Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

The biosensors developed in the authors' laboratory have been based on different designs, each imparting specific strengths and weaknesses. Here we describe detailed protocols for the application of three biosensors exemplifying different designs?first, a design in which an environmentally sensitive dye is used to report the activation of endogenous Cdc42, followed by two biosensors based on FRET, one using intramolecular and the other intermolecular FRET. The design differences lead to the need for different approaches in imaging and image analysis. Curr. Protoc. Cell Biol. 46:14.11.1?14.11.26. © 2010 by John Wiley & Sons, Inc.

Keywords: biosensors; Rho family GTPases; FRET; live cell imaging

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Introduction
Basic Protocol 1: Production and Use of meroCBD, Dye‐Based Biosensor for Cdc42
Support Protocol 1: Labeling CBD‐EGFP with Reactive Fluorophore
Basic Protocol 2: Imaging meroCBD in Living Cells
Basic Protocol 3: Expressing the RhoA Single‐Chain Biosensor
Basic Protocol 4: Imaging the RhoA Biosensor
Basic Protocol 5: Expression of Rac1 Flair, Dual‐Chain Fret Biosensor for Rac1
Basic Protocol 6: Imaging Rac1‐Flair
Basic Protocol 7: Image Processing Procedures
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Production and Use of meroCBD, Dye‐Based Biosensor for Cdc42

Materials

Transformation‐competent BL21(DE3) E. coli (e.g., Stratagene)
pET23‐CBD‐EGFP (Addgene)
LB medium and plates ( appendix 2A ) containing 100 µg/ml carbenicillin
1 M IPTG (in water), store at −20°C
Lysis buffer (see recipe )
Talon resin (Co²⁺ affinity, Clontech)
Lysis buffer (see recipe but omit 2‐ME and PMSF) containing 5 mM and 150 mM imidazole
50 mM Tris⋅Cl, pH 7.5 to 8.0 ( appendix 2A )
50 mM sodium phosphate buffer, pH 7.5 ( appendix 2A )
Storage buffer (see recipe )

2000‐ml Erlenmeyer flask
Incubator with shaker
250‐ml centrifuge bottles
Beckman centrifuge with JA‐10 and JA‐20 rotors (or equivalent)
50‐ml conical polypropylene centrifuge tubes (Falcon)
Centrifuge with swinging‐bucket rotor
End‐over‐end rotator
Ultrafree Centrifugal Filtration Device (MWCO, 5000; Fisher Scientific, cat. no. UFV5BCC25)
Slide‐A‐Lyzer cassettes (MWCO 3500; Pierce)

Additional reagents and equipment for transformation of bacteria and other basic molecular biological techniques (e.g., Sambrook et al., ; Ausubel et al., ), SDS‐PAGE (unit 6.1 ), and dialysis ( appendix 3H )

NOTE: Use the buffers suggested in this unit. Apparently small changes have proven to greatly reduce yield.

Support Protocol 1: Labeling CBD‐EGFP with Reactive Fluorophore

Materials

Dye: ISO‐IAA (Toutchkine et al., , , b )
Dimethylsulfoxide (DMSO)
CBD‐EGFP ( protocol 1 )
2‐mercaptoethanol (2‐ME)
50 mM sodium phosphate buffer, pH 7.5 ( appendix 2A )
12% SDS‐PAGE gel (unit 6.1 )
50 mM Tris⋅Cl, pH 8.0 ( appendix 2A )

Spectrophotometer
2‐ml microcentrifuge tubes
End‐over‐end rotator
0.5 cm × 6 to 8 cm column packed with Sephadex G15 gel‐filtration resin (GE Healthcare)

Additional reagents and equipment for SDS‐PAGE (unit 6.1 ) and spectrophotometric determination of protein concentration ( appendix 3B )

Basic Protocol 2: Imaging meroCBD in Living Cells

Materials

Tet‐off stable MEF/3T3 cell system (Clontech)
MEF/3T3 cells transduced with the appropriate construct
10 mg/ml doxycycline stock solution
10 mg/ml puromycin stock solution

10‐cm tissue culture dishes
Coverslips coated with fibronectin: immerse glass coverslips 30 to 60 min in 10 µg/ml fibronectin (e.g., Sigma), then rinse three times with PBS ( appendix 2A ) and leave immersed in PBS until use

Additional reagents and equipment for cell culture techniques including trypsinization (unit 1.1 ), flow cytometric cell sorting (Robinson et al., ), and imaging the RhoA biosensor ( protocol 5 )

Basic Protocol 3: Expressing the RhoA Single‐Chain Biosensor

Materials

Mouse embryo fibroblast (MEF/3T3) cells (Clontech; tet‐OFF MEF/3T3)
Rac1 biosensor vector system (see above)
Fugene6 transfection reagent (Roche)
10 mg/ml doxycycline stock solution
Coverslips coated with fibronectin: immerse glass coverslips 30 to 60 min in 10 µg/ml fibronectin (e.g., Sigma), then rinse three times with PBS ( appendix 2A ) and leave immersed in PBS until use

Additional reagents and equipment for cell culture techniques (unit 1.1 ), flow cytometric cell sorting (Robinson et al., ), and imaging the Rac1‐FLAIR biosensor ( protocol 7 )

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Figures

Figure 14.11.1 Fluorescent biosensor designs. (A ) MeroCBD, biosensor of endogenous Cdc42 activation. Here, a fragment of Wiskott‐Aldrich syndrome protein (WASP) that binds only to activated Cdc42 is covalently derivatized with an environmentally sensitive dye. When the WASP fragment encounters and binds to activated Cdc42, the solvation of the dye by water is reduced, leading to a fluorescence change. Advantages of this design include the ability to study endogenous protein, and enhanced sensitivity due to direct excitation of a bright, long‐wavelength dye (as opposed to indirect excitation via FRET). The disadvantage is the need for microinjection, electroporation, or some other means to load the covalently tagged protein biosensor into cells. (B ) RhoA biosensor. Here a fragment of Rhotekin that binds only to activated RhoA is attached to RhoA as part of the same protein chain. Two different fluorescent proteins undergoing FRET are in the chain between RhoA and the Rhotekin fragment, such that binding of the fragment to activated RhoA alters the distance and/or orientation between the fluorescent proteins, affecting FRET. This biosensor is fully genetically encoded, greatly simplifying loading into the cell. Because the Rhotekin fragment is attached to the RhoA, image processing is simplified relative to the dual‐chain sensor shown in (C) (see text). (C ) Rac1 FLAIR, biosensor of Rac1 activation. This design is similar to that of the RhoA biosensor, but here the PAK fragment used to bind activated Rac1 is not part of the same chain as the Rac1. The use of a dual‐chain, intermolecular FRET design enhances sensitivity because, unlike the single‐chain design, there is no FRET when the biosensor is in the off state. Additional image processing (bleed‐through correction) is required because the biosensor components can distribute differently throughout the cell.

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Figure 14.11.2 Transmittance spectra for the “Scripps Custom” dichroic mirror (Chroma Technology, lot no. 511111886).

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Figure 14.11.3 x ‐ y translational image registration artifacts generate features on ratio images. In panel (A ), a correctly registered ratio image of MeroCBD is shown. Panel (B ) shows the ratio image from the same cell without registration. In the latter case, the dye image was misaligned by −5 pixels in both x and y directions. The white arrows in both figures point to regions where misalignment produces edge artifacts and other artifactual features. In (B), a lower‐ratio rim can be seen on one side of the nucleus, and a higher ratio on the opposite side. Similarly, edge artifacts appear as higher ratio on predominantly one side of the cell, with an artifactually low ratio on the opposite side.

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Figure 14.11.4 Transmittance spectra for the “Quad Custom” dichroic mirror (Chroma Technology, lot no. 511112038).

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Figure 14.11.5 A representative histogram from a shade‐corrected and background‐subtracted image, prior to masking. The prominent histogram peak at zero intensity is followed by a continuous distribution of pixels at a range of positive intensity values. Threshold masking to remove pixels of too low an intensity is performed using this histogram. One maximizes selection of pixels from within the cell while removing pixels outside the cell, including noise around the periphery.

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Figure 14.11.6 Image shear between two cameras creates artifacts in ratio images. In the upper panels, calibration grid images from two cameras are overlaid with and without shear correction (red arrow: two channels aligned relative to the corner indicated). In the lower panels, the ratios of RhoA biosensor readouts from the two camera channels are shown with and without shear correction. A peripheral ruffle shows a large artifactual feature when correction is not applied (white arrow).

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Figure 14.11.7 Effect of the multiplication factor during ratiometric calculation. In panel (A ), using a scaling factor of 1000 produced a smooth histogram distribution in a ratio image. Panel (B ) shows the same ratio image with the scaling factor of 25. Though the general distribution pattern is similar, the histogram distribution in (A) retains higher bit‐depth resolution.

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Videos

Literature Cited

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Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells

Abstract

Table of Contents

Materials

Basic Protocol 1: Production and Use of meroCBD, Dye‐Based Biosensor for Cdc42

Support Protocol 1: Labeling CBD‐EGFP with Reactive Fluorophore

Basic Protocol 2: Imaging meroCBD in Living Cells

Basic Protocol 3: Expressing the RhoA Single‐Chain Biosensor

Figures

Videos

Literature Cited