Time-Resolved Single Cell, Sub-Cellular Compartmentalized Proteomics, Combining Precise Microfluidics, Deconvolution and Ultrasensitive Single-Molecule Microscopy

Abstract

Optical microscopy is emerging as a powerful tool in quantified in vivo proteomics, allowing protein copy number determination in live cells and even sub-cellular compartments, but to date without dynamic information. Here, we have combined a novel microfluidics technology, Fluicell(1) with cell tracking and deconvolution software(2), to create a new high-throughput dynamic proteomics platform, capable of quantifying copy number changes in sub-cellular compartments in live cells in response to extracellular micro-environmental changes. We used a canonical signal transduction pathway in eukaryotic budding yeast cells as a test system to observe the localisation changes of the Mig1 transcription factor between nucleus and cytoplasm in response to extra-cellular glucose concentration changes. Combined with single-molecule Slimfield measurements of the dynamics of individual Mig1 molecular clusters, we find that clusters of Mig1 molecules translocate across the nuclear envelope and bind target genes, enabling a tuneable response to the glucose concentration signal. By combining microfluidics control of the input signal, with molecular quantification of the output on a single cell level, this new platform generates novel insight into the precise mechanisms of cellular processes.1. A. Ainla, G. D. Jeffries, R. Brune, O. Orwar, A. Jesorka, A multifunctional pipette. Lab Chip. 12, 1255-1261 (2012).2. A. J. M. Wollman, M. C. Leake, Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss.184, 401-24 (2015).