engleski

TALINs, KANKs and actin-microtubule crosstalk in integrin αvβ5 focal adhesions in melanoma cell line MDA-MB-435s

Integrins are heterodimeric adhesion molecules that bind cells to extracellular matrix and upon clustering form multimolecular integrin adhesion complexes (IACs), reorganising the cytoskeletal networks and regulating cell proliferation, migration and survival. Motivated by observation of increased sensitivity to microtubule (MT) poisons, paclitaxel (PTX) and vincristine, upon integrin αV knockdown, in the melanoma cell line MDA-MB-435S, we analysed the IAC composition and showed that cells predominantly use integrin αVβ5 for adhesion. The IAC analysis revealed components of αVβ5 IACs that included talins (TLN) 1 and 2, and KANKs 1 and 2. Since KANK2 knockdown mimicked changes observed upon αV knockdown, i.e. increased sensitivity to MT poisons and decreased migration, we concluded that KANK2 is a key molecule linking αVβ5 focal adhesions (FAs) to MTs. To distinguish the mutual binding of TLN and KANK isoforms, we performed immunofluorescent analyses of TLN1, TLN2, KANK1, KANK2 and MTs as well as measured sensitivity to PTX in MDA-MB-435S cells upon knockdown of each TLN or KANK isoform. TLN1 knockdown eliminated integrin αVβ5 FAs indicating that it is the central adapter protein for FA formation, unlike TLN2 knockdown which slightly changed FA size but didn’t affect its number. Knockdown of TLN1 or TLN2 didn’t change the localization of KANK1. As expected, KANK1 knockdown didn’t affect sensitivity to PTX. Although knockdown of TLN2 didn’t affect KANK2 localisation, it increased sensitivity to PTX and altered the MT appearance in the cell suggesting changes of recruitment of MTs to integrin αVβ5 FAs. Currently, we are performing co- immunoprecipitations of TLNs and KANKs as well as microtubule dynamics measurements 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 ; actin-microtubule crosstalk ; paclitaxel

nije evidentirano