of 44
Affinity requirements for control of synaptic targeting and neuronal cell survival
by heterophilic IgSF cell adhesion molecules
Shuwa Xu
1
*
, Alina P Sergeeva
2
,
Phi
nikoula S. Katsamba
3
,
Seetha Mannepalli
3
, Fabiana
Bahna
3
,
Jude Bimela
3
,
S. L Zipursky
4
, Lawrence
Shapiro
3,5
, Barry Honig
2,
3
,5,6
, Kai Zinn
1
*
1
California Institute of Technology, Biology and Biological Engineering, Pasadena, CA,
2
Department of Systems Biology, Columbia University Medical Center, New York, NY 10032,
USA
3
Zuckerman Mind
Brain and Behavior Institute, Columbia University, New York, NY 10027, USA
4
Department of Biological Chemistry, HHMI, David Geffen School of Medicine, University of
California, Los Angeles, Los Angeles, CA 90095, USA
5
Department of Biochemistry and
Molecular Biophysics, Columbia University, New York, NY
10032, USA
6
Department of Medicine, Columbia University, New York, NY 10032, USA
*
Corresponding authors
:
Shuwa Xu
(
shuwaxu@caltech.edu
)
,
Kai Zinn
(
zinnk@caltech.edu
).
SUMMARY:
Neurons in the developing brain express many different cell adhesion molecules
(CAMs) on their surface
s
, and CAM interactions are essential for
the
determination of
synaptic
connectivity patterns. CAM binding af
finities can vary by more than 2
00
-
fold,
but the significance of
affinity differences among
CAMs
is unknown. Here we provide
a
systematic characterization of the
in vivo
consequences of altering CAM affinity.
Interactio
ns between DIP
-
α
and its binding partners Dpr6 and Dpr10 control synaptic
targeting and cell survival for
Drosophila
optic lobe neurons. We generated mutations
that
change
DIP
-
α
::Dpr10 binding
affinity
and introduced these into the endogenous
loci
. We show
that cell survival and synaptic targeting have different affinity
requirements
, and that there is a
threshold affinity required for targeting
.
Reducing
affinity causes
graded
loss
-
of
-
function phenotypes, while i
ncreasing affinity rescues
cells that would
normally die. Affinity reduction can be compensated for by increasing
gene copy number
.
.
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INTRO
DUCTION
:
Synapses in the central nervous systems of both vertebrates and invertebrates reside
within dense and complex neuropils
. During the development of “hard
-
wired” neural
systems such as the
Drosophila
brain, axonal and dendritic processes choose
genetically specified synaptic targets within environments where they have access to
the surfaces of many non
-
target neurons. Roger
Sperry’s chemoaffinity hypothesis
proposed that individual neurons in such systems are labeled by molecules that give
them unique identities. The modern version of this hypothesis is that cell adhesion
molecule (CAM)
-
like cell surface proteins (CSPs) expre
ssed on
interacting neuro
nal
surfaces bind to each other and trigger
downstream events
that can cause
establish
ment of
synaptic connections
between appropriate partners. CAM
-
like CSPs
involved in synaptic targeting in both mammals and
Drosophila
include im
munoglobulin
superfamily (IgSF) proteins, cadherin superfamily proteins, leucine
-
rich repeat proteins,
and teneurins. Some of these proteins bind homophilically, some have unique
heterophilic partners, and still others bind to a variety of partners with di
fferent affinities
(Honig and Shapiro, 2020; Sanes and Zipursky, 2020)
.
One interaction networ
k of particular interest was discovered in an
in vitro
“interactome”
screen of all ~130 IgSF CSPs in
Drosophila
(Özkan et al., 2013)
. In this network, the
“Dpr
-
ome”,
21
Dpr (Defective Proboscis Retraction) proteins interact in a complex
pattern with 11 DIPs (Dpr Interaction Proteins)
(Carrillo et al., 2015; Cosmanescu et al.
,
2018; Tan et al., 2015)
. Most DIPs bind to multiple Dprs, and vice versa. Some DIPs
and Dprs also bind homophilically. In the pupal brain, neurons expressing a particular
DIP are often postsynaptic to neurons expressing a Dpr to which that DIP binds
i
n vitro
(Carrillo et al., 2015; Cosmanescu et al., 2018; Tan et al., 2015)
. Loss of these Dprs
and DIPs can alter synaptic connectivity and cause neuronal death (revie
wed by Sanes
& Zipursky 2020
)
(Ashley et al., 2019; Baris
h et al., 2018; Bornstein et al., 2019; Carrillo
et al., 2015; Cheng et al., 2019; Courgeon and Desplan, 2019;
Menon
et al., 2019;
Venkatasubramanian et al., 2019; Xu et al., 2018
;
Xu et al., 2019
)
.
.
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Compared to binding between secreted ligands and their receptors, CAM interactions
such as those within the Dpr
-
ome network usually have much lower affinities,
with
dissociation con
s
tants (K
D
s) in the μM range. In the
Drosophila
pupal brain, sequencing
stu
dies show that each neuron can express 100 or more different CAM genes
(Barish et
al., 201
8; Konstantinides et al., 2018; Kurmangaliyev et al., 2019, 2020; Li et al., 2020;
Özel et al., 2021; Tan et al., 2015)
. The number in vertebrates is comparable
(Sarin
et
al., 2018)
. Collectively, these CAM interactions
can form stable junctions between cells
due to avidity effe
cts. In
development, neurons form transient interactions with many
other
cells during axon/dendri
te outgrowth and synaptogenesis. Some transie
nt
interactions, such as those with guidepost cells and intermediate targets, are genetically
specified and help to determine the correct pattern of synaptic connections. Others
probably occur randomly as a consequence of the dense packing of the developin
g
neuropil. Only a small fraction of the interactions experienced by a cell during
development are with its final synaptic partners. The ability of a cell to form and break
transient interactions may be facilitated by having many different CAMs on its surf
aces
that bind with low affinity. This allows the cell to manipulate the strength of its adhesive
interactions with a particular target by modulating the types and levels of multiple CAMs
that have partners on that target.
By contrast, if
cells interacted
with each other
via
a
small number of high affinity CAMs,
these
might have to be completely removed in
order to allow a neuron to break a transient interacti
on with an intermediate target.
Within each CAM family,
K
D
s for homophilic and heterophilic bind
ing can vary by as
much as 100
-
fold. For example, the
vertebrate clustered protocadherin (Pcdh)
genes
encode ~60 different isoforms, which interact homophilically with K
D
s varying from ~
1
μ
M to ~100
μ
M. Type I and type II cadherins interact both
homophilically and
heterophilically, and their K
D
s can vary over a similar range (reviewed by Shapiro &
HOnig 2020)
.
In vitro
studies have shown that differences in cadherin affinities and
expression levels can program the organization of cell clusters and
the localization of
junctions within collections of tissue culture cells
(Brasch et al., 2018; Toda et al., 2018)
.
Similar principles may a
pply
in vivo
for other CAMs
.
For example, hair cells and
.
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supporting cells in the mouse cochlea
r
epithelia in the auditory system are arranged in
checkerboard
-
like patterns. Each cell type selectively expresses either Nectin
-
1 or
Nectin
-
3. Heterophilic Nec
tin
-
1::Nectin
-
3 interactions are stronger than homophilic
Nectin
-
1 or Nectin
-
3 interactions. Removing either protein disrupts the checkerboard
arrangement of the two cell types
(Togashi et al., 2011)
. Another example is in the
lamina region of the
Drosophila
optic lobe (OL), where different cell types all express a
single cadherin, CadN, and the concentric organization of the lamina cartridge is
controlled by the relative l
evel of CadN. The L1 and L2 neurons at the center of the
cartridge express the highest levels of CadN, and the R cells at the periphery of the
cartridge express low levels of CadN
.
Manipulating the relative levels of CadN
expression in different cells can
change the relative positions of the neurons within the
cartridge and alter its pattern of synapses
(Schwabe et al., 2014)
.
Many studies have analyzed the effects of changing CAM expression levels on synaptic
connectivity, but the significance of single protein
-
protein interaction affinity variation is
not well understood. A study by Ozkan,
et. al.
in
C. elegans
examined this i
ssue for the
SYG
-
1 and SYG
-
2 CAMs. The SYG
-
1::SYG
-
2 interaction affects the formation of
synapses between the HSN neuron and vulval muscles. HSN presynaptic elements are
positioned by complexes between SYG
-
1 on HSN and SYG
-
2 on guidepost vulval
epithelial
cells.
syg
-
1
null mutations disrupt HSN synapse localization, and this
phenotype can be rescued by overexpressing SYG
-
1 in HSN neurons. SYG
-
1 mutant
proteins with
modestly
reduced affinity for SYG
-
2 are impaired in their ability to rescue
the
syg
-
1
null mu
tant, suggesting that SYG
-
1::SYG
-
2 binding affinity is important for
function
(Ozkan reference)
.
To determine the role of affinity for a CAM binding pair
in vivo
, it is important to alter
both members of the pair and to separate the effects of affinity alt
erations from those of
expression level.
Here, we
systematically
alter
ed
the affinity of heterophilic
interaction
s
between DIP
-
α
and Dpr10 in a context where both proteins were expressed at normal
levels, and examined effects
on neuronal wiring during
OL
d
evelopment.
Binding
affinities within the Dpr
-
ome vary from 1
μ
M to ~200
μ
M. DIP
-
α
binds to its two Dpr
partners, Dpr6 and Dpr10, with relatively high affinity (K
D
s of 2
μ
M and 1.36
μ
M,
.
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respectively
(Cosmanescu et al., 2018; Sergeeva et al., 2020)
. DIP
-
α
is expressed in
several classes of neurons in the medulla of the
pupal OL, including Dm1, Dm4 and
Dm12, which arborize in the M1, M3 and M3 medulla layers respectively (Figure 2A).
The L3 lamina neuron is presynaptic to both Dm4 and Dm12, and it expresses both
Dpr6 and Dpr10
(Davis et al., 2020; Kurmangaliyev et al., 2020; Tan et al., 2015; Xu et
al., 2018)
. The loss of DIP
-
α
::Dpr10 interactions causes several phe
notypes during
development, including apoptosis
-
mediated cell loss
of
Dm1, Dm4 and Dm12, ectopic
projections of Dm12 processes to the M8 layer, and alteration of synapse number in
Dm12. In addition, overexpression of Dpr10 in the M10 layer of the medulla c
auses both
Dm4 and Dm12 neurons to arborize in M10
(Xu et al., 2018)
. This p
rovides an ideal
system in which to study the significance of affinity for a heterophilic binding pair
that
controls several developmental
processes.
In order to separate the effects of binding affinity alterations from those of expression
levels, we intro
duced a series of designed affinity mutations into the endogenous
DIP
-
α
and
dpr10
genes. We found that two functions of Dpr10::DIP
-
α
interactions, control of
cell survival and of targeting specificity, have different affinity requirements. There is no
incr
ease in cell death unless affinity is reduced by 20
-
fold or more. Remarkably,
however, more cells survive than in wild
-
type when affinity is increased by 2
-
fold.
Synaptic targeting defects are observed when affinity is reduced by ~8
-
fold (to ~11
μ
M),
and t
he penetrance of these defects reaches a plateau with a ~20
-
fold reduction (to ~28
μ
M). This transition between 11
μ
M and 28
μ
M is also observed when targeting is
assessed by ectopic expression of Dpr10 in M10. In addition, we observe that adding or
subtra
cting copies of affinity mutant genes affects their phenotypes, indicating that
subtle changes in expression level can compensate for alterations in affinity
. These
results suggest that the affinities between DIP
-
α
and its Dpr partners are finely tuned to
allow the correct number of DIP
-
α
expressing neurons to survive and form the
appropriate synaptic connections.
.
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RESULTS:
Generation and selection
of DIP
-
α
and Dpr10
mutations
that change DIP
-
α
::
Dpr10
affinity
DIP
-
α
binds to Dpr6 and Dpr10 with affinities of 2.0 μM and 1.
3
6 μM, respectively. DIP
-
α
also binds to itself with an affinity of 24 μM, and
heterophilic
and
homophilic
binding
use the same interface residues
(Cheng et al., 2019; Cosmanescu et al., 2018;
Sergeeva et al., 2020)
.
We
developed
computational approaches
that allowed the
design
of
DIP
-
α
and Dpr10 mutants that
changed
DIP
-
α
::
Dpr10 binding affinity
in vitro
(Sergeeva et al., 2020)
. To determine how changes in
affinity
affect neuron
-
neuron
recognition events, we selected
a set of
DIP
-
α
and Dpr10 mutations for
in vivo
stud
ies
based on the following criteria: 1) the mutation
s
should alter binding affinity between
DIP
-
α
and Dpr10 in a graded fashion
, so as to generate a set of proteins with affinities
varying
over a
wide
range
; 2)
the mutations should not change their specificity for
binding to
other DIP/Dpr proteins;
3) the mutation
s
should not
have strong effects on
homophilic binding affinity.
Based on these criteria, we chose DIP
-
α
mutants G74A (K
D
=0.9
μ
M
;
when expressed
in
v
ivo
, this mutant is designated as
DIP
-
α
+2F
), K81Q (K
D
=31.8
μ
M,
DIP
-
α
-
20F
), and K81Q
G74S (K
D
=68.0
μ
M,
DIP
-
α
-
5
0F
); and Dpr10 mutants V144K (K
D
=11.3
μ
M,
d
pr10
-
8F
) and
Q138D (K
D
=27.6
μ
M,
d
pr10
-
2
0
F
)
(Sergeeva et al., 2020)
. To generate a comparable
Dpr10 affinity reduction range as that for DIP
-
α
, we designed an additional
d
pr10
mutant
,
V144K Q142E G99D (K
D
=50.0
μ
M,
d
pr10
-
4
0F
)
(Fig
ure
s
1B,
C)
. The location of
the designed mutations in the DIP
-
α
::
Dpr10 interface between N
-
terminal Ig1 domains
is indicated in Figure 1A
.
These
Dpr10
and
DIP
-
α
mutations did not change
the
specificity
of
the
ir
binding to
other
DIP/Dpr
protein
s
. Fig
ure
1B
shows
binding isotherms for interactions
of Dpr10 wild
-
type
and mutant proteins
with
DIP
-
α
, DIP
-
β
and DIP
-
γ
(see
Figure
S
1 for corresponding
.
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sensorgrams)
. DIP
-
β
is closest to DIP
-
α
among all other DIPs in sequence, and is also
a Dpr10 binding protein,
but with a much weaker affinity (K
D
=33
μ
M).
DIP
-
γ
is not a
Dpr10 binding protein (K
D
of >1000
μ
M).
Like
wild
-
type Dpr10, a
ll three mutant Dpr10
proteins
interact more strongly with
DIP
-
α
than with
D
IP
-
β
(Fig
ure
1B).
None of the
mutants displays detectable b
inding to DIP
-
γ
(
Figure 1B
)
. Fig
ure
1C
shows
binding
isotherms for DIP
-
α
wild
-
type and mutant proteins to
Dpr10, Dpr4, Dpr7 and Dpr12
(see
Figure
S
1 for corresponding sensorgrams)
.
Like
w
ild
-
type
DIP
-
α
, none of the
DIP
-
α
mutants
exhibits measurable binding to
Dpr4, Dpr7, or Dpr12, which are
members of
non
-
cognate
Dpr
subgroups
(Figure 1C).
Dpr10 is a monomer, while DIP
-
α
is a dimer with a K
D
of 23.9
μ
M
(Cosmanescu et al.,
2018)
.
The
DIP
-
α
/DIP
-
α
and Dpr10/DIP
-
α
interfaces are very similar (RMSD of 0.6Å),
and hence changes in the heterophilic interface would be expected
to change the
homophilic DIP
-
α
/
DIP
-
α
interface. To ensure that the DIP
-
α
mutants retained the ability
to homodimerize, we measured homophilic binding affinities of all DIP
-
α
mutants using
analytical ultracentrifugation (AUC).
The homophilic binding K
D
for
the three DIP
-
α
mutant
proteins are:
DIP
-
α
G74A
(
DIP
-
α
+2F
,
in vivo
),
K
D
=
50
μ
M
;
DIP
-
α
K81Q (
DIP
-
α
-
20F
)
K
D
=
19.6
μ
M
;
DIP
-
α
K81Q G74S
(
DIP
-
α
-
50F
) K
D
=46
μ
M
.
We confirmed that all mutants
remain dimeric, and th
e changes of homophilic binding between
DIP
-
α
in all mutants are
no more than 2
-
fold
(
Table S1).
In summary
,
these
results indicate that we have
successfully created Dpr10
an
d DIP
-
α
mutants with a wide affinity range that do not
affect cognate binding preferences of DIPs and Dprs relative to non
-
cogn
ate partners.
DIP
-
α
and Dpr10 affinity muta
nts are expressed normally in vivo
We introduced the chosen mutations into the endogenous
DIP
-
α
and
d
pr10
genomic
loci by a precise CRISPR mediated knock
-
in strategy
(Zhang et al., 2014)
, and
sequence verified the generated transgenic animals. We tested expression of
the
mutant pr
oteins
in vivo
using antibodies specific for DIP
-
α
and Dpr10 (Fig
ure
s
2
B, 2C
)
(
Xu et al., 2018)
.
Wild type DIP
-
α
is expressed in three neuropil layers
in the
medulla region
of
the
OL
.
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during mid
-
pupal development (48h after puparium formation/APF)
(Xu et al., 2018)
.
All
DIP
-
α
affinity mutants were expressed in the same pattern as the wild t
ype (Fig
ure
s
2B’’
-
B
’’’). Changing cell surface protein sequences sometimes causes proteins to fail to
transport to neuronal terminals, but all of our mutants localized to the medulla neuropil,
where axonal and dendritic endings are located
(Fig
ures
2B’’
-
B
’’’)
.
The
DIP
-
α
-
expressing
Dm1
neuron
, and
Dm4 and Dm12
neurons
,
project to
the
first
(M1)
and
second (M3)
DIP
-
α
expressing
layer
s
at 48h APF
, respectively
(
Figure 2B’
).
A
large fraction of these neurons undergo
cell death
during pupal development i
n
DIP
-
α
null mutants
(see below)
(Xu et al., 2018)
.
If our introduced mutations w
ere loss
-
of
-
function (LOF), they would be expected to cause a reduction in staining intensity in the
first two
layers, even if they do not alter the levels of expression in individual cells.
Therefore, since
another set of
DIP
-
α
expressing neurons that pro
ject to
the third layer
(
M10;
yellow dotted line) do not exhibit detectable cell death in null mutants, we
quantitated the expression levels of
mutant
DIP
-
α
in this layer. All three alleles showed
similar expression levels
to
wild
-
type DIP
-
α
in this layer
(Fig
ure
2D). When
anti
-
DIP
-
α
staining was quantitated across
the whole neuropil, DIP
-
α
+2F
was
present at
similar
levels
as
wild
-
type.
Staining intensity for
DIP
-
α
-
20F
and DIP
-
α
-
5
0F
was
lower than for
wild
-
type, consistent with the idea that some DIP
-
α
-
expr
essing neurons undergo cell death in
loss
-
of
-
function
mutants (Fig
ure
S
2, and see below).
Dpr10 is expressed in two major medulla layers in the 48h APF optic lobe (Figure
s
2C’
-
C’’’’). All three Dpr10 affinity mutant proteins were expressed at the same patt
erns
as
the wild type
, showing no defects in neuronal terminal localization. Since no Dpr10
-
expressing OL neurons are known to exhibit cell death, we quantified Dpr10 expression
levels in the whole neuropil. Two Dpr10 alleles
were expressed at the same
level
as
the
wild
-
type (Dpr10
-
8F
and Dpr10
-
4
0F
), while one was expressed at
slightly
higher
level
than
wild
-
type (Dpr10
-
20
F
).
Reducing DIP
-
α
::Dpr10 affinity causes graded mistargeting
of Dm12 neurons
Null
DIP
-
α
mutation
s
,
or
dpr6
,
dpr10
double mutations
,
caus
e
disruption of several
.
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cellular processes during
OL
development
including
mistargeting of
Dm12 neurons
to
M8 layer
, and
death of
Dm4
and
Dm12
neuron
s
(Xu et al., 2018)
. To determine
how
changing binding affinity between DIP
-
α
and Dpr10 affects t
hese processes, we first
analyzed neuronal targeting in Dm12 neurons.
Dm12 neurons arborize in the M3 layer
(Figure 2A)
.
We have previously reported that
DIP
-
α
null
Dm12 clones in a wild
-
type
background target to a more proximal medulla layer, M8
(Xu et al., 2018)
.
We have now
developed methodologies to quantitate
mistarget
ing
in whole
-
animal mutants, allowing
us to examine much larger numbers of neurons. In
DIP
-
α
null whole
-
animal mutants,
about one
-
third (43) of Dm12 neurons mistarget
ed
to M8 (Fig
ures
3
E
,
F
). In
DIP
-
α
-
20F
,
which has a ~20
-
fold reduction in DIP
-
α
::Dpr10 bindin
g affinity, ~5 (4%) Dm12 neurons
per OL mistarget
ed
to M8 (Fig
ures
3
C
,
3
F
,
3
F
inset). This number increased to ~20
(17%) in
DIP
-
α
5
0F
, which reduces affinity by ~
5
0 fold
(Figure 3D, 3F)
. The mutation that
increased DIP
-
α
/Dpr10 binding affinity by 2 fold
(DIP
-
α
+2F
)
produced
no change in
Dm12 targeting (
Figure
3B, 3F
).
Dm12 cell death and mistargeting are differentially affected by
DIP
-
α
affinity
mutations
In
DIP
-
α
null mutants, about 25 Dm12 neurons (~22%) die during development,
reducing the total Dm12 complement
to
~
90
(Fig
ure
3F)
(Xu et al., 2018)
. We observed
that reducing DIP
-
α
::
Dpr10 binding affinity by ~20 fold did not
cause any cell loss (
DIP
-
α
-
20F
). A small number of Dm12 neurons were lost (~
8,
5%), when affinity was reduced
by
5
0
-
fold
in
DIP
-
α
-
5
0F
mutants (Fig
ure
3G)
. These results indicate that different affinity
thresholds control Dm12 targeting and cell survival. B
oth the
DIP
-
α
-
20F
and
DIP
-
α
-
5
0F
mutations cause
substantial
mistarget
ing but have little or no effect on cell survival
(Fig
ure
s
3E
, 3F
). The mutation that increased DIP
-
α
/Dpr10 binding affinity by 2 fold
(DIP
-
α
+2F
)
produced
a slight increase of Dm12 cell
number (~7) with p value <0
.
018
(Fig
ure
3
G
).
Differential effects on targeting and cell survival are also observed for
d
pr10
affinity mutants
.
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https://doi.org/10.1101/2021.02.16.431482
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