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Role of talins and KANKs in integrin αvβ5 focal adhesions, actin-microtubule crosstalk and response to paclitaxel treatment in MDA-MB-435S cells

Multiple signalling pathways that can control cytoskeletal network organisation, as well as cell proliferation, differentiation, survival and motility are triggered by cell binding to the extracellular matrix (ECM) via integrins. Upon binding to the ECM, these heterodimeric cell surface receptors cluster and form multimolecular integrin adhesion complexes (IACs). IAC composition analysis in MDA-MB-435s, previously described that integrin αVβ5 is the predominant integrin used in long term culture and showed increased sensitivity to microtubule (MT) poisons, paclitaxel (PTX) and vincristine, upon integrin αV knockdown. The analysis also revealed that αVβ5 IACs contain talins (TLN) 1 and 2, and KANKs 1 and 2. Out of these, KANK2 has already been shown to have a key role in connecting the αVβ5 focal adhesions (FAs) to MTs, and influencing cell sensitivity to PTX. Since talins and KANKs bind, our goal was to investigate their localisation, distinguish the mutual binding of their isoforms as well as elucidate their role in formation of IACs and MT dynamics, and subsequently their effect on cell sensitivity to PTX. We performed immunofluorescence and western blot analyses of TLN1, TLN2, KANK1, KANK2 localisation/ expression, actin and MTs visualisation as well as survival and proliferation assay upon knockdown of each TLNs and KANKs. TLN1 knockdown resulted in decreased expression and changed localisation of KANK2 in cell, eliminated integrin αVβ5 FAs, changed cell morphology and proliferation as well actin and MT appearance ; however, it did not increase cell sensitivity to PTX. On the other hand, knockdown of TLN2 did not affect cell morphology and proliferation, nor KANK2 expression and localisation, but it slightly changed FA size, increased sensitivity to PTX and altered the MT appearance. Interestingly, knockdown of TLN1 or TLN2 did not change the localisation/ expression of KANK1, and KANK1 knockdown does not affect sensitivity to PTX. We are currently performing microtubule dynamics measurements upon each TLN and KANK protein using fluorescently labelled microtubule end-binding protein 3. These data will elucidate the differential role of TLN and KANK isoforms in αVβ5 FAs which contribute to actin-MT crosstalk and consequential response to MT poisons in the melanoma

TLN ; KANK ; microtubule ; paclitaxel

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