Relocation sensors to quantify signaling dynamics in live single cells

Wosika, Victoria; Pelet, Serge

doi:10.1016/j.copbio.2016.12.005

Cited by 4 publications

(2 citation statements)

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“…The characterization, insights and solutions provided in this work will allow yeast researchers to adapt this powerful tool for single cell perturbation experiments. We expect that - fueled by recent developments in microfluidics setups 26–31 and in novel single cell reporters 52–54 , as well as the need to zoom into the single cell level – these types of experiments will gain importance.…”

Section: Discussionmentioning

confidence: 99%

Quantitative characterization of the auxin-inducible degron: a guide for dynamic protein depletion in single yeast cells

Papagiannakis

Jonge

Zhang

et al. 2017

Sci Rep

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Perturbations are essential for the interrogation of biological systems. The auxin-inducible degron harbors great potential for dynamic protein depletion in yeast. Here, we thoroughly and quantitatively characterize the auxin-inducible degron in single yeast cells. We show that an auxin concentration of 0.25 mM is necessary for fast and uniform protein depletion between single cells, and that in mother cells proteins are depleted faster than their daughters. Although, protein recovery starts immediately after removal of auxin, it takes multiple generations before equilibrium is reached between protein synthesis and dilution, which is when the original protein levels are restored. Further, we found that blue light, used for GFP excitation, together with auxin results in growth defects, caused by the photo-destruction of auxin to its toxic derivatives, which can be avoided if indole-free auxin substitutes are used. Our work provides guidelines for the successful combination of microscopy, microfluidics and the auxin-inducible degron, offering the yeast community an unprecedented tool for dynamic perturbations on the single cell level.

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Section: Discussionmentioning

confidence: 99%

Quantitative characterization of the auxin-inducible degron: a guide for dynamic protein depletion in single yeast cells

Papagiannakis

Jonge

Zhang

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…3 Primary among these approaches has been the development of peptides and proteins that exhibit an increase in fluorescence as a function of phosphorylation by a specific protein kinase. [4][5][6][7][8][9][10][11][12][13][14] We previously described an approach to phosphorylation-dependent protein design, termed protein kinase-inducible domains (pKID). 15,16 In this approach, we re-engineered an EF-Hand calcium-binding domain to be responsive to protein phosphorylation, with phosphorylation leading to terbium binding and terbium luminescence.…”

Section: Introductionmentioning

confidence: 99%