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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

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Abstract

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.

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Abbreviations

BSA:

Bovine serum albumin

CARD:

Catalyzed reporter deposition

DAB:

3,3′-Diaminobenzidine

DMSO:

Dimethylsulfoxide

DMEM:

Dulbecco’s modified Eagle medium

DTT:

Dithreitol

EM:

Electron microscopy

FBS:

Fetal bovine serum

FFPE:

Formalin-fixed paraffin-embedded

IF:

Immunofluorescence

IHC:

Immunohistochemistry

MEM:

Minimal essential medium

PEX13:

Peroxisome biogenesis protein 13

PEX14:

Peroxisome biogenesis protein 14

PBS:

Phosphate-buffered saline

PBSA:

Bovine serum albumin in phosphate-buffered saline

PFA:

Depolymerized paraformaldehyde

QDots:

QuantumDots™

ROS:

Reactive oxygen species

RT:

Room temperature

SOD2:

Mitochondrial superoxide dismutase

TBS:

Tris-buffered saline

TBST:

Tris-buffered saline plus 0.5 % Tween 20

WB:

Western blotting

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Acknowledgments

We thank Bianca Pfeiffer, Magdalena Gottwald, and Elke Rodenberg-Frank for excellent technical assistance in the morphology laboratory as well as Dr. Wiebke Möbius, Max-Planck-Institute for Experimental Medicine, Göttingen, for the donation of rat liver tissue for cryo-immuno-electron microscopy for catalase and Jessica and Michelle Woods for proofreading the manuscript. The data of this manuscript have been published as part of the doctoral thesis of Phillip Grant in electronic form (Online library JLU, 2011).

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  1. Phillip Grant

    Present address: Personality Psychology and Individual Differences, Justus Liebig University, Otto-Behaghel-Str. 10F, 35394, Giessen, Germany

  2. Klaus Kuchelmeister

    Present address: Institute for Neuropathology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany

  3. Anca Nenicu

    Present address: Institute for Clinical and Experimental Surgery, University Hospital Homburg-Saar, 66421, Homburg, Germany

Authors and Affiliations

  1. Institute for Anatomy and Cell Biology II, Division of Medical Cell Biology, Justus Liebig University, Aulweg 123, 35385, Giessen, Germany

    Phillip Grant, Barbara Ahlemeyer, Srikanth Karnati, Timm Berg, Ingra Stelzig, Anca Nenicu & Eveline Baumgart-Vogt

  2. Eskitis Institute for Drug Discovery, School of Biomolecular and Physical Sciences, Griffith University, Nathan, Brisbane, QLD, 4111, Australia

    Denis I. Crane

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418_2013_1133_MOESM1_ESM.jpg

Supplemental Figure 1: A-H. Comparison of antibodies against mouse full-length PEX14 (self-created) and human full-length PEX14 protein (commercially available) in mouse and human cell lines and tissues. Parallel immunostainings for PEX14 were performed on mouse (Hepa 1-6; A, B) and human (HepG2; C, D) hepatoma cells, mouse neocortex (E, F) and autolytic submandibular gland tissue from human donors (H, I) using the anti-PEX14 antibody from Denis Crane (DC) and Millipore (MP) at the indicated dilutions. I-J. Comparison of staining patterns using the anti-catalase antibody from Denis Crane (DC, I) and the anti-ABCD3 antibody from Alfred Völkl (AV, J) to that for PEX14 (G, H) in paraffin sections of the autolytic human submandibular gland. As with the PEX14 antigen, the peroxisomal catalase antigen is conserved and peroxisomes can be distinguished clearly. The autolysis of the tissue is most obvious in a capillary with leaky autofluorescent erythrocytes (asterisks). In contrast to the PEX14 and catalase antigens, the ABCD3 antigen is degraded during progressive autolysis. Arrows indicate cells with TOTO-3-iodide-labeled DNA in leaky mitochondria. A few peroxisomes with a weak staining can be identified at a higher magnification. (JPEG 2921 kb)

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Grant, P., Ahlemeyer, B., Karnati, S. et al. 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 (2013). https://doi.org/10.1007/s00418-013-1133-6

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