A Microfluidic Device for Temporally Controlled Gene Expression and Long-Term Fluorescent Imaging in Unperturbed Dividing Yeast Cells

Abstract

BackgroundImaging single cells with fluorescent markers over multiple cell cycles is a powerful tool for unraveling the mechanism and dynamics of the cell cycle. Over the past ten years, microfluidic techniques in cell biology have emerged that allow for good control of growth environment. Yet the control and quantification of transient gene expression in unperturbed dividing cells has received less attention.Methodology/Principal FindingsHere, we describe a microfluidic flow cell to grow Saccharomyces Cerevisiae for more than 8 generations (≈12 hrs) starting with single cells, with controlled flow of the growth medium. This setup provides two important features: first, cells are tightly confined and grow in a remarkably planar array. The pedigree can thus be determined and single-cell fluorescence measured with 3 minutes resolution for all cells, as a founder cell grows to a micro-colony of more than 200 cells. Second, we can trigger and calibrate rapid and transient gene expression using reversible administration of inducers that control the GAL1 or MET3 promoters. We then show that periodic 10–20 minutes gene induction pulses can drive many cell division cycles with complete coherence across the cell cluster, with either a G1/S trigger (cln1 cln2 cln3 MET3-CLN2) or a mitotic trigger (cdc20 GALL-CDC20).Conclusions/SignificanceIn addition to evident cell cycle applications, this device can be used to directly measure the amount and duration of any fluorescently scorable signal-transduction or gene-induction response over a long time period. The system allows direct correlation of cell history (e.g., hysteresis or epigenetics) or cell cycle position with the measured response.

Interallelic interaction and gene regulation in budding yeast

“…2A). GFP and Venus have comparable brightness and maturation rate in yeast, allowing us to directly compare their intensity (27,28). Using a flow cell device that allows rapid media exchange, we pregrew the cells in glucose, switched to galactose + raffinose + 0.05% glucose, and monitored the GFP and YFP intensity during the induction process ( Fig.…”

“…Briefly, live yeast cells were placed into CellAsic flow-cells, pregrown in 2% glucose for 4 h, and induced in 3% galactose, 2% raffinose, and 0.05% glucose for 8 h. In the meantime, we took GFP and YFP fluorescent images every 5 min. The movies were analyzed by using semiautomated data analysis software written in Matlab (27) to obtain the fluorescent intensity of individual cells at each movie frame. We then eliminated the cross-talk between the GFP and YFP signals through linear subtraction (28), which resulted in the single cell activation curves shown in Fig.…”

Dynamic analysis of MAPK signaling using a high-throughput microfluidic single-cell imaging platform

“…Single-pulse experiments. Microfluidics offers unique opportunities for measuring cellular response to precisely controlled time-varying stimulation and with high temporal resolution (19,20,38). We used this temporal control to investigate differences in network memory between mutants.…”

Déplétion des nucléosomes dans les régions promotrices

“…Dans la plupart des études précédentes, les données transcriptionnelles étaient exprimées comme la moyenne de mesures faites dans des populations de cellules. Au contraire, nous avons travaillé sur des cellules individuelles de levures et mesuré in vivo, dans des expériences de vidéomi-croscopie de fluorescence, la transcription de gènes spécifiques en fonction du temps [12][13][14]. Deux promoteurs de gènes régulés au cours du cycle cellulaire, CLN2 (une cycline active lors de la transition G1/ S) et HO 1 , ont été sélectionnés pour étudier l'influence des NDR sur l'activation transcriptionnelle.…”