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Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line.
Loncaric, Marija; Stojanovic, Nikolina; Rac-Justament, Anja; Coopmans, Kaatje; Majhen, Dragomira; Humphries, Jonathan D; Humphries, Martin J; Ambriovic-Ristov, Andreja.
Afiliação
  • Loncaric M; Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia.
  • Stojanovic N; Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia.
  • Rac-Justament A; Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia.
  • Coopmans K; Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia.
  • Majhen D; Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia.
  • Humphries JD; Department of Life Science, Manchester Metropolitan University, Manchester, United Kingdom.
  • Humphries MJ; Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
  • Ambriovic-Ristov A; Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia. Andreja.Ambriovic.Ristov@irb.hr.
Cell Mol Biol Lett ; 28(1): 56, 2023 Jul 17.
Article em En | MEDLINE | ID: mdl-37460977
BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVß5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVß5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. CONCLUSION: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Talina / Melanoma Tipo de estudo: Diagnostic_studies / Guideline Limite: Humans Idioma: En Revista: Cell Mol Biol Lett Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Croácia

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Talina / Melanoma Tipo de estudo: Diagnostic_studies / Guideline Limite: Humans Idioma: En Revista: Cell Mol Biol Lett Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Croácia