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Composition of focal and reticular adhesions in melanoma cell lines MDA-MB-435S and RPMI-7951

Cell adhesion mediated by integrins, cell surface heterodimers composed of α and β subunits, integrate signalling between the extracellular matrix (ECM) and cells and control many aspects of normal and tumour cells behaviour. Molecular mechanisms related to cell adhesion have been extensively studied, especially in cancer in which mutations and/or changes in expression of these proteins have been observed contributing to its proliferation, migration and invasion. Through binding to the ECM and clustering, integrins form multimolecular adhesion complexes (IACs) which are connected to, and can regulate the cell cytoskeleton and are termed nascent adhesions, focal adhesions (FAs), fibrillar adhesions and hemidesmosomes. A new class of IACs, named reticular adhesions (RAs) were firstly described as clathrin lattices composed of hexagonal clathrin structures, enriched in dynamin and AP2 (adaptor protein complex 2), both involved in clathrin-mediated endocytosis. They are formed by integrin αVβ5, lack association with actin and are devoid of vinculin which is marker of FAs. There is only a limited evidence for other integrins than αVβ5 being localized to these sites as well as presence of other components especially talins whose binding is necessary for integrin activation. While FAs are completely depleted during mitosis, RAs are maintained to enable effective mitosis and also transmit spatial memory from pre-mitotic to post-mitotic daughter cells. However, the better understanding of contribution of RAs to normal and tumour cell physiology is needed. We have previously analysed IACs of two melanoma cell lines MDA-MB-435S and RPMI-7951, grown in long term culture, using biochemical isolation and mass spectrometry (MS)-based proteomics, and demonstrated that both cell lines use preferentially integrin αVβ5 for adhesion forming FAs. Immunofluorescent analysis has shown that integrin αVβ5, in both cell lines, was localized in vinculin positive FAs and vinculin negative, ring-like or reticular structures thus resembling RAs. To determine their composition, we exposed MDA-MB-435S and RPMI-7951 cells grown in long term culture to inhibitor of actin polymerisation Cytochalasine D to disrupt FAs, thus enabling us to isolate only RAs. The composition of RAs was then analysed by (MS)-based proteomics. Our results show the absence of FA components such as Talin1, filamins and alpha actinins and enrichment of proteins such as AP-2, disabled homolog 2 (DAB2) and Numb. Interestingly, in RAs isolates we observed presence of Talin2. To validate MS results, we preformed Western blot analysis of RA isolates and immunofluorescence analysis to demonstrate localisation of TLN2, DAB2 and Numb proteins. The comprehensive analysis of FA and RA compositions in two cell lines, sharing the same integrin αVβ5, will enable us to analyse their regulatory interplay/relationships.

integrins, adhesome, reticular adhesions, focal adhesions

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