title: Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins.
abstract: The formation of protein complexes or clusters in the plasma membrane is essential for many biological processes, such as signaling. We develop a tool, based on single-molecule microscopy, for following cluster formation in vivo. Detection and tracing of single autofluorescent proteins have become standard biophysical techniques. The determination of the number of proteins in a cluster, however, remains challenging. The reasons are (i) the poor photophysical stability and complex photophysics of fluorescent proteins and (ii) noise and autofluorescent background in live cell recordings. We show that, despite those obstacles, the accurate fraction of signals in which a certain (or set) number of labeled proteins reside, can be determined in an accurate an robust way in vivo. We define experimental conditions under which fluorescent proteins exhibit predictable distributions of intensity and quantify the influence of noise. Finally, we confirm our theoretical predictions by measurements of the intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in living cells. Quantification of the average number of EYFP-C10HRAS chimeras in diffraction-limited spots finally confirm that the membrane anchor of human Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of living Chinese hamster ovary cells.
authors.affiliation: Technische Universität Darmstadt, Membrane Dynamics, Department of Biology, Darmstadt, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Meckel
authors.forename: Stefan
authors.initials: S
authors.lastname: Semrau
authors.forename: Marcel J M
authors.initials: MJ
authors.lastname: Schaaf
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D055592:Biophysical Phenomena; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005453:Fluorescence; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D055620:Optical Phenomena; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)