1
2
3
4
5
6
7
8
A
n
extracellular vesicle targeting
ligand
that binds to Arc proteins
and facilitates Arc
9
transport
in vivo
10
11
12
Peter H.
Lee
*
, Michael Anaya,
Mark S. Ladinsky,
Justin
M.
Reitsma
1
, and Kai Zinn
*
13
14
15
16
17
Division of Biology and Biological
Engineering, California Institute of Technology, Pasadena, CA
18
91125 USA
19
20
1
Current address:
AbbVie
, 1 N Waukegan Rd, North Chicago, IL 60064
21
22
*To whom correspondence should be addressed (
hlee@caltech.edu
, zinnk@caltech.edu)
23
24
25
26
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint
27
28
Abstract
29
Communication between distant cells can be mediated by e
xtracellular vesicles (EVs)
that
30
deliver
proteins and RNAs
to recipient cells. Little is known about how EVs are targeted to
31
specific cell types.
Here we identify the
Drosophila
cell
-
surface protein Stranded at second
32
(Sas) as a targeting
ligand
for EVs.
Full
-
length Sas is present in EV preparations from
33
transfec
ted
Drosophila
Schneider 2 (S2 cells).
Sas is a
binding partner
for
the Ptp10D receptor
34
tyrosine phosphatase
, and
Sas
-
bearing EVs preferentially
target
to
cells expressing Ptp10D. We
35
used co
-
immunoprecipitation
and peptide binding to show that
the cytoplas
mic domain (ICD) of
36
Sas binds to dArc1. dArc1 and mammalian Arc
are related to retrotransposon Gag proteins.
37
They
form virus
-
like capsids
which
encapsulate
Arc
and other
mRNAs and are transported
38
between cells via EVs. The Sas
ICD
contains a motif required
for dArc1 binding that is shared
39
by the mammalian and
Drosophila
amyloid precursor protein (APP) orthologs, and the Sas and
40
APP ICDs
also
bind to mammalian Arc. Sas facilitates
delivery
of dArc1
capsids bearing
dArc1
41
mRNA
in
to
d
i
s
t
a
n
t
Ptp10D
-
expressing recipient
cells
in vivo
.
42
43
44
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint
I
NT
R
O
D
U
C
T
I
ON
45
Extracellular vesicles
(EVs) are mediators of cell
-
cell communication that transport
specific
46
protein and RNA
cargoes
. They are a heterogeneous collection of vesicular structures that are
47
exported from cells by a variety of mechanisms. Exosomes are
3
0
-
150 nm in diameter
and are
48
released into cell supernatants
via
fusion of multivesicular bodies (MVBs) with the plasma
49
membrane. Exosomes and other EVs carry specific proteins and RNAs, and EVs derived from
50
different cell types contain different cargoes. EV cargoes are biomarkers for specific diseases.
51
Because EVs can
encapsulate RNAs and protect them from degradation, and then deliver those
52
RNAs to recipient cells, they represent a promising new type of therapeutic agent
(O'Brien et al.,
53
2020; Teng and Fussenegger, 2020)
.
54
55
While the biogenesis of EVs is comparatively well understood, much less is known about
56
mechanisms involved in their targeting to specific cell types. EVs c
an directly activate
57
intracellular signaling by interacting with cell surface receptors
. They are
internalized into cells
58
after receptor binding using a variety of endocytic mechanisms, resulting in the delivery of their
59
cargoes into the recipient cells. In this paper, we identify
Stranded at second (
Sas
)
, a large
60
Drosophila
cell surface protein (
CSP
)
(Schonbaum et al., 1992)
, as an EV t
argeting
ligand
. Sas
61
has an
extracellular domain (
ECD
)
containing
a signal peptide, a unique N
-
terminal region, four
62
v
on Willebrand factor C (VWFC) domains
,
and
three
Fibronectin Type III (FN
-
III) repeats (Fig.
63
1a).
It has a single transmembrane (TM) domai
n and a short (37 amino acids (aa)) cytoplasmic
64
domain (ICD).
Sas is commonly used as a marker for the apical surfaces of epithelially
-
derived
65
cells, including tracheal cells in the respiratory system.
sas
mutant larvae die at or before
66
second instar (henc
e the name
stranded at second, which is derived from baseball terminology
)
67
and have tracheal phenotypes
(Schonbaum
et al.
, 1992)
. A tyrosine motif in the Sas ICD binds
68
to the PTB domain of Numb
(Chien et al., 1998)
, an endocytic protein that is a negative regulator
69
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint
of Notch. Sas has no mammalian orthologs, but there are many mammalian CSPs that contain
70
VWFC and FN
-
III domains.
71
72
We identified the receptor tyrosine phosphatase (RPTP) Ptp10D
as a binding partner for Sas,
73
and showed that Sas::Ptp10D interactions regulate embryonic axon guidance, as well as glial
74
migration and proliferation
(Lee et al., 2013)
. Ptp10D
is one of the two
Drosophila
R3
subfamily
75
RPTP
s, which have ECDs composed of long chains o
f FN
-
III repeats.
Sas::Ptp10D interactions
76
also control the elimination of neoplastic epithelial clones by surrounding normal tissue. Sas
is
77
on normal epithelial cells, and it
relocalizes to the
parts of their cell
surface
s that are adjacent
to
78
the neoplas
tic clone and binds to Ptp10D on the neoplastic cells. Ptp10D in turn relocalizes and
79
dephosphorylates the EGF receptor tyrosine kinase, leading to death of the neoplastic
80
cells
(Yamamoto et al., 2017)
. The Sas ECD
probably has
other binding partners
as well
,
81
because it interacts with cells that do not express Ptp10D in live embryo staining assays
(Lee
et
82
al.
, 2013)
.
83
84
Sas localizes to EVs,
as demonstrated by immuno
-
electron microscopy (immuno
-
EM) and
85
Western blotting of EV preparations. These EVs preferentially target to cells expressing Ptp10D,
86
and expression of Numb further increases incorporation of EV contents into recipient cell
87
lysates
. We used mass spectrometry to identify proteins associated with Sas in EVs, and found
88
that dArc1 is the most highly enriched protein. We then used co
-
immunoprecipitation (co
-
IP)
89
and peptide binding to show that dArc1 binds directly to the short Sas ICD.
90
91
Arc was originally identified in mammals as a locally translated dendritic protein that regulates
92
synaptic plasticity, in part by modulating
endocytosis of AMPA receptors
(
Chowdhury et al.,
93
2006; Shepherd et al., 2006)
.
The
Drosophila
genome encodes two Arc
-
related proteins, dArc1
94
and dArc2. The
dArc2
gene, which encodes a truncated protein, was likely generated by a gene
95
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint
duplication, and the
dArc1
and
dArc2
genes are adj
acent
(Mattaliano et al., 2007)
.
dArc1
96
functions in larval and adult brain neurons to regulate aspects of metabolism
(Keith et al., 2021;
97
Mattaliano
et al.
, 2007; Mosher et al., 2015)
.
Arc
and dArc1
evolved independently from
98
retrotransposon
Gag proteins
(Shepherd, 2018)
.
Remarkably, they
were both recently shown to
99
form virus
-
like capsids that
can
encapsulate
Arc
mRNAs and are transported between cells via
100
EVs
(Ashley et al., 2018; Hantak et al., 2021; Pastuzyn et al., 2018)
.
dArc1, but not dArc2, has a
101
C
-
terminal Zn
2+
finger that might be involved in nucleic acid binding
(
Erlendsson et al., 2020;
102
Pastuzyn
et al.
, 2018)
. Mammalian Arc lacks Zn
2+
fingers, but RNA is required for normal capsid
103
assembly
(Pastuzyn
et al.
, 2018)
.
Drosophila
dArc1
capsids bearing
dArc1
mRNA
move from
104
neurons to muscles across larval neuromuscular junction (NMJ) synapses, and dArc1 transfer is
105
required for activity
-
induced induction of morphological synaptic plasticity
(Ashley
et al.
, 2018)
.
106
107
The
short Sas
ICD
contains a
tyrosine
motif required for dArc1 binding
. Appl, the ortholog of
108
amyloid precursor protein (APP), is the only other Drosophila CSP that shares this motif, and its
109
ICD also binds to dArc1. The motif is conserved in human APP, and the APP and Sas ICDs also
110
bind to mammalian Arc. The interaction bet
ween APP and Arc is of interest because several
111
studies have implicated Arc in control of
b
-
amyloid accumulation and
Alzheimer’s disease
112
(AD)
(Bi et al., 2018; Landgren et al., 2012; Wu et al., 2011)
, and APP also localizes to
113
EVs
(Laulagnier et al., 2018; Perez
-
Gonzalez et al., 2020)
.
114
115
To determine whether Sas can target dArc1 to Ptp10D
-
expressing recipient
cells
in vivo
, we
116
expressed dArc1 with and without Sas
in embryonic salivary glands (SGs). We observed that
117
expression of dArc1 protein from a cDNA construct induces expression of the endogenous
118
dArc1
gene in SGs. When Sas and dArc1 are expressed together in SGs, high levels of
119
endogenous
dArc1
mRNA appear in
distant tracheal cells, which express Ptp10D. The data
120
suggest that Sas EVs bearing dArc1 capsids that contain
dArc1
mRNA travel within the embryo
121
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint
and are internalized into tracheal cells, which then also turn on expression of the endogenous
122
dArc1
gene.
123
124
125
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint
R
E
S
U
L
T
S
A
ND
D
IS
C
U
S
SI
ON
126
Sas is an EV targeting
ligand
127
Sas
exists as two isoforms generated by alternative splicing. Full
-
length Sas (PB/PD
isoform,
128
denoted here as
Sas
FL
)
is a 1693 aa protein. It
contains a 345
aa
region (EVT)
between the
129
VWFC
and FN
-
III domains that is lacking in the
PA/PC isoform (Sas
short
)
(Fig. 1a)
.
We expressed
130
Sas
FL
tagged with an N
-
terminal V5 epitope tag (inserted immediately after the signal sequence)
131
in
embryonic
late stage 16
Apterous (Ap) neurons, which consist of pai
red neurons (one per
132
hemisegment) in the ventral nerve cord (VNC) and scattered neurons in the brain lobes. W
e
133
noted that
V5
-
Sas
FL
move
d
away from the expressing cells
and accumulate
d
in sheaths around
134
brain lobes and
around
axons in the VNC
, as well as
in puncta throughout the VNC and brain
135
(Fig. 1b)
. This
was surprising, since Sas
FL
is a transmembrane CSP. It was expressed together
136
with mCD8
-
GFP, which is also a transmembrane CSP, and the GFP signal was
restricted
to the
137
Ap neuron cell bodies
,
with
fain
t staining on the axons
(Fig. 1b)
.
138
139
Movement of
V5
-
Sas
FL
, and presumably of endogenous Sas
FL
,
away from its source
could occur
140
through cleavage of the Sas ECD from the cell surface
or by release of intact Sas in EVs. To
141
distinguish between these
possibil
ities
, we expressed
V5
-
tagged
Sas
FL
and Sas
short
in
142
Drosophila
Schneider 2 (S2) cells in culture,
prepared
EVs from cell supernatants
using the
143
Invitrogen Exosome Isolation Kit
,
and
analyzed their contents by Western blotting
.
EV
144
preparations generated
with this kit have been shown to
have similar characteristics
to
those
145
generated
by ultracentrifugation
(Skottvoll et al., 2019)
.
Both preparations contain p
rimarily
146
exosomal proteins
. However,
they also contain
similar
levels of
proteins annotated as
147
components of other compartments,
esp
e
cially
nuclear proteins. Thus, both methods should be
148
regarded as enrichments rather than purifications. The kit has the advantage of requiring mu
ch
149
less material, making it
suitable for gene
ration of EVs from small populations of transfected
150
cells.
EVs
generated from S2 cells
contain
the
Evi
protein
,
which is
a common
ly used EV
151
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint
marker. Evi is also present
in
cell
lysate
s
, however
.
The EV
pre
parations
lack
b
-
actin, showing
152
that they are not
heavily
contaminated by
cytosol
(Fig. 1c)
.
We found that
most of the
V5
-
Sas
FL
153
localize
d
to EVs, while
V5
-
Sas
short
was
retained in the cell
lysate
(Fig. 1c)
.
We
did not observe
154
any proteolytic cleavage products in EVs or
unpurified supernatants.
Endogenous Sas is
155
expressed at almost undetectable levels in S2 cells.
156
157
The commonly used
rabbit
antiserum against Sas primarily recognizes the EVT region, so
cell
158
staini
ng
reveals the localization o
f
Sas
FL
.
(Schonbaum
et al.
, 1992)
To visualize Sas
short
, we
159
made an anti
-
peptide antibody against a sequence spanning
an
exon junction
in the PA/PC
160
isoforms
.
This
selectively
recognizes Sas
short
(Supp. Fig.
1). Double
-
staining
of the
fore
gut
and
161
hindgut
with the two
Sas
antibodies shows that Sas
FL
localizes to apical cell surfaces, while
162
Sas
short
is
distributed across
the entire cell membrane
(Fig. 1d)
. These data
imply
that the EVT
163
sequence lacking in
Sas
short
is required for both
apical localization and
targeting to EVs
.
164
Polarized cells
can
release EVs with different cargoes from their apical and basolateral
165
surfaces
(Matsui et al., 2021)
, so EV targeting could be downstream of apical localization
in vivo
.
166
S2 cells are unpolarized,
however,
so this mechanism
is unlikely to
a
pply to localization of Sas
FL
167
to EVs
in culture
d S2s
.
168
169
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 8, 2022.
;
https://doi.org/10.1101/2022.09.06.506798
doi:
bioRxiv preprint