engleski

Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration

Integrins are heterodimeric adhesion molecules that bind cells to extracellular matrix, and upon clustering, form multimolecular focal adhesions (FAs), reorganising cytoskeletal networks and regulating cell proliferation, migration and survival. Microtubules (MT) are stabilised in the vicinity of FAs through interaction with the components of the cortical microtubule stabilising complex (CMSC). KANK family proteins within the CMSC, KANK1 or KANK2, bind talin1 or talin2 within FA and thus mediate actin-MT crosstalk. A comparative analysis between the melanoma cell line MDA-MB-435S and cell clones with decreased expression of integrin αV showed that these cells preferentially use integrin αVβ5 for adhesion, and identified key components of integrin αVβ5 adhesion complexes, including talin1 and talin2. The data also revealed decreased levels of several components of the CMSC including KANK2. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin αV knockdown and resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine, and decreased migration, thus showing that KANK2 is a key molecule enabling the actin-MT crosstalk important for both sensitivity to MT poisons and cell migration. Here, we aimed to distinguish which talin isoform bound KANK2. Immunofluorescence analysis showed that talin1 knockdown led to disruption of integrin αVβ5 FAs along with reduction of KANK2, indicating that talin1 is the central adapter protein for FA formation. Conversely, talin2 knockdown slightly increased integrin αVβ5 FA size but did not affect the αVβ5, talin1 or KANK2 localisation. However, knockdown of either talin1, talin2 or KANK2 increased the velocity of MT growth, as shown by live cell microscopy of fluorescent EB3-Dendra2, indicating that talin2 might link αVβ5 FAs to the CMSC. Biochemical isolation and western blotting of crosslinked FA proteins upon knockdown of talin2 showed a reduced amount of KANK2 with unchanged levels of integrin β5 and talin1. Finally, talin2 knockdown mimicked changes observed upon KANK2 knockdown i.e. increased sensitivity to PTX and reduced migration. These data elucidate the cell- type-specific role of talin2 and KANK2 isoforms which contribute to actin-MT crosstalk and the consequential response to MT poison PTX and cell migration.

talin1 ; talin2 ; KANK2 ; aktin ; microtubules

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