Membrane to cytosol redistribution of αII‐spectrin drives extracellular vesicle biogenesis in malignant breast cells

Abstract

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. K E Y W O R D S calcium, cancer, cytoskeleton, extracellular vesicles, spectrin 1 INTRODUCTION Microvesicles (MV) are extracellular vesicles (EVs) measuring ∼0.1-1 μm that are shed from the plasma membrane of cells following loss of phospholipid asymmetry and cytoskeletal reorganisation [1].Plasma membrane-derived EVs are characteristic of their cell of ori-

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“…The relationship between membrane and cytoskeleton provides structural stability and one of the proteins involved in this stability is Spectrin. A reduction of Spectrin may be an indicator of cells with a higher propensity to vesiculate ( Taylor et al, 2021 ).…”

Decreased MARCKS Protein Expression in Kidney Cortex Membrane Fractions of Cathepsin B Knockout Mice Is Associated with Reduced Lysophosphatidylcholine and Protein Kinase C Activity

“…Cho et al showed the cell-permeable methyl ester derivative of the negatively charged cathepsin B inhibitor CA074ME reduces α-spectrin cleavage, similar to the effect of calpain inhibition [27]. Moreover, Taylor et al showed proteolysis of spectrin in erythrocytes results in the biogenesis of extracellular vesicles from the plasma membrane [28].…”