Marc Fouet, Postdoctoral Researcher
University of California, Berkeley
The Rine lab at the University of California, Berkeley is working towards understanding mechanisms underlying establishment, maintenance, and epigenetic inheritance of gene silencing in yeast. The lab has developed a genetic strategy to capture transient losses of gene silencing of heterochromatin in S. cerevisiae, and translating these dynamic processes as a permanent modification of fluorescence expression. By using the CRASH (cre-reported altered states of heterochromatin) reporter, red fluorescent yeast cells become green once gene silencing by Sir proteins has been lost.1 Monitoring fluorescence of yeast colonies can be used to describe dynamic epigenetic phenomena quantitatively. Recently, the Rine lab began using high-throughput yeast aging analysis (HYAA) chips2 with high-resolution time-lapse microscopy to monitor the relative life span of yeast and understand better the relationship between gene silencing and aging.3 Dr. Marc Fouet, a postdoctoral researcher in the Rine lab, is working on solutions to detect events leading to a lower signal of fluorescence and on the microfluidic process intensification to image an increasing number of single cells.
Rare transcription events within heterochromatin occur in approximately 1/1000 cell divisions and cells divide every 90 minutes. In order to capture these rare events, 40 images are taken of 5,000 yeast cell traps for each sample. Images are captured every 5 to 10 minutes to measure cell division and fluorescent protein levels. Phototoxicity and photobleaching should be minimized, and resolution is increasingly important when machine learning algorithms are used to segment fluorescence data.
The bigger our field of view and the lower the exposure times are, the more cells we can image overall as less images are needed and the acquisition is faster [using the Prime 95B Scientific CMOS camera].
- Dodson AE, Rine J. Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae. Elife (2015), doi: 10.7554/eLife.05007.
- Jo MC, Liu W, Gu L, Dang W, Qin L. High-throughput analysis of yeast replicative aging using a microfluidic system. Proc Natl Acad Sci U S A (2015), doi: 10.1073/pnas.1510328112.
- Schlissel G, Krzyzanowski MK, Caudron F, Barral Y, Rine J. Aggregation of the Whi3 protein, not loss of heterochromatin, causes sterility in old yeast cells. Science (2017), doi: 10.1126/science.aaj2103.