Reversible Chemical Dimerization by rCD1

M. Schifferer, S. Feng, F. Stein, Carsten Schultz

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

An optimal tool to unravel the role of a specific player within a cellular network or process requires its spatiotemporally resolved perturbation. Chemically induced dimerization (CID) by the rapamycin system has proven useful to induce protein dimerization or translocation with high spatiotemporal precision. Recently, we and others have added reversibility of the dimerization event as a novel feature to CID approaches. Among those, our reversible chemical dimerizer (rCD1) shows the fastest release kinetics observed, comparable to optogenetic methods. Induction and termination of enzyme activities, including phosphatidylinositol 3-kinase (PI3K) and 5-phosphatase (5Ptase), therefore allowed us to monitor the relaxation of the downstream effectors within living cells by imaging and traditional biochemical methods. Because switching off the rCD1-induced enzyme activity is sufficiently fast, it is possible to estimate kinetic parameters for enzyme activity and metabolism. Fast reversible CIDs are therefore unique tools for performing semiquantitative biochemistry in intact cells. In this chapter, we discuss advantages and constraints for the design of reversible CID applications. We provide detailed protocols for rCD1 synthesis, CID component expression in and delivery to mammalian cells and the determination of enzyme kinetics inside intact cells by a specially designed image acquisition and data analysis method.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages173-195
Number of pages23
Volume583
DOIs
StatePublished - 2017
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume583
ISSN (Print)00766879
ISSN (Electronic)15577988

    Fingerprint

Keywords

  • Cell biology
  • Enzyme kinetics
  • Imaging
  • Translocation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Schifferer, M., Feng, S., Stein, F., & Schultz, C. (2017). Reversible Chemical Dimerization by rCD1. In Methods in Enzymology (Vol. 583, pp. 173-195). (Methods in Enzymology; Vol. 583). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2016.10.035