CD63 sorts cholesterol into endosomes for storage and distribution via exosomes.

Extracellular vesicles such as exosomes are now recognized as key players in intercellular communication. Their role is influenced by the specific repertoires of proteins and lipids, which are enriched when they are generated as intraluminal vesicles (ILVs) in multivesicular endosomes. Here we report that a key component of small extracellular vesicles, the tetraspanin CD63, sorts cholesterol to ILVs, generating a pool that can be mobilized by the NPC1/2 complex, and exported via exosomes to recipient cells. In the absence of CD63, cholesterol is retrieved from the endosomes by actin-dependent vesicular transport, placing CD63 and cholesterol at the centre of a balance between inward and outward budding of endomembranes. These results establish CD63 as a lipid-sorting mechanism within endosomes, and show that ILVs and exosomes are alternative providers of cholesterol.

Lipoprotein particles exhibit distinct mechanical properties.

Lipoproteins (LPs) are micelle-like structures with a similar size to extracellular vesicles (EVs) and are therefore often co-isolated, as intensively discussed within the EV community. LPs from human blood plasma are of particular interest as they are responsible for the deposition of cholesterol ester and other fats in the artery, causing lesions, and eventually atherosclerosis. Plasma lipoproteins can be divided according to their size, density and composition into chylomicrons (CM), very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Here, we use atomic force microscopy for mechanical characterization of LPs. We show that the nanoindentation approach used for EV analysis can also be used to characterize LPs, revealing specific differences between some of the particles. Comparing LPs with each other, LDL exhibit a higher bending modulus as compared to CM and VLDL, which is likely related to differences in cholesterol and apolipoproteins. Furthermore, CM typically collapse on the surface after indentation and HDL exhibit a very low height after surface adhesion both being indications for the presence of LPs in an EV sample. Our analysis provides new systematic insights into the mechanical characteristics of LPs.

Active probing of the mechanical properties of biological and synthetic vesicles.

BACKGROUND: The interest in mechanics of synthetic and biological vesicles has been continuously growing during the last decades. Liposomes serve as model systems for investigating fundamental membrane processes and properties. More recently, extracellular vesicles (EVs) have been investigated mechanically as well. EVs are widely studied in fundamental and applied sciences, but their material properties remained elusive until recently. Elucidating the mechanical properties of vesicles is essential to unveil the mechanisms behind a variety of biological processes, e.g. budding, vesiculation and cellular uptake mechanisms. SCOPE OF REVIEW: The importance of mechanobiology for studies of vesicles and membranes is discussed, as well as the different available techniques to probe their mechanical properties. In particular, the mechanics of vesicles and membranes as obtained by nanoindentation, micropipette aspiration, optical tweezers, electrodeformation and electroporation experiments is addressed. MAJOR CONCLUSIONS: EVs and liposomes possess an astonishing rich, diverse behavior. To better understand their properties, and for optimization of their applications in nanotechnology, an improved understanding of their mechanical properties is needed. Depending on the size of the vesicles and the specific scientific question, different techniques can be chosen for their mechanical characterization. GENERAL SIGNIFICANCE: Understanding the mechanical properties of vesicles is necessary to gain deeper insight in the fundamental biological mechanisms involved in vesicle generation and cellular uptake. This furthermore facilitates technological applications such as using vesicles as targeted drug delivery vehicles. Liposome studies provide insight into fundamental membrane processes and properties, whereas the role and functioning of EVs in biology and medicine are increasingly elucidated.

Mechanical Characterization of Liposomes and Extracellular Vesicles, a Protocol.

Both natural as well as artificial vesicles are of tremendous interest in biology and nanomedicine. Small vesicles (<200 nm) perform essential functions in cell biology and artificial vesicles (liposomes) are used as drug delivery vehicles. Atomic Force Microscopy (AFM) is a powerful technique to study the structural properties of these vesicles. AFM is a well-established technique for imaging at nanometer resolution and for mechanical measurements under physiological conditions. Here, we describe the procedure of AFM imaging and force spectroscopy on small vesicles. We discuss how to image vesicles with minimal structural disturbance, and how to analyze the data for accurate size and shape measurements. In addition, we describe the procedure for performing nanoindentations on vesicles and the subsequent data analysis including mechanical models used for data interpretation.

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms.

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.

The fluid membrane determines mechanics of erythrocyte extracellular vesicles and is softened in hereditary spherocytosis.

Extracellular vesicles (EVs) are widely studied regarding their role in cell-to-cell communication and disease, as well as for applications as biomarkers or drug delivery vehicles. EVs contain membrane and intraluminal proteins, affecting their structure and thereby likely their functioning. Here, we use atomic force microscopy for mechanical characterization of erythrocyte, or red blood cell (RBC), EVs from healthy individuals and from patients with hereditary spherocytosis (HS) due to ankyrin deficiency. While these EVs are packed with proteins, their response to indentation resembles that of fluid liposomes lacking proteins. The bending modulus of RBC EVs of healthy donors is ~15 kbT, similar to the RBC membrane. Surprisingly, whereas RBCs become more rigid in HS, patient EVs have a significantly (~40%) lower bending modulus than donor EVs. These results shed light on the mechanism and effects of EV budding and might explain the reported increase in vesiculation of RBCs in HS patients.

Atomic Force Microscopy: An Introduction.

Imaging of nano-sized particles and sample features is crucial in a variety of research fields. For instance in biological sciences, where it is paramount to investigate structures at the single particle level. Often two-dimensional images are not sufficient and further information such as topography and mechanical properties are required. Furthermore, to increase the biological relevance, it is desired to perform the imaging in close to physiological environments. Atomic force microscopy (AFM) meets these demands in an all-in-one instrument. It provides high-resolution images including surface height information leading to three-dimensional information on sample morphology. AFM can be operated both in air and in buffer solutions. Moreover, it has the capacity to determine protein and membrane material properties via the force spectroscopy mode. Here we discuss the principles of AFM operation and provide examples of how biomolecules can be studied. By including new approaches such as high-speed AFM (HS-AFM) we show how AFM can be used to study a variety of static and dynamic single biomolecules and biomolecular assemblies.