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

2. Cellular signaling is fundamentally dynamic.
Cells exist in dynamically changing environments.

3. Prototypical Signaling Pathway
Haploid (1N)
Diploid (2N)

4. 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

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

6. Non-Adherent Cell Trapping
Microchamber
Sieve Valve

7. Fully Automated Live Cell Microscopy

8. Genetic PerturbationsX
Kinetic Stimuli
Genetic Perturbations
PRE::GFP
INPUT
OUTPUT

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

10. a-factor concentration dependent cell response

11. a-factor concentration dependent cell response

12. a-factor concentration dependent cell responseWT
increasing a-factor

13. Over 3,000 Live Cell Imaging ExperimentsΔw
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

14. 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