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Role of α-actinins in αVβ5 FA maturation, actin organisation and migration in melanoma cell line RPMI-7951

Changes in expression of integrins, heterodimeric transmembrane cell adhesion molecules used by cells to bind to the extracellular matrix and control cytoskeletal network organization, are frequently associated with cancer. Integrins foster tumour cell survival, proliferation and migration during invasion and metastasis through bidirectional signalling via multimolecular integrin adhesion complexes (IACs). Our previously published data in melanoma cell line RPMI-7951 showed that knockdown of integrin αV decreased migration and invasion. Mass spectrometry (MS) analysis of biochemically isolated IACs revealed that this cell line preferentially uses integrin αVβ5 for adhesion forming FAs, and we confirmed its exclusive role in regulating cell migration. MS also revealed that αVβ5 IACs contain actin cross-linking proteins α- actinin 1 (ACTN1) and α- actinin 4 (ACTN4). These are ubiquitously expressed cytoskeleton proteins that cross-link actin filaments, and through interaction with a number of IAC proteins and integrins anchor them to focal adhesions at the leading edge of migrating cells. As such, α- actinins can influence actin cytoskeleton organization and dynamics, focal adhesion maturation and cell migration. To investigate their localisation, elucidate their individual role in formation of IACs, actin dynamics and migration, we performed immunofluorescence and western blot analyses of ACTN1 and ACTN4 localisation/ expression, actin and FAs visualisation as well as migration assay upon knockdown of each ACTNs. Both knockdown of ACTN1 and ACTN4 resulted in diminished cell size and decreased expression of integrin αVβ5 FAs. However, the integrin αVβ5 FAs size only changed upon ACTN1 knockdown, indicating its possible role in maturation of αVβ5 FAs. Additional data pointing to the role of ACTN1 in FAs maintenance was the increased expression of ACTN1 upon ACTN4 knockdown. Also, we observed a transition from dorsal actin fibers with the transverse arc to ventral stress fibers upon ACTN4 knockdown. We are currently performing migration assays and live cell imaging upon each ACTN knockdown using fluorescently labelled ACTN4. These data will elucidate the differential role of ACTN isoforms in αVβ5 FAs which contribute to actin organisation and migration in melanoma.

actinin, actin cytoskeleton, migration

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