Comparative interactomics analysis reveals potential regulators of α6β4 distribution in keratinocytes.

Arnoud Sonnenberg,Maaike Kreft,Lisa te Molder,Onno Bleijerveld,Liesbeth Hoekman

doi:10.1242/bio.054155

Arnoud Sonnenberg, Maaike Kreft + Show 3 more

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https://doi.org/10.1242/bio.054155

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Abstract

ABSTRACTThe integrin α6β4 and cytoskeletal adaptor plectin are essential components of type I and type II hemidesmosomes (HDs). We recently identified an alternative type II HD adhesion complex that also contains CD151 and the integrin α3β1. Here, we have taken a BioID proximity labeling approach to define the proximity protein environment for α6β4 in keratinocytes. We identified 37 proteins that interacted with both α6 and β4, while 20 and 78 proteins specifically interacted with the α6 and β4 subunits, respectively. Many of the proximity interactors of α6β4 are components of focal adhesions (FAs) and the cortical microtubule stabilizing complex (CMSC). Though the close association of CMSCs with α6β4 in HDs was confirmed by immunofluorescence analysis, CMSCs have no role in the assembly of HDs. Analysis of the β4 interactome in the presence or absence of CD151 revealed that they are strikingly similar; only 11 different interactors were identified. One of these was the integrin α3β1, which interacted with α6β4 more strongly in the presence of CD151 than in its absence. These findings indicate that CD151 does not significantly contribute to the interactome of α6β4, but suggest a role of CD151 in linking α3β1 and α6β4 together in tetraspanin adhesion structures.

Highlights

The cytoskeleton of epithelial cells is an integrated network of actin microfilaments, keratin intermediate filaments and microtubules, which helps the cell to maintain its shape and internal organization
Identification of the β4 associated proteins by proximitydependent biotinylation To identify proteins that interact with integrin α6β4 in keratinocytes, we applied the BioID method (Roux et al, 2012), which has been successfully used to identify proteins that reside in close proximity of a specific protein of interest, including proteins that are only transiently associated
None of the identified proteins were components of HDs, cortical microtubule stabilizing complex (CMSC) or focal adhesions (FAs) (Fig. 3B; Table S4). These results show that despite the fact that CD151 forms a complex with α6β4, this protein does not substantially contribute to the α6β4 interactome. These findings suggest that the CD151-containing adhesions in the central region of the cells hardly contribute to the interaction between HDs and CMSCs or FAs

Summary

Introduction

The cytoskeleton of epithelial cells is an integrated network of actin microfilaments, keratin intermediate filaments and microtubules, which helps the cell to maintain its shape and internal organization. The actin and keratin networks are linked to the cytoskeletons of adjacent cells and the extracellular matrix by specialized junctional complexes and, thereby, contribute to tissue integrity and intracellular communication (Kirfel et al, 2003; Blanchoin et al, 2014). Hemidesmosomes (HDs) are integrin-based adhesion complexes that mediate stable anchorage of epithelial cells to the underlying basement membrane and serve as anchoring sites for the keratin. In addition to α6β4, these two structures share the tetraspanin CD151 and the cytoskeletal linker protein plectin, which through binding to β4 and keratin establishes a linkage between the extracellular matrix (ECM) and the keratin intermediate filament (IF) cytoskeleton. In type I HDs an additional linkage between the plasma membrane and keratin filaments through BP180 and BP230 enhances the stability of these adhesion structures. Besides its role in mediating stable cell-matrix adhesion, the HD integrin α6β4 cooperates with growth factor receptors to promote pro-tumorigenic signaling (Ramovs et al, 2017)

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