The Application of KillerRed for Acute Protein Inactivation in Living Cells

Timothy S. Jarvela1, Adam D. Linstedt1

1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.35
DOI:  10.1002/0471142956.cy1235s69
Online Posting Date:  July, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Generating loss of protein function is a powerful investigatory tool particularly if carried out on a physiologically relevant timescale in a live‐cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live‐cell imaging of cultured cells. Curr. Protoc. Cytom. 69:12.35.1‐12.35.10. © 2014 by John Wiley & Sons, Inc.

Keywords: KillerRed; CALI; acute inactivation; protein inactivation; ROS

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic planning and Controls for KillerRed Experiments
  • Basic Protocol 1: Inactivation of KillerRed Using Epifluorescent Light
  • Alternate Protocol 1: Inactivation of KillerRed Constructs Using a Scanning Laser Capable of Photobleaching a Region of Interest
  • Support Protocol 1: Construct Generation and Cell Transfection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Inactivation of KillerRed Using Epifluorescent Light

  Materials
  • Cells expressing KillerRed and reporter constructs (see the protocol 3Support Protocol)
  • Inverted confocal microscope
  • High numerical aperture objective
  • Lasers for desired fluorophores—561 nm or 535 nm for KillerRed imaging
  • Epifluorescent light source (e.g., Mercury arc lamp, high‐powered LEDs)
  • Excitation filter for KillerRed—between 540 to 580 nm (e.g., Cy3, TRITC, or TexasRed filter sets)
  • Stage incubator with gas control by injection of CO 2 or flow of 95% air, 5% CO 2 mixture through the incubator (see Critical Parameters)
  • Gas tank (100% CO 2 or 95% air/5% CO 2 depending on setup)

Alternate Protocol 1: Inactivation of KillerRed Constructs Using a Scanning Laser Capable of Photobleaching a Region of Interest

  Additional Materials (also see protocol 1Basic Protocol)
  • Steerable focused laser system [e.g., FRAPPA (Andor), Direct FRAP (Zeiss), or cellˆfrap (Olympus)]

Support Protocol 1: Construct Generation and Cell Transfection

  Materials
  • Cell line of interest (e.g., HeLa cells)
  • Cell culture medium (e.g., MEM with 10% fetal bovine serum) for cell line used
  • Expression vector containing KillerRed fused in frame with the coding sequence of the protein of interest (see Commentary for designing fusion proteins)
  • Vector containing a relevant reporter construct (e.g., encoding GFP‐tagged Golgi protein for Golgi experiments]
  • Imaging medium (see recipe)
  • Additional reagents for tissue culture maintenance
  • Transfection reagent [e.g., JetPRIME (PolyPlus), Oligofectamine, LipofectAMINE2000 (Invitrogen)]
  • Additional reagents for knockdown of endogenous protein (if desired)
  • 60‐mm tissue culture dishes
  • Imaging chamber [metal chamber with 12‐mm glass coverslips or glass‐bottom dishes (MaTek)]
  • 35‐mm tissue culture dish lid
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Beck, S., Sakurai, T., Eustace, B.K., Beste, G., Schier, R., Rudert, F., and Jay, D.G. 2002. Fluorophore‐assisted light inactivation: A high‐throughput tool for direct target validation of proteins. Proteomics 2:247‐255.
  Bulina, M.E., Chudakov, D.M., Britanova, O.V., Yanushevich, Y.G., Staroverov, D.B., Chepurnykh, T.V., Merzlyak, E.M., Shkrob, M.A., Lukyanov, S., and Lukyanov, K.A. 2006. A genetically encoded photosensitizer. Nat. Biotechnol. 24:95‐99. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16369538.
  Buytaert, E., Dewaele, M., and Agostinis, P. 2007. Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim. Biophys. Acta 1776:86‐107. Available at: http://dx.doi.org/10.1016/j.bbcan.2007.07.001.
  Carpentier, P., Violot, S., Blanchoin, L., and Bourgeois, D. 2009. Structural basis for the phototoxicity of the fluorescent protein KillerRed. FEBS Lett. 583:2839‐2842. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19646983.
  Halliwell, B. 1989. Oxidants and the central nervous system: some fundamental questions. Is oxidant damage relevant to Parkinson's disease, Alzheimer's disease, traumatic injury or stroke? Acta Neurol. Scand. 126:23‐33.
  Jarvela, T. and Linstedt, A.D. 2012. Irradiation‐induced protein inactivation reveals Golgi enzyme cycling to cell periphery. J. Cell Sci. 125:973‐980. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22421362.
  Jarvela, T. and Linstedt, A.D. 2014. Isoform‐specific tethering links the Golgi ribbon to maintain compartmentalization. Mol. Biol. Cell 25:133‐144.
  Jay, D.G. 1988. Selective destruction of protein function by chromophore‐assisted laser inactivation. Proc. Natl. Acad. Sci. U.S.A. 85:5454‐5458. Available at: http://www.pnas.org/cgi/doi/10.1073/pnas.85.15.5454.
  Liao, J.C., Roider, J., and Jay, D.G. 1994. Chromophore‐assisted laser inactivation of proteins is mediated by the photogeneration of free radicals. Proc. Natl. Acad. Sci. U.S.A. 91:2659‐2663.
  Lin, J.Y., Sann, S.B., Zhou, K., Nabavi, S., Proulx, C.D., Malinow, R., Jin, Y., and Tsien, R.Y. 2013. Optogenetic inhibition of synaptic release with chromophore‐assisted light inactivation (CALI). Neuron 79:241‐253. Available at: http://dx.doi.org/10.1016/j.neuron.2013.05.022.
  Linden, K.G., Liao, J.C., and Jay, D.G. 1992. Spatial specificity of chromophore assisted laser inactivation of protein function. Biophys. J. 61:956‐962. Available at: http://dx.doi.org/10.1016/S0006‐3495(92)81902‐1.
  Marek, K.W. and Davis, G.W. 2002. Transgenically encoded protein photoinactivation (FlAsH‐FALI): Acute inactivation of synaptotagmin I. Neuron 36:805‐813.
  Muthiah, M., Park, S.‐H., Nurunnabi, M., Lee, J., Lee, Y., Park, H., Lee, B., Min, J.‐J., and Park, I.‐K. 2014. Intracellular delivery and activation of the genetically encoded photosensitizer Killer Red by quantum dots encapsulated in polymeric micelles. Colloids Surf. B Biointerfaces 116C:284‐294.
  Pletnev, S., Gurskaya, N.G., Pletneva, N.V., Lukyanov, K.A., Chudakov, D.M., Martynov, V.I., Popov, V.O., Kovalchuk, M.V., Wlodawer, A., Dauter, Z., and Pletnev, V. 2009. Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. J. Biol. Chem. 284:32028‐32039. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2797274&tool=pmcentrez&rendertype=abstract.
  Roy, A., Carpentier, P., Bourgeois, D., and Field, M. 2010. Diffusion pathways of oxygen species in the phototoxic fluorescent protein KillerRed. Photochem. Photobiol. Sci. 9:1342‐1350. Available at: http://dx.doi.org/10.1039/c0pp00141d.
  Serebrovskaya, E.O., Gorodnicheva, T.V., Ermakova, G.V., Solovieva, E.A., Sharonov, G.V., Zagaynova, E.V., Chudakov, D.M., Lukyanov, S., Zaraisky, A.G., and Lukyanov, K.A. 2011. Light‐induced blockage of cell division with a chromatin‐targeted phototoxic fluorescent protein. Biochem. J. 435:65‐71.
  Serebrovskaya, E.O., Ryumina, A.P., Boulina, M.E., Shirmanova, M.V., Zagaynova, E.V., Bogdanova, E.A., Lukyanov, S.A., and Lukyanov, K.A. 2014. Phototoxic effects of lysosome‐associated genetically encoded photosensitizer KillerRed. J. Biomed. Optics 19:71403. Available at: http://dx.doi.org/10.1117/1.JBO.19.7.071403.
  Shu, X., Lev‐Ram, V., Deerinck, T.J., Qi, Y., Ramko, E.B., Davidson, M.W., Jin, Y., Ellisman, M.H., and Tsien, R.Y. 2011. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9:e1001041. Available at: http://dx.doi.org/10.1371/journal.pbio.1001041.
  Surrey, T., Elowitz, M.B., Wolf, P.E., Yang, F., Nédélec, F., Shokat, K., and Leibler, S. 1998. Chromophore‐assisted light inactivation and self‐organization of microtubules and motors. Proc. Natl. Acad. Sci. U.S.A. 95:4293‐4298. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=22482&tool=pmcentrez&rendertype=abstract.
  Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., Kotera, I., Arai, Y., Horiuchi, M., Fukui, K., Ayabe, T., Inagaki, F., Suzuki, H., and Nagai, T. 2013. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore‐assisted light inactivation. Sci. Rep. 3:2629.
  Tour, O., Meijer, R.M., Zacharias, D.A., Adams, S.R., and Tsien, R.Y. 2003. Genetically targeted chromophore‐assisted light inactivation. Nat. Biotechnol. 21:1505‐1508. Available at: http://dx.doi.org/10.1038/nbt914.
  Waldeck, W., Mueller, G., Wiessler, M., Tóth, K., and Braun, K. 2011. Positioning effects of KillerRed inside of cells correlate with DNA strand breaks after activation with visible light. Int. J. Med. Sci. 8:97‐105. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3030142&tool=pmcentrez&rendertype=abstract.
  Yadav, S., Puthenveedu, M.A., and Linstedt, A.D. 2012. Golgin160 recruits the dynein motor to position the Golgi apparatus. Dev. Cell 23:153‐165. Available at: http://dx.doi.org/10.1016/j.devcel.2012.05.023.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library