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Book ChapterDOI

Chemogenetic Control of Protein Localization and Mammalian Cell Signaling by SLIPT.

01 Jan 2021-Methods of Molecular Biology (Humana, New York, NY)-Vol. 2312, pp 237-251
TL;DR: In this article, a self-localizing ligand-induced protein translocation (SLIPT) system was proposed to control protein localization in living mammalian cells using synthetic SLs.
Abstract: Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.
References
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Journal ArticleDOI
TL;DR: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis that facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system.
Abstract: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.

43,540 citations

Journal ArticleDOI
TL;DR: P piggyBac transposon–based reprogramming may be used to generate therapeutically applicable iPSCs and could be identified by negative selection.
Abstract: Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.

613 citations

Journal ArticleDOI
TL;DR: An optimized backbone for the rapid development of a highly sensitive intramolecular fluorescence resonance energy transfer (FRET) biosensor is reported, which includes an optimized pair of fluorescent proteins and a long flexible linker ranging from 116 to 244 amino acids in length.
Abstract: We report an optimized backbone for the rapid development of a highly sensitive intramolecular fluorescence resonance energy transfer (FRET) biosensor, which includes an optimized pair of fluoresce...

535 citations

Journal ArticleDOI
19 Jun 2014-Cell
TL;DR: This work describes an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells that converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy and allows for calculation of active kinase concentrations via a mathematical model.

438 citations