Regulation of reticular adhesions by KANK2 and talin2 in two melanoma cell lines

BACKGROUND: Integrins bind extracellular matrix proteins and, upon clustering, form multimolecular integrin adhesion complexes (IACs) that connect to and regulate the cell cytoskeleton, influencing various aspects of cell behaviour. Alongside well-characterized focal adhesions (FAs) and fibrillar adhesions (FBs), a new class of IACs, reticular adhesions (RAs), have been identified. RAs, formed by integrin αVβ5, lack actin association and classical FAs markers and their physiological role in cells remains poorly understood. Previously, we showed that two melanoma cell lines, MDA-MB-435S and RPMI-7951, preferentially use integrin αVβ5 (which can form FAs or RAs) for adhesion under long-term culture conditions. Here we investigate the composition of RAs in these two melanoma cell lines. METHODS: Cells were treated with the actin polymerisation inhibitor cytochalasin D to disrupt FAs and enable isolation and mass spectrometry–based analysis of RAs components. Western blotting, immunofluorescence microscopy and proximity ligation assays (PLA) were performed to assess protein expression and localization within RAs. The effects of talin2 and KN motif and ankyrin repeat domains protein 2 (KANK2) knockdown on RA composition were examined in both melanoma cell lines. RESULTS: Known RA-associated proteins, including the Adaptor protein 2 (AP2) complex, disabled homolog 2 (DAB2), Numb and talin2 were identified in both lines, along with a new protein, KANK2. PLA following actin disruption confirmed the proximity of KANK2 and talin2 in RAs. Knockdown experiments demonstrated that although both talin2 and KANK2 are located in RAs, neither is essential for RA formation. Talin2 knockdown resulted in reduced abundance of RA components in both cell lines. In MDA-MB-435S cells, KANK2 knockdown produced a similar effect. However, in RPMI-7951 cells, KANK2 knockdown had no significant effect on RA components abundance, reflecting the differential localization and role of KANK2 in this cell line. CONCLUSION: We provide a comprehensive analysis of RAs in two melanoma cell lines and identify KANK2 as a novel RA-associated protein. The differential effects of KANK2 knockdown on the abundance of RAs underscores its distinct role in αVβ5-mediated adhesions, FAs and RAs, across the two cell lines. These findings emphasize the importance of adhesion crosstalk in regulating integrin αVβ5–mediated cell adhesions.