Doug Auld, Ph.D. Novartis Institutes for BioMedical Research

High-Throughput Image Analysis of Proteome Dynamics in Live Cells

Assays for measuring dynamic changes in cells often involve the use of imaging techniques such as FRAP- or FRET-based methods. A promising technique to measure single molecule fluorescent intensity changes is fluorescence correlation spectroscopy (FCS), which measures fluctuations in fluorescent intensity over a microvolume (∼1 μm3). FCS can measure molecular properties such as size, diffusion properties, and brightness of labeled molecules. However, FCS is typically a manual procedure requiring both tedious data collection and analysis routines. In this paper, a plate-based high-throughput workflow for FCS is described (see figure). To automate the image collection process, the authors used the software Micropilot (see ADDT Literature Search & Review, June 2011, p. 199). This software uses a machine-learning algorithm to detect a region of interest based on desired parameters automatically. Following a low-resolution scan, regions meeting preselected criteria are then subjected to high-resolution analysis. In the HT-FCS workflow, the cells in the microtiter plate wells are automatically scanned at regular grid positions, and FCS data is then collected on suitable cells (see figure). Single-molecule fluctuation data are then processed in a software module called the FluctuationAnalyzer, which automatically corrects for photobleaching and cross-talk artifacts and fits the data to diffusion models. The workflow was applied to two biological processes. One involved measuring chromatin interactions using 53 different EYFP-tagged proteins in live HeLa cells that stably co-expressed a histone H2B-mCherry reporter for chromatin. In total, >60,000 images were collected over more than 10,000 cells in 69 overnight sessions where data analysis was automatically coupled to image collection. A total of 18 different parameters per protein were calculated, which were then clustered to describe diffusion and chromatin binding characteristics. In a second case, the HT-FCS platform was applied in a time series to measure cell cycle dynamics. The HT-FCS requires labeled proteins, which may alter the function/kinetics in cells and analysis is currently only applied to complexes of two proteins; multiprotein complex will have much more complicated analysis. However, this paper describes a workflow to enable measurement of biophysical parameters of proteins in live cells on a proteomic scale using an automated process, which should complement proteomic methods based on LC/MS analysis of cell lysates.

HT-FCS workflow. (a) “Screening” and “time-lapse” are two instances of HT-FCS workflows that employ automated confocal imaging to control FC(C)S measurements of large sets of proteins or for detailed time-resolved analysis of smaller sets of proteins. In screening mode, a regular grid of positions in one or several wells of a plate is imaged in the confocal laser scanning microscope (CLSM). Position by position, an automatically focused high-resolution image is acquired, segmented and classified by Micropilot to identify cells suitable for FCS and automatically position FCS measurement points (marked with cross-hairs) in nucleus and cytoplasm. If no suitable cell is found, the position is skipped. In time-lapse mode, several positions with suitable cells are first selected; the system then tracks the cells over time at defined intervals. Also here, Micropilot automatically determines the subcellular coordinates of FCS measurement points. The system continues with the next position and, after all positions are measured, proceeds after the specified interval to the next time point. Scale bars, 5 μm. (b) Multistep automated processing of FCS data. The FluctuationAnalyzer module collects and processes the raw FCS single-molecule fluctuation data. For each measurement, correction factors for photobleaching and other slow fluctuations, background signal and spectral cross-talk are extracted from the raw data. Then the photobleaching-corrected correlation functions are computed and fitted with appropriate diffusion and interaction models. The resulting parameters are adjusted by the correction factors to yield physical parameters such as concentrations and diffusion coefficients. These are analyzed statistically for each protein in a screening application and for each time point in a time-lapse application, yielding tables, histograms and time-series of the parameters of interest.

* Abstract from Nat Biotech 2015; Vol. 33, p. 384–389

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/INCENP5 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.

Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Nat Biotech titled "High-throughput fluorescence correlation spectroscopy enables analysis of proteome dynamics in living cells." Authors of the paper are Wachsmuth M, Conrad C, Bulkescher J, Koch B, Mahen R, Isokane M, Pepperkok R, Ellenberg J

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