Rac, A.; Loncaric, M.; Stojanovic, N.; Fatima, M.; Resetar, M.; Hrsak, D.; Humphries, J. D.; Humphries, M. J.; Ambriovic-Ristov, A.

Integrins bind to extracellular matrix proteins and, upon clustering, form multimolecular integrin adhesion complexes (IACs) that connect to and regulate the cell cytoskeleton, influencing various aspects of normal and tumour cell behaviour. Alongside well-characterized nascent adhesions, focal adhesions (FAs), fibrillar adhesions (FBs) and hemidesmosomes, a new class of IACs, reticular adhesions (RAs), have been identified. RAs, initially described as flat clathrin lattices formed by integrin V{beta}5, lack association with actin and are devoid of FAs markers. The physiological role of RAs in normal and tumor cells is still incompletely understood and requires further investigation. Previously, we analysed IACs of two melanoma cell lines, MDA-MB-435S and RPMI-7951, grown under long term culture conditions, and demonstrated that both cell lines preferentially use integrin V{beta}5 for adhesion. Here we present a comprehensive analysis of RAs in these two melanoma cell lines that differ in their ability to form FBs. To determine RAs composition, we treated cells with actin polymerisation inhibitor cytochalasin D (CytoD) which disrupts FAs, allowing isolation of RAs, which were analysed by MS-based proteomics, Western blotting and immunofluorescence. Known RA-associated proteins, including the AP-2 adaptor complex, disabled homolog 2 (DAB2) and Numb were identified in both lines, along with talin2. Notably, we also detected the presence of KN motif and ankyrin repeat domains protein (KANK2) in RA isolates. Proximity ligation analysis following CytoD-induced actin disruption confirmed the proximity of KANK2 and talin2 in RAs. We then investigated the effect of talin2 or KANK2 knockdown on RAs composition. While both talin2 and KANK2 are located in RAs, neither is essential for RA formation. Talin2 knockdown led to a reduction in RA components abundance in both cell lines. In MDA-MB-435S cell line, KANK2 produced a similar effect, mirroring the functional interaction of talin2 and KANK2 in FAs. However, in RPMI-7951 cells, KANK2 knockdown had no significant effect on RA components abundance. This discrepancy likely reflects the preferential localization of KANK2 in FBs and underscores the differing roles of talin2 and KANK2 in V{beta}5-mediated FAs across the two cell lines. These findings underscore the complexity of adhesion signalling and highlight the importance of adhesion crosstalk in regulating cellular function.