continuous-high-resolution-microscopic-observation-replicative-aging

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

ETH Zurich

Apoptosis is regulated by several signaling pathways which are extensively linked by crosstalks. Boolean or logical modeling has become a promising approach to capture the qualitative behavior of such complex networks. Here we built a large-scale literature-based Boolean model of the central intrinsic and extrinsic apoptosis pathways as well as pathways connected with them. The model responds to several external stimuli such as Fas ligand, TNF-alpha, UV-B irradiation, interleukin-1beta and insulin. Timescales and multi-value node logic were used and turned out to be indispensable to reproduce the behavior of the apoptotic network. The coherence of the model was experimentally validated. Thereby an UV-B dose-effect is shown for the first time in mouse hepatocytes. Analysis of the model revealed a tight regulation emerging from high connectivity and spanning crosstalks and a particular importance of feedback loops. An unexpected feedback from Smac release to RIP could further increase complex II formation. The introduced Boolean model provides a comprehensive and coherent description of the apoptosis network behavior. It gives new insights into the complex interplay of pro- and antiapoptotic factors and can be easily expanded to other signaling pathways.

ON/OFF and beyond--a boolean model of apoptosis.

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform.

This protocol describes the production and operation of a microfluidic dissection platform for long-term, high-resolution imaging of budding yeast cells. At the core of this platform is an array of micropads that trap yeast cells in a single focal plane. Newly formed daughter cells are subsequently washed away by a continuous flow of fresh culture medium. In a typical experiment, 50-100 cells can be tracked during their entire replicative lifespan. Apart from aging-related research, the microfluidic platform can also be a valuable tool for other studies requiring the monitoring of single cells over time. Here we provide step-by-step instructions on how to fabricate the silicon wafer mold, how to produce and operate the microfluidic device and how to analyze the obtained data. Production of the microfluidic dissection platform and setting up an aging experiment takes ~7 h.

Article	Year
ON/OFF and beyond--a boolean model of apoptosis. PLoS computational biology\| PubMed ID: 20011108	2009
Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22421136	2012
Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast. Nature protocols\| PubMed ID: 23640166	2013

Ima Avalos Vizcarra

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