RESUMO
Spectrin is a ubiquitous cytoskeletal protein that provides structural stability and supports membrane integrity. In erythrocytes, spectrin proteolysis leads to the biogenesis of plasma membrane extracellular vesicles (EVs). However, its role in non-erythroid or cancer-derived plasma membrane EVs biogenesis is unknown. This study aims to examine the role of αII-spectrin in malignant and non-malignant plasma membrane vesiculation. We developed a custom, automated cell segmentation plugin for the image processor, Fiji, that provides an unbiased assessment of high resolution confocal microscopy images of the subcellular distribution of αII-spectrin. We show that, in low vesiculating non-malignant MBE-F breast cells, prominent cortical spectrin localises to the cell periphery at rest. In comparison, cortical spectrin is diminished in high vesiculating malignant MCF-7 breast cells at rest. A cortical distribution of spectrin correlates with increased biomechanical stiffness as measured by Atomic Force Microscopy. Furthermore, cortical spectrin can be induced in malignant MCF-7 cells by treatment with known vesiculation modulators including the calcium chelator, BAPTA-AM or the calpain inhibitor II (ALLM). These results demonstrate that the subcellular localisation of spectrin is distinctly different in malignant and non-malignant cells at rest and shows that the redistribution of cortical αII-spectrin to the cytoplasm supports plasma membrane-derived EV biogenesis in malignant cells.