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Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08 Page 7

limit (< 20 nm), i.e., essentially co-localized. When using labeled tetraspanins, they determined that there
is an offset in the overlay of the membrane protein and the membrane dye, which they hypothesized to be
the localization of the tetraspanins in lipid raft microdomains. The biophysical characteristics shown were
consistent with those found from other methodologies (e.g., nanoparticle tracking analysis, resistive pules
sensing, and surface plasmon resonance).

Frederik Verweij (INSERM, France) rounded out the session with a talk on in vitro and in vivo exosome
tracking. He described a novel live cell imaging technique using pH sensitive reporters visualized by total
internal reflection fluorescence (TIRF) microscopy. With tagged tetraspanins (CD63, CD81) or other
endosomal markers, they could visualize which endosomal sub-compartments fuse with the plasma
membrane to release exosomes. They have extended these studies to in vivo settings using zebrafish models.
The remarkable resolution of their technology allows tracking of release of exosomes, as well as recipient
cell uptake throughout the organism; e.g., macrophage and endothelial cell scavenging, and internalizing
exosomes released by cells of the yolk syncytial layer. Following the fate of such internalized exosomes, it
seems that many exosomes are degraded in the endo-lysosomal system. Further, inhibiting the biogenesis of
exosomes in zebrafish embryo caudal vein plexus (CVP) led to reduced growth of that tissue, implying a role
for exosomes in trophic support of the CVP.

ASEMV2020 Day 2 began with an EV Function session, chaired by Michael Graner (University of Colorado
Anschutz, US) and Leonid Margolis (NIH, US). It started with a joint presentation by Jay Debnath and
Andrew Leidal (University of California San Francisco, US) on secretory autophagy and EVs. Jay introduced
the biologic processes of autophagy, particularly secretory autophagy, and their relationships to EVs and
EV release. Given that knockdowns/knockouts of autophagy components had pleiotropic effects, Drew had
developed a technique called proximity-specific biotinylation to identify new targets of autophagy-dependent
secretion. This allowed for proteomic identification of > 200 such targets, many of which were exosomal/
EV components and RNA-binding proteins, suggesting an intersection between autophagy and exosome
protein-related secretion. Additional work showed the dependency of autophagy machinery on secretion of
diverse RBPs as well as certain small RNAs (including snoRNAs) in small EVs/exosomes. They believe this
represents a sub-routine of the autophagy pathway involving component (LC3) processing and lipidation,
“LC3 dependent EV loading and secretion” (LDELS). Drew inhibited lysosomal activity (baflomycin,
chloroquine), which revealed a re-direction of endo-lysosomal cargo released as EVs outside the cell in
an autophagy-dependent manner, with autophagy proteins, including cargo receptors, prominent in those
vesicles. Further genetic studies indicated that inhibition of autophagosome-lysosome fusion enhances cargo
receptor release in EVs, in contrast to the requirements for LDELS.

Sarah Andres (Oregon Health and Science University, US) continued the autophagy theme, this time focusing
on IGF2PB1/IMP1, an RNA-binding protein whose expression peaks during embryonic development in
mammals, and decreases as the organism matures. In the intestine, damage causes upregulation of IGF2PB1/
IMP1; this is also seen in GI cancers, where it acts in tumor progression and metastasis. The hypothesis was
that IMP1 plays a role in endosomal and autophagic pathways (secretion vs. degradation) in colon cancers.
IMP1 overexpression increased EV release, and modulated endosomal and autophagy pathways at least in
some colon cancer cell lines. Using an enteroid (organoid) culture system (from crypt regions of intestinal
epithelium), IMP1 overexpression in these non-transformed cells did not alter EV production, but had some
impact on cargo, such as TSG101; these results are an on-going work. Thus, IMP1’s influence on EV biology
via endosomal and autophagy pathways appears to be cell-type and context dependent.

Inge Zuhorn (University of Groningen, The Netherlands) presented next on exosomal cargo fate upon uptake
by recipient cells. Their work originated from challenges that accompanied nanoparticle-based drug delivery
to the brain; subsequently leading to EV-based delivery into the brain with functional release of cargo into
target cells. Inge presented four hypothetical exosome cargo release scenarios (direct fusion to plasma