Cellular signaling is fundamentally dynamic.

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Presentation transcript:

High-Throughput Microfluidic Technologies for Systems Studies of Protein Signaling in Yeast

Cellular signaling is fundamentally dynamic. Cells exist in dynamically changing environments. www.ambion.com

Prototypical Signaling Pathway http://fig.cox.miami.edu Haploid (1N) Diploid (2N)

Experimental System for Quantitative Dynamic Cellular Signaling Modulate the chemical environment precisely over time. Resolve clonal population heterogeneity. Track and measure individual cells over time. Gene expression & phenotype High-throughput to obtain a system level understanding. Microfluidic System for Live Cell Fluorescent Microscopy

µFluidics - Multilayer Soft Lithography BLUE – cell flow lines RED – control lines 8 strains x 32 experimental conditions x time

Non-Adherent Cell Trapping Microchamber Sieve Valve

Fully Automated Live Cell Microscopy

Genetic Perturbations X Kinetic Stimuli Genetic Perturbations PRE::GFP INPUT OUTPUT

a-factor concentration dependent cell response Δh time Single step function 32 different a-factor concentrations in range 0nM to 100nM Exponential increments (ci=1.16i), i is the row number

a-factor concentration dependent cell response

a-factor concentration dependent cell response

a-factor concentration dependent cell response WT increasing a-factor

Over 3,000 Live Cell Imaging Experiments Δw Single pulse function 4 different a-factor concentrations 8 different pulse widths Δh time Δw Repeated pulse function 4 different a-factor concentrations 4 different pulse delays Δh time

Acknowledgements: Paper: Didier Falconnet Timothy Galitski Carl Hansen Antti Niemisto Susi Prinz Stephen Ramsey Ilya Shmulevich Galitski Lab: Greg Carter, Song Li Hansen Lab: Milenko Despotovic, Mark Homenuke Hieter Lab: Phil Hieter, Kirk McManus, Ben Montpetit, Jan Stoepel, Karen Yuen