Abstract
Here, we present the full integration of a proximity ligation assay (PLA) on a microfluidic chip for systematic cell signaling studies. PLA is an in situ technology for the detection of protein interaction, post-translational modification, concentration, and cellular location with single-molecule resolution. Analytical performance advances on chip are achieved, including full automation of the biochemical PLA steps, target multiplexing, and reduction of antibody consumption by 2 orders of magnitude relative to standard procedures. In combination with a microfluidic cell-culturing platform, this technology allows one to gain control over 128 cell culture microenvironments. We demonstrate the use of the combined cell culture and protein analytic assay on chip by characterizing the Akt signaling pathway upon PDGF stimulation. Signal transduction is detected by monitoring the phosphorylation states of Akt, GSK-3β, p70S6K, S6, Erk1/2, and mTOR and the cellular location of FoxO3a in parallel with the PLA. Single-cell PLA results revealed for Akt and direct targets of Akt a maximum activation time of 4 to 8 min upon PDGF stimulation. Activation times for phosphorylation events downward in the Akt signaling pathway including the phosphorylation of S6, p70S6K, and mTOR are delayed by 8 to 10 min or exhibit a response time of at least 1 h. Quantitative confirmation of the Akt phosphorylation signal was determined with the help of a mouse embryonic fibroblast cell line deficient for rictor. In sum, this work with a miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry.
Highlights
From the ‡Microfluidic and Biological Engineering, IMTEK - Department of Microsystems Engineering, University of Freiburg, GeorgesKoehler-Allee 103, 79110 Freiburg, Germany; §Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; ¶BIOSS - Centre for Biological Signalling Studies, University of Freiburg, 79108 Freiburg, Germany; ʈBiozentrum, University of Basel, CH 4056, Basel, Switzerland; **Max Planck Institute of Immunobiology and Epigenetics, D-79108 Freiburg, Germany; ‡‡Biology III, Faculty of Biology, University of Freiburg, 79108 Freiburg, Germany
We integrated 128 cell cultures together with the proximity ligation assay in a high parallel fashion on a microfluidic chip
We used the technological advantages of integrated microfluidic flow logics to obtain temporal and spatial resolution of protein phosphorylation and translocation changes during growth factor activation of the Akt signaling pathway in multiple fibroblast cell lines
Summary
Antibody; iRicKO, rictor inducible deficient mouse embryonic fibroblast cell line; MEF, mouse embryonic fibroblast; mTORC, mTOR complex; PDGF, platelet-derived growth factor; PDMS, polydimethylsiloxane; PIP3, phosphatidylinositol[3,4,5]-trisphosphate; PLA, proximity ligation assay. Chip platforms combining time-lapsed microscopy with automated cell culturing or with fully integrated workflows of immunofluorescence assays [11] are the first steps toward complete analysis systems. The microfluidic chip is made of multilayered polydimethylsiloxane (PDMS) and combines a perfusion system for cell culturing and stimulation with a multiplexed PLA. Fixation of the cell cultures with paraformaldehyde at different time points after stimulation maintained the signaling state of the cell on chip and made it accessible for subsequential analysis with the PLA. Multiplexing of the PLA along row elements of the matrix design with primary Abs allowed the measurement of systematic and quantitative signal dynamics of six different phosphorylation sites and one protein translocation event in the Akt pathway in response to PDGF stimulation. We first describe the functionalities of the microfluidic chip platform and demonstrate its use for general signaling research
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