Supplemental Material:
The NAP-motif of Activity-Dependent Neuroprotective Protein (ADNP)
Regulates Dendritic Spines through Microtubule End Binding Proteins
Saar Oz1, Oxana Kapitansky1, Yanina Ivashco-Pachima1, Anna Malishkevich1,
Eliezer Giladi1, Nir Skalka2, Rina Rosin-Arbesfeld2, Leonid Mittelman3, Oshik
Segev4,5, Joel A. Hirsch4 and Illana Gozes1,5*
1
Departments of Human Molecular Genetics and Biochemistry and 2Anatomy,
Interdepartmental
Services,
Sackler
Faculty of
Medicine;
4
Department
3
of
Biochemistry and Molecular Biology, George Wise Faculty of Life Sciences, 5Sagol
School of Neuroscience and Adams Super Center for Brain Studies, Tel Aviv
University, Israel
*Corresponding Author:
Illana Gozes, Ph.D.
Professor of Clinical Biochemistry
The Lily and Avraham Gildor Chair for the Investigation of Growth Factors
Director, The Adams Super Center for Brain Studies and The Edersheim Levie-Gitter
fMRI Institute
Head, the Dr. Diana and Zelman Elton (Elbaum) Laboratory for Molecular
Neuroendocrinology
Sackler Faculty of Medicine, Tel Aviv University
Tel Aviv 69978, Israel, Phone: 972-3-640-7240, Fax: 972-3-640-8541
e-mail: igozes@post.tau.ac.il
Running Title: Molecules that make our minds
Supplemental Materials and Methods
P19 Cells
Mouse embryonic teratocarcinoma cells (P19 cells) were obtained from the American
Type Culture Collection (ATCC, Bethesda, MD, USA) and grown in minimal
essential medium (alpha-MEM, Biological Industries, BeitHaemek, Israel) containing
5% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Biological
Industries) in a 5% CO2 incubator at 37°C.
Neuronal or cardiomyocyte induced P19 differentiation
For induction of differentiation, 1×106 P19 cells were cultured in 90 mm
bacteriological grade dishes with their usual growth medium and supplemented with
1μM all-trans retinoic acid (RA, Sigma, St. Louis, MO, USA) to induce neuronal and
astroglial differentiation 1, or 0.8% dimethylsulfoxide (DMSO, Sigma) to induce
cardiac and skeletal muscle differentiation, as previously described
2-4
. Four days
later, cell aggregates were suspended with trypsin-C (Biological Industries) and
transferred to poly-L-lysine (Sigma) coated tissue culture dishes. The cells were
grown in RA/DMSO-free Dulbecco's modified Eagle's medium (DMEM) containing
2.5% fetal calf serum, 4mM L-glutamine and antibiotics (Biological Industries) for
additional four days to induce neuronal and astroglial, or cardiac and skeletal muscle
phenotype. As controls to the differentiated conditions, both non-differentiated cells
as well as cells that went through the differentiation process in the absence of the
inducer were evaluated.
Rat cerebral cortical astrocyte cell cultures
Newborn rats (Harlan, Jerusalem, Israel) were sacrificed by decapitation, and the
brain was removed. The cortex was dissected, and the meninges - removed. The tissue
was minced with scissors and placed in Hank’s balanced salts solution 1 (S1),
containing HBSS (Biological Industries, BeitHaemek, Israel), 15 mM HEPES buffer,
pH 7.3 (Biological Industries, BeitHaemek, Israel), and 0.25% trypsin (Biological
Industries), and incubated at 37 °C 5% CO2 for 20 min. The cells were then placed in
5 mL of solution 2 (S2) containing 10% heat inactivated fetal serum (Biological
Industries), 0.1% gentamycin sulphate solution (Biological Industries), and 0.1%
penicillin-streptomycin-nystatin solution (Biological Industries) in Dulbecco’s
modified Eagle’s medium (DMEM, Sigma, Rehovot, Israel). The cells were allowed
to settle and were then transferred to a new tube containing 2.5 mL of S2 and
triturated using a Pasteur pipette. The process was repeated twice more. Once all the
cells were suspended, cell density was determined using a hemocytometer (Neubauer
Improved, Germany) and 15 × 106 cells/15 mL S2 were inoculated into a 75-cm2
flask (Corning, Corning, NY, USA). Cells were incubated at 37 °C 10% CO2. The
medium was changed after 24 hours, and cells were grown until confluence 5.
Rat cerebral cortical astrocyte cell subcultures
The flasks were shaken to dislodge residual neurons and oligodendrocytes that may be
present. Flasks were then washed with 10 mL cold HBSSx1, HEPES15mM. 5 mL of
versene-trypsin solution (BioLab, Jerusalem, Israel) were added to each flask, and the
flasks were incubated at room temperature for 5 min to remove astrocytes. The flasks
were shaken to dislodge the cells. The versene trypsin solutions were neutralized with
5 mL of S2. The cell suspension was collected and centrifuged at 100 g for 10 min.
The supernatant was removed and the cells resuspended in S2. Cells were inoculated
into 75-cm2 flasks or plated in 35-mm dishes (Corning, Corning, NY, USA) and
incubated until confluent at 37 °C 10% CO2.
NIH3T3 cells
NIH3T3 (mouse fibroblasts; ATCC, Bethesda, MD, USA) were cultured in DMEM
containing 10% heat inactivated fetal calf serum, 2 mM L-Glutamine, 100 units/mL
penicillin, and 0.1 mg/mL streptomycin (Biological Industries) in 5% CO2 at 37 °C.
Cells were split every 3-4 days using trypsin-EDTA solution B (Biological
Industries).
PC-12 cells
PC12 cells (Pheochromocytoma cells; ATCC, Bethesda, MD, USA) were seeded at
3x104 cells/cm2 on poly-L-Lysine-coated plastic tissue culture dishes (Corning,
Lowell, MA, USA) to form an adherent monolayer. Cells were maintained in DMEM
supplemented with 10% horse serum, 5% fetal calf serum, 2 mM glutamine, 100 U/ml
penicillin and 100 mg/ml streptomycin (Biological Industries, BeitHaemek, Israel).
When stated, PC12 differentiation was induced by nerve growth factor (NGF; Sigma)
at concentrations of 50ng/ml by replacing half of the medium every other day until the
cells acquire a differentiated morphology. We defined differentiated cells as bearing
two or more neurites with lengths equal to, or more than twice the diameter of the cell
body 6. The cells were incubated in 5% CO2 in a humidified incubator at 37°C. The
cells were sub-cultured every 5 days at a 4:1 split ratio by pipetting, using trypsinEDTA solution B (Biological Industries). The medium was changed every 2 or 3 days
after adhesion.
For RNA silencing and cell protection, PC12 cells (Cat. No. CRL-1721TM, ATCC,
Bethesda, MD, USA) were maintained as above. Cells of passage number 20 were
used in the present study for RNA silencing (see main text).
Poly-L-lysine coating of dishes
One mL of 10 μg/mL poly-L-lysine hydrobromide (Sigma) in sterile double-distilled
water was added to 35-mm dishes. After 1 h, dishes were washed three times with
sterile double-distilled water and kept at room temperature.
RNA Extraction
Total RNA from cells was extracted using the RNeasyPlus Mini Kit (Qiagen, Hilden,
Germany) according to the manufacturer’s protocol.
RNA integrity was determined by fractionation on 1% agarose gel and staining with
ethidium bromide (Sigma). RNA quantity and quality were analyzed by the Nanodrop
ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Each sample measurement was performed in duplicates.
Reverse Transcription and Quantitative Real-Time PCR
Procedures were carried out essentially as before 7. Samples containing equal amount
of total RNA were used to synthesize single-strand cDNA using SuperScript III
Reverse Transcriptase (RT, Invitrogen) or High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems), with random hexamers according to the
manufacturer's instructions. In each RT-PCR run, two negative controls were
included: sterile water without RNA to rule out contamination in the reaction
components, and total RNA without the RT enzyme to test for genomic
contamination. Real-Time polymerase chain reaction (PCR) was performed using the
SYBR® Green PCR Master Mix or Fast SYBR® Green PCR Master Mix and ABI
PRISM 7900 or ABI Step One Plus Sequence Detection Systems instrument and
software (Applied Biosystems), accordingly using the default thermocycler program
for all genes
Primers
Primer pairs (Supplemental Table S1) were designed using the primer 3 web interface
(http://frodo.wi.mit.edu/primer3/) or by using the NCBI primer designing tool
(http://www.ncbi.nlm.nih.gov/tools/primer-blast/)
and
synthesized
by
Sigma-
Genosys. To avoid amplification of contaminating genomic DNA, primers for
quantitative real time PCR were designed to target exon-exon junction.
RNA expression levels were normalized to mouse/rat hypoxanthine-guanine
phosphoribosyltransferase (HPRT1) as endogenous control.
Whole cell homogenate preparations
Cells were homogenized on ice in a buffer (Tris 50mM, EDTA 10mM, NaCl 100mM,
pH7.4) containing protease and phosphatase inhibitors (1mM phenylmethylsulphonylfluoride (PMSF), leupeptin 25μg/ml, pepstatin 25μg/ml, Na3VO4 1mM, NaF 20mM).
The amount of proteins in homogenates was estimated with the Pierce® BCA Protein
assay (Thermo Scientific). Homogenates were then subjected to SDS polyacrylamide
gel electrophoresis and western blotting as described in the main text.
Antibody list
Rat monoclonal mapre1 antibody [KT51] (ab53358, Abcam, MA) binds to the Cterminal part of rodent and human EB1. Rat monoclonal mapre2 antibody [K52]
(ab45767, Abcam) recognizes EB2 protein in the same species as the EB1 antibody.
Rabbit polyclonal EB3 antibody (ab13859, Abcam) reacts as above. Rabbit polyclonal
actin antibody (ab1801, Abcam, MA, USA) recognizes beta and gamma actin in
human, rodent, chicken and drosophila samples. The secondary antibodies used were
peroxidase-conjugated AffiniPure goat anti–mouse, anti-rat and anti-rabbit IgG
(Jackson ImmunoResearch, USA).
Additional antibodies
Primary antibody list: monoclonal Tyr-α-tubulin(YL1/2) (VMA1864, Abcys, Paris,
France), polyclonal Glu-α-tubulin (L4)(AbC0101, Abcys), monoclonal PSD-95(ab2723, Abcam), actin(ab1801, Abcam) and -α-tubulin(1:500; Sigma, T9026). Rabbit
anti-mouse SNIP/p140cap Antibody (Cell Signaling, Technology, Inc., Beverly, MA),
dilution 1:500. Secondary antibodies: Peroxidase AffiniPure Goat anti–mouse, Cy3conjugated Goat Anti-Rat IgG, Cy5-conjugated goat anti-rabbit IgG, Rhodamine RedX-AffiniPure Fab Fragment goat anti-rabbit IgG(Jackson ImmunoResearch, Suffolk,
UK) and goat anti-mouse far red(1:500; Alexa, Molecular Probes, Grand Island, NY).
DyLight 488-labeled secondary goat anti-mouse IgG, DyLight 633-labeled secondary
goat anti-rabbit IgG(KPL, Gaithersburg, MD).
RNA silencing
The siRNA against rat EB3 (MAPRE3, NM_001007656) was obtained as an OnTarget plus smart pool L-099082-01-0005-0010 (Dharmacon, Thermo Fisher
Scientific, Lafayette, CO). Dharmacon ON-TARGETplus Non-targeting siRNA (D001810-01-05) was used as a negative control. To control transfection efficiency, a
Dharmacon siGLO RISC-free siRNA (D-001600-01-05), was used. Further studies
included Dharmacon ON-TARGETplus SMARTpool siRNA (Thermo Scientific)
targeting EB1 (Rat MAPRE1, Gene ID: 114764) and EB2 (Rat MAPRE2, Gene ID:
679221).
Transfections
were
performed
using
Lipofectamine
RNAiMAX
(Invitrogen). For rat MAPRE3 knockdown, PC12 cells or neurons were plated in
growth medium without antibiotics at a concentration of 104cells/0.32 cm2 such that
they were 30-50% confluent at the time of transfection. To obtain the highest
transfection efficiency and low non-specific effects, siRNA transfection was
performed in the following optimized conditions: 50nM for neurons and 25nM for
PC12 of each siRNA construct with 0.2µl Lipofectamine RNAiMAX (Invitrogen)
added per well of the 96-well plate and scaled up for different 24 or 6well plates. The
medium was changed one day after transfection. 48 and 72h later, cells were
harvested to assay for gene knockdown at the RNA and protein levels.
Legends:
Supplemental Figure S1. EB1,2,3 protein sequence alignment: The SxIP binding
site.
The original paper described EB1 binding to the SxIP motif in +TIPS 8. Here, we
compared the protein sequence of EB1,2 and 3 by multiple sequence alignment in
order to assess the EB1 binding motif SxIP for EB2 and EB3. Protein sequence
alignment of EB1,2 and 3 (MAPRE1,2,3) from mouse, rat and human origin showed
high similarity. Alignment was done using UniProtClustalW 9. Uniprot identifiers are
shown on the left. Colored rectangular indicate the binding cavity interacting residues
as reported 8.
In detail, the binding domain is conserved among the EBs in rat, human and mouse.
EB3 is slightly more similar to EB1 than EB2 to EB1. The main (SxIP) motifinteracting residues are highly conserved among EB1, EB2, and EB3, (yellow
rectangle), while the other residues involved in the cavity, which are likely to have
interactions with the other residues of the binding peptide, are less conserved,
especially with EB2 (red rectangle). This analysis indicates an extensive homology in
the SxIP binding groove among the EB’s, suggesting it may be also be a binding
motif for EB2, and EB3. The differences between the sequences may explain the
specificity of SxIP motif containing proteins with their EB partner.
Supplemental Figure S2. EB1,2,3 mRNA expressions in cell cultures.
Reverse transcription and quantitative real time polymerase chain reaction (PCR)
analysis of mRNA expression using the relative standard curve method. (a) Rat cell
cultures: rat non-differentiated PC12 cell line, differentiated PC12 cells treated with
NGF and primary cultures of astrocytes and neurons grown for 4DIV or 19DIV. RNA
silencing (in the indicated concentration) of EB3 in PC12 cells and primary neuronal
cultures compared with cells treated with non-targeting siRNA. (b) Mouse cell
cultures: mouse fibroblasts NIH3T3 cell line, non-differentiated (nd) P19
teratocarcinoma cell line, P19 differentiated into neuro-glial-like cells by retinoic acid
(RA) and to cardiomyocytes-like cells by DMSO. Neuronal differentiated cells
exhibit relatively high expression levels of EB3.
Description of the results:
RT and Q-PCR analysis of mRNA expression in rat non-differentiated PC12 cells,
differentiated PC12 cells treated with NGF, and primary cultures of cortical astrocytes
and neurons grown for 4 days in vitro (DIV) or 19DIV, showed that EB3 is highly
enriched in cortical neurons. RNA silencing of EB3 in PC12 cells and in primary
neuronal cultures resulted in up to 50% mRNA expression inhibition of EB3
compared with cells treated with non-targeting siRNA and with no effect on the other
EB’s (Supplemental Fig. S1a). Further analysis of mouse cell lines, showed that EB3
mRNA is preferentially expressed in mouse P19 cells subjected to neuro-glial
differentiation by retinoic acid (RA) compared with non- (Supplemental Fig. S1b).
Furthermore, the NIH3T3 and cardiac and skeletal muscle-differentiated P19 cells
(DMSO) showed low levels of EB1,2, and 3 mRNA species, indicating tissue
specificity as expected.
Supplemental Figure S3. NAP affinity column specificity.
When one performs peptide affinity chromatography, it is required to have a spacer
arm to avoid the possibility of steric hindrance. This is one of the basic principles of
affinity chromatography as originally established by the group led by the Nobel Prize
winner, CB Anfinsen and the leaders in affinity chromatography, P. Cuatrecasas and
M. Wilchek10.
In our case, the sequence of the spacer peptide is: CKKKGG, with the following
characteristics. 1] The first C (cysteine) is required for binding to the column material.
2] The sequence of NAP (NAPVSIPQ) in ADNP (human and mouse) is preceded by
GG = …GGNAPVSIPQ…..11. Thus, the GG sequence is shared by the extended
NAP and ADNP, the parent protein which binds EB1-3. 3] The addition of KKK
(Lysine-Lysine-Lysine) is often used as a spacer peptide also in protein expression
systems (e.g. Protein expression system, patent number: US 5935824 A).
To ascertain specificity to the NAP sequence, we have competed NAP-bound EB3
with NAPVSIPQ (the NAP sequence itself), and the analogue NAPVSKIPQ, but not
with NAPVSAIPQ or NAPVAAAAQ, despite elution in a very acidic pH. ).
Importantly, the displacing peptides did not include the spacer peptide, and still were
able to displace indicating that the spacer peptide is not involved in the binding.
Lanes a-d are described in the main text, only here the full blots are shown including
molecular weight markers (M).Supplemental Figure S4. NAP specifically increases
individual puncta area, while NAPVAAAAQ did not.
2h treatment with 10-12M NAP increased PSD-95 puncta average area significantly.
Control peptide in which the SxIP motif was substituted with AAAA
(NAPVAAAAQ) showed no effect compared to vehicle. (ANOVA, p<0.001, n=12
dendrites per treatment).
Supplemental Figure S5. EB1, EB2 and EB3 silencing results in protein reduction of
>50%.
Western blot analysis of EB1,EB2 and EB3 protein levels in extracts from PC12 cell
cultures under different transfection conditions: – EB protein levels in PC12 cells
without any manipulations (positive control, without transfection); “+ trans. regent ” –
EB's protein levels in PC12 cells that were exposed to the transfection reagent
Lipofectamine RNAiMAX only without any siRNA;; “siRNA-EB1”, “siRNA-EB2”
"siRNA EB3 – EB1, EB2 and EB3 protein levels in PC12 cells exposed to
transfection condition as described in the materials and methods section,
Description of the results
At least a 50% specific reduction in the expression of EB1, EB2 and EB3 is seen. The
western analysis was repeated thrice and representative pictures are shown. For EB3,
the results were confirmed at the RNA level (Supplemental Fig. S2a).
Supplemental Table S1: Quantitative Real-Time PCR: primer pairs and EB1
and EB2, cloning pairs.
Primer
Mouse
Sequence
MAPRE1 5’- GCGTTGACAAAATAATTCCT-3’
5’- TGGCAGCTACAGGATCATAC-3’
(NM_007896)
Mouse
MAPRE2 5’- ATACAGCTCAACGAGCAGGTACAT-3’
(NM_001162941)
5’- CAGCAGCTCAATCTCTCTCAACTTC-3’
Mouse MAPRE3
5’- GCTGTGTTCACTTGAGGAAG -3’
(NM_133350)
5’- GAATGATTTTGTCAACACCC-3’
Mouse HPRT1
5’- GGATTTGAATCACGTTTGTGTC -3’
(NM_013556)
5’- CAGGACTCCTCGTATTTGCAG -3’
Rat MAPRE1
5’- GAAGAAAGTGAAATTCCAGG -3’
(NM_138509)
5’- AGGAATTATTTTGTCAACGC -3’
Rat MAPRE2(NM_001101000)
Rat MAPRE3(NM_001007656)
5’- GGGCGTTTCCAAGACAACCT -3’
5’-CTTGTCGAGCCTCAACAGGAT-3’
5’- GGACAAAATCATTCCCGTAG -3’
5’- GGTTGTAATCCTTTCCATCA -3’
Rat HPRT1
5’- AGGCCAGACTTTGTTGGATT -3’
(NM_012583)
5’- GCTTTTCCACTTTCGCTGAT -3’
Human MAPRE1
Human MAPRE2
5’ACGTAAGCTTGCATGGCAGTGAACGTATAC3’
5’ACGTCTCGAGTCAATACTCTTCTTGCTCCTC3’
5’ACGTCTCGAGTCAGTACTCTTCCTGCTGCGG’
5’ACGTAAGCTTGCATGGCGGTCAATGTGTAT3’
Supplemental Table S2: EB3 binds SIP and SKIP containing sequences.
Supplemental Table S2 described the amounts of protein load, flow through (FT),
wash (W) and eluted (E) protein as calculated from the gels on Fig. 3B.
Load
Load
Load
Load
(EB3+NAPVSIP
(EB3+NAPVSKIP
(EB3+NAPVAAAA
(EB3+NAPVSAIP
Q)
=10μl
0.16μg
FT1
=10μl
~ Q) =10μl ~ 1μg
Q) =10μl ~ 0.3μg
=10μl FT1 =10μl ~0.06μg
FT1 =10μl ~0.7μg
=10μl FT2 =10μl ~0.6μg
FT2 =10μl ~0.7μg
0.151μg
=10μl FT1
~0.06μg
FT2
~ Q)
~0.119μg
=10μl FT2
~0.1μg
~0.065μg
W1 =45μl ~0μg
W1 =45μl ~0.2μg
W2 =45μl ~0μg
W2
W1 =45μl ~1.6μg
=45μl W2 =45μl ~0.3μg
W1 =45μl ~0.08μg
W2 =45μl ~0.2μg
~0.045μg
E1=40μl ~0μg
E1=40μl ~0μg
E1=40μl ~0μg
E1=40μl ~0μg
E2=40μl ~0μg
E2=40μl ~0.05μg
E2=40μl ~0.4μg
E2=40μl ~0.5μg
E3=40μl ~0.3μg
E3=40μl ~0.6μg
E3=40μl ~1μg
E3=40μl ~0.3μg
Supplemental Figure S1
n
no
PC
PC 12
ta
12
rg
N
G
PC etin
F
g
12
(2
si
5n
R
N
M
N
eu
)
A
ro
(
25
ns
nM
A
(4
st
)
D
ro
IV
N
cy
)n
eu
N
te
ro
eu
on
s
ns
ro
t
a
ns
rg
(4
N
D
et
(4
eu
IV
in
D
)
ro
g
IV
(5
)s
ns
0
iR
nM
(1
N
9D
)
A
N
IV
(5
eu
N
)
0
no
ro
eu
nM
ns
ro
n
)
ta
ns
(1
rg
9D
(1
e
9D
tin
IV
)
g
IV
(
)s
50
iR
nM
N
)
A
(5
0n
M
)
12
PC
Normalized mRNA quantity
Supplemental Figure S2
(a)
EB's in rat cell cultures
4
3
EB1
EB2
EB3
2
1
0
Cell culture
Supplemental Figure S2
(b)
8
EB1
EB2
6
EB3
4
2
SO
R
A
P1
9-
D
M
9P1
P1
9-
nd
3
0
3T
Normalized mRNA quantity
EB's in mouse cell cultures
Cell culture
Supplemental Figure S3
Supplemental Figure S4
Supplemental Figure S5
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