of 37
Affinity requirements for control of synaptic targeting and
neuronal cell survival by heterophilic IgSF cell adhesion
molecules
Shuwa Xu
1,7,*
,
Alina P. Sergeeva
2
,
Phinikoula S. Katsamba
3
,
Seetha Mannepalli
3
,
Fabiana
Bahna
3
,
Jude Bimela
3
,
S. Lawrence Zipursky
4
,
Lawrence Shapiro
3,5
,
Barry Honig
2,3,5,6
,
Kai
Zinn
1,*
1
California Institute of Technology, Division of Biology and Biological Engineering, Pasadena, CA
91125, USA
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
7
Lead contact
SUMMARY
Neurons in the developing brain express many different cell adhesion molecules (CAMs) on their
surfaces. CAM-binding affinities can vary by more than 200-fold, but the significance of these
variations is unknown. Interactions between the immunoglobulin superfamily CAM DIP-
α
and
its binding partners, Dpr10 and Dpr6, control synaptic targeting and survival of
Drosophila
optic
lobe neurons. We design mutations that systematically change interaction affinity and analyze
function
in vivo
. Reducing affinity causes loss-of-function phenotypes whose severity scales with
the magnitude of the change. Synaptic targeting is more sensitive to affinity reduction than is
cell survival. Increasing affinity rescues neurons that would normally be culled by apoptosis. By
manipulating CAM expression together with affinity, we show that the key parameter controlling
This is an open access article under the CC BY-NC-ND license (
http://creativecommons.org/licenses/by-nc-nd/4.0/
).
*
Correspondence: shuwaxu@gmail.com (S.X.), zinnk@caltech.edu (K.Z.).
AUTHOR CONTRIBUTIONS
S.X., K.Z., and S.L.Z. conceived the project and wrote the majority of the paper. A.P.S., L.S., and B.H. wrote the section on mutant
design and edited the remainder of the paper. S.X. and K.Z. designed the experiments. S.X. conducted the
in vivo
experiments and
analyzed data. A.P.S., L.S., and B. H. designed the protein mutations and analyzed
in vitro
data. P.S.K. performed and analyzed
surface plasmon resonance (SPR) experiments, and S.M., F.B., and J.B. produced and purified proteins.
DECLARATION OF INTERESTS
The authors declare no competing interests.
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at
https://doi.org/10.1016/j.celrep.2022.110618
.
HHS Public Access
Author manuscript
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. Author manuscript; available in PMC 2022 May 07.
Published in final edited form as:
Cell Rep
. 2022 April 05; 39(1): 110618. doi:10.1016/j.celrep.2022.110618.
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circuit assembly is surface avidity, which is the strength of adherence between cell surfaces. We
conclude that CAM binding affinities and expression levels are finely tuned for function during
development.
Graphical Abstract
In brief
Xu et al. demonstrate that altering the affinity of transsynaptic interactions between DIP-
α
and
Dpr10 affects multiple aspects of circuit assembly in the
Drosophila
visual system.
INTRODUCTION
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)
expressed on interacting neuronal surfaces bind to each other and trigger downstream events
that cause establishment of synaptic connections between appropriate partners.
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CAM-like CSPs involved in synaptic targeting, which are denoted cell-recognition
molecules (CRMs), include immunoglobulin superfamily (IgSF) proteins, cadherin
superfamily proteins, leucine-rich repeat proteins, teneurins, and others. Each
Drosophila
neuron can express 100 or more different CAM genes during development (
Barish et al.,
2018
;
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
).
There are large variations in binding affinity even among CAMs in the same family (
Honig
and Shapiro, 2020
). Loss or misexpression of CAMs can change synaptic connectivity, but
the
in vivo
functions of CAM affinity variation have not been previously investigated.
The “Dpr-ome” interaction network was discovered in an
in vitro
“interactome” screen of
IgSF CSPs (
Özkan et al., 2013
). Twenty-one Dpr proteins interact in a complex pattern with
11 DIPs (
Carrillo et al., 2015
;
Cosmanescu et al., 2018
;
Tan et al., 2015
). Most DIPs bind to
multiple Dprs and vice versa. In the pupal optic lobe (OL), neurons expressing a particular
DIP are often postsynaptic to neurons expressing a Dpr to which that DIP binds
in vitro
(
Carrillo et al., 2015
;
Cosmanescu et al., 2018
;
Tan et al., 2015
). Loss of DIPs and Dprs
can alter synaptic connectivity and cause neuronal death, indicating that these proteins are
CRMs (
Ashley et al., 2019
;
Barish et al., 2018
;
Bornstein et al., 2021
;
Carrillo et al., 2015
;
Cheng et al., 2019
;
Courgeon and Desplan, 2019
;
Menon et al., 2019
;
Venkatasubramanian
et al., 2019
;
Xu et al., 2018
,
2019
). The binding affinities of all interacting DIP/Dpr pairs
have been measured using biophysical methods, and K
D
s for homophilic and heterophilic
binding vary by more than 100-fold, ranging from <2 to >200 μM (
Cosmanescu et al.,
2018
). The Dpr-ome provides an attractive system in which to examine if and how affinity
variation contributes to the determination of synaptic-connectivity patterns.
DIP-
α
and Dpr10, the binding pair examined here, have one of the highest-affinity
interactions in the network, with a K
D
of 1.4 μM. The high-resolution structures of DIP-
α
complexed with itself and with its Dpr partners have been determined, and binding
in
vitro
has been characterized in detail using biophysical methods (
Carrillo et al., 2015
;
Cosmanescu et al., 2018
). DIP-
α
and Dpr10 are expressed in synaptically connected visual
system neurons, and their interactions regulate synaptic targeting and cell survival for
several neuronal types (
Xu et al., 2018
). Thus, the study of DIP-
α
::Dpr10 interactions allows
us to connect an analysis of
in vivo
function to
in vitro
measurements of binding affinity.
To understand how affinity affects CAM function
in vivo
, it is important to separate the
effects of affinity alterations from those of the protein expression level. We introduced
a series of designed mutations that either decrease or increase binding affinity into the
DIP-
α
and
dpr10
loci, so that the mutant proteins would be expressed under the control of
endogenous regulatory elements. We observed that reducing affinity causes loss-of-function
(LOF) phenotypes, and that the severity of these phenotypes scales with the magnitude of
affinity reduction. Synaptic targeting and cell survival have different affinity requirements.
Increasing affinity does not alter targeting, but it does affect cell survival. Our results
suggest that DIP-
α
::Dpr10 engagement controls a trophic-support pathway that counteracts
cell-death pathways. When DIP-
α
::Dpr10 interactions are sufficiently strengthened, the
culling of visual system neurons by apoptosis that occurs during wild-type development
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is eliminated. Changes in gene expression levels can compensate for alterations in protein
binding affinity, indicating that avidity is the key parameter that determines the outcome of
interactions between neurons mediated by these CAMs. The affinities and expression levels
of DIP-
α
and its Dpr partners appear to be tuned so that the correct number of neurons
survive and form appropriate synaptic connections.
RESULTS
Generation and selection of DIP-
α
and Dpr10 mutations that change DIP-
α
::Dpr10 binding
affinity
DIP-
α
binds to two Dprs, Dpr10 and Dpr6, with affinities of 1.4 and 2.0 μM, respectively.
DIP-
α
also binds to itself with an affinity of 24 μM (
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
studies based
on the following criteria: (1) the mutations 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, and (3) the mutations should not have strong effects on the homophilic
binding affinity of DIP-
α
.
Based on these criteria, we chose the designed DIP-
α
mutants K81Q (K
D
= 31.8 μM,
DIP-
α
20F
; the superscripts in mutant names indicate the direction and fold change in
affinity relative to wild type), K81Q G74S (K
D
= 68.0 μM,
DIP-
α
50F
), and G74A (K
D
=
0.9 μM,
DIP-
α
+
2F
) and Dpr10 mutants V144K (K
D
= 11.3 μM,
dpr1
8F
) and Q138D (K
D
=
27.6 μM,
dpr10
20F
). To achieve a greater affinity range, we designed two additional Dpr10
mutants, V144K Q142E G99D (K
D
= 50.0 μM,
dpr10
40F
) and Q142M (K
D
= 0.19 μM,
Dpr10
+10F
) (Figures 1B and 1C). K
D
is further decreased to 0.10 μM (a ~20-fold increase
in affinity relative to wild type) when DIP-
α
+2F
binds to Dpr10
+10F
. The locations of the
designed mutations in the DIP-
α
::Dpr10 interface are indicated in Figure 1A.
These Dpr10 and DIP-
α
mutations did not change the specificity of their binding to other
DIP/Dpr proteins. Figure 1B shows binding isotherms for interactions of Dpr10 wild-type
and mutant proteins with DIP-
α
, DIP-
β
, and DIP-
γ
(see Figure S1 for corresponding
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 lower affinity (K
D
= 33 μM). DIP-
γ
is not a Dpr10
binding protein (K
D
>1,000 μM). Like wild-type Dpr10, all three mutant Dpr10 proteins
interact more strongly with DIP-
α
than with DIP-
β
and do not bind to DIP-
γ
(Figure 1B).
Figure 1C shows binding isotherms for DIP-
α
wild-type and mutant proteins to Dpr10,
Dpr4, Dpr7, and Dpr12 (see Figure S1 for corresponding sensorgrams). Like wild-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
can form dimers
in vitro
with a K
D
of 24 μM
(
Cosmanescu et al., 2018
). The DIP-
α
/DIP-
α
and Dpr10/DIP-
α
interfaces are very
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similar (root-mean-square deviation [RMSD] of 0.6 Å), and hence, changes in the
surface used for heterophilic binding by DIP-
α
would be expected to also alter 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
s for the three DIP-
α
mutant proteins are DIP-
α
G74A (
DIP-
α
+
2F
), K
D
= 50 μM, DIP-
α
K81Q (
DIP-
α
20F
) K
D
=
19.6 μM, and DIP-
α
K81Q G74S (
DIP-
α
50F
) K
D
= 46 μM (Table S1). All mutants remain
dimeric, and homophilic binding affinity is changed by no more than 2- fold. In summary,
we have successfully created Dpr10 and DIP-
α
mutants with a wide affinity range that do
not affect cognate binding preferences of DIPs and Dprs relative to non-cognate partners.
DIP-
α
and Dpr10 affinity mutants are expressed normally
in vivo
We introduced the chosen mutations into the endogenous
DIP-
α
and
dpr10
genomic loci by
a precise CRISPR-mediated knockin strategy (
Zhang et al., 2014
). We tested expression
in
vivo
using antibodies specific for DIP-
α
and Dpr10 (Figures 2B and 2C). Wild-type DIP-
α
is expressed in three neuropil layers in the medulla region of the OL during mid-pupal
development (48 h after puparium formation [APF]) (
Xu et al., 2018
) (Figure 2B’). All
DIP-
α
affinity mutants were localized to the medulla neuropil and expressed in the same
layers as wild type (Figures 2B”–2B””). The DIP-
α
-expressing Dm1, Dm4, and Dm12
neurons project to the first (M1) and second (M3) DIP-
α
expressing layers (Figures 2A
and 2B’). A large fraction of these neurons undergo cell death during pupal development in
DIP-
α
null mutants (
Xu et al., 2018
). If our introduced mutations caused LOF phenotypes,
they would be expected to cause a reduction in staining intensity in these two layers due to
cell death even if they do not alter the levels of expression in individual cells. Another set
of DIP-
α
-expressing neurons that project to a third layer (M10; yellow dotted line in Figure
2B’) do not exhibit detectable cell death in null mutants, so we quantitated the expression
levels of DIP-
α
mutant proteins in this layer. All three alleles showed similar expression
levels to wild-type DIP-
α
in M10 (Figure 2D).
Dpr10 is expressed in two major medulla layers in the 48 h APF OL (Figure 2C’). All three
Dpr10 affinity mutant proteins were expressed in the wild-type pattern (Figures 2C”–2C””).
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
−40F
), while one was expressed at a slightly higher
level than wild-type (Dpr10
−20F
) (Figure 2E).
Reducing DIP-
α
::Dpr10 affinity causes graded mistargeting of Dm12 neurons
Null
DIP-
α
mutations or
dpr6 dpr10
double-null mutations disrupt targeting of Dm12
neurons and reduce survival of Dm1, Dm4, and Dm12 neurons (
Xu et al., 2018
). To
determine how changing binding affinity between DIP-
α
and Dpr10 affects these processes,
we first analyzed targeting of Dm12 neurons. Previously, we showed that
DIP-
α
null
Dm12 clones in a wild-type background target to a more proximal medulla layer, M8
(Figures 3B and 3G) (
Xu et al., 2018
). To better quantitate and visualize mistargeting in
affinity mutants, we modified our methodologies by changing the genetic background and
altering permeabilization, which allowed us to examine much larger numbers of neurons
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using whole-animal mutants (see Method details and Immunohistochemistry). In
DIP-
α
null
mutants, about one-third of Dm12 neurons (~43 per OL) mistargeted to M8 (Figures 3B, 3G,
and 3M). In
DIP-
α
20F
, which has a ~20-fold reduction in DIP-
α
::Dpr10 binding affinity,
~5 (4%) of Dm12 neurons per OL mistargeted to M8 (Figures 3C and 3M). This number
increased to ~20 (17%) in
DIP-
α
50F
, which reduces affinity by ~50 fold (Figures 3D and
3M).
Dpr10 and Dpr6 are both expressed in the L3 lamina neuron, which forms synapses with
Dm12 and Dm4 (Figure 2A). Loss of both Dprs (in a double-null mutant) causes the same
mistargeting and cell-death phenotypes seen in
DIP-
α
null mutants.
dpr10
or
dpr6
single
mutants have much weaker phenotypes, indicating that the Dprs have partially redundant
functions (
Xu et al., 2018
). Thus, to facilitate the analysis of the relationships between
Dpr10 affinity and function, we knocked
dpr10
affinity mutations into the endogenous
dpr10
locus in a
dpr6
null mutant background. We analyzed Dm12 neurons in the three
dpr10
affinity mutants described above (
dpr10
8F
, dpr10
20F
, and
dpr10
40F
). Animals
expressing only wild-type Dpr10 but not Dpr6 (
dpr6
null
single mutant), displayed a mild
mistargeting phenotype in Dm12 neurons, with on average ~8 mistargeted Dm12 neurons
per OL (Figures 3H and 3M). When Dpr10 affinity to DIP-
α
was reduced by 8-fold (in
dpr6
null
dpr1
8F
), ~14 mistargeted Dm12 neurons were observed (Figures 3I and 3M). A
further reduction in affinity to 20-fold less than wild type (
dpr6
null
dpr10
20F
) caused a
doubling of the number of mistargeted neurons, to ~29 cells (Figures 3J and 3M). Thus, in
both
dpr10
and
DIP-
α
mutants, the severity of Dm12 mistargeting scales with DIP-
α
::Dpr10
affinity reduction.
In these experiments, we observed that
dpr10
mutations that reduce affinity by 20- or
40-fold had stronger phenotypes than
DIP-
α
mutations that reduce affinity by 20- or 50-fold
(Figure 3M). However, as described above,
dpr10
affinity mutations were knocked into a
dpr6
null background, while
DIP-
α
affinity mutant phenotypes were analyzed in a wild-type
dpr6
background. Because Dpr6 and Dpr10 are partially redundant (
Xu et al., 2018
), to
accurately assess the relationships between phenotypic severity and DIP-
α
::Dpr10 binding
affinity, we analyzed Dm12 neurons in
DIP-
α
affinity mutants that also lacked Dpr6. The
presence of the
dpr6
null
mutations increased the extent of Dm12 mistargeting observed in
DIP-
α
affinity mutants. For example, in the
DIP-
α
mutant that decreases DIP-
α
::Dpr10
affinity by 20-fold, the number of mistargeted Dm12 neurons per OL increased from ~5 in
DIP-
α
20F
to ~24 in
DIP-
α
20F
dpr6
null
(Figures 3E and 3M). This number is similar to
that observed in the
dpr10
affinity mutant that also reduces DIP-
α
::Dpr10 affinity by 20-fold
(
dpr6
null
dpr10
20F
: ~29). In conclusion, in the absence of Dpr6, comparable phenotypic
severity is observed for
DIP-
α
or
dpr10
mutants that change DIP-
α
::Dpr10 affinity to a
similar extent (Figure 3M).
Different affinity thresholds control Dm12 targeting and cell survival
There are ~115 Dm12 neurons in a wild-type OL. In
DIP-
α
or
dpr6 dpr10
null mutants,
about 25 of these (~22%) die during development, reducing the total Dm12 complement to
~90 (Figure 3N) (
Xu et al., 2018
). Reducing DIP-
α
::Dpr10 binding affinity by 20-fold in a
wild-type
dpr6
background did not cause any cell loss (
DIP-
α
20F
). However, in the absence
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of Dpr6 (
DIP-
α
20F
dpr6
null
), ~9 Dm12 cells were lost. Removing Dpr6 from
DIP-
α
50F
mutants produced a further increase in Dm12 cell loss, from ~9 to ~17 (Figure 3N).
dpr10
affinity mutants were examined in a
dpr6
null mutant background, and ~8 Dm12 cells
were lost in
dpr6
single mutants. This number was not significantly changed by addition
of
dpr10
8F
or
dpr10
20F
affinity mutations. However, in
dpr6
null
dpr10
40F
mutants, ~24
cells were lost (Figure 3N). Thus, in the absence of Dpr6, a 40- to 50-fold reduction in
DIP-
α
::Dpr10 binding affinity produced a null or near-null cell-loss phenotype.
These results indicate that mistargeting and cell loss have different dependencies on DIP-
α
::Dpr10 binding affinity. We first observed a significant increase in mistargeting when
affinity (K
D
) was reduced to 11.3 μM in
dpr6
null
dpr10
8F
. When affinity was reduced by
~20-fold, in either
dpr6
null
dpr10
20F
(K
D
= 27.8 μM) or
DIP-
α
20F
dpr6
null
(K
D
= 31.8
μM), the penetrance of the mistargeting phenotype was about 50% of that observed in
null mutants (Figures 3M and 3O). For cell loss, no increase above the
dpr6
null mutant
penetrance was observed for
DIP-
α
20F
or
dpr6 dpr10
20F
mutants. A threshold of ~50% of
the null penetrance was first exceeded in
dpr6
null
dpr10
40F
(K
D
= 50.0 μM) or
DIP-
α
50F
dpr6
null
(K
D
= 68.0 μM) (Figures 3N and 3O). Thus, we conclude that the 50% threshold
for mistargeting is reached at a K
D
of ~30 μM, while the threshold for cell loss occurs at
~50 μM. Note that there is more cell death in
dpr6
null
dpr10
40F
than in
DIP-
α
50F
dpr6
null
(Figure 3N). Perhaps in a
dpr6
null
background, the number of accessible DIP-
α
molecules
on Dm12 processes is in excess of the number of Dpr10 molecules on the axons of L3 and
other presynaptic neurons in the M3 layer. This could account for cell-survival phenotypes
being more sensitive to perturbation of Dpr10.
A reduction in cell survival with decreasing affinity is also observed for Dm4 and Dm1
neurons
Dm4 and Dm1 processes do not mistarget in
DIP-
α
null or
dpr6 dpr10
double-null mutants
(Figures 4A, 4D, and S2) (
Xu et al., 2018
). This suggests that DIP-
α
::Dpr10 interactions
are redundant with other cues in directing the arbors of Dm4 and Dm1 neurons to the
correct layers. However, survival of Dm4 and Dm1 neurons is affected by null
DIP-
α
or
dpr6 dpr1
0 mutations, so we were able to examine the effects of affinity mutations on cell
survival for Dm4 and Dm1. We observed that Dm4 and Dm1 cell survival is also decreased
in affinity mutants and that their survival is more sensitive to affinity reduction than that
of Dm12 neurons. In
DIP-
α
20F
/DIP-
α
null
(null) mutants (wild type for
dpr6
), cell loss
was seen for Dm4 and Dm1 but not for Dm12 (Figures 3N, 4B, 4I, and S2). The stronger
affinity mutant
DIP-
α
50F
/DIP-
α
null
exhibited as much cell loss as the null allele for Dm4
and Dm1 but had a weaker phenotype than the null for Dm12 (Figures 3N, 4C, 4I, and S2).
Survival of Dm4 neurons was also more sensitive than survival of Dm12 neurons to affinity
reduction by
dpr10
mutations. Dm4 cell loss is observed in
dpr6
null
dpr10
20F
mutants
(Figures 4G and 4J), which have wild-type numbers of Dm12 neurons (Figure 3N). As
for Dm12,
dpr10
affinity mutants have stronger phenotypes than the corresponding
DIP-
α
mutants, presumably because Dpr6 is still expressed in these
DIP-
α
mutant backgrounds.
These data suggest that different cell types have different affinity thresholds for regulation of
cell survival. This could be due to different levels of expression of DIP-
α
in Dm4 and Dm1
neurons compared with Dm12 and/or to the use of alternative cell-death pathways.
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Increasing DIP-
α
::Dpr10 affinity rescues Dm4 cell death
The analysis of affinity-reduction mutants described above shows that affinity thresholds
exist for neuronal targeting and survival. When affinity is reduced to below these thresholds,
neurons exhibit LOF phenotypes. We wished to also examine the consequences of increasing
the affinity between DIP-
α
and Dpr10. Because DIP-
α
::Dpr10 is already one of the
highest-affinity interactions in the Dpr-ome, it is difficult to design mutants that increase
affinity based on comparisons among DIP::Dpr binding interfaces. However, using position
scanning mutagenesis via the FoldX computational algorithm (see STAR Methods), we were
able to predict and experimentally validate two mutants that passed the selection criteria
discussed above: DIP-
α
G74A
(K
D
= 0.90 μM, DIP-
α
+2F
) and Dpr10
Q142M
(K
D
= 0.19 μM,
Dpr10
+10F
). When DIP-
α
+2F
binds to Dpr10
+10F
, K
D
is further decreased to 0.10 μM, which
represents a ~20-fold increase in affinity compared with wild type (Figures 5A and 5B).
Dm4, Dm12, and Dm1 exhibited normal layer targeting in both of the two increased affinity
mutants (Figures 5C–5H and S3). We then analyzed cell-death phenotypes in Dm4 neurons.
We have shown previously that Dm4 neurons are produced in excess in wild-type animals
(~55 cells/OL) and that their number is reduced to ~40 cells/OL by apoptotic cell death
during early pupal development (Figures 5I and 5J) (
Xu et al., 2018
). When apoptosis
inhibitors (either baculovirus p35 protein or the
Drosophila
death-associated inhibitor of
apoptosis 1 protein) are expressed in Dm4, the number of Dm4 neurons in adults remains at
~55 per OL (Figures 5I and 5J) (
Xu et al., 2018
). Loss of DIP-
α
or its Dpr partners causes
increased Dm4 cell death, but it was unknown whether the natural Dm4 cell death that
occurs in wild type is also regulated by DIP::Dpr interactions. To evaluate this, we counted
adult Dm4 cell numbers in flies with increased affinity between DIP-
α
and Dpr10. In flies
carrying two copies of
DIP-
α
+
2F
, or one copy of
dpr10
+
10F
over wild-type
dpr10
, there was
a small increase in Dm4 numbers, to ~45–48 cells/OL. Note that, because
dpr10
+
10F
was
knocked into a
dpr6
null background, we analyzed
dpr10
+
10F
over a wild-type chromosome
(
dpr6
WT
,
dpr10
WT
) to eliminate effects caused by loss of Dpr6, which in theory would
counteract the effects of increasing affinity between DIP-
α
and Dpr10. In flies expressing
both DIP-
α
+2F
and Dpr10
+10F
(
DIP-
α
+
2F
/DIP-
α
WT
; dpr10
+
10F
/dpr10
WT
), there were ~55
Dm4 cells per OL, which is equal to the number observed when apoptosis inhibitors are
expressed in wild type (Figures 5I and 5J). These results suggest that Dm4 cell death is
entirely controlled by DIP-
α′
s interactions with its Dpr partners. Apoptosis in wild type
is partially suppressed by a trophic-support pathway controlled by transsynaptic DIP::Dpr
interactions. Decreasing trophic support by reducing DIP-
α
::Dpr10 affinity leads to excess
cell death, and increasing trophic support to above wild-type levels by increasing affinity
suppresses normal cell death. The affinity and expression levels of DIP-
α
and its partners,
and therefore the extent of apoptosis suppression, may have been adjusted by evolution to
produce the desired number of adult Dm4 cells.
An affinity threshold for induction of Dm4 mistargeting by ectopic Dpr10
Ectopic expression of Dpr10 in M10 is sufficient to induce mistargeting of the processes
of DIP-
α
-expressing neurons (
Xu et al., 2018
). To analyze if this instructional role is also
dependent on affinity, we made UAS lines for Dpr10 affinity mutants and tested their
abilities to induce mistargeting. Both Dm4 and Dm12 neurons displayed misexpression-
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induced mistargeting (
Xu et al., 2018
). Because of Dm4’s smaller cell number (40 Dm4 s
versus 115 Dm12 s) and thicker mistargeting axon branches, it is easier to quantitatively
assess Dm4 mistargeting events. Thus, we used Dm4 neurons for analysis of mistargeting by
Dpr10 affinity mutants.
T4 is not a synaptic partner of Dm4, but T4 processes come into contact with neurons that
project to other medulla layers during early pupal development. They later segregate into
the M10 layer of the medulla (Figure 2A). When endogenous Dpr10 is knocked out, and
ectopic Dpr10 is misexpressed at high levels in T4 cells, it causes Dm4 neurons to send their
processes to M10, bypassing their normal M3 target layer (
Xu et al., 2018
). We expressed
Dpr10 affinity mutants in M10 using T4-Gal4. Dpr10 antibody staining revealed that M10
layer expression of UAS-Dpr10
−8F
and UAS-Dpr10
−20F
is comparable to that of wild-type
UAS-Dpr10, while UAS-Dpr10
−40F
is expressed at 1.6-fold higher levels than wild type
(Figure S4).
We analyzed each affinity mutant protein’s ability to induce mistargeting of Dm4 neurons in
a
dpr6 dpr10
double-null mutant background (Figure 6A–A” and 6B–B”). About half of the
Dm4 neurons undergo cell death due to the lack of Dpr6 and Dpr10, and as a result, there
are fewer Dm4 processes in M3. The remaining ~20 Dm4 cells all target to the correct layer,
so there must be other cues that direct Dm4 processes to M3 in the absence of DIP-
α
and
its Dpr partners (Figures 4A–4H) (
Xu et al., 2018
). When wild-type Dpr10 was expressed
in M10, Dm4 cell death was partially rescued (see below), and most of the Dm4 terminals
were in the M10 layer, leaving few terminals in M3 (Figures 6C–C”). We counted Dm4
processes leaving M3 and targeting to M10 (Figures 6C–6F, yellow arrows) and divided that
number by the total number of Dm4 cell bodies in the same sample, which we defined as the
percentage of mistargeted Dm4 neurons. It sometimes exceeded 100%, indicating that some
Dm4 neurons send out more than one process to the M10 layer (Figure 6G). Expressing
Dpr10
−8F
in M10 caused about half as much Dm4 mistargeting as wild-type Dpr10, while
expressing Dpr10
−20F
or Dpr10
−40F
produced almost no mistargeting (Figures 6D–6H).
We also tested the three Dpr10 affinity variants’ abilities to induce Dm4 mistargeting in a
wild-type animal, in which endogenous Dpr6 and Dpr10 in M3 compete with exogenous
Dpr10 in M10. The percentage of mistargeted Dm4 neurons was reduced in this genetic
background, but the three variants’ relative ability to induce mistargeting remained the same
(Figure 6G). The gain-of-function (GOF) mistargeting phenotype has more stringent affinity
requirements than the LOF Dm12 targeting phenotype because Dpr10
−8F
(K
D
= 11.3 μM)
induces mistargeting about half as well as wild-type Dpr10, while Dpr10
−20F
(K
D
= 27.8
μM) produces no mistargeting. This places the 50% penetrance threshold for this phenotype
around 11 μM, while for the LOF phenotype it is at ~30 μM (Figure 3O).
Cell-surface avidity is a key parameter controlling circuit assembly
The targeting and trophic-support functions of DIP-
α
::Dpr10 interactions are both mediated
through contact between neuronal cell surfaces. The contacting regions can be considered
apposed two-dimensional sheets, each of which contains many DIP or Dpr molecules. The
overall affinity of the cell surfaces for each other, known as avidity, is a combination
of individual protein-protein binding affinity and protein density. It is likely that avidity
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determines whether or not the strength of interaction between the two cells is sufficient
to produce correct targeting and/or to suppress cell death. To examine this issue, we
manipulated the expression levels of DIP and Dpr affinity mutants and examined the
resulting phenotypes.
In the first set of experiments, we analyzed cell-number control in Dm4 neurons, comparing
flies carrying one versus two copies of an affinity mutant
DIP-
α
gene. Although ~6 Dm4
cells were rescued from cell death in
DIP-
α
+
2F
/DIP-
α
+
2F
flies (Figure 5I), there was no
change in the Dm4 cell number in
DIP-
α
+
2F
/DIP-
α
animals (Figure 7A). In mutants that
reduce affinity (
DIP-
α
20F
and
DIP
50F
), there were significant differences in the Dm4
number between animals with two copies of a mutant gene versus those with one mutant
copy over a null (Figure 7A). Thus, the Dm4 cell number is affected by
DIP-
α
gene
expression level, implying that avidity determines trophic-support levels. Removing a copy
of wild-type
DIP-
α
did not alter the Dm4 cell number (Figure 7A), suggesting that the
phenotype is buffered around the wild-type condition. This may be a general phenomenon
that explains why haploinsufficiency is rare in
Drosophila
and why most LOF mutations are
recessive.
To examine whether expression levels affect neuronal targeting as well, we analyzed Dm12
mistargeting phenotypes in animals bearing one or two copies of wild-type or affinity
mutant
dpr10
genes. Removing one copy of wild-type
dpr6
and one copy of wild-type
dpr10
produced no phenotypes (Figure S5A). In
dpr6
null
/dpr6
null
(null) animals (bearing
two copies of wild-type
dpr10
), there are on average ~8 Dm12 neurons that mistarget to
M8. Loss of one copy of wild-type
dpr10
in the
dpr6
null mutant background produced
a significant increase in mistargeting, with ~20 Dm12 cells mistargeting to M8 (Figure
S5A). In
dpr10
null
/dpr10
null
(null) animals (bearing two copies of wild-type
dpr6
), ~31
Dm12 neurons mistarget to M8, so mistargeting is more sensitive to the loss of Dpr10
than to the loss of Dpr6. Loss of one copy of wild-type
dpr6
in the
dpr10
null mutant
background did not further increase mistargeting (Figure S5A). For affinity mutants,
dpr6
null mutant animals with one copy of
dpr10
8F
had stronger phenotypes than those with
two copies (22 versus 14 mistargeted axons, respectively) (Figure S5A). However, there
were no copy-number effects for
dpr10
20F
or
dpr10
40F
. In Figure 3M, we showed that
there is a large (~2-fold) change in phenotypic penetrance between
dpr10
8F
/dpr10
8F
and
dpr10
20F
/dpr10
20F
, so mistargeting is likely sensitive to avidity alteration within
that affinity range. However, Dpr10
−20F
is at the 50% penetrance affinity threshold, and
Dpr10
−40F
is below that threshold, so copy-number changes for these alleles may have little
effect. In conclusion, for both Dm4 cell-number control and Dm12 targeting, 2-fold changes
in gene copy number can have strong effects on the phenotype in certain mutants, suggesting
that the avidity of the interactions between the L3 and Dm4/Dm12 cell surfaces determines
the functional consequences of these interactions.
Overexpression of Dpr10 mutants can compensate for reduced protein binding affinity
The results described above indicate that
dpr10
LOF phenotypes are affected both by
altering binding affinity and by changing avidity through alteration of gene expression
levels through copy-number changes. To further examine this issue, we asked whether
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overexpressing Dpr10 affinity mutants could compensate for a reduction in individual
protein-protein binding affinity.
dpr10
20F
and
dpr10
40F
mutants both showed significant
Dm4 cell loss. We overexpressed these mutant Dpr10 proteins in Dm4’s synaptic partner,
the L3 neuron, and analyzed rescue of Dm4 cell loss. We had previously shown that
overexpressing wild-type Dpr10 in L3 fully rescues Dm4 cell loss in
dpr6
null
dpr10
null
mutants (
Xu et al., 2018
). When we overexpressed Dpr10
−20F
or Dpr10
−40F
in L3, they fully
rescued Dm4 cell loss in
dpr6
null
dpr10
20F
or
dpr6
null
dpr10
40F
, respectively, and were
also able to rescue the
dpr6
null
dpr10
null
phenotype (Figures 7B and S5B). These results
show that increasing protein amounts can indeed compensate for reductions in individual
protein-protein binding affinity (Figure 7C). Interestingly, they also indicate that our affinity
mutants would have been classified as fully functional using conventional Gal4 rescue.
Finally, to test whether these results are specific to the Dm4 synaptic partner L3, we
overexpressed different UAS-Dpr10 variants in T4 neurons, which project to M10. When
wild-type Dpr10 was expressed in T4 cells, it was able to rescue cell loss due to the
dpr6 dpr10
double mutation but not fully to the wild-type number (34 versus 40) (
Xu et
al., 2018
). Dpr10
−20F
or Dpr10
−40F
were able to rescue the Dm4 cell number to similar
extents as wild-type Dpr10 (Figure S5C). The fact that rescue of Dm4 neuronal survival by
T4-Gal4 is not complete even for wild-type Dpr10 could be due to the fact that the T4-Dm4
interaction is transient. Alternatively, there may be other proteins on the L3 surface that
contribute to cell survival that are missing from T4 neurons. Note also that induction of
Dm4 mistargeting by expression of Dpr10 in T4 cells has stringent affinity requirements
(Dpr10
−20F
cannot induce mistargeting) (Figure 6H), while T4>Dpr10
−20F
works as well as
wild type to rescue Dm4 cell death. This is likely because, in order to induce mistargeting,
T4>Dpr10 must override the other cues that cause Dm4 to arborize in M3. By contrast,
to rescue cell death, it is only necessary to supply enough Dpr10 signaling to replace that
provided by the normal interaction between L3 and Dm4, and this can be done by increasing
the expression levels of low-affinity mutants.
DISCUSSION
In this paper, we systematically explore the impact of CAM affinity and avidity on
synaptic connectivity in the
Drosophila
brain, focusing on the Dm4 and Dm12 medulla
neurons, which are postsynaptic to the L3 lamina neuron in medulla layer M3. Dm4 and
Dm12 express DIP-
α
, and L3 expresses its binding partners Dpr10 and Dpr6. The loss of
interactions between DIP-
α
and its Dpr partners causes death of Dm4 and Dm12 neurons
and mistargeting of Dm12 processes from layers M3 to M8 (
Xu et al., 2018
). To examine
how alterations in DIP-
α
::Dpr10 binding affinity affect targeting and cell survival, we
introduced designed affinity mutations into the endogenous
DIP-
α
and
dpr10
loci in the
background of a
dpr6
null mutation so that the mutant proteins would be expressed at
endogenous levels. We made fly lines expressing DIP-
α
mutants that bound to Dpr10 with
~20- and ~50-fold decreases in affinity relative to wild type, as well as a mutant with a
~2-fold increase in affinity. Dpr10 lines expressed mutants that bound to DIP-
α
with ~8-,
~20-, and ~40-fold decreases and a ~10-fold increase in affinity (Figures 1 and 2).
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