Abstract
Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile β3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation.
Highlights
Identified at the inner surface of the erythrocyte membrane, spectrins are the central components of a universal and complex spectrin-actin scaffold, called the spectrin-based skeleton [1]
Other plasmids were used to transfect mouse embryonic fibroblasts (MEFs) v-Src Y527F cells: recombinant full-length of αII-spectrin fused to GFP from pCep4 plasmid, and plasmids expressing the LifeAct peptide fused with fluoro-Ruby (Ruby-LifeAct, a red marker visualising F-actin in living cells), β3-integrin tagged with RFP, actin tagged with RFP (RFP-actin), cortactin tagged with RFP, or paxillin tagged with RFP, these sixth last allowing detecting invadosome structures
We determined the localization of endogenous αII-spectrin in different invadosome models such as in human endothelial cells (HMEC-1 cell line), primary human endothelial cells (HUVEC) after different treatments (PMA or EGF) leading to invadosome formation, and in mouse embryonic fibroblasts expressing a constitutively activated mutant of the non-receptor tyrosine kinase Src (SrcY527F)
Summary
Identified at the inner surface of the erythrocyte membrane, spectrins are the central components of a universal and complex spectrin-actin scaffold, called the spectrin-based skeleton [1]. As flexible large/long tetramers (200 nm length) composed of α and β subunits that associate side by side head to head constitute the filaments of this network. Spectrins are encoded by seven genes, including two genes for the α-spectrin subunits (αI and αII), and five genes for the β-spectrin subunits (βI to βV). The αβ heterodimers display distinct tissue-specific cellular and subcellular patterns of expression: red-blood-cell spectrin consists.
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