A Monoclonal Antibody for <i>in vivo</i> Detection of Peroxisome-associated PTS1 Receptor

Cizmowski, Christian; Jung, Martin; Erdmann, Ralf; Schliebs, Wolfgang

doi:10.1089/hyb.2011.0038

Cited by 6 publications

(4 citation statements)

References 21 publications

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“…At the membrane, PEX5 is interacting with PEX14, triggering the translocation of the cargo protein [15–17] . The monoclonal PEX5 antibody used here, only binds to PEX5 when it is located at the peroxisomal membrane and not to the cytosolic pool of the shuttling receptor [18] . Here, we obtained good STED images using PEX14 and PEX5 in a dual colour STED colocalization study [8] but the signal with the PEX5 antibody was too weak for ExM‐STED (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Challenges of Using Expansion Microscopy for Super‐resolved Imaging of Cellular Organelles

et al. 2020

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Expansion microscopy (ExM) has been successfully used to improve the spatial resolution when imaging tissues by optical microscopy. In ExM, proteins of a fixed sample are crosslinked to a swellable acrylamide gel, which expands when incubated in water. Therefore, ExM allows enlarged subcellular structures to be resolved that would otherwise be hidden to standard confocal microscopy. Herein, we aim to validate ExM for the study of peroxisomes, mitochondria, nuclei and the plasma membrane. Upon comparison of the expansion factors of these cellular compartments in HEK293 cells within the same gel, we found significant differences, of a factor of above 2, in expansion factors. For peroxisomes, the expansion factor differed even between peroxisomal membrane and matrix marker; this underlines the need for a thorough validation of expansion factors of this powerful technique. We further give an overview of possible quantification methods for the determination of expansion factors of intracellular organelles, and we highlight some potentials and challenges.

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Section: Resultsmentioning

confidence: 99%

Challenges of Using Expansion Microscopy for Super‐resolved Imaging of Cellular Organelles

et al. 2020

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“…1 A ). For studying membrane-bound PEX5, only an antibody specifically recognizing the membrane-bound conformation ( 17 ) was used.…”

Section: Resultsmentioning

confidence: 99%

“…Samples were incubated with primary antibodies in blocking buffer for 1 h at room temperature. To stain peroxisomal proteins, antibodies recognizing PEX14 ( 38 ), PEX5 ( 17 ), and PEX11B (Abcam, Cambridge, UK) were used. Mitochondria were visualized using an antibody against TOM20 (FL-145, Santa Cruz Biotechnology, Dallas, TX).…”

Section: Methodsmentioning

confidence: 99%

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

Galiani

Waithe

Reglinski

et al. 2016

Journal of Biological Chemistry

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Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.

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“…To study interactions between PEX5 and PEX13 dynabeads were coupled with a mouse PEX5 antibody 65 , using the dynabeads™ Antibody Coupling Kit (Invitrogen, USA) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif

Gaussmann,

Peschel,

Ott

et al. 2024

Nat Commun

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Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.

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A Monoclonal Antibody for in vivo Detection of Peroxisome-associated PTS1 Receptor

Cited by 6 publications

References 21 publications

Challenges of Using Expansion Microscopy for Super‐resolved Imaging of Cellular Organelles

Challenges of Using Expansion Microscopy for Super‐resolved Imaging of Cellular Organelles

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif

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