ARHGAP32 as a novel RhoGAP interacting with desmoplakin is required for desmosomal organization and assembly

Abstract Desmosomes are specialized cell-cell junctions that play a critical role in maintaining tissue barrier integrity, particularly in mechanically stressed tissues. The assembly of desmosomes is regulated by the cytoskeleton and its regulators, and desmosomes also function as a central hub for regulating F-actin. However, the specific mechanisms underlying the crosstalk between desmosomes and F-actin, particularly involving RhoGAP or RhoGEF proteins, remain unclear. In our study, we identified that ARHGAP32, a Rho GTPase-activating protein, is located in desmosomes through its interaction with DSP via its GAB2-interacting domain. Using CRISPR-Cas9 gene knockout system, we confirmed that ARHGAP32 is required for proper desmosomal organization, maturation, and length regulation. Notably, the loss of ARHGAP32 resulted in increased formation of F-actin stress fibers and phosphorylation of MYOSIN at T18/S19, indicating enhanced actomyosin contractility. Furthermore, inhibition of ROCK1 kinase activity using Y27632 effectively restored desmosomal organization. Moreover, we demonstrated that the regulation of desmosomes by ARHGAP32 is crucial for maintaining the integrity of epithelial cell sheets. Collectively, our study unveils ARHGAP32 as a RhoGAP present at desmosomes, potentially facilitating the crosstalk between desmosomes and F-actin. Its presence is indispensable for desmosomal assembly and the preservation of epithelial cell sheet integrity by regulating actomyosin contractility.