Integrins form focal adhesions (FAs) at the cell edge and fibrillar adhesions (FBs) located centrally. Talin1 is essential to FAs, while talin2 is found in FAs and FBs. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics and are recruited to adhesions through interaction with talins. Previously, we showed in MDA-MB-435S melanoma cells that KANK2 is part of integrin αVβ5 FAs, that its interaction with talin2 regulates actin-microtubule (MT) crosstalk and that KANK2 knockdown mimics the effect of integrin αV or β5 knockdown by reducing cell migration. Here, in another melanoma cell line RPMI-7951 we observed that KANK2 is part of FAs and FBs and that KANK2 knockdown increases cell migration. Therefore, we analyse integrin adhesion complexes in RPMI-7951 cells, explore the localization and role of KANK2 in FAs and FBs. Knockdown in human melanoma RPMI-7951 cells was achieved by transfection with gene-specific siRNAs. Integrin adhesion complexes were isolated and analysed by mass spectrometry. Immunofluorescence analysis, live cell imaging and the proximity ligation assays were done using confocal microscopy. Cell migration was assessed using Transwell Cell Culture Inserts. MTT assays were performed to determine cell sensitivity to paclitaxel. Data were statistically evaluated using one-way or two-way ANOVA or unpaired Student’s t-tests in GraphPad Software. We demonstrate that RPMI-7951 melanoma cells use integrin αVβ5 FAs and integrin α5β1 FBs for adhesion, and that KANK2 is part of both structures. KANK2 is predominantly in proximity to talin1 at the cell edge (FAs) and in proximity to talin2 in the cell center (FBs). KANK2 in FAs functionally interacts with talin1 to maintain FAs, and with talin2 to regulate their dynamics. KANK2 is a component of FBs, and its knockdown mimics integrin α5 knockdown by increasing MT-dependent cell migration. Our study reveals the distinct roles of KANK2 in FAs and FBs. We show that KANK2 is a component of FBs, linking them to MTs and promoting their stabilisation. Loss of integrin α5 or KANK2 from FBs increases cell migration, a process that relies on the MT cytoskeleton. Cells adhere to their surroundings using specialized structures called adhesions, which are formed by different types of integrins and have different cell localisations: focal adhesions at the cell edge and fibrillar adhesions in the cell center. Talin1 is essential for focal adhesions, while talin2 is present in focal and fibrillar adhesions. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics by connecting talins to the microtubule cytoskeleton. Our study explored the different roles of KANK2 in focal or fibrillar adhesions, by focusing on its interactions with integrins and talins. We show that in RPMI-7951 cells, which form αVβ5 focal and α5β1 fibrillar adhesions, KANK2 is present in both of these adhesions. KANK2 supports αVβ5 focal adhesion maintenance via talin1 and regulates their dynamics via talin2. KANK2 is also a component of α5β1 fibrillar adhesions, and its depletion mimics integrin α5 knockdown and enhances cell migration, a process dependent on the microtubule cytoskeleton. In conclusion, our study demonstrates that KANK2 plays distinct roles in different adhesion structures, contributing to both focal and fibrillar adhesion function.
Stojanović et al. (Tue,) studied this question.