title: High-throughput microfluidics to control and measure signaling dynamics in single yeast cells.
abstract: Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. By using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, while collecting data for thousands of single cells. Compared with other protocols, the present protocol is relatively easy to adopt and of higher throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms.
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA. [3] Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA.
authors.forename: Anders S
authors.identifier: 0000000175407858
authors.initials: AS
authors.lastname: Hansen
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Hao
authors.affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA. [3] Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA. [4] Department of Molecular and Cellular Biology, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA.
authors.forename: Erin K
authors.initials: EK
authors.lastname: O'Shea
mesh_terms: D004268:DNA-Binding Proteins / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D046210:Microfluidic Analytical Techniques; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction; D059008:Time-Lapse Imaging; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C081935:MSN2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors