors.initials: J
authors.lastname: Beaudouin
authors.affiliation: Department for Bioinformatics and Functional Genomics, Bioquant and Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, and Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 267, 69120 Heidelberg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Eils
authors.affiliation: Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
mesh_terms: D000465:Algorithms; D018122:B7-1 Antigen / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008853:Microscopy / Q000379:methods*; D008962:Models, Theoretical; D055503:Protein Multimerization; D015203:Reproducibility of Results; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D018122:B7-1 Antigen; D008565:Membrane Proteins; D014764:Viral Proteins