Probing Peroxisome Dynamics and Biogenesis by Fluorescence Imaging

Miluska Jauregui1, Peter K. Kim1

1 Department of Biochemistry, University of Toronto, Toronto, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.9
DOI:  10.1002/0471143030.cb2109s62
Online Posting Date:  March, 2014
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Abstract

Peroxisomes are the most recently discovered classical organelles, and only lately have their diverse functions been truly recognized. Peroxisomes are highly dynamic structures, changing both morphologically and in number in response to both extracellular and intracellular signals. This metabolic organelle came to prominence due to the many genetic disorders caused by defects in its biogenesis or enzymatic functions. There is now growing evidence that suggests peroxisomes are involved in lipid biosynthesis, innate immunity, redox homeostasis, and metabolite scavenging, among other functions. Therefore, it is important to have available suitable methods and techniques to visualize and quantify peroxisomes in response to various cellular signals. This unit includes a number of protocols that will enable researchers to image, qualify, and quantify peroxisome numbers and morphology—with both steady‐state and time‐lapse imaging using mammalian cells. The use of photoactivatable fluorescent proteins to detect and measure peroxisome biogenesis is also described. Altogether, the protocols described here will facilitate understanding of the dynamic changes that peroxisomes undergo in response to various cellular signals. Curr. Protoc. Cell Biol. 62:21.9.1‐21.9.20. © 2014 by John Wiley & Sons, Inc.

Keywords: peroxisome; organelle biogenesis; organelle dynamics; organelle morphology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fluorescent Immunolabeling for Peroxisome Imaging
  • Basic Protocol 2: Quantification of Peroxisome Numbers
  • Alternate Protocol 1: Total Fluorescence Intensity
  • Basic Protocol 3: Photo Pulse‐Chase Assay: Visualizing Peroxisome Movement from the ER to Peroxisomes
  • Basic Protocol 4: In Cellulo Pulse Chase
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescent Immunolabeling for Peroxisome Imaging

  Materials
  • Culture cells or stably transfected cells expressing a fluorescently tagged peroxisomal molecule of interest
  • 4.0% (w/v) paraformaldehyde (PFA) in PBS
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.1% (v/v) Triton X‐100 in PBS
  • Blocking buffer: 10% (v/v) FBS/0.02% (w/v) sodium azide in PBS
  • Antibody towards a peroxisomal protein (see Table 21.9.1)
  • Fluorescently tagged secondary antibody against species from which primary antibody was obtained
  • Fluorescent friendly mounting solution: e.g., DAKO fluorescent mounting medium (cat. no. S3023)
  • Microscopy‐grade coverslip sealant (optional)
  • Coverslip (1.0 or 1.5 borosilicate glass at #1.0 or #1.5 thickness—acid cleaned)
  • Microscope slides (VWR, cat. no. CA16004‐368)
  • Confocal fluorescent microscope, laser scanning or spinning disk (see below; also see unit 4.5)
  • High‐NA objective: e.g., 63× NA 1.4, 60× NA 1.4, or 100× NA 1.45
  • Additional reagents and equipment for live‐cell imaging (Coling and Kachar, )
Table 1.9.1   MaterialsImmunofluorescent Probes for Peroxisome Imaging

Immunofluorescent probes Location
Rabbit anti‐PMP70 Peroxisome transmembrane protein
Rabbit anti‐catalase Peroxisome matrix
Rabbit anti‐PEX14 Peroxisome membrane. Exposed to cytosol.

Basic Protocol 2: Quantification of Peroxisome Numbers

  Materials
  • 3‐dimensional fluorescent images of cells that have been immunostained with a peroxisome‐specific antibody ( protocol 1)
  • Specialized 3‐D Image analysis software, such as Volocity (PerkinElmer); alternatively ImageJ (NIH) with 3D Object Counter plugin

Alternate Protocol 1: Total Fluorescence Intensity

  Materials
  • COS‐7 cells (ATCC no. CRL‐1651)
  • PEX16‐PaGFP plasmid (made in the Kim lab; contact if interested in obtaining the plasmid) or any ER‐targeted membrane protein tagged with PaGFP (Addgene, cat. no. 18697)
  • RFP‐SKL or ubiquitin‐tagged RFP‐SKL plasmid (made in the Kim lab; contact if interested in obtaining the plasmid)
  • Lipofectamine 2000 Transfection Reagent (Life Technologies, cat. no. 11668‐019)
  • Confocal fluorescent microscopy (see unit 4.5) setup for live‐cell imaging (Coling and Kachar, )
  • High‐NA objective: e.g., 63× NA 1.4 or 60× NA 1.4
  • Additional reagents and equipment for transfection using Lipofectamine 2000 (see manufacturer's instructions and unit 4.5)

Basic Protocol 3: Photo Pulse‐Chase Assay: Visualizing Peroxisome Movement from the ER to Peroxisomes

  Materials
  • Culture cells of interest, e.g., COS‐7 cells (ATCC no. CRL‐1651)
  • Ubiquitin‐tagged RFP‐SKL, RFP, or CFP plasmid (made in the Kim lab; contact if interested in obtaining the plasmid)
  • 4.0% (w/v) paraformaldehyde (PFA) in PBS
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010023)
  • Grid coverslip (Electron Microscopy Sciences, cat. no. 72265‐50; or MatTek, cat. no. P35G‐2‐14‐CGRD)
  • Confocal fluorescent microscopy (see unit 4.5) setup for live‐cell imaging (Coling and Kachar, )
  • 405‐nm laser light or mercury lamp with a CFP filter (436/455LP)
  • Additional reagents and equipment for transfection using Lipofectamine 2000 (see manufacturer's instructions and unit 4.5), cell cycle synchronization (unit 8.3), and setting scanning parameters ( protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
  Bolte, S. and Cordelieres, F.P. 2006. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224:213‐232.
  Bonekamp, N.A. and Schrader, M. 2012. Transient complex peroxisomal interactions: A new facet of peroxisome dynamics in mammalian cells. Commun. Integr. Biol. 5:534‐537.
  Coling, D. and Kachar, B. 1998. Principles and application of fluorescence microscopy. Curr. Protoc. Mol. Biol. 44:14.10.1‐14.10.11.
  Dean, P.N. 1998. Confocal microscopy: Principles and practices. Curr. Protoc. Cytom. 5:2.8.1‐2.8.12.
  De Duve, C. and Baudhuin, P. 1966. Peroxisomes (microbodies and related particles). Physiol. Rev. 46:323‐357.
  Deosaran, E., Larsen, K.B., Hua, R., Sargent, G., Wang, Y., Kim, S., Lamark, T., Jauregui, M., Law, K., Lippincott‐Schwartz, J., Brech, A., Johansen, T., and Kim, P.K. 2012. NBR1 acts as an autophagy receptor for peroxisomes. J. Cell Sci. 126:939‐952.
  Dixit, E., Boulant, S., Zhang, Y., Lee, A.S., Odendall, C., Shum, B., Hacohen, N., Chen, Z.J., Whelan, S.P., Fransen, M., Nibert, M.L., Superti‐Furga, G., and Kagan, J.C. 2010. Peroxisomes are signaling platforms for antiviral innate immunity. Cell 141:668‐681.
  Faust, P.L., Banka, D., Siriratsivawong, R., Ng, V.G., and Wikander, T.M. 2005. Peroxisome biogenesis disorders: The role of peroxisomes and metabolic dysfunction in developing brain. J. Inherit. Metab. Dis. 28:369‐383.
  Fransen, M., Nordgren, M., Wang, B., and Apanasets, O. 2012. Role of peroxisomes in ROS/RNS‐metabolism: Implications for human disease. Biochim. Biophys. Acta 1822:1363‐1373.
  Grant, P., Ahlemeyer, B., Karnati, S., Berg, T., Stelzig, I., Nenicu, A., Kuchelmeister, K., Crane, D.I., and Baumgart‐Vogt, E. 2013. The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies. Histochem. Cell Biol. 140:423‐442.
  Huybrechts, S.J., Van Veldhoven, P.P., Brees, C., Mannaerts, G.P., Los, G.V., and Fransen, M. 2009. Peroxisome dynamics in cultured mammalian cells. Traffic 10:1722‐1733.
  Kim, P.K., Hailey, D.W., Mullen, R.T., and Lippincott‐Schwartz, J. 2008. Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. Proc. Natl. Acad. Sci. U.S.A. 105:20567‐20574.
  Koch, J., Pranjic, K., Huber, A., Ellinger, A., Hartig, A., Kragler, F., and Brocard, C. 2010. PEX11 family members are membrane elongation factors that coordinate peroxisome proliferation and maintenance. J. Cell Sci. 123:3389‐3400.
  Kovacs, W.J. and Krisans, S. 2003. Cholesterol biosynthesis and regulation: Role of peroxisomes. Adv. Exp. Med. Biol. 544:315‐327.
  Kovacs, W.J., Olivier, L.M., and Krisans, S.K. 2002. Central role of peroxisomes in isoprenoid biosynthesis. Prog. Lipid Res. 41:369‐391.
  Lametschwandtner, G., Brocard, C., Fransen, M., Van Veldhoven, P., Berger, J., and Hartig, A. 1998. The difference in recognition of terminal tripeptides as peroxisomal targeting signal 1 between yeast and human is due to different affinities of their receptor Pex5p to the cognate signal and to residues adjacent to it. J. Biol. Chem. 273:33635‐33643.
  Lepe‐Zuniga, J.L., Zigler, J.S., and Gery, I. 1987. Toxicity of light‐exposed Hepes media. J. Immunol. Methods 103:145.
  Lizard, G., Rouaud, O., Demarquoy, J., Cherkaoui‐Malki, M., and Iuliano, L. 2012. Potential roles of peroxisomes in Alzheimer's disease and in dementia of the Alzheimer's type. J. Alzheimers Dis. 29:241‐254.
  Mellman, W.J., Schimke, R.T., and Hayflick, L. 1972. Catalase turnover in human diploid cell cultures. Exp. Cell Res. 73:399‐409.
  Patterson, G.H. 2011. Photoactivation and imaging of optical highlighter fluorescent proteins. Curr. Protoc. Cytom. 57:12.23.1‐12.23.12.
  Schrader, M. and Fahimi, H.D. 2006. Peroxisomes and oxidative stress. Biochim. Biophys. Acta 1763:1755‐1766.
  Schrader, M., Reuber, B.E., Morrell, J.C., Jimenez‐Sanchez, G., Obie, C., Stroh, T.A., Valle, D., Schroer, T.A., and Gould, S.J. 1998. Expression of PEX11beta mediates peroxisome proliferation in the absence of extracellular stimuli. J. Biol. Chem. 273:29607‐29614.
  Smith, J.J. and Aitchison, J.D. 2013. Peroxisomes take shape. Nat. Rev. Mol. Cell Biol. 14:803‐817.
  Wanders, R.J. 2004. Peroxisomes, lipid metabolism, and peroxisomal disorders. Mol. Genet. Metab. 83:16‐27.
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