title: Single molecule analysis reveals coexistence of stable serotonin transporter monomers and oligomers in the live cell plasma membrane.
abstract: The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.
authors.affiliation: From the Institute of Applied Physics, Vienna University of Technology, Getreidemarkt 9, A-1060 Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Anderluh
authors.forename: Enrico
authors.initials: E
authors.lastname: Klotzsch
authors.forename: Alexander W A F
authors.initials: AW
authors.lastname: Reismann
authors.forename: Mario
authors.initials: M
authors.lastname: Brameshuber
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
authors.forename: Amy Hauck
authors.initials: AH
authors.lastname: Newman
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins
keywords: Membrane Proteins; Oligomerization; Protein Dynamics; Protein-Protein Interactions; Serotonin Transporters; Single Molecule Biophysics