La proteína 4.1r regula la organización del citoesqueleto de tubulina y la migración celular

Resumo

Protein 4.1R was identified in red blood cells as a multifunctional protein that stabilizes the actin and spectrin network and anchors it to the plasma membrane. In nonerythroid cells, 4.1R binds to actin and tubulin, two of the major components of the cytoskeleton which play an essential role in cell migration. To contribute to the characterization of functional roles of 4.1R in nonerythroid cells, I have analyzed the participation of protein 4.1R in cell motility. Confocal microscopy of endothelial cells revealed that endogenous 4.1R accumulates at the leading edge of migrating cells. Exogenous 4.1R isoforms containing a complete membrane-binding domain consistently localized to plasma membrane extensions enriched in F-actin. Time-lapse microscopy analysis showed that silencing of 4.1R expression causes loss of persistence of migration in subconfluent cells and a reduction of directional migration in cells moving into a wound. Moreover, 4.1R knock-down cells show altered cell polarization and defects in the organization and dynamics of the microtubule network. By coimmunoprecipitation and pull-down assays I have identified the scaffold protein involved in cell migration, IQGAP1, as a new partner for protein 4.1R. Interestingly, the 4.1R membrane-binding domain is involved in binding IQGAP1. Importantly, I also show that protein 4.1R is necessary for the correct localization of IQGAP1 to the leading edge of cells migrating into a wound, while IQGAP1 is not required for protein 4.1R localization. In addition, proteomic analysis of 4.1R has allowed the identification of CLASP2 as another binding partner for 4.1R. By combining time-lapse and TIRF microscopy I show that 4.1R modulates CLASP2 distribution as a +TIP protein and its dynamics as a component of cortical platforms, multiprotein complexes which are essential for MT anchorage to the cell cortex. Collectively, my results suggest a crucial role for protein 4.1R in cell migration. 4.1R is a novel scaffold protein that regulates localization and dynamics of CLASP2 and IQGAP1 thereby regulating microtubule dynamics and cell migration.