Plakophilin 1 but not plakophilin 3 regulates desmoglein clustering

Abstract

Plakophilins (Pkp) are desmosomal plaque proteins crucial for desmosomal adhesion and participate in the regulation of desmosomal turnover and signaling. However, direct evidence that Pkps regulate clustering and molecular binding properties of desmosomal cadherins is missing. Here, keratinocytes lacking either Pkp1 or 3 in comparison to wild type (wt) keratinocytes were characterized with regard to their desmoglein (Dsg) 1- and 3-binding properties and their capability to induce Dsg3 clustering. As revealed by atomic force microscopy (AFM), both Pkp-deficient keratinocyte cell lines showed reduced membrane availability and binding frequency of Dsg1 and 3 at cell borders. Extracellular crosslinking and AFM cluster mapping demonstrated that Pkp1 but not Pkp3 is required for Dsg3 clustering. Accordingly, Dsg3 overexpression reconstituted cluster formation in Pkp3- but not Pkp1-deficient keratinocytes as shown by AFM and STED experiments. Taken together, these data demonstrate that both Pkp1 and 3 regulate Dsg membrane availability, whereas Pkp1 but not Pkp3 is required for Dsg3 clustering.

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Abbreviations

AFM:

Atomic force microscopy

ARVC:

Arrhythmogenic cardiomyopathy

DP:

Desmoplakin

Dsc:

Desmocollin

Dsg:

Desmoglein

EC:

Extracellular domains

FRAP:

Fluorescence recovery after photobleaching

HaCaT:

Cultured human keratinocytes

IF:

Intermediate filament

MAPK:

Mitogen-activated protein kinase

MKZ:

Murine keratinocytes

PEG:

Polyethylenglycol

Pg:

Plakoglobin

Pkp:

Plakophilins

PV:

Pemphigus vulgaris

QI:

Quantitative imaging

STED:

Stimulated emission depletion

Sulfo-EGS:

Ethylene glycolbis (sulfosuccinimidylsuccinate)

UFs:

Unbinding forces

UP:

Unbinding position

wt:

Wild type

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Acknowledgements

We thank Andrea Wehmeyer and Sabine Mühlsimer for excellent technical assistance and JPK instruments and Sunil Yeruva for constructive technical and scientific discussion. The project is funded by Else-Kröner-Fresenius-Stiftung 2016_AW157 to FV and JW and DFG SPP1782 to JW.

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MFu, MF, MR, DK: methodology, data acquisition and analysis; FV and JW: funding acquisition, conceptualization and supervision; MFu, FV, JW: writing and editing, RK, MH, VS: methodology, review.

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Correspondence to Jens Waschke or Franziska Vielmuth.

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Fuchs, M., Foresti, M., Radeva, M.Y. et al. Plakophilin 1 but not plakophilin 3 regulates desmoglein clustering. Cell. Mol. Life Sci. 76, 3465–3476 (2019). https://doi.org/10.1007/s00018-019-03083-8

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Keywords

  • Desmosome
  • Cell adhesion
  • Desmosomal clustering
  • Atomic force microscopy
  • STED