Nanomechanics of Extracellular Vesicles Reveals Vesiculation Pathways

Abstract

Extracellular vesicles (EVs) are emerging as important mediators of cell-cell communication as well as potential disease biomarkers and drug delivery vehicles. However, the mechanical properties of these vesicles are largely unknown, and processes leading to microvesicle-shedding from the plasma membrane are not well understood. Here an in depth atomic force microscopy force spectroscopy study of the mechanical properties of natural EVs is presented. It is found that several natural vesicles of different origin have a different composition of lipids and proteins, but similar mechanical properties. However, vesicles generated by red blood cells (RBC) at different temperatures/incubation times are different mechanically. Quantifying the lipid content of EVs reveals that their stiffness decreases with the increase in their protein/lipid ratio. Further, by maintaining RBC at "extreme" nonphysiological conditions, the cells are pushed to utilize different vesicle generation pathways. It is found that RBCs can generate protein-rich soft vesicles, possibly driven by protein aggregation, and low membrane-protein content stiff vesicles, likely driven by cytoskeleton-induced buckling. Since similar cortical cytoskeleton to that of the RBC exists on the membranes of most mammalian cells, our findings help advancing the understanding of the fundamental process of vesicle generation.

Vesiculation of Red Blood Cells in the Blood Bank: A Multi-Omics Approach towards Identification of Causes and Consequences

“…These observations require more detailed investigation but suggest that during storage, when microvesicles are not removed as in the circulation, vesiculation may proceed within existing vesicles. Together with the biphasic patterns of many of our present observations, these data support the hypothesis that various vesiculation mechanisms become active with increasing storage time, with a critical period around three weeks of storage [1,14,16,19,22,48].…”

“…The changes in vesicle morphology (Figure 1) provide further evidence for the presence of progressive alterations in the interaction between the membrane and the cytoskeleton. Mechanical investigations, showing that vesicles from fresh RBCs are softer than vesicles from stored RBCs and that this is mainly dependent on the protein/lipid ratio [48], support the hypothesis that small changes in protein organization are the main triggers of vesicle generation. Our findings on the content of the microdomain-associated protein stomatin and the GPI-linked proteins (Figure 2, Figure 5) indicate that small, local changes may trigger secondary changes in lipid organization.…”

“…Previously, we have observed an increase in the lyso-PC content of the RBC membrane in patients with sepsis, together with increased microvesicle numbers and increased phospholipase A2 activity in the blood [34]. Additionally, we have found that lyso-PC is present in vesicles, but not in RBC membranes [48]. These findings all suggest that small changes in phospholipid composition of the RBC membrane, resulting in changes in organization, may be involved in the generation of microvesicles in affected, susceptible membrane regions.…”

Synaptotagmin-1 and Doc2b exhibit distinct membrane remodeling mechanisms

“…As it disturbs the packing of the lipid acyl chains, this might result in a change in the mechanical properties of the membrane. To quantitatively study how Doc2b and Syt1 C2AB fragment insertion into the negatively charged membrane influences its mechanical properties, we used a recently developed method based on atomic force microscopy (AFM) (36)(37)(38). By first imaging liposomes ( Fig.…”

Mechanical properties of plasma membrane vesicles correlate with lipid order and viscosity and depend on cell density

“…This might be one reason why cells invest energy into the synthesis and repair of these oxidation-prone lipids (65). Another contribution to the low values of bending rigidities of GPMVs might be the high membrane protein content (about 40% by mass is contained in GPMVs (66)), and proteins and peptides are often observed to reduce membrane bending rigidity (20,(67)(68)(69). GPMV membrane softening by transmembrane proteins is also consistent with lower bending rigidity values (κ≈10 ) found in GPMVs derived from HEK cells overexpressing membrane protein (15).…”