Using a Kinase‐Inducible Bimolecular Switch to Control Enzyme Activity in Living Cells

Sohum Mehta1, Jin Zhang1

1 The Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130090
Online Posting Date:  October, 2013
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Abstract

Molecular switches have been instrumental in the development of powerful and versatile genetic tools for directly probing biochemical processes, such as intracellular signaling, within their native contexts. A molecular switch can be broadly defined as a molecular system capable of existing in either of two states (e.g., conformations), which can be converted from one state to the other by a specific input stimulus. This protocol outlines a method for using a kinase‐inducible bimolecular switch, along with live‐cell fluorescence microscopy, to directly control and monitor the activity of a specific enzyme in living cells. Curr. Protoc. Chem. Biol. 5:227‐237 © 2013 by John Wiley & Sons, Inc.

Keywords: live‐cell imaging; biochemical perturbation; biosensor; fluorescence

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Stimulating and Monitoring Enzymatic Activity Controlled by a Kinase‐Inducible Biomolecular Switch
  • Support Protocol 1: Maintenance and Preparation of Mammalian Cells for Live‐Cell Fluorescence Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Stimulating and Monitoring Enzymatic Activity Controlled by a Kinase‐Inducible Biomolecular Switch

  Materials
  • Plasmid DNA encoding the KIBS components and biosensor (available upon request): CFP‐FHA1‐Inp54p, PKA substrate‐RFP‐PM, YFP‐PH(PLCδ1) (Sample et al., )
  • Opti‐MEM I reduced serum medium (Gibco)
  • Lipofectamine 2000 (Invitrogen)
  • COS7 cells plated on 35‐mm glass‐bottom dishes (see Support Protocol)
  • Immersion oil
  • HBSS imaging buffer (see recipe)
  • Fsk stock solution: 50 mM forskolin (Calbiochem) in DMSO
  • IBMX stock solution: 100 mM 3‐isobutyl‐1‐methylxanthine (Sigma) in DMSO
  • H89 stock solution: 10 mM H89 (Sigma) in DMSO
  • Microcentrifuge tubes, size according to volume prepared
  • Inverted fluorescence microscope with appropriate objective, filters, mirrors, detector, and image acquisition software, e.g.,
    • Light source (e.g., XBO 75W xenon arc lamp, Carl Zeiss)
    • Zeiss Axiovert 200M microscope (Carl Zeiss)
    • 40×/1.3‐NA oil‐immersion objective lens
    • Dichroic mirror, excitation filters (CFP, YFP, RFP), emission filters (CFP, YFP, RFP)
    • Cooled charge‐coupled device (CCD) camera (e.g., MicroMAX BFT512, Roper Scientific)
    • Lambda 10‐2 filter changer (Sutter Instruments)
    • METAFLUOR 7.7 imaging software (Molecular Devices)
  • Computer to run microscope
  • Spreadsheet application (e.g., Microsoft Excel)

Support Protocol 1: Maintenance and Preparation of Mammalian Cells for Live‐Cell Fluorescence Microscopy

  Materials
  • COS‐7 cells (American Type Culture Collection) in 25‐cm2 cell culture flasks
  • COS‐7 cell culture medium (see recipe)
  • 70% (v/v) ethanol
  • Dulbecco's phosphate‐buffered saline (DPBS, Gibco)
  • 0.25% (w/v) trypsin/EDTA solution (Gibco)
  • 37°C, 5% CO 2 humidified tissue culture incubator
  • Dissecting microscope
  • Tissue culture hood
  • 25‐cm2 tissue culture flasks (e.g., BD Falcon)
  • 35‐mm glass‐bottom imaging dishes (MatTEK)
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Figures

Videos

Literature Cited

Literature Cited
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