Microdomains form on the luminal face of neuronal extracellular vesicle membranes

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology.

Results neurons release small-and medium-sized eVs. An important question in EV biology is whether EVs released by specific cell types vary in size or morphology 7 . EVs typically exhibit a range of sizes from 30-250 nm [11][12][13] and various morphologies 11,14 . Since different EV concentration protocols can affect the mean size distribution of vesicles and, in some cases, induce morphological changes 15 , we first sought to characterize the size distribution of neuronal EVs concentrated using two different methods: differential ultracentrifugation (dUC) and ultrafiltration (UF). We grew primary cortical neurons from P0 rat pups for 10 days to allow neurons to differentiate and form functional connections [16][17][18] (Fig. 1A). Cultured neurons are routinely grown in serum-free media thereby www.nature.com/scientificreports/ eliminating the risk of extracellular vesicles present in serum to influence subsequent analyses 16 . Neurons were grown in AraC to prevent growth of contaminating glial cells. Neuron-conditioned medium was collected and subjected to low-speed centrifugation (10,000×g for 20 min) to remove the cell debris and some large vesicles. EVs in the supernatant were then purified by ultra-high-speed centrifugation (300,000×g for 3 h) (Fig. 1D). This ultra-high speed allows a greater proportion of vesicles to be pelleted, as a considerable number of EVs remain in the supernatant following 100,000×g centrifugation 19,20 . We verified the presence of vesicles by negative staining with TEM imaging (Fig. 1B). We next turned to cryoEM to obtain high-resolution images of EVs in their native hydrated state. Based on our cryoEM imaging, we found that neuronal EVs separated by dUC exhibited a broad range of sizes, from 25-450 nm in diameter ( Fig. 1C-E). A bimodal distribution consisting of small (sEV; < 100 nm) and medium-sized (mEV; 100-500 nm) vesicles was observed (Fig. 1E) with a mean diameter of 99.07 nm.
Western blotting of dUC vesicles reveals an enrichment of the EV marker syntenin (Fig. 1F) 10,21,22 . Consistent with proteomics characterization of EVs 10,22 , flotillin-1 was present but not enriched in the dUC EVs (Fig. 1F). Neuron-specific β3-tubulin was not detected in our concentrated EVs, nor was the endoplasmic reticulumresident marker gp96 (Fig. 1F) 10,22 . In addition to exosomes and microvesicles, unhealthy cells release large vesicles called apoptotic bodies 23 . The lack of gp96 detected suggests that apoptotic bodies are unlikely to be present in EVs concentrated by dUC 10,22,24 .
We also concentrated EVs using UF as a second approach (Fig. 1G). This approach does not subject vesicles to ultra-high g-force and eliminates the need to mechanically resuspend vesicles from a pellet 25,26 . Neuronconditioned medium was collected and centrifuged at 2,000×g to remove dead cells. The supernatant was concentrated using a centrifugal filter unit with a 100 kDa molecular weight cut-off by centrifuging for 20 min at 4,000×g (Fig. 1G) 26 . Based on our cryoEM imaging, we found that neuronal EVs concentrated by UF were larger than those concentrated by dUC, with a mean diameter of 147.82 nm (Fig. 1H). This is likely due to the removal of many mEVs by the 10,000×g centrifugation step used when concentrating EVs by dUC (Fig. 1D,E). Like the dUC EVs, UF EVs contained syntenin and flotillin-1 but not the organelle marker gp96 by Western blot, indicating that apoptotic bodies are unlikely to be present in EVs concentrated by ultrafiltration (Fig. 1I).
As expected, our data shows that cortical neurons release a range of EV sizes. The difference in average EV diameter from 99 to 148 nm illustrates the ability of different concentration methods to selectively enrich different sized vesicles. Although the identity of a vesicle cannot be determined based on size alone, the distribution of EV sizes suggests that neurons may release exosomes and microvesicles.

Macromolecular clustering occurs on luminal membranes of meVs.
We next sought to characterize the biophysical properties of neuronal EVs by cryoEM. We discovered dense macromolecular clustering on the luminal face of EV membranes ( Fig. 2A,B, black arrows). Reconstructed 3-dimensional tomograms confirm that these clusters are within vesicles as opposed to on the surface and are often present where the membrane bulged outward (Fig. 2C, cluster in yellow, Movie S1). Clusters were found predominantly in mEVs with an average vesicle size of 188.45 nm and were observed in 9.2% (21/229) of dUC EVs and 12.6% (63/499) of UF EVs (Fig. 2D). No difference in the size of EVs containing the clusters was observed between the two methods of EV concentration (Fig. 2E). The slightly higher abundance of clusters in UF EVs is consistent with the larger size distribution of vesicles by UF (Fig. 1). There was no difference in the mean 2-dimensional area of the clusters imaged from dUC EVs (403 nm 2 ) and UF EVs (432 nm 2 ) (Fig. 2F). The mean maximum length of the clusters was larger in dUC EVs (67 nm) compared to UF EVs (33 nm) (Fig. 2G).
Taking advantage of the fact that different macromolecules show varying susceptibility to radiation damage allowed us to probe the composition of these clusters 27 . Clusters were more resistant than the EV lumen to radiation damage as assessed by long electron exposure and gas/bubble formation (Fig. 3, black arrow). This suggests that the macromolecular environment within the cluster is distinct from the aqueous lumen. This also suggests that the clusters may be rich in phosphate, as phosphate-containing molecules are more resistant to radiation damage 27 . The clusters were more resistant to radiation damage than large regions of the phospholipid-rich plasma membrane (Fig. 3, red arrow). We speculate that these clusters are largely protein-based, may contain phosphate-rich molecules such as RNA, and likely form a distinct microdomain. neuronal eVs are largely homogenous in morphology. We next characterized the morphology of EVs released from primary neurons to identify any similarities or differences to EVs reported from nonneuronal cell types. EVs have a variety of reported morphologies, including round, tubular, and pleomorphic/ irregular 11 . EVs released from neurons were predominantly round with 98.2% (715/728 EVs) having a roundness of greater than 0.5 ( Fig. 4A-C). 2/229 (0.9%) of dUC EVs and 11/499 (2.2%) UF EVs displayed an oval morphology (roundness of less than 0.5) (Fig. 4A,B). We did not detect any coated vesicles or vesicles exhibiting electron-dense protrusions 11,28 . We detected a single vesicle (1/728 EVs) containing filamentous structures, suggesting that this is not common for EVs concentrated from neuron-conditioned medium by either method. This is consistent with undetectable levels of cellular markers gp96 and tubulin by Western blotting (Fig. 1F,I). Collectively, our imaging data suggest that EVs released from neurons are largely homogenous in morphology. neuronal eVs contain internal vesicles. Similar to EVs from non-neuronal cells 11 , many neuronal EVs contained what appears to be one or more internal vesicles (Fig. 4C-D). Vesicles containing one or more internal vesicles were observed in 20/229 (8.7%) of dUC EVs and 48/499 (9.6%) of UF EVs. The mean diameter of EVs containing internal vesicles did not differ by method of concentration (Fig. 4E). Based on imaging of a single plane alone, it is not possible to tell whether the smaller vesicles are within the larger vesicle, or whether they are above or below the large vesicle. Using 3-dimensional cryo-electron tomography (cryoET), we confirmed that neuronal eVs make membrane contact with other eVs. Finally, we noted that several neuronal EVs formed contacts with other EVs (Fig. 4C,D,F). This phenotype has previously been described as an artefact caused of high-speed centrifugation 15 . We observed EV-EV contacts more frequently when concentrated by UF (95/499 EVs or 19.0%) compared to those concentrated by dUC (16/229 or 7.0%). While our data does not preclude the possibility that EV-contacts are caused by the procedures used to concentrate them, it does reveal that EV contacts are not caused solely by high-speed centrifugation 15 .

Discussion
Here, we used high-resolution cryoEM to image EVs released from cultured neurons in their native hydrated state. We found that neuronal EVs exhibit a broad range of sizes ranging from 25 to 600 nm. As microvesicles are typically larger than exosomes, we speculate that in addition to release of exosomes by the fusion of multivesicular bodies with the plasma membrane 29  www.nature.com/scientificreports/ macromolecules between neighboring cells 30 . The role of microvesicles versus exosomes released from neurons is not well characterized. Neuronal EVs may be more homogenous in morphology compared to those released from other cell types 11 . We found that EVs released by neurons are largely round with few detectable vesicles exhibiting irregular phenotypes such as tubular vesicles or containing filamentous structures. Like EVs from other cell types, we observed vesicles containing internal vesicles and EVs contacting each other. The later of these phenotypes has been attributed to high centrifugal forces used to concentrate vesicles from solution 15 ; however, we also observe these phenotypes in vesicles concentrated by ultrafiltration where high g-forces are not used.
The discovery of macromolecular clustering on the luminal face of EV membranes is a potentially important phenomenon for EV physiology by acting as a functional microdomain. However, to begin to understand the function of these structures, several questions remain. First, are these microdomains specific to an EV subtype? Since we observed clusters more frequently on medium-sized EVs, we speculate that this phenotype may be associated with microvesicles as opposed to exosomes, which are typically smaller.
Determining when these clusters form might shed insight into their function. We anticipate three scenarios of when these clusters might form: (1) at the time of EV formation (2) extracellularly to facilitate docking and fusion or (3) under pathological conditions that favor macromolecular aggregation. Microvesicle formation involves recruitment of lipids, RNAs and proteins to the plasma membrane resembling that of viral particle formation 31 . If assembly of this platform drives EV budding, then determining the molecular composition within the cluster could reveal the machinery used to generate EVs. This clustering might also serve to recruit cargo into the vesicle for selective packaging.
If the clusters assemble extracellularly, they could function in the communication of the EV with recipient cells. Clustering does not appear to extend to the extracellular face of the membrane, so it is unclear whether this clustering would affect transmembrane receptors. However, it could affect membrane lipids that would allow for recognition by the recipient cell 32 . The clusters frequently occur where membranes bulge outward, suggesting that they may induce membrane curvature that could be important for recognition by recipient cells. If protein clustering generates a signaling platform, then proteins or lipids within this platform could determine cell-type specific recognition of EVs 33 .
Lastly, if clusters form under conditions that favor aggregation, they may have the potential to disrupt normal EV-cellular function. In fact, EVs transmit pathological proteins from one cell to another in the brain. For instance, EVs transfer alpha-synuclein and hyper-phosphorylated tau in Parkinson's disease and Alzheimer's disease models, respectively 34 . They may contribute to the spread of pathogenic proteins in the brain during neurodegenerative disease. Alternatively, they may help neurons transport macromolecules (such as lipids) damaged by oxidative stress to glial cells for support 35 . An important future direction will be to identify the composition of molecules in the clusters and whether pathogenic proteins susceptible to aggregation are present. Recent advances in cryo-correlative light and electron microscopy offer a promising strategy to address this outstanding question. Given the involvement of EVs in pathologies such as neurodegenerative disease, the proteins within these clusters may be useful targets for treating disease. www.nature.com/scientificreports/ In summary, we performed a detailed characterization of neuronal EVs by cryoEM and discovered macromolecular clusters on the luminal membrane. We speculate that these clusters constitute a functional microdomain within vesicles and play an important role in EV physiology.

Materials and methods
primary culture of hippocampal neurons. All animal work was approved by and performed in accordance with the Institutional Animal Care and Use Committee at Janelia Research Campus (IACUC 16-146) and the Canadian Council of Animal Care at the University of Alberta (AUP#3,358). Sprague-Dawley timed pregnant rats were purchased from Charles River Laboratories and housed in our facility for one week prior to birth. Primary cortical neurons were prepared as previously described 35 . Briefly, the cortices were dissected from P0 Sprague-Dawley rat pups, digested with papain (Worthington Biochemical), triturated, and filtered with a cell strainer. Neurons were grown for 10 days on poly-d-lysine coated 10-cm plastic culture plates in serum-free NbActiv4 medium (BrainBits) containing antibiotic-antimycotic (Gibco) at 37 °C. 2 µM cytosine-beta-d-arabinofuranose (AraC, ThermoFisher Scientific) was added on DIV 2. Half the media was replaced every 3 days with fresh NbActiv4 without AraC. fluorescence microscopy. Neurons were plated on poly-d-lysine coated glass coverslips. After 10 days in culture neurons were washed 3 times in PBS, fixed in 4% paraformaldehyde, blocked with PBS containing 1% BSA plus 0.02% TritonX-100, immunostained with mouse monoclonal anti-β3-tubulin (Biolegend, 801201, RRID:AB_2313773) and goat anti-mouse alexafluor488 (Invitrogen, A11001) and stained with DAPI (Abcam, ab228549). Neurons were routinely monitored for viability and mycoplasma contamination by imaging nuclei stained with DAPI. Imaging was performed using a Laser Scanning Confocal Microscope (LSM880, Zeiss) equipped with a plan-apochromat 63 × oil objective (Zeiss, NA = 1.4) and ZEN software (Zeiss).
Purification of extracellular vesicles. Media of DIV 9 neurons was reduced to 5 mL and 16 h later, neuron-conditioned medium was collected. EV Centrifugation: Neuron-conditioned medium from a 10-cm plate was centrifuged at 2,000×g at 4 °C for 10 min to remove dead cells or cell debris followed by centrifugation at 10,000×g at 4 °C for 20 min. The supernatant containing EVs was centrifuged at 300,000×g at 4 °C for 3 h in a TLA-110 fixed angle rotor (k-factor 13) (Beckman Coulter). The pellet containing EVs was resuspended in 10 µL PBS. EV Ultrafiltration: media from three 10-cm plates was pooled and centrifuged at 2,000×g at 4 °C for 10 min, then added to the top chamber of an Amicon Ultra centrifugal filter unit with a 100 kDa molecular weight cutoff (Millipore-Sigma) and centrifuged at 4,000×g at 4 °C for 20 min until final volume reached approximately 200 µL.

Macromolecular bubbling assay.
Images were recorded on the same Titan Krios that was used for tomography (see above). Multiple 60 frame-images were recorded at a dose rate of ~ 15 e − /px/s and pixel size of 1.07 Å/px using super-resolution counting mode (0.535 Å/px), accumulating ~ 60 e − /Å 2 per image. A defocus of − 3.5 µm was applied.
Analysis of extracellular vesicles. EVs were identified by the presence of a bilayer membrane. EVs and clusters were manually traced, and the area, diameter, and roundness were calculated using ImageJ software. For roundness, only vesicles entirely within the field of view were quantified. Vesicles more than 20% out of the field of view were excluded from quantification of vesicle diameter. Statistical analysis was performed using SPSS Statistics 17.0 (IBM) or GraphPad Prism 8. Student's t-test was used for comparison of groups. Since no difference (t-test, p = 0.743) was detected in dUC EV diameter from cells stimulated with 10 µM NMDA for 18 h (n = 32) versus unstimulated cells (n = 227), this data was combined in the histograms showing distribution of diameters.