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Focal and Reticular Adhesion Composition in the Melanoma Cell Lines MDA-MB-435S and RPMI-7951

Through binding to the extracellular matrix (ECM) and clustering, integrins form multimolecular adhesion complexes (IACs) which are connected to, and can regulate, the cell cytoskeleton as well as control many aspects of normal and tumour cells behaviour. In addition to the already known nascent adhesions, focal adhesions (FAs), fibrillar adhesions and hemidesmosomes, a new class of IACs, named reticular adhesions (RAs) have been identified. First described as clathrin lattices composed of hexagonal clathrin structures and enriched in dynamin and AP2 (adaptor protein complex 2), RAs are formed by integrin αVβ5, lack association with actin and are devoid of markers of FAs, including vinculin. While FAs are 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 contribution of RAs to normal and tumour cell physiology is still not completely understood and needs to be investigated further. 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. Immunofluorescence analysis has shown that integrin αVβ5, in both cell lines, is localised 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 an inhibitor of actin polymerisation, cytochalasin D, which disrupts 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 major FA components and enrichment of clathrin adaptor proteins, disabled homolog 2 (DAB2), Numb and AP-2. In addition to proteins described as part of RAs by other authors, we provide an additional list of enriched proteins whose involvement in RAs needs to be determined. 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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