Fast Relaxation Imaging in Living Cells

Apratim Dhar1, Martin Gruebele1

1 University of Illinois, Urbana, Illinois
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.1
DOI:  10.1002/0471140864.ps2801s65
Online Posting Date:  August, 2011
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Abstract

This protocol describes the technique of Fast Relaxation Imaging (FReI) as applied to protein folding inside living cells. The required modifications of a fluorescence microscope by addition of a diode laser temperature jump source, a yellow/blue switchable light‐emitting diode source, and a two‐color CCD camera to collect movies of protein dynamics inside cells are discussed. A description of how proteins are labeled for imaging, how cells are prepared for imaging, and how imaging of kinetics inside cells with millisecond time resolution is obtained, along with the complementary in vitro experiments, is also provided. The ability to carry out comparative in vitro and “in‐cell” measurements on the same setup allows for direct comparison of the features distinguishing cellular protein folding (or other biomolecular processes) from studies performed in dilute solution. Curr. Protoc. Protein Sci. 65:28.1.1‐28.1.19. © 2011 by John Wiley & Sons, Inc.

Keywords: protein folding; fluorescence microscopy; FRET; FReI; GFP; kinetics; thermal denaturation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Setup of Microscope, Camera, and Illumination Source
  • Basic Protocol 2: Setup of the Temperature‐Controlled Sample Stage
  • Alternate Protocol 1: Building a Custom Temperature‐Controllable Microscope Stage
  • Basic Protocol 3: Setup of Infrared Laser for Temperature Jump
  • Basic Protocol 4: Measurement of Protein Kinetics In Vivo
  • Alternate Protocol 2: In Vitro FReI Reference Measurements
  • Basic Protocol 5: Measurement of Protein Thermodynamics In Vivo
  • Basic Protocol 6: Kinetic, Thermodynamic, and Image Analysis
  • Basic Protocol 7: Temperature Downward Jump Measurements
  • Support Protocol 1: Design of Protein Vector, Choice of Cells, and Transfection of AcGFP1‐Protein‐mCherry Fusion Construct for In Vivo FReI Studies
  • Support Protocol 2: Expression and Purification of AcGFP1‐PGK‐mCherry Fusion Protein for In Vitro Fret Studies
  • Support Protocol 3: Measurement of PGK Fusion Protein Activity
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Setup of Microscope, Camera, and Illumination Source

  Materials
  • Illumination sources and optics:
    • Blue LED for FRET imaging, 470 nm (Luxeon V Star)
    • Yellow LED for direct acceptor imaging, 590 nm (Thorlabs, M590L2)
    • Power supply for LEDs (Elenco Precision)
    • Dichroic mirror #3 to combine blue and yellow LEDs (Chroma, T550lpxr)
    • Threaded Cube to mount LEDs (Thorlabs, SM1C6)
    • Threaded Lens Tube (Thorlabs, SM1L03; SM1L10; SM1L30)
    • Lens, Aspheric (focal length f = 50 mm); this is relay lens #1
    • Lens, Plano‐convex (f = 150 mm); this is relay lens #2
  • The microscope's filter box:
    • Filter cube to mount objective filters (available from the microscope manufacturer)
    • Filter set for blue LED‐Excitation Filter (Chroma, ET470/40×); Dichroic Mirror #1 (Chroma, T495LP); Emission Filter (Omega, XF3108/25)
    • Filter set for yellow LED‐Excitation Filter (Chroma, ET572/35×); Dichroic Mirror #2 (Chroma, T585LP); Emission Filter (ET630/75m)
  • For fluorescence imaging by the CCD:
    • Right Angle Prism Mirror (Thorlabs, MRA25‐E02)
    • Collimating lens (achromat, ca. f = 150 mm)
    • Silver Mirrors (Thorlabs, PF10‐03‐P01; two of these mirrors are required)
    • Dichroic Mirrors #2 (Chroma, T585LP; two of these mirrors are required); these are the same as the dichroic used in the filter set for the yellow LED
    • Mirror Mounts for the Silver Mirrors (Thorlabs, KM100‐E02)
    • 45° mirror mounts for Dichroic Mirrors #2
    • CCD Camera (Lumenera Lm075; the camera should be capable of acquiring images at 60 fps at 640×480 resolution, or better)
    • C‐mount camera Lens (f = 16 mm, comes with Lumenera Lm075)
  • To test performance of the imaging system:
    • Microscope slide with fixed cells expressing fluorescently labeled protein—either prepare a slide with cells expressing the protein of interest or for general testing of the system, buy a set of slides from Chroma (Fluor‐o‐4 Slide Set # 92000)

Basic Protocol 2: Setup of the Temperature‐Controlled Sample Stage

  Materials
  • 1 M HCl
  • FCS2 chamber (Bioptechs)
  • FCS2 stage adapter (Bioptechs); this allows the use of the FCS2 chamber with the existing microscope stage
  • FCS2 controller (Bioptechs); this controls the temperature of the FCS2 chamber
  • Objective heater system (Bioptechs); this allows the use of immersion objectives for imaging, by controlling the temperature of the objective

Alternate Protocol 1: Building a Custom Temperature‐Controllable Microscope Stage

  Materials
  • Microscope stage, fabricated (by a machine shop) from heat‐resistant plastic, with a cutout for standard 1‐in. × 3‐in. microscope slides (see Fig. )
  • Aluminum plate (with the same dimensions as the cutout) with two 4‐Ω resistors and a cutout in the middle to allow the IR pulse from the T‐jump laser to pass through a small hole in the side of the aluminum plate to allow temperature measurement using a thermocouple (also fabricated by a machine shop)
  • Thermocouple with readout (Omega)
  • Programmable power supply (e.g., from BK Precision or Agilent)
NOTE: If immersion objectives are used for imaging, the fact that the objective acts as a heat sink and conducts a lot of heat away from the microscope stage should be taken into account. This heat loss can be offset by simply passing more current to ensure that the cells being imaged reach the desired temperature.

Basic Protocol 3: Setup of Infrared Laser for Temperature Jump

  Materials
  • T‐jump laser, controller and optics
    • IR diode laser, 2200 nm (m2k laser)
    • Diode power supply (Thorlabs, LDC 340)
    • Diode temperature controller (Thorlabs, ITC 510)
    • Diode holder (Thorlabs, TCLDM9)
    • Collimating lens (Thorlabs, C036TME‐D)
    • Achromatic near‐infrared transmitting lens (focal length f = 25 mm)
    • Silver mirror (Thorlabs, PF10‐03‐P01)
  • Computer control of T‐jump laser
  • PCI card (National Instruments, PCI 6221)
  • Connector Block (National Instruments, BNC 2110)
  • Shielded Cable (National Instruments, SHC68‐58‐EPM)
  • Slide with purified AcGFP1‐PGK‐mCherry (also known as PGK‐FRET) solution for imaging ( protocol 11)

Basic Protocol 4: Measurement of Protein Kinetics In Vivo

  Materials
  • Coverslip with cells expressing PGK‐FRET (also referred to as AcGFP1‐PGK‐mCherry; see protocol 10)
  • Liebovitz L‐15 medium (Invitrogen) supplemented with 30% (v/v) fetal bovine serum (FBS)
  • Microscope slide (Fisher)
  • 100‐µm spacer (Grace Bio Labs)
  • FReI microscope (assembled following Basic Protocols protocol 11, protocol 22, and protocol 43)

Alternate Protocol 2: In Vitro FReI Reference Measurements

  • Purified AcGFP1‐PGK‐mCherry (also referred to as PGK‐FRET) fusion protein ( protocol 11)
  • Slides

Basic Protocol 5: Measurement of Protein Thermodynamics In Vivo

  Materials
  • LabVIEW (National Instruments)
  • Mathematica (Wolfram Research) or Matlab

Basic Protocol 6: Kinetic, Thermodynamic, and Image Analysis

  Materials
  • Microscope slide with purified protein solution ( protocol 11)
  • Additional reagents and equipment for setting up the IR laser for T‐jump ( protocol 4)

Basic Protocol 7: Temperature Downward Jump Measurements

  Materials
  • PGK sequence, synthesized by Genscript (http://www.genscript.com/)
  • pAcGFP1 vector (Clontech)
  • pmCherry vector (Clontech)
  • U2OS cells (ATCC)
  • Complete DMEM medium with 10% FBS ( appendix 3B)
  • Complete DMEM medium with 10% FBS, without antibiotics
  • OptiMEM I medium (Invitrogen)
  • Lipofectamine 2000 (Invitrogen)
  • 35‐mm Petri dishes (Corning)
  • Coverslips (Corning)
  • Additional reagents and equipment for basic molecular biology techniques (Ausubel et al., )
NOTE: All of the molecular biology techniques mentioned in the steps below are described in Ausubel et al. ( ).

Support Protocol 1: Design of Protein Vector, Choice of Cells, and Transfection of AcGFP1‐Protein‐mCherry Fusion Construct for In Vivo FReI Studies

  Materials
  • BL21 CodonPlus (DE3)‐RIPL cells (Stratagene)
  • pDream 2.1 vector with PGK‐FRET (constructed as described in protocol 10)
  • Luria‐Bertani (LB) medium ( appendix 4A)
  • IPTG (Inalco, http://www.inalcopharm.com/)
  • Ni‐NTA column (Qiagen)
  • 23°C bacteriological incubator
  • French press or sonicator
  • Additional reagents for growth of E. coli ( appendix 4A), lysis of E. coli using French press (unit 6.2), metal‐chelate affinity chromatography (unit 9.4), dialysis ( appendix 3B), electrospray ionization mass spectrometry (ESI‐MS; unit 16.9), and SDS‐PAGE (unit 10.1).

Support Protocol 2: Expression and Purification of AcGFP1‐PGK‐mCherry Fusion Protein for In Vitro Fret Studies

  Materials
  • EDTA [H 4EDTA; ethylenediaminetetraacetic acid]
  • Tris base [tris(hydroxymethyl)aminomethane]
  • HCl [hydrochloric acid]
  • DTT [DL‐dithiothreitol]
  • MgCl 2 [magnesium chloride hexahydrate]
  • 3PG [D‐(–)‐3‐phosphoglyceric acid disodium salt]
  • ATP [adenosine 5′‐triphosphate disodium salt]
  • NaOH [sodium hydroxide]
  • NADH [β‐nicotinamide adenine dinucleotide, reduced]
  • GAPDH [glyceraldehyde 3‐phosphate dehydrogenase]
  • PGK with fluorescent label (can be obtained as described in Support Protocols protocol 101 and protocol 112)
  • Wild‐type PGK
  • 225‐ml Erlenmeyer flask
  • Disposable cuvettes (Sigma‐Aldrich, Plastibrand, PMMA)
  • UV‐vis spectrometer
NOTE: All chemicals and wild‐type PGK can be purchased from Sigma‐Aldrich.
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Figures

  •   FigureFigure 28.1.1 Optical layout of the epi‐fluorescence microscope modified for FReI experiments. The LED assembly consists of donor excitation LED (blue) and acceptor excitation LED (yellow) mounted to the threaded cube. Dichroic #3 combines the blue and yellow lasers, or one LED can be turned on at a time. From there, relay lenses collimate the excitation lasers into the filter cube provided by the microscope manufacturer located below the objective. At the same time that sample fluorescence is excited and collected from below, the IR laser pulse induces a T‐jump from above (see Fig. B). The fluorescence passes to the bottom of the microscope, and is turned sideways by the prism mirror. A collimating lens passes the beam into the red/green splitting rectangle, whose output is combined (with an offset) onto the lens of the CCD camera, where a red and a green image are obtained. The CCD camera transmits a movie of the fluorescence to a computer via a USB connection for analysis.
  •   FigureFigure 28.1.2 (A) Absorption (dotted) and emission (solid) spectra of the AcGFP donor and mCherry fluorescence acceptor. (B) transmission of the filters housed in the filter cube: B=blue LED; Y=yellow LED; ExF=excitation filter; EmF=emission filter; DM=dichroic mirror.
  •   FigureFigure 28.1.3 Output side of the epifluorescence microscope. The red and green fluorescence is directed by the right angle prism mirror (RAPM) to the first dichroic #2. There, the beam is split into red and green, and the two beams are directed onto the CCD by the second dichroic #2. The CCD images two side‐by‐side red and green images through the camera lens, producing a movie of the fluorescence such as that in Video 28.1.1 (where green and red channels have been superimposed). (B) The other side of the microscope shows the T‐jump diode laser mounted on a stage above the main microscope stage. A silver mirror directs the infrared T‐jump laser pulse (purple beam) down through the focusing lens towards the objective, where the sample stage would sit. Below the main stage, the threaded lens tube can be seen that directs the LED excitation light (blue or yellow, see Fig. ) to the filter cube housed just below the objective.
  •   FigureFigure 28.1.4 Laser diode power, temperature profile, and protein folding. (A) The rapid initial power rise produces a fast temperature rise in (B). When the desired temperature is reached in (B) (here: 43°C), the power is dropped in (A) to a level which holds the temperature constant at the final desired temperature. Panel (C) shows the resulting unfolding and refolding profile of PGK. The ratio D/A (donor fluorescence intensity over acceptor fluorescence intensity) plotted on the y axis is unitless, but not necessarily equal to 1 at the beginning of the experiment. In this panel, the ratio was normalized, so it equals 1 just before the temperature jump is applied.
  •   FigureFigure 28.1.5 Schematic sequence of the plasmid used for expression of a fluorescent labeled protein (here: PGK) with a donor (AcGFP1) and acceptor (mCherry).

Literature Cited

   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., (eds). 2011. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Dhar, A., Ebbinghaus, S., Shen, Z., Mishra, T., and Gruebele, M. 2010a. The diffusion coefficient for PGK folding in eukaryotic cells. Biophys. J. 99:L69–L71.
   Dhar, A., Samiotakis, A., Ebbinghaus, S., Nienhaus, L., Homouz, D., Gruebele, M., and Cheung, M.S. 2010b. Structure, function and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding. Proc. Natl. Acad. Sci. U.S.A. 107:17586‐17591.
   Ebbinghaus, S., Dhar, A., McDonald, J.D., and Gruebele, M. 2010. Protein folding stability and dynamics imaged in a living cell. Nat. Methods 7:319‐323.
   Ghaemmaghami, S. and Oas, T.G. 2001. Quantitative protein stability measurement in vivo. Nat. Struct. Biol. 8:879‐882.
   Ignatova, Z., Krishnan, B., Bombardier, J.P., Marcelino, A.M.C., Hong, J., and Gierasch, L.M. 2007. From the test tube to the cell: Exploring the folding and aggregation of a beta‐clam protein. Biopolymers 88:157‐163.
   Kim, S.A., Heinze, K.G., and Schwille, P. 2007. Fluorescence correlation spectroscopy in living cells. Nat. Methods 4:963‐973.
   Mas, M.T., Chen, C.Y., Hitzeman, R.A., and Riggs, A.D. 1986. Active human‐yeast chimeric phosphoglycerate kinases engineered by domain interchange. Science 233:788‐790.
   Schoen, I., Krammer, H., and Braun, D. 2009. Hybridization kinetics is different inside cells. Proc. Natl. Acad. Sci. U.S.A. 106:21649‐21654.
   Verkman, A.S. 2002. Solute and macromolecule diffusion in cellular aqueous compartments. Trends Biochem. Sci. 27:27‐33.
Key References
  Ebbinghaus et al., 2010. See above.
  Describes the first measurement of protein folding thermodynamics and kinetics in single living cells and illustrates application of the protocols described in this unit.
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