title: High-throughput fluorescence correlation spectroscopy enables analysis of proteome dynamics in living cells.
abstract: To understand the function of cellular protein networks, spatial and temporal context is essential. Fluorescence correlation spectroscopy (FCS) is a single-molecule method to study the abundance, mobility and interactions of fluorescence-labeled biomolecules in living cells. However, manual acquisition and analysis procedures have restricted live-cell FCS to short-term experiments of a few proteins. Here, we present high-throughput (HT)-FCS, which automates screening and time-lapse acquisition of FCS data at specific subcellular locations and subsequent data analysis. We demonstrate its utility by studying the dynamics of 53 nuclear proteins. We made 60,000 measurements in 10,000 living human cells, to obtain biophysical parameters that allowed us to classify proteins according to their chromatin binding and complex formation. We also analyzed the cell-cycle-dependent dynamics of the mitotic kinase complex Aurora B/INCENP and showed how a rise in Aurora concentration triggers two-step complex formation. We expect that throughput and robustness will make HT-FCS a broadly applicable technology for characterizing protein network dynamics in cells.
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Wachsmuth
authors.affiliation: 1] Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Heidelberg, Germany. [2] Theoretical Bioinformatics, German Cancer Research Center/BioQuant, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Conrad
authors.affiliation: Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Bulkescher
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Birgit
authors.identifier: 0000000296108854
authors.initials: B
authors.lastname: Koch
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Mahen
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Isokane
authors.affiliation: 1] Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany. [2] Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Pepperkok
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
mesh_terms: D057166:High-Throughput Screening Assays / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism*
chemical_list: D020543:Proteome