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Related Experiment Videos

Reversible Chemical Dimerization by rCD1.

M Schifferer1, S Feng2, F Stein1

  • 1European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

Methods in Enzymology
|January 9, 2017
PubMed
Summary
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This study introduces a fast, reversible chemical inducer of dimerization (rCD1) for precise control of cellular processes. This tool enables semiquantitative biochemical analysis within living cells by rapidly switching enzyme activity on and off.

Area of Science:

  • Cellular biology
  • Biochemistry
  • Molecular pharmacology

Background:

  • Precise spatiotemporal perturbation of cellular players is crucial for understanding cellular networks.
  • Chemically induced dimerization (CID) offers high spatiotemporal control over protein interactions.
  • Existing CID systems lack rapid reversibility, limiting kinetic analysis.

Purpose of the Study:

  • To develop and validate a fast-acting, reversible chemical inducer of dimerization (rCD1).
  • To demonstrate the utility of rCD1 for monitoring cellular responses and estimating kinetic parameters.
  • To provide protocols for implementing rCD1 in mammalian cells.

Main Methods:

  • Synthesis and application of the reversible chemical dimerizer (rCD1).
  • Induction and termination of enzyme activities (e.g., PI3K, 5Ptase) using rCD1.
Keywords:
Cell biologyEnzyme kineticsImagingTranslocation

Related Experiment Videos

  • Monitoring downstream effector relaxation via live-cell imaging and biochemical assays.
  • Development of specialized image acquisition and data analysis for kinetic determination.

    • Main Results:

    • rCD1 exhibits the fastest release kinetics among reversible CID systems, comparable to optogenetics.
    • Rapid termination of enzyme activity allows for the estimation of kinetic parameters.
    • Demonstrated successful application in monitoring enzyme activity and downstream effects in living cells.

    Conclusions:

    • Fast reversible CIDs, like rCD1, are powerful tools for semiquantitative biochemistry in intact cells.
    • rCD1 provides unprecedented temporal control for studying cellular dynamics.
    • The developed protocols facilitate the application of rCD1 in various biological systems.