Neuron, Volume 69

Supplemental Information

Postsynaptic TrkC and Presynaptic PTP

Function as a Bidirectional Excitatory

Synaptic Organizing Complex

Hideto Takahashi, Pamela Arstikaitis, Tuhina Prasad, Thomas E. Bartlett, Yu Tian Wang, Timothy H. Murphy, and Ann Marie Craig

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Supplemental Figures and Legends

Supplemental Figure S1. Isolation of Non-catalytic TrkC as a Novel Synaptogenic Protein

from an Unbiased Expression Screen and Similar or Higher Surface Expression Levels of

Non-Synaptogenic Constructs Compared to TrkCTK-, Related to Figure 1

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(A) Cocultures of primary neurons with COS cells expressing candidate genes such as

neuroligins have been used to test ability to trigger presynaptic differentiation in contacting axons.

We recently generated a custom full-length size-selected cDNA library from P11 rat brain and

developed an unbiased screen for synaptogenic factors (Linhoff et al., 2009). cDNA pools are

expressed in COS cells, grown together with hippocampal neurons for 24 hr, and cocultures are

screened for ability to induce clusters of synapsin presynaptic marker in contacting axons,

lacking immunoreactivity for postsynaptic markers PSD-95 and gephyrin to distinguish these

induced hemi-synapses from endogenous bona fide synapses. From a screen of >105 cDNAs in

pools of ~250, three positive pools that were PCR negative for known synaptogenic factors were

isolated. Here, we subdivided pool PB270 into smaller pools and rescreened, isolating positive

sub-pool PB270-46 containing ~50 clones. This sub-pool was arrayed in two 384-well plates of

individual clones and these were screened in pooled then individual format to isolate two single

active clones. Each active clone, PB270-46-2-3H and PB270-46-17-9M, encodes a non-catalytic

form of the neurotrophin receptor tyrosine kinase TrkC.

(B) Representative images of neuron-COS cell cocultures transfected with the indicated positive

cDNA pool, sub-pool, and isolated clone and immunolabeled for synapsin, PSD-95, gephyrin and

DAPI. The “C” in the phase contrast images indicates COS cells that induce significant synapsin

clustering.

(C) Measurement of the sensitivity of the antibodies that recognize the extracellular part of TrkC,

TrkA or p75NTR. We performed surface immunolabeling of two coverslips of COS cells

expressing TrkCTK- -CFP, TrkA-CFP and p75NTR-CFP, fixed them and performed

immunolabeling of one of two coverslips after permeabilization with PBST (PBS + 0.2% Triton

X-100) to immunolabel total CFP-tagged proteins using anti-TrkC, anti-TrkA and anti-p75NTR

antibody, respectively. The ratio of the average intensity of total immunolabeling signal to CFP

signal for each antibody was calculated as its antibody sensitivity.

(D) Representative images of surface immunolabeled COS cells expressing the indicated CFP-

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tagged proteins on another coverslip.

(E) To compare the surface protein expression level of CFP-tagged proteins labeled by different

kinds of antibodies, we calculated the ratio of the average intensity of surface immunolabeling

signal to antibody sensitivity. TrkA-CFP, and TrkCTK- Ig1-CFP have a similar surface

expression level as TrkCTK- -CFP. P75NTR-CFP has higher surface expression level than

TrkCTK- -CFP. These data indicate that the lack of synaptogenic activities of TrkA, p75NTR and

TrkCIg1 are not due to insufficient surface expression.

(F) Coculture images of COS cells expressing the indicated extracellular HA-tagged proteins

labeled by surface HA, synapsin and Tau. Although each COS cell has significant surface HA

signal, only HA-TrkCTK- induces synapsin clustering on COS cells.

(G-H) Quantification of the average intensity of surface HA (G) and total integrated intensity of

synapsin divided by the axon contact area (H) for the indicated HA-tagged protein-expressing

COS cells. Although all constructs generate similar surface expression, only HA-TrkCTK- has

synaptogenic activity.

Scale bars: 20 m. All error bars are SEM.

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Supplemental Figure S2. TrkC or PTP Do Not Show High Affinity Homophilic Binding,

Related to Figure 2

(A) List of candidate molecules expressed in COS cells that were screened for binding of soluble

TrkC-Fc protein. Only PTP-CFP bound TrkC-Fc.

(B-C) Binding assay of neurotrophin receptors expressed in COS cells with soluble TrkC

ectodomain containing NT-3 binding-dead mutations (N366AT369A) fused to human Ig Fc

fragment, TrkCN366AT369A-Fc. Like TrkC-Fc, TrkCN366AT369A-Fc also binds to PTP,

indicating that NT-3 binding is not important for TrkC-PTP binding. On the other hand,

TrkCN366AT369A-Fc did not bind to either TrkCTK-, TrkCTK+ or any other type of

neurotrophin receptor tested. ANOVA p<0.0001, n>20 cells each; *p<0.01 compared with N-

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cadherin by Dunnett's test.

(D-E) Binding assay of type IIa receptor tyrosine phosphatases expressed in COS cells with

soluble PTP-Fc. PTP-Fc did not bind to either PTP, PTP or LAR, nor to negative control

membrane-associated CFP (mCFP) whereas PTP-Fc bound to positive control TrkCTK-.

ANOVA p<0.0001, n>20 cells each; *p<0.01 compared with mCFP by Dunnett's test.

Scale bars: 20 m. All error bars are SEM.

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Supplemental Figure S3. Overexpression of TrkC in Cultured Neurons Increases

Excitatory but Not Inhibitory Presynaptic Inputs, Related to Figure 3

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(A-C) Cultured hippocampal neurons were transfected with ECFP alone (A), HA-TrkCTK- and

ECFP (B), or HA-TrkCTK+ and ECFP (C) at DIV 9-10 and immunostained for synapsin at DIV

14-15.

(D-F) Cultured hippocampal neurons were transfected with ECFP alone (D), HA-TrkCTK- and

ECFP (E), or HA-TrkCTK+ and ECFP (F) at DIV 9-10 and immunostained for VGLUT1 at DIV

14-15.

(G-I) Cultured hippocampal neurons were transfected with ECFP alone (G), HA-TrkCTK- and

ECFP (H), or HA-TrkCTK+ and ECFP (I) at DIV 9-10 and immunostained for VGAT at DIV 14-

15.

(J) Left: High magnification image showing dendrites co-expressing HA-TrkCTK- plus ECFP

and dendrites of the neighboring untransfected neurons. Right panel shows synapsin

immunolabeling of the same region as the left panel. Synapsin signals along HA-TrkCTK-

expressing dendrites are significantly stronger than those along the untransfected neighboring

dendrites.

(K-M) Quantification of average integrated cluster intensity of synapsin (K), VGLUT1 (L) and

VGAT (M) along the dendrites of transfected neurons with the indicated constructs and the

neighboring untransfected neurons. ANOVA p<0.0001 for synapsin and VGLUT1, p>0.1 for

VGAT, n≥30 neurons each; *p<0.01 compared with ECFP-transfected neurons by Dunnett's test.

Scale bars: 20 m. All error bars are SEM.

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Supplemental Figure S4. TrkC-Fc-Coated Beads Induce Excitatory Presynaptic

Differentiation in Contacting Axons; PTP-CFP Expressed in COS Cells Induces PSD-95

Clustering in Contacting Dendrites, Related to Figure 4

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(A) We plated the inert beads coated with human immunoglobulin Fc fragment (Fc), TrkB

ectodomain fused to Fc (TrkB-Fc), or TrkC ectodomain fused to Fc (TrkC-Fc) onto hippocampal

neurons at DIV 8 and immunolabeled with synapsin and tau at DIV9. TrkC-Fc, but not Fc or

TrkB-Fc, induces clustering of synapsin in contacting axons.

Scale bars: 10m, 5m in inset.

(B) TrkC-Fc induces clustering of VGLUT1 excitatory presynaptic marker but not VGAT

inhibitory presynaptic marker in contacting axons. TrkB and Fc control were inactive. Scale bars:

10m, 5m in inset.

(C-F) Quantification of the average intensity of synapsin (C), Bassoon (D), VGLUT1 (E), and

VGAT (F) around beads coated with the indicated Fc-fusion proteins and in apparent contact with

axons. ANOVA p<0.0001 for synapsin, VGLUT1, and Bassoon, p>0.1 for VGAT, n≥45 beads

each; *p<0.01 compared with Fc by Dunnett's test. All error bars are SEM.

(G) COS cells expressing PTP-CFP or membrane-bound CFP (mCFP) were cocultured with

hippocampal neurons at DIV14 and immunostained for PSD-95, gephyrin, synapsin and MAP2 at

DIV15. COS cells expressing PTP-CFP induced the formation of PSD-95 clusters not apposed

to synapsin (arrows) in contacting dendrites (Upper middle panel). PTP-CFP-expressing COS

celld did not induce gephyrin clustering (Upper right panel). COS expressing negative control

mCFP did not induce either PSD-95 or gephyrin clustering (Lower panels). The synaptogenic

activity of PTP-CFP-expressing COS cells to induce PSD-95 clustering was lower than that of

PTP-Fc-coated beads, so we used PTP-Fc-coated beads for quantitative analysis (Figures 4F-

4I and 6D-E)Scale bar: 20 m.

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Supplemental Figure S5. The Efficiency and Specificity of sh-RNA Constructs for TrkC

Knockdown in Culture, Related to Figure 7

(A) Knockdown of TrkC in heterologous cells by shRNA. HEK cells were co-transfected with the

indicated shRNA vectors plus the indicated CFP-tagged Trk constructs for 24 hours. We used

pLL(CMV)mcherry as the shRNA vector, driving shRNA from the U6 promoter and mcherry

from the CMV promoter. TrkCTK-*-CFP bears silent mutations rendering it RNAi-resistant

against both sh-TrkC#1 and sh-TrkC#2.

(B) Quantification of knockdown efficiency and specificity of shRNA. sh-TrkC#1 and sh-TrkC#2

significantly knock down the expression of TrkCTK-/TK+-CFP and have no effects on the

expression of TrkBTK-/TK+-CFP and TrkCTK-*-CFP. ANOVA p<0.0001, n>20 images each

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(approximately 20-30 transfected cells in a single image); *p<0.01 compared with sh-vec or sh-

con by Dunnett's test.

(C) Knockdown of TrkC in cultured hippocampal neurons by shRNA. Cultured hippocampal

neurons at 9-10 DIV were transfected with the indicated shRNA vectors or co-transfected with

sh-TrkC#1 or sh-TrkC#2 plus non-tagged TrkCTK-* (plasmid ratio: 4:1), and immunostained for

TrkC. Transfection with sh-TrkC#1 or sh-TrkC#2 significantly reduces TrkC

immunofluorescence on dendrites. Co-transfection of TrkCTK-* with sh-TrkC#1 or #2 rescued

the TrkC immunosignal on dendrites.

(D) Quantification of knockdown efficiency of shRNA and rescue efficiency of TrkCTK-* in

hippocampal neurons. sh-TrkC#1 and sh-TrkC#2 significantly knock down the neuronal

expression of TrkC to around 35% of the endogenous expression level. Co-transfection of

TrkCTK-* rescues total TrkC expression to around 120%-150% of the endogenous expression

level with a larger variation. ANOVA p<0.0001, n>20 cells each; *p<0.01 compared with sh-con

by Dunnett's test.

Scale bars: 20 m. All error bars are SEM.

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Supplemental Figure S6. Effectiveness of In Vivo Knockdown of TrkC Based on In Utero

Electroporation, Related to Figure 8

(A) Confocal images of coronal brain slices showing GFP-positive P32 neurons transfected with

the indicated constructs by in utero electroporation at E15.5-16 in layer II/III of cingulate cortex

area 1 and 2 (Cg1 and Cg2) positioned at Bregma 0.0±0.2 mm. In each transfection condition,

many GFP-positive neurons were located in layer II/III of cortex, and some of their dendrites

reached into layer I. Scale bar: 100 m

(B) Knockdown of TrkC in vivo by shRNA. Transfection with sh-TrkC#1 significantly reduced

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TrkC immunofluorescence around the nucleus labeled by DAPI compared to the neighboring

untransfected neurons. Co-transfection of TrkCTK-* with sh-TrkC#1 rescued the TrkC

immunofluorescence around the nucleus. The “T” and “U” in the images indicate the transfected

neurons and the untransfected neighboring neurons, respectively. Scale bar: 20 m.

(C) Quantification of knockdown efficiency of shRNA and rescue efficiency of TrkCTK-* in

cortical layer II/III neurons in vivo. sh-TrkC#1 significantly knocks down TrkC expression to

around 35% of the endogenous expression level of the neighboring untransfected neurons. Co-

transfection of TrkCTK-* rescues total TrkC expression to around 120% of the endogenous

expression level of the neighboring untransfected neurons with a larger variation. ANOVA

p<0.0001, n>30 cells from two mice each; *p<0.01 and #p<0.05 compared with sh-con by

Dunnett's test.

All error bars are SEM.

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Supplemental Experimental Procedures

DNA constructs

Constructs for full-length mouse PTP that possesses four fibronectin III-like domains

(FNIII) (BC052462), rat PTP that possesses eight FNIII domains (BC105753), and human

PTP (BC106714) were purchased from Open Biosystems. For cloning C-terminal CFP fusion

proteins, full-length cDNAs lacking stop codons were subcloned into pECFP-N1 (Clontech). The

following deletion constructs and point mutations for TrkC-CFP were made by inverse PCR and

DpnI digestion: ICD (amino acids (aa) 493-612 deleted), ECD (aa 33-383 deleted), LRRCC

(aa 33-208 deleted), Ig1 (aa 227-288 deleted), Ig2 (aa 319-382 deleted), LRRNT (aa 33-63

deleted) and N366AT369A. SWAP chimaeras TrkCECD/TrkB, TrkC LRRCC+Ig1/TrkB and

TrkC LRRCC/TrkB were made by fusing aa 1-428 of TrkC to aa 405-476 of TrkB, aa 1-318 of

TrkC to aa 300-476 of TrkB and aa 1-208 of TrkC to aa 196-476 of TrkB, respectively. The

following deletion constructs and splice variants were made for mouse PTP-CFP: Ig1-3 (aa

42-306 deleted), FNⅢ1-4 (aa 310-690 deleted), meA+, meB+ and meA+meB+.

For expressing extracellular YFP-tagged Trks and PTP, cDNA encoding the mature form

of each transmembrane protein was subcloned into spYFP-C1, a vector that expresses YFP with

an N-terminal signal sequence derived from the NMDA receptor NR2B cDNA. For expressing

extracellular HA-tagged proteins, cDNA encoding the mature form of each protein was subcloned

into spHA-C1, a vector that expresses HA with an N-terminal signal sequence derived from TrkC

cDNA. spYFP-C1 and spHA-C1 both express from the CMV promoter. For constructs YFP-

PTPICD and HA-PTPICD, the mature form of mouse PTP lacking aa 945-1501 was

subcloned into spYFP-C1 and spHA-C1, respectively.

Amigo-CFP, N-cadherin-CFP, neuroligin 2-CFP (NLG2-CFP), neurexin1�(-S4)-CFP

(Nrx1-CFP), Nrx1β(+S4)-CFP, Nrx1(-S4)-CFP, Nrx1(+S4-CFP) and membrane bound CFP

(mCFP) were previously described (Graf et al., 2004; Linhoff et al., 2009; Siddiqui et al., 2010).

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The other constructs used in the candidate screen for the TrkC-Fc binding partner (Figure S2A)

were also previously described (Siddiqui et al., 2010). The following plasmids were kind gifts:

LAR-CFP from Dr. Eunjoon Kim (Korea Advanced Institute of Science and Technology, Korea),

Nrx1-Fc from Dr. Peter Scheiffele (University of Basel, Switzerland), pLentiLox3.7 vectors

from Dr. Alaa El-Husseini (University of British Columbia), pCAG-EGFP (Addgene plasmid

11150) from Dr. Connie Cepko (Harvard Medical School, Boston, MA), contactin-GFP from Dr.

Stephen G. Waxman (Yale University, New Haven, CT), neural cell adhesion molecule (NCAM)-

140-GFP and NCAM-180-GFP from Dr. Brigitte Schmitz (University of Bonn, Bonn, Germany),

CD166-CFP from Dr. A. M. Carmo (Universidade do Porto, Portugal), CD81-GFP from Dr.

Francisco Sánchez-Madrid (Universidad Autónoma de Madrid, Madrid, Spain), NgCAM-YFP

from Dr. Peter Sonderegger (University of Zurich, Zurich, Switzerland), CASPR1-HA and

CASPR2-HA from Dr. Elior Peles (Weizmann Institute of Science, Rehovot, Israel) and CD4-

GFP from Dr. Chen Gu (Ohio State University). For the construct of extracellular YFP-tagged

CD4 (YFP-CD4), the mature form of CD4 was subcloned into spYFP-C1 vector.

For plasmid-based RNA inhibition of all isoforms of TrkC, the complementary

oligonucleotides encoding inverted repeats that target nucleotides 113–131 of rat and mouse TrkC

(GCAGCAAGACTGAGATCAA) for sh-TrkC#1 and nucleotides 144–162 of rat TrkC

(GGACGATGGGAACCTCTTT) for sh-TrkC#2 were annealed. Annealed oligonucleotides were

ligated into the HpaI/XhoI sites of the U6 promoter-driven short hairpin RNA expression vector

LenLox3.7. Variants pLL(CMV)mcherry and pLL(syn)CFP were used, expressing mcherry under

the CMV promoter or ECFP under the synapsin promoter, respectively. As a control shRNA, we

used shMORB that mediates knockdown of MORF4L1 involved in chromatin regulation but has

no effects on neuronal morphology including spine density (Alvarez et al., 2006), and does not

induce any interferon response (Bridge et al., 2003). The construct for expressing TrkCTK-*

resistant against both sh-TrkC#1 and sh-TrkC#2 was generated by making the following five

point mutations, indicated by underlines, in each of the shRNA-targeting sites:

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GCAGTAAAACGGAAATTAA for sh-TrkC#1 resistance and GGATGACGGAAATCTGTTT

for sh-TrkC#2 resistance.

For in vivo RNAi experiments, we first ligated the annealed oligonucleotides for sh-

TrkC#1 or sh-con (shMORB) into the HpaI/XhoI sites of a LentiLox3.7 variant,

pLL(CMV)EGFP vector expressing EGFP under the CMV promoter. We then replaced the CMV

promoter with a CAG promoter (chicken β-actin promoter and cytomegalovirus immediate-early

enhancer) derived from the pCAG-EGFP vector. For the expression vector for TrkCTK-* under

the CAG promoter (pCAG-TrkCTK-*), TrkCTK-* was subcloned into the pCAG-EGFP vector

by replacing the EGFP coding region. For rescue electroporation, a plasmid mixture of sh-

TrkC#1 and TrkCTK-* was prepared at a ratio of 4:1.

All constructs were verified by DNA sequencing.

Cell Culture and Transfection

Hippocampal neurons from E18 rat embryos were cultured at low density on poly-L-lysine

coated glass coverslips inverted over a feeder layer of astrocytes (Goslin et al., 1998; Kaech and

Banker, 2006). For all experiments described here, the serum-free media was supplemented with

100 µM APV (Research Biochemicals) starting at 7 DIV to limit excitotoxicity. Hippocampal

neurons were transfected at 14 DIV for YFP-TrkC localization analysis, or at 8-10 DIV for other

experiments, using the ProFection Mammalian Transfection System (Promega). Analysis of

localization of endogenous TrkC and PTP (Figure 3) was performed at 15 DIV.

COS-7 and HEK293T cells were cultured in DMEM-H supplemented with 10% fetal

bovine serum (FBS).

Isolation of TrkC from the Coculture Screen

Pool PB270 able to induce presynaptic differentiation in the coculture screen (Figure S1)

was PCR negative for known synaptogenic proteins (data not shown). Thus, a glycerol stock was

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used to prepare subpools of ~50 cDNAs each. The glycerol stock corresponding to the most

active subpool in coculture, PB270-46, was arrayed by the Michael Smith Genome Sciences

Centre. Two 384 well plates of individual colonies and plasmid DNA were prepared. cDNA from

sets of 32 wells was pooled, transfected, and screened. Positive sets of pooled cDNAs were then

tested individually and the two active isolated clones sequenced.

Antibodies, Immunocytochemistry and Culture Imaging

Rabbit monoclonal anti-TrkC (1:500; clone C44H5; Cell Signaling) and the mouse

monoclonal anti-PTP (1:500; clone 17G7.2; MediMabs) were used. The following additional

polyclonal antibodies were used: rabbit anti-synapsin I (1:2000; Millipore; AB1543P), rabbit

anti-VGLUT1 (1:1000; Synaptic Systems; 135 302), gunia pig anti-VGLUT1 (1:2000; Millipore;

AB5905), rabbit anti-VGAT (1:2000; Synaptic Systems; 131 003). The following mouse

monoclonal antibodies were used: anti-synapsin (IgG1; 1:1000; clone 46.1, Synaptic Systems), ,

anti-VGLUT1 (IgG1; 1:1000; clone 317G6, Synaptic Systems), anti-VGAT (IgG3; clone 117G4,

Synaptic Systems), anti-PSD-95 family (IgG2a; 1:500; clone 6G6-1C9; Thermo Scientific;

recognizes PSD-95, PSD-93, SAP102 and SAP97), anti-gephyrin (IgG1; 1:1000; mAb7a;

Synaptic Systems), anti-Tau-1 (IgG2a; 1:2000; clone PC1C6; Millipore; recognizes

dephosphorylated tau), anti-bassoon (IgG2a; 1:1000; Stressgen; VAM-PS003), anti-NMDAR1,

(IgG; 1:1000; Millipore; 05-4328), anti-HA (IgG2b; clone 12CA5; Roche). For labeling dendrites,

we used anti-MAP2 (chicken polyclonal IgY;1:8000; Abcam; ab5392).

For most of the experiments, we used highly cross-adsorbed, Alexa-dye conjugated

secondary antibodies generated in goat towards the appropriate species and monoclonal isotype

(1:500; Invitrogen; we used exclusively Alexa-488, Alexa-568, and Alexa-647 labeled secondary

antibodies for these experiments). For the coculture screen, Texas Red conjugated goat anti-

mouse (1:500; Invitrogen) was used to detect gephyrin and PSD-95 in the same channel. AMCA

conjugated anti-chicken IgY (donkey IgG; 1:200; Jackson ImmunoResearch; 703-155-155) was

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used for visualizing dendrites.

Cultures were fixed either with parafix solution (4% paraformaldehyde and 4% sucrose in

PBS (pH 7.4)) for 12 min followed by permeabilization with PBST (PBS + 0.2% Triton X-100)

or with -20°C methanol for 10 min. They were incubated with blocking solution (PBS + 3% BSA

and 5% normal goat serum) for 30 min at 37°C, then with primary antibodies in blocking solution

(overnight, 20°C) and secondary antibodies (45 min, 37°C). Coverslips were mounted in elvanol

(Tris-HCl, glycerol, and polyvinyl alcohol with 2% 1,4-diazabi-cyclo[2,2,2]octane).

For the synaptotagmin I antibody uptake assay, neurons were incubated live with

antibodies to the synaptotagmin luminal domain (IgG1; clone 604.2; Synaptic Systems) at the

dilution of 1:200 for 30 min in culture media at 37°C in a 5% CO2 incubator.

For surface labeling of TrkA-CFP, TrkCTK--CFP, TrkCTK-ICR-CFP and p75NTR-CFP,

the following antibodies that recognize the ectodomain of each protein were added into culture

media at 1:500 and incubated for 30 min on ice in a 5% CO2 incubator: rabbit polyclonal anti-

TrkA (Millipore), rabbit monoclonal anti-TrkC (C44H5; Cell Signaling) and mouse monoclonal

anti-p75NTR (MC-192; Santa Cruz Biotechnology). For surface labeling of extacellular HA-

tagged proteins, mouse monoclonal anti-HA (IgG2b; clone 12CA5; Roche) was added into

culture media at 1:500 and incubated for 30 min on ice in a 5% CO2 incubator. After antibody

incubation, coverslips were washed in PBS three times, fixed with parafix solution, incubated

with blocking solution, and immuno-labeled with appropriate secondary antibodies without

permeabilization.

Images were acquired on a Zeiss Axioplan2 microscope with a 40x 1.30 NA oil objective

or a 63X 1.4 NA oil objective and Photometrics Sensys cooled CCD camera using Metamorph

imaging software (Molecular Devices) and customized filter sets. Controls lacking specific

antibodies confirmed no detectable bleed-through between channels AMCA, CFP, YFP or Alexa

488 (imaged through a YFP filter set), Alexa 568, and Alexa 647. Images were acquired as 12 bit

grayscale and prepared for presentation using Adobe Photoshop. For quantification, sets of cells

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were stained simultaneously and imaged with identical settings.

Production of Soluble Fc-fusion Proteins

Soluble human IgG Fc (Fc), TrkC ectodomain fused to Fc (TrkC-Fc) and TrkB ectodomain

fused to Fc (TrkB-Fc) were purchased from R&D Systems. Additional Fc fusion proteins were

custom generated. To make a stable cell line for the effective production of Fc-fusion proteins, we

first made the cloning vector, pc4-sp-Fc, pcDNA4 with the following sequences inserted between

HindIII and XhoI: neurexin1 signal sequence followed by� a multiple cloning site (EcoRV-

EcoRI-NotI) and next the human IgG Fc cDNA containing a stop codon. The pc4-sp-Fc itself can

express soluble Fc proteins. For constructs of PTP-Fc (pc4-sp-PTP-Fc) and

TrkCN366AT369A-Fc (pc4-spTrkCN366AT369A-Fc), the PTP ectodomain lacking its signal

sequence and the ectodomain of TrkCTK-N366AT369A-CFP lacking its signal sequence were

subcloned into pc4-sp-Fc, respectively.

For production of soluble PTP-Fc, TrkCN366AT369A-Fc and Fc as the negative control

protein, HEK-293 cells were transfected with FuGENE 6 (Roche) with pc4-sp-PTP-Fc, pc4-

spTrkCN366AT369A-Fc and pc4-sp-Fc, respectively, and cultured in DMEM medium containing

10% FBS and 0.5 mg/ml Zeocin (Invitrogen). After a 14 day-long selection with Zeocin, medium

was replaced with serum-free AIM V synthetic medium (Invitrogen). The conditioned medium

was collected every 2-3 days for two weeks, and frozen at -80°C. For purification, about 500 ml

of conditioned medium was concentrated using Centricon Plus-70 ultrafiltration units (30 kDa

cutoff; Millipore). Concentrated proteins were purified using 500 l protein G–Sepharose (GE

Healthcare) and eluted with 5 ml glycine 0.1M pH 2.7. Fractions of 500 l were collected into

tubes containing 37.5 l Tris 1M, pH 9.0, to neutralize. Glycine was removed using Spectra/Por6

dialysis membrane (10 kDa cutoff; Spectrum Laboratories). Purified recombinant proteins were

confirmed by performing SDS-PAGE and staining the gel in Sypro Ruby gel stain (Invitrogen).

The protein concentration was measured by DC protein Assay (BioRad).

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TrkC Neutralizing Antibody Experiments

The rabbit C44H5 monoclonal antibody for TrkC (Cell Signaling, custom produced free of

azide and glycerol), which recognizes the LRR region of the ectodomain of TrkC (company

datasheet and confirmed experimentally, data not shown), was used for all TrkC neutralizing

antibody experiments. For quantification of neutralizing effects of TrkC antibody on TrkC-PTP

binding, 20 nM soluble TrkC-Fc protein in ECS/BSA was incubated with serial concentrations

(0-100 g/ml) of TrkC antibody or negative control rabbit polyclonal anti-TrkA antibody

(Millipore) for 1 hr at room temperature. COS cells expressing PTP-CFP were then incubated

with the TrkC-Fc antibody mixture for 1 hour at room temperature in a binding assay. Cells were

then fixed and biotin-conjugated human IgG (H+L) and Alexa568-conjugated streptavidin were

used to visualize bound TrkC-Fc. GraphPad Prism was used for calculating the neutralizing dose

50% (ND50). To investigate the neutralizing effects on synaptogenic activities of TrkC and

PTPwe applied ~10 g/ml TrkC antibody to the culture media one hour after COS cells

expressing TrkCTK- -CFP or PTP-Fc-coated beads had attached to the neurons, respectively.

Non-immunized rabbit IgG (~10 g/ml) was used as the negative control for TrkC antibody. To

investigate the effects of TrkC neutralizing antibody on endogenous synapse formation, ~10

g/ml TrkC antibody or non-immunized rabbit IgG was added into culture media on each of DIV

9, 10, 11 and 12. The treated neuronal cultures were fixed by parafix solution at DIV 12.5.

Supplemental References

Alvarez, V. A., Ridenour, D. A., and Sabatini, B. L. (2006). Retraction of synapses and dendritic

spines induced by off-target effects of RNA interference. J Neurosci 26, 7820-7825.

Bridge, A. J., Pebernard, S., Ducraux, A., Nicoulaz, A. L., and Iggo, R. (2003). Induction of an

interferon response by RNAi vectors in mammalian cells. Nat Genet 34, 263-264.

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Goslin, K., Asmussen, H., and Banker, G. (1998). Rat hippocampal neurons in low-density

culture. In: Banker G, Goslin K, editors. In Culturing Nerve Cells. (Cambridge: MIT Press),

pp. 339-370.

Kaech, S., and Banker, G. (2006). Culturing hippocampal neurons. Nat Protoc 1, 2406-2415.

Siddiqui, T. J., Pancaroglu, R., Kang, Y., Rooyakkers, A., and Craig, A. M. (2010). LRRTMs and

neuroligins bind neurexins with a differential code to cooperate in glutamate synapse

development. J Neurosci 30, 7495-7506.