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Neuron
Supplemental Information
Anterograde C1ql1 Signaling Is Required in Order
to Determine and Maintain a Single-Winner
Climbing Fiber in the Mouse Cerebellum
Wataru Kakegawa, Nikolaos Mitakidis, Eriko Miura, Manabu Abe, Keiko Matsuda, Yukari
H. Takeo, Kazuhisa Kohda, Junko Motohashi, Akiyo Takahashi, Soichi Nagao, Shin-ichi
Muramatsu, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, and Michisuke
Yuzaki
SUPPLEMENTAL FIGURES
Figure S1 (Related to Figure 1). Normal Gross Anatomy and Immunoreactivities of
vGluT1 and vGAT in C1ql1-Null Cerebella
(A) Schematic representation of the C1ql1 genome. WT (C1ql1+), targeted genome before
(C1ql1GFP) and after FLP-mediated recombination (C1ql1GFP(Neo)) are shown.
(B) Southern blot analysis of genomic DNA from ES clones. Positions of DNA size
markers (kb) are indicated to the left.
(C) Representative immunogold EM images of endogenous C1ql1 (red arrows) in adult
WT (left; taken from Figure 1B) and C1ql1-null (right) mouse cerebella. C1ql1 and
vGluT2 (a marker for CF terminals) were immunolabeled with 10-nm and 20-nm gold
particles, respectively. sp, Purkinje cell spine. Scale bar, 200 nm.
(D) Cresyl violet-stained sagittal sections of adult WT (left) and C1ql1-null (right) mouse
cerebella. Scale bar, 1 mm.
(E and F) IHC images of vGluT1 (E) and vGAT (F) in WT (top) and C1ql1-null (bottom)
cerebella from at P14 (E, F) and adult (E) mice. Dotted lines and asterisks represent pial
surface of the molecular layer and Purkinje cell soma, respectively. Scale bar, 20 m.
Figure S2 (Related to Figure 1). Normal Voltage-Dependent Ca2+ Channel Responses
between WT and C1ql1-Null Purkinje Cells at P5
(A) Representative traces of Ca2+ currents evoked by the 100-ms depolarization from Vh
= 60 mV to +40 mV every 10-mV steps in P5 Purkinje cells.
(B) IV curve of Ca2+ currents. Peak amplitudes are plotted against the voltage steps.
Data are represented as mean  SEM.
Figure S3 (Related to Figure 3). Normal Gross Anatomy and Immunoreactivities of
vGluT1 and vGAT in PC-Bai3-Null Cerebella
(A) Schematic representation of the Bai3 genome. WT (Bai3+), targeted genomes
(Bai3flox), and knockout genomes (Bai3) after a Purkinje cell-specific Cre-mediated
recombination by crossing with the pcp2-Cre transgenic mice are shown.
(B) Southern blot analysis of genomic DNA from ES clones. Positions of DNA size
markers (kb) are indicated to the left.
(C) Representative immunogold EM images of endogenous Bai3 (red arrows) in adult
WT (left; taken from Figure 3C) and PC-Bai3-null (right) mouse cerebella. Bai3 and
vGluT2 were immunolabeled with 10-nm and 20-nm gold particles, respectively. sp,
Purkinje cell spine. Scale bar, 200 nm.
(D) Cresyl violet-stained sagittal sections of adult WT (left) and PC-Bai3-null (right)
mouse cerebella. Scale bar, 1 mm.
(E and F) IHC images of vGluT1 (E), and vGAT (F) in PC-Bai3-null (bottom) and its
control littermates (CTL, top) cerebella from P14 (E, F) and adult (E) mice. Dotted lines
and asterisks represent pial surface of the molecular layer and Purkinje cell soma,
respectively. Scale bar, 20 m.
Figure S4 (Related to Figures 3 and 4). SPR and In Vitro Binding Assay for
C1lq1Bai3
(A to E) Representative SPR data for the binding between C1ql1WT and eBai3WT (A),
C1ql1WT and eBai3CUB (B), C1ql1WT and nBai3CUB (C), gC1ql1WT and eBai3WT (D), and
C1ql1GW and eBai3WT (E) in various concentrations of eBai3. Kd values are shown in
insets.
(F) In vitro binding assay between HA-tagged C1ql1 (C1ql1WT-HA [top] or C1ql1GW-HA
[bottom]) and full-length Bai subfamily (Bai1WT, Bai2 WT or Bai3WT) or Bai3 mutant
lacking CUB domain (Bai3CUB) expressed at the surface of HEK293 cells. hBai and
mBai stand for human and mouse-derived ones, respectively. Scale bar, 50 m.
(G) Quantification of bound HA signal intensity per GFP-positive area of the cells in each
condition.
###
p < 0.001 by KruskalWallis test followed by Scheffe post-hoc test. ***p < 0.001 by
Mann–Whitney U test. Data are represented as mean  SEM.
Figure S5 (Related to Figure 3). vGluT2 Immunoreactivity in Adult PC-Bai2-Null
Cerebella
(A) Schematic representation of the Bai2 genome. WT (Bai2+), targeted genomes
(Bai2flox), and knockout genomes (Bai2) after a Purkinje cell-specific Cre-mediated
recombination by crossing with the pcp2-Cre transgenic mice, are shown.
(B) Southern blot analysis of genomic DNA from ES clones. Positions of DNA size
markers (kb) are indicated to the left.
(C) IHC images of vGluT2 in adult PC-Bai2-null (right) and its control littermate (CTL,
left) mouse cerebella. Dotted lines and asterisks represent pial surface of the molecular
layer and Purkinje cell soma, respectively. Scale bar, 20 m.
(D and E) Quantification of the density (D) and the height (E) of vGluT2 puncta on the
molecular layer of each mouse. ns, no significance by Mann–Whitney U test. Data are
represented as mean  SEM.
Figure S6 (Related to Figures 3 and 4). Detection of Exogenous C1ql1 and Bai3 in
Null-Mutant Cerebella
(A) Immunohistochemical images of virally expressed C1ql1 (C1ql1WT (top) and
C1ql1GW (bottom)) in C1ql1-null mouse CFs. Area surrounded by white squares in left
panels are magnified in the middle and right ones. GFP (green) and vGluT2 (blue) were
co-stained with C1ql1 (red). Scale bars, 20 m and 5 m.
(B) Immunohistochemical images of Bai3 (Bai3WT (top) and Bai3CUB (bottom)), which
were introduced by IUE into PC-Bai3-null mouse Purkinje cells. Area surrounded by
white squares in left panels are magnified in the middle and right ones. GFP (green) was
co-stained with Bai3 (red). Scale bars, 20 m and 3 m.
Figure S7 (Related to Figure 5). Bai3 Knockdown in Adult WT Purkinje Cells
Impairs C1ql1 Localization and CF Synapse Integrity
(A) Cartoon showing the lentivirus injection into adult WT mouse cerebellum for the
introduction of miR-Bai3 or miR-SCR. Experiments were performed at 1421 dpi.
(B and C) IHC images (B) and the quantitative data (C) showing Bai3 is knocked down
by miR-Bai3 (right) but not miR-SCR (left) in GFP-expressing Purkinje cells. Scale bar,
5 m.
(D to F) IHC images of C1ql1 around Purkinje cell dendrites introducing miR-SCR (left
in D) or miR-Bai3 (right in D), and the quantitative data of the density of C1ql1 puncta
(E) and the percentage of the number of C1ql1 colocalized on vGluT2 immunoreactivity
(F). Scale bar, 3 m.
(G) IHC images of vGluT2 in adult WT mouse cerebella with GFP-positive Purkinje cells
expressing miR-SCR (left) and miR-C1ql1 (right). vGluT2 immunoreactivities are
binarized in the bottom. Dotted lines and asterisks represent pial surface of the molecular
layer and Purkinje cell soma, respectively. Scale bar, 20 m.
(H and I) Quantification of the density (H) and the height (I) of vGluT2 puncta on the
molecular layer of each mouse.
(J) CF-EPSCs from WT Purkinje cells introducing miR-SCR, miR-Bai3, miR-Bai3 plus
a resistant form of Bai3 (rBai3WT) or miR-Bai3 plus a rBai3CUB.
(K and L) Histograms showing the percentage of the number of CFs innervating single
Purkinje cells (K) and the strongest CF-EPSC amplitudes (L) in each condition.
*p < 0.05, ***P < 0.001 by Mann–Whitney U test in (C), (E), (F), (H) and (I). *p < 0.05,
***p < 0.001 by KruskalWallis test followed by Scheffe post-hoc test in (L). ns, no
significance. Data are represented as mean  SEM.
Figure S8 (Related to Figure 6). Normal Motor Coordination and No Nystagmus in
Adult Mutant Mice
(A and B) Results of rotor-rod test (550-rpm accelerating mode) in adult WT vs.
C1ql1-null (A) and PC-Bai3-null vs. its control littermate (CTL, B) mice.
(C and D) Representative traces of eye movements in WT (C and D) and other mouse
lines (D) monitored for 15 s. Scale bar, 0.5 mm in (C).
(E) Quantification of the total distance of eye movements for 15 s in each mouse line.
*p < 0.05, **p < 0.01 by KruskalWallis test followed by Scheffe post-hoc test. Data
are represented as mean  SEM.
SUPPLEMENTAL MOVIES
Movie S1 (Related to Figure 6). Walking Patterns and Beam Test Performance of
C1ql1-Null and PC-Bai3-Null Mice
C1ql1-null mice were compared with wild-type (WT) mice and PC-Bai3-null mice were
compared with its littermate Bai3flox mice (CTL).
Movie S2 (Related to Figure 6). hOKR Tests in C1ql1-Null and PC-Bai3-Null mice
C1ql1-null mice were compared with wild-type (WT) mice and PC-Bai3-null mice were
compared with its littermate Bai3flox mice (CTL). Eye movements are played at 3×
speed.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Animals
We isolated C57BL/6 bacterial artificial chromosome (BAC) genomic clones (Advanced
GenoTechs); RP23-117E15, RP23-462I17 and RP23-347F2 containing the C1ql1, Bai2
and Bai3 genes, respectively. The Quick and Easy BAC modification Kit (Gene Bridges)
was used for vector construction. To produce C1ql1-null mice, we designed a genetargeting vector in which GFP gene was inserted immediately after the translational
initiation site of the C1ql1 in frame. The fragments containing GFP and simian virus 40
polyadenylation (SV40 poly(A)) signal sequence were amplified by polymerase chain
reaction
(PCR)
from
pEGFP-N1
(Clontech),
and
combined
with
pDTMC/FRTpgkgb2Neo (modified from pgkgb2Neo (Gene Bridges)) containing
pgk/gb2-Neo cassette flanked by two FLP recognition target (frt) sites. This construct was
used as a PCR template for the amplification of fragment composed of GFP-Neo cassette.
By using two primers C1ql1GFPKI-GFPF1 (5’-AGG CTC CGC GCC GGC CGG AAG
ACC CTG CTG GCT GCC GCC GCG GGC GTG GTG ATG GTG AGC AAG GGC
GAG G-3’) and C1ql1GFPKI-NeoR1 (5’-GGG GGC TCC CTG GGA GCC CTG CCC
CCA GGT CAG CCC CAC GCC CCC TCA TGC ATC ACT ATA GGG CTC GAG G3’), the cassette attached by 5’ and 3’ homology arms was amplified by PCR and then
recombined into the translation initiation site of C1ql1 in RP23-117E15. The
approximately 14.1 kb fragment containing the exon1 of the C1ql1 and GFP-Neo cassette
was subcloned into pMC-DT#3 vector including a MC1 promoter-driven diphtheria toxin
gene. The targeting vector contained the translational initiation site of the C1ql1 inserted
by GFP-Neo cassette, 6.5 kb upstream and 4.9 kb downstream genomic sequences, and
4.4 kb pMC-DT#3. To establish the C1ql1GFP mouse line, we introduced the linearized
targeting vector into the C57BL/6N-derived embryonic stem (ES) line, RENKA and then
selected recombinant clones under the medium containing 175 g/mL G418. Culture of
ES cells was performed as described previously (Mishina and Sakimura, 2007). The
targeted clones were confirmed by Southern blot analysis using the 5’, 3’ and Neo probes
under the following conditions; NdeI-digested DNA hybridized with 5’ probe, 17.1 kb for
WT and 19.2 kb for targeted allele; EcoT22-I-digested DNA hybridized with Neo probe,
13.3 kb for targeted allele; EcoT22-I-digested DNA hybridized with 3’ probe, 23.1 kb for
WT and 9.1 kb for targeted allele. Generation of chimeric mice was performed as
described previously (Mishina and Sakimura, 2007). Briefly, targeted clones were
microinjected into 8 cell-stage embryos of CD-1 mouse strain. Resulting chimeric
embryos were developed to the blastocyst stage by incubation for more than 24 h and
then transferred to pseudopregnant CD-1 mouse uterus. Germline chimeras were crossed
with C57BL/6N female mice and the heterozygous offspring were crossed with a FLP
deleter mouse line to establish the C1ql1GFP(Neo) mouse line. Genotyping of mice tail
DNA was determined by PCR with the following specific primers: (forward), 5’-CTT
ACC CGC CGG CAT CAT TG-3’; (reverse), 5’-TGA TCC AAT ACG CAT TCT CC-3’.
We used the homozygous C1ql1GFP(Neo) mice as C1ql1-null mice in this study.
To construct Bai2 targeting vector, a 1.19 kb DNA fragment carrying exon 58 of
the Bai2 was amplified by PCR, and inserted to the KpnI/SacI sites of middle entry clone
(pDME-1) in reverse orientation. In this clone, a DNA fragment of pgk promoter-driven
Neo-poly(A) (pgk-Neo) flanked by two frt sites and loxP sequence was located at the site
173 bp downstream of the exon 8, while the other loxP sequence was placed at the site
147 bp upstream of the exon 5. The 6.55 kb upstream and 6.71 kb downstream
homologous genomic DNA fragments were retrieved from the BAC clone, and then
subcloned to 5’ entry clone (pD3UE-2) and 3’ entry clone (pD5DE-2), respectively. For
targeting vector assembly, the three entry clones were recombined to a destination vector
plasmid (pDEST-DT; containing a cytomegalovirus enhancer/chicken  actin (CAG)
promoter-driven diphtheria toxin gene) by using MultiSite Gateway Three-fragment
Vector construction Kit (Invitrogen). Culture of ES cells and generation of chimeric mice
(Bai2flox mice) were carried out as described above. Homologous recombinant ES clones
were identified by Southern blot analysis. KpnI-digested DNA hybridized with 5’ probe,
22.8 kb for WT and 13.7 kb for targeted allele; KpnI-digested DNA hybridized with neo
probe, 12.4 kb for targeted allele; HincII-digested DNA hybridized with 3’ probe, 12.5 kb
for WT and 14.4 kb for targeted allele. Genotyping of mice tail DNA was determined by
PCR with the following specific primers: (forward), 5’-GTA TAG CTG CCA GCA GTC
AAT GG-3’; (reverse), 5’-CAT TCT AGC CAC TGG CCT TCC AC-3’. To generate PC-
Bai2-null mice, the Bai2flox mice were crossed with the pcp2-Cre transgenic mice
(Jackson Laboratory), in which the Cre gene was expressed in cerebellar Purkinje cells
under the control of the L7/pcp2 promoter (Barski et al., 2000).
To construct Bai3 targeting vector, a 0.92 kb DNA fragment carrying exon 8 and 9
of the Bai3 gene was amplified by PCR, and inserted into the SacI/KpnI sites of pDME1. In this clone, a DNA fragment of loxP sequence and the pgk-Neo cassette flanked by
two frt sites was located at the site 172 bp upstream of the exon 8, while the other loxP
sequence was placed at the site 164 bp downstream of the exon 9. The 6.32 kb upstream
and 6.16 kb downstream homologous genomic DNA fragments were retrieved from the
BAC clone, and then subcloned to pD5UE-2 and pD3DE-2, respectively. Targeting vector
assembly, culture of ES cells and generation of chimeric mice (Bai3flox mice) were carried
out as described above. Homologous recombinant ES clones were identified by Southern
blot analysis. BglI-digested DNA hybridized with 5’ probe, 19.8 kb for WT and 13.6 kb
for targeted allele; NheI-digested DNA hybridized with neo probe, 13.0 kb for targeted
allele; EcoRV-digested DNA hybridized with 3’ probe, 11.2 kb for WT and 7.7 kb for
targeted allele. Genotyping of mice tail DNA was determined by PCR with the following
specific primers: (forward), 5’-TCT CAG TCT TCA TGG AGT GG-3’; (reverse), 5’-TCT
GTG GTG CAA GGA ACC AT-3’. Bai3flox mice were crossed with the pcp2-Cre
transgenic mice to generate PC-Bai3-null mice.
DNA Constructs
A series of constructs for Mus musculus proteins C1ql1 (Genbank AF095155; C1ql1WT,
residues 1258; gC1ql1WT, residues 125258), and Bai3 (Genbank AY168406; eBai3WT,
residues 1868; eBaiCUB, residues 152868; nBai3CUB, residues 1291) were amplified
by PCR and cloned into the pHLSec vector (Aricescu et al., 2006), between the
EcoRI/KpnI or AgeI/KpnI restriction sites (for constructs not containing the native signal
sequence), introducing an N-terminal secretion signal sequence and a C-terminal
hexahistidine (His) tag. Based on the structure of gC1ql1, two N-linked glycosylation
sites were introduced to the gC1ql1 domain (Q211N + N212S + Y213T + S244N +
K246S) to generate a glycan wedge mutant C1ql1 (C1ql1GW); the attachment of a bulky
N-glycan moiety to Asn residues has been shown to prevent specific proteinprotein
interactions (Luo et al., 2003; Rondard et al., 2008). Bai3CUB and C1ql1GW mutants were
generated by two-step overlapping PCRs. In some experiments, C1ql1 or Bai3 were
coexpressed with the fluorescent proteins (GFP, Venus or DsRed2) linked by a selfcleaving P2A peptide from foot-and-mouth-disease virus. Human protein Bai3 was
utilized for in vitro binding assay. Nucleotide sequences of the amplified open reading
frames were confirmed by bidirectional sequencing and the cDNAs were cloned into
expression vectors, either pTracer (Invitrogen) or pCAGGS (provided by Dr. J. Miyazaki,
Osaka University, Japan)..
In Utero Electropolation (IUE)
For introduction of Bai3 constructs into cerebellar Purkinje cells in PC-Bai3-null mice,
IUE was performed as previously described (Nishiyama et al., 2012). Fertilized eggs for
PC-Bai3-null mice were introduced into the uterus of the surrogate mother mouse (ICR
mouse) to perform IUE at E11.5 when most Purkinje cells are located on the surface of
the cerebellar ventricular zone. 13 l of cDNA solution (pCAGGS vectors, 15 mg/ml)
together with Fast Green (0.01%) was injected into the 4th cerebral ventricle to apply the
electrical shocks five times at an intensity of 33 V for 30 ms, at intervals of 970 ms per
pulse). To identify the transfected Purkinje cells, cDNA for GFP was mixed into cDNA
solution as the IUE shows a high ability to co-transfect several genes into single cells
(over 99%) (Nishiyama et al., 2012).
Virus Preparation and in vivo Injection
To express genes of interest into neurons in vivo, AAV type 5 (AAV5) or lentivirus
vectors were utilized. AAV5 vector plasmids contained an expression cassette with a
human CAG promoter followed by the target cDNA, and woodchuck hepatitis virus
posttranscriptional regulatory element (WPRE) and SV40 poly(A), all positioned
between the inverted terminal repeats of the AAV5 genome. AAV5 vectors were produced
using the AAV5 plasmid, the AAV5 helper plasmid containing the rep and cap sequences
from AAV5, as well as the pHelper plasmid from the AAV Helper-Free System containing
the E2A, E4, and VA RNA genes of the adenovirus genome (Agilent Technologies).
Human embryonic kidney 293 (HEK293) cells were co-transfected with the AAV5
plasmid and two helper plasmids by the calcium phosphate method. AAV particles were
then harvested and purified by two sequential continuous CsCl ultracentrifugations. The
vector titer was determined by quantitative PCR of the DNase-I-treated vector stocks and
was estimated at 101213 vector genome (vg) copies.
To produce the lentivirus vectors, the plasmids for the VSV-G (G glycoprotein of
vesicular stomatitis virus)-psuedotyped vectors were used. pCL36-C1L-CMp (kindly
provided by St. Jude Children’s Research Hospital) carrying a gene of interest was
transfected to HEK293T cells together with helper plasmids by the calcium phosphate
method. Eighteen hours after transfection, cells were washed with fresh culture medium
(DMEM with 10% fetal bovine serum) and allowed to produce virus particles for 24 h.
The culture supernatant was gently applied over the 20% sucrose solution and centrifuged
at 6,000 x g for 16 h at 4 °C for concentrating the lentivirus vector at the level of 1089
titer unit (TU).
For in vivo virus infection into cerebellar Purkinje cells, lentivirus solution (2 l; 1089
TU) were injected into the cerebellar vermis of adult mice, and for the virus infection into
the IO neurons, AAV (1 l; 101213 vg/ml) or lentivirus (1 l; 1089 TU) solutions were
injected into the ventral medial portion of the medulla in adult and P01 mice,
respectively.
microRNA Experiments
microRNAs targeted to the following sequences were designed according to the BLOCKiT Pol II miR RNAi Expression Vector kit guidelines (Invitrogen): 5’-CAG CGG CAA
GTT TAC ATG CAA-3’ for miR-C1ql1 and 5’-AGA TCG CGT TGT GAT TCC AAA3’ for miR-Bai3. Scrambled control microRNA (miR-SCR) for C1ql1 (5’-GCT CAG
GAC ATG AAG CTC TAA-3’) or Bai3 (5’-GTC AGC ACT TAT ACG GTA GAT-3’)
was designed by shuffling the recognition region sequences. A BLAST search confirmed
that the miR-SCR had no target gene. Each microRNA was subcloned after cDNA
encoding GFP or DsRed2 in the proper vectors. For the rescue experiment, microRNAresistant forms of C1ql1 (rC1ql1; 5’-GAG TGG TAA ATT CAC GTG TAA-3’) and Bai3
(rBai3; 5’-AGG TCC CGG TGC GAC TCT AAG-3’), which harbor sense mutations
without alteration of amino acid codons, were generated by two-step overlapping PCRs.
After confirming the sequence by bidirectional sequencing, constructs were introduced
into virus vectors.
Antibodies
Antibodies against C1ql1 and Bai3 were produced by injecting glutathione S-transferase
(GST) fusion proteins into guinea pigs. The cDNA fragment encoding mouse C1ql1
(4465 amino acid residues) or eBai3 was subcloned into the BamHI/EcoRI site of
pGEX4T-2 plasmid (GE Healthcare). Immunization and affinity purification were
performed as previously described (Watanabe et al., 1998). The following antibodies
were obtained commercially: ant-vGluT1 (rabbit, Frontier Institute), anti-vGluT2 (guinea
pig, rabbit or goat, Frontier Institute), anti-calbindin (rabbit, Millipore, mouse, SWANT,
or goat, Frontier Institute), anti-vGAT (guinea pig or goat, Frontier Institute), anti-GFP
(rabbit or goat, Frontier Institute) and anti-HA (mouse, COVANCE) antibodies.
Immunohistochemistry
Under deep anesthesia with a pentobarbital, mice were fixed by cardiac perfusion with
0.1 M sodium phosphate buffer (PB, pH7.4) containing 4% paraformaldehyde (4%
PFA/PB); the brain was then removed and soaked in 4% PFA/PB for 2h. After rinsing the
specimens with PB, parasagittal slices (50-m thickness) were prepared using a
microslicer (DTK-2000; D.S.K.). To unmask the antigens in the slice preparations, we
used antigen-exposing methods (Watanabe et al., 1998), i.e., microslicer sections were
treated with 1 mg/ml of pepsin in 0.2 N HCl at 37C for 3 min, and then were
permeabilized with 0.1% Triton X-100 in PB with 10% normal donkey serum or 2%
normal goat serum/2% bovine serum albumin (BSA) for 20 min. IHC staining was
performed using selective primary antibodies overnight at room temperature, followed by
incubation with the proper fluorescent dye-conjugate secondary antibodies (1: 200,
Jackson ImmunoResearch or Invitrogen) for 2 h. Photographs were taken with a confocal
laser-scanning microscope (FLUOVIEW, FV1000; Olympus) and a super-resolution SIM
(ELYRA; Zeiss). Quantification of the fluorescence signals was performed using an
ImageJ and its Add-in software.
Postembedding Immunogold EM Analysis
Microslicer sections (300-m thick) were cryoprotected with 30% sucrose/PB and frozen
rapidly with liquid propane in an EM CPC unit. Frozen sections were immersed in 0.5%
uranyl acetate in methanol at 90°C in an AFS freeze-substitution unit, infiltrated at
45°C with Lowicryl HM-20 resin, and polymerized with UV light. After etching with
saturated sodium-ethanolate solution for 3 s, ultrathin sections on nickel grids were
treated successively with 2% BSA/0.1% Tween 20 in Tris-buffered saline (pH 7.5; TBST)
for 30 min, either guinea pig C1ql1 antibody (20 g/ml) or guinea pig Bai3 antibody (20
g/ml) and rabbit vGluT2 antibody (10 g/ml) in 0.5% BSA/TBST overnight, and
colloidal gold-conjugated anti-guinea pig IgG (1:100; 10-nm particle) and anti-rabbit IgG
(1:100; 20-nm particle) in TBST for 1 h, respectively. Finally, the grids were stained with
2% uranyl acetate for 5 min and mixed lead solution for 2 min. Photographs were taken
with an electron microscope (JEM-1400Plus; JEOL). The density of the immunogold
particles on the electron micrographs was quantitatively analyzed using ImageJ software.
Gross Cerebellar Anatomy
To observe gross structure in adult mouse cerebella, cresyl violet-staining was performed.
Bright-field images were captured using a CCD camera (DP70, Olympus) attached to a
stereomicroscope (SMZ1000, Nikon).
Surface Plasmon Resonance (SPR) Experiments
SPR analysis were performed with a Biacore T200 instrument (GE Healthcare) at 25oC
in (mM): 20 HEPES (pH 7.5), 150 NaCl, 2 CaCl2, 2 MgCl2 , 0.005% v/v Tween 20. Bai3
constructs were used as analytes and were purified by analytical size exclusion
chromatography in the SPR buffer before use. C1ql1 constructs were C-terminally
biotinilated and were immobilized on CM5 BIAcore sensor chips (Biacore Life Sciences),
pre-coated with streptavidin (control protein, Thermo Fisher Scientific). For
measurements of Bai3 binding to immobilized proteins, samples were injected at 20
l/min. Signal derived from experimental flow cells was corrected by subtraction of
signal derived from buffer and reference from a control protein coupled flow cell. Each
injection was followed by a regeneration step with 10 mM EDTA (30 l/min, 1 min). All
experiments were performed in duplicates. Kd values were calculated using
BIAevaluation 3.0 software suite (Biacore Life Sciences) and a 1:1 Langmuir binding
isotherm model.
In Vitro Binding Assay
Constructs for Flag-tagged full-length Bai3 proteins (human Bai13, mouse Bai3 or
Bai3CUB) were transfected into HEK293 cells using the CellPhect transfection kit
(Amersham Pharmacia) and the cells were incubated with C1ql1WT-HA or C1ql1GW-HA
for 6 h, fixed with 4% PFA, and immunostained with anti-HA antibody (mouse) without
permeabilization to selectively stain C1ql1-HA on the cell surface. After treatment with
secondary antibodies conjugated with Alexa546-anti-mouse IgG and Alexa488-anti
rabbit IgG (1:1,000), fluorescence images were captured using a confocal laser scanning
microscope (FLUOVIEW, FV1000; Olympus). Quantification of the signal intensity was
performed as described previously (Matsuda et al., 2009).
Electrophysiology
Parasagittal cerebellar slices (200-m thick) were prepared from WT, C1ql1-null, PCBai3-null and its control littermate mice, as described previously (Kakegawa et al., 2011).
Briefly, whole-cell patch-clamp recordings were made from visually identified Purkinje
cells using a 60 water-immersion objective attached to an upright microscope (BX51WI,
Olympus) at room temperature. The solution used for slice storage and recording
consisted of the following (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4,
26 NaHCO3 and 10 D-glucose, bubbled continuously with a mixture of 95% O2 and 5%
CO2. Picrotoxin (100 M, Sigma-Aldrich) was always present in the saline to block the
inhibitory inputs. For the Ca2+ current recordings, tetrodotoxin (0.5 M, Alomone labs)
was added and 75 mM NaCl was substituted with tetraethyl ammonium (TEA)-Cl.
Intracellular solutions were composed of (in mM): 150 Cs-gluconate, 10 HEPES, 4
MgCl2, 4 Na2ATP, 1 Na2GTP, 0.4 EGTA and 5 lidocaine N-ethyl bromide (QX-314) (pH
7.25, 292 mOsm/kg) for the CF-EPSC recordings, 60 CsCl, 10 Cs-gluconate, 20 TEA-Cl,
20 BAPTA, 4 MgCl1, 4 Na2ATP, 30 HEPES (pH 7.3, 295 mOsm/kg) for the Ca2+ current
recordings, and 65 Cs-methanesulfonate, 65 K-gluconate, 20 HEPES, 10 KCl, 1 MgCl2,
4 Na2ATP, 1 Na2GTP, 5 sucrose, and 0.4 EGTA (pH 7.25, 295 mOsm/kg) for the complex
spike recordings and LTD experiments. The patch pipette resistance was 2−4 M when
filled with each intracellular solution.
To evoke CF- and PF-EPSCs, square pulses were applied through a stimulating
electrode placed on the granular layer (10 s, 0−200 A) and the molecular layer (~50
m away from the pial surface; 10 s, 0−200 A), respectively. Selective stimulation of
CFs and PFs was confirmed by the paired-pulse depression (PPD) and paired-pulse
facilitation (PPF) of EPSC amplitudes at a 50-ms interstimulus interval, respectively. As
the time course for the maturation of the CF inputs onto Purkinje cells is somewhat
different at the lobules and the subdivisions (that is bank, sulcus or gyrus) (Nishiyama
and Linden, 2004), we restricted to the recording from cells positioned at the bank regions
of the lobules IVVII.
To record the complex spikes, whole-cell current-clamp recordings were performed
from Purkinje cell soma and CF was stimulated at a frequency of 1 Hz for 2 min (120
times) after checking the selective CF stimulation in voltage-clamp mode. The
occurrences of the complex spikes, CF-evoked after-hyperpolarization and suppression
of spontaneous spikes were analyzed (Davie et al., 2008).
In the LTD experiments, PF-EPSCs were first recorded successively at a frequency
of 0.1 Hz from Purkinje cells voltage-clamped at –80 mV. After stable PF-EPSCs were
observed for at least 10 min, a conjunctive stimulation (CJ-stim) composed of 120 single
CF stimuli followed by PF stimuli (20 ms later) was applied at a frequency of 1 Hz in
current-clamp mode to induce LTD (see Figure 6E). Access resistances were monitored
every 10 s by measuring the peak currents in response to 2-mV, 50-ms hyperpolarizing
steps throughout the experiments; the measurements were discarded if the resistance
changed by more than 20% of its original value.
For the Ca2+ current recordings, P5 Purkinje cells were voltage-clamped at Vh = –60
mV and 100-ms depolarizing step pulses were applied from –60 mV to +40 mV every
10-mV step. Ca2+ currents were estimated by subtraction of the leak currents (measured
by the 100-ms hyperpolarizing step pulses before depolarization) from depolarizationinduced currents.
Current responses were recorded with an Axopatch 200B amplifier (Molecular
Devices), and pClamp software (version 9.2, Molecular Devices) was used for data
acquisition and analysis. Signals were filtered at 1 kHz and digitized at 4 kHz for the
evoked EPSCs and Ca2+ currents, and 10 kHz for the complex spike recordings.
Protein Purification, Crystallization and X-Ray Crystallography
Secreted gC1ql1 constructs expressed in HEK293T cells, were purified from 0.2 m
filtered cell culture media by immobilized nickel affinity chromatography (His-Trap Fast
Flow, GE Healthcare) followed by analytical size exclusion chromatography (Superdex
S200 HiLoad HR 16/60, GE Healthcare) in (mM): 20 HEPES (pH 7.5), 150 NaCl, 2
CaCl2 and 2 MgCl2. Crystallization trials, using 100 nl gC1ql1 solution (5.46 mg/ml) plus
100 nl reservoir solution were set up in 96-well plates in a sitting drop vapor diffusion
format, using Cartesian Technologies robots (Walter et al., 2005). Crystallization plates
were stored at 20.5oC in a TAP Homebase storage vault and imaged using a Veeco
visualization system (Mayo et al., 2005).
Crystals of gC1ql1 were obtained in a condition comprising of (M): 0.02 NiCl
hexahydrate, 0.02 MgCl2 hexahydrate, 0.02 CdCl2 hydrate, 0.1 Na-Acetate trihydrate (pH
4.5) and 24% w/v polyethylene glycol monomethyl ether 2000. Crystals were mounted
on LithoLoops (Molecular Dimensions), cryo-protected in 25% v/v ethylene glycol
(added to the reservoir solution) and flash frozen at 100K. X-ray diffraction images of
(0.15°) oscillation were collected at the Diamond Light Source beamline I04, on a Pilatus
6M-F detector, wavelength 0.97625 Å, and were indexed, integrated, scaled and merged
using xia2 (Winter et al., 2010). The gC1ql1 structure was solved by molecular
replacement using the M.musculus ACRP30 adipocyte complement-related protein or
AdipoQ (Shapiro and Scherer, 1998) (37% sequence identity, Protein Data Bank (PDB)
accession code 1C28) as a search model in Phaser (McCoy et al., 2005). The molecular
replacement solution was then subjected to iterative rounds of model building in Coot
(Emsley et al., 2010), and refinement in REFMAC (Murshudov et al., 2011) and PHENIX
(Afonine et al., 2012). Electron density features consistent to the presence of four metal
ions have been observed along the central three-fold gC1ql1 symmetry axis. In a
physiological environment, based on the coordination geometry, these will likely be
occupied by calcium ions. However, in our structure, these correspond to cadmium
(peripheral) and nickel/cadmium (partial occupancy) atoms, present in excess in the
crystallization condition. Stereochemical properties of the model were assessed in Coot
(Emsley et al., 2010) and MolProbity (Chen et al., 2010). Protein geometry analysis
revealed no Ramachandran outliers, with 95.52% residues in favored regions and 4.48%
residues in allowed regions. Molprobity clash score after adding hydrogens is 0.5 (100th
percentile) and the overall Molprobity score is 0.98 (99th percentile). Crystallographic
statistics are presented in Table 1. Figures of the final model were rendered in PyMOL
Molecular Graphics System (Schrodinger) and panels assembled using the CorelDraw
Graphics Suite X6 (Corel Corporation).
Behaviors
To examine motor coordination in the mice, the beam test and the accelerating rotor-rod
test were performed from adult mice as previously described (Kakegawa et al., 2011).
Briefly, six trials were continuously performed at 550 rpm and a 30-s interval, and the
time that each mouse stayed on the rod was measured (maximum score, 120 s). A series
of trials were performed on 3 successive days.
Spontaneous Eye Movements and Horizontal Optokinetic Response (hOKR)
Over 8-week male mice were anesthetized with an intraperitoneal injection of
ketamine/xylazine (80/20 mg/kg; Sigma) and a 1-cm flat-head screw was attached to the
cranial bone with synthetic resin cement (Super-Bond, Sun Medical). Three or more days
later, mice were placed on a table with their head fixed by the screw and the body loosely
strained in a plastic cylinder. Spontaneous eye movements were continuously monitored
in the resting state under room light conditions (300400 lux) with an infrared camera
and the traces were recorded for 15 s for 5 times and averaged every mouse.
hOKR experiments were performed by sinusoidal oscillation of the checked-pattern
screen (screen height, 55 cm from the eye of the mouse; check size, 2-cm square) by 15
(peak-to-peak) at 0.33 Hz in light (300400 lux; see Figure 6A). Over 10 cycles of the
evoked eye movements free from blinks and saccades were averaged, and the mean
amplitude were calculated by a modified Fourier analysis as previously described (Nagao,
1990). The gain of the eye movement was defined as the ratio of the peak-to-peak
amplitude of eye movements to that of the screen oscillation. The adaptabilities of the
hOKR were examined by exposing the mouse to 1 h of sustained sinusoidal screen
oscillation.
Data Analysis and Statistics
Results are presented as the means ± s.e.m.. Statistical analyses were performed using the
Excel statistics 2012 add-in software (Social Survey Research Information Co.) and
significant differences were defined as *P < 0.05, **P < 0.01 and ***P < 0.001. For the
comparison between two groups, we used MannWhitney U test.
For the multiple
comparisons among various groups, we used KruskalWallis test followed by Scheffe
post-hoc test, and for the multiple comparisons with a control group, we used
KruskalWallis test followed by Steel post-hoc test. When multiple comparisons were
performed between two groups in various time points, we used two-way ANOVA
followed by Bonferroni post-hoc test.
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