Mapping the NPHP-JBTS-MKS
Protein Network Reveals Ciliopathy
Disease Genes and Pathways
Liyun Sang,1,16Julie J. Miller,2,16Kevin C. Corbit,3Rachel H. Giles,4Matthew J. Brauer,1Edgar A. Otto,5Lisa M. Baye,6
Xiaohui Wen,1Suzie J. Scales,1Mandy Kwong,1Erik G. Huntzicker,1Mindan K. Sfakianos,1Wendy Sandoval,1
J. Fernando Bazan,1Priya Kulkarni,1Francesc R. Garcia-Gonzalo,3Allen D. Seol,3John F. O’Toole,5Susanne Held,5
Heiko M. Reutter,8William S. Lane,9Muhammad Arshad Rafiq,10Abdul Noor,10Muhammad Ansar,11
Akella Radha Rama Devi,12Val C. Sheffield,7,15Diane C. Slusarski,6John B. Vincent,10,13Daniel A. Doherty,14
Friedhelm Hildebrandt,5,15Jeremy F. Reiter,3and Peter K. Jackson1,*
1Genentech Inc., South San Francisco, CA 94080, USA
2Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
3Department of Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, San Francisco,
CA 94158, USA
4Department of Medical Oncology and Departmentof Nephrology and Hypertension, University Medical Center Utrecht, Heidelberglaan 100,
3584CX Utrecht, The Netherlands
5Departments of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA
6Department of Biology
7Department of Pediatrics,
University of Iowa, Iowa City, IA 52242, USA
8Institute of Human Genetics and Department of Neonatology, Children’s Hospital, University of Bonn, D-53111 Bonn, Germany
9Mass Spectrometry and Proteomics Resource Laboratory, Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA
10Molecular Neuropsychiatry and Development Lab, Neurogenetics Section, Centre for Addiction and Mental Health, Toronto,
ON M5T 1R8, Canada
11Department of Biochemistry, Quaid-e-Azam University, Islamabad 45320, Pakistan
12Rainbow Children’s Hospital, Hyderabad 500 034, India
13Department of Psychiatry, University of Toronto, Toronto, ON M5T 1R8, Canada
14Department of Pediatrics, University of Washington, Seattle 98195, WA
15Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
16These authors contributed equally to this work
Nephronophthisis (NPHP), Joubert (JBTS), and
Meckel-Gruber (MKS) syndromes are autosomal-
retinal degeneration, and cerebellar/neural tube
malformation. Whether defects in kidney, retinal, or
neural disease primarily involve ciliary, Hedgehog,
or cell polarity pathways remains unclear. Using
interactors copurifying with nine NPHP/JBTS/MKS
proteins and discovered three connected modules:
‘‘NPHP1-4-8’’ functioning at the apical surface,
‘‘NPHP5-6’’ at centrosomes, and ‘‘MKS’’ linked to
Hedgehog signaling. Assays for ciliogenesis and
epithelial morphogenesis in 3D renal cultures link
renal cystic disease to apical organization defects,
whereas ciliary and Hedgehog pathway defects lead
to retinal or neural deficits. Using 38 interactors as
candidates, linkage and sequencing analysis of 250
patients identified ATXN10 and TCTN2 as new
NPHP-JBTS genes, and our Tctn2 mouse knockout
shows neural tube and Hedgehog signaling defects.
Our study further illustrates the power of linking pro-
teomic networks and human genetics to uncover
critical disease pathways.
Ciliopathies are a heterogeneous group of diseases that present
with a broad constellation of clinical phenotypes, including renal
cysts, retinal degeneration, polydactyly, mental retardation, and
obesity (reviewed by Hildebrandt et al., 2009a; Zaghloul and
Katsanis, 2009). Studies of these diseases suggest that their
pathogenesis relates to dysfunction of the microtubule-based
primary cilium. It is hypothesized that the primary cilium is a
sensory organelle, acting as a mechanosensor in the kidney
and organizing sensory receptors, including rhodopsin, in the
retina. Cilia are also key components of the Hedgehog (Hh)
signaling pathway (Corbit et al., 2005; Huangfu et al., 2003).
The consistent finding of kidney, retinal, liver, limb, and brain
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 513
NPHP2 / INVS
NPHP5 / IQCB1
NPHP9 / NEK8
AHI1 / JOUBERIN
NPHP6 / CEP 290
NPHP8 / RPGRIP1L
Figure 1. Mapping the NPHP-JBTS-MKS Disease Protein Network Using G-LAP-Flp Strategy
(A) List of genes mutated in NPHP-JBTS-MKS ciliopathies.
(B)HeatmapsummarizingMS/MS interactionsamong NPHP proteins discovered usingG-LAP-Flp strategy. Horizontal axis,LAP-tagged‘‘bait’’proteins;vertical
axis, interacting proteins. Identified interactions are shown in black. The NPHP1-4-8 (1-4-8), NPHP5-6 (5-6), and MKS modules are color coded in blue, orange,
514 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.
defects among the ciliopathies in turn suggests that cilia-depen-
dent sensory and signaling functions are critical in the develop-
ment, tissue organization, and physiological function of multiple
organ systems. However, whether all ‘‘ciliopathies’’ are simply
caused by the absence of cilia themselves remains unclear.
Specific ciliary signaling pathways, ciliary receptors, additional
ciliary effectors, or even centrosomes, may be at the root of
have remained elusive.
bert syndrome (JBTS), and Meckel-Gruber syndrome (MKS) are
autosomal-recessive disorders, initially described as distinct
entities but recently found to share phenotypic overlap, notably
in cystic kidney disease. Nephronophthisis is the least severe of
the group, characterized primarily by renal cysts but sometimes
involving retinal degeneration (called Senior-Loken syndrome),
situs inversus, and mental retardation (Hildebrandt et al.,
2009a). Joubert syndrome involves both renal and nonrenal
manifestations of NPHP disease but is distinguished by cere-
that is linked to ataxia (Parisi et al., 2007). As a perinatal lethal
ized by occipital encephalocele, polydactyly, liver fibrosis, and
severe renal cysts (Salonen and Paavola, 1998).
To date, mutations in 16 genes have been linked to this group
of disorders: NPHP1–9, AHI1/Jouberin, ARL13B, INPP5E,
TMEM216, MKS1, MKS3/TMEM67, and MKS6/CC2D2A (Tallila
et al., 2008; Hildebrandt et al., 2009a; Lee and Gleeson 2010;
Supplemental References). Interestingly, different alleles of the
same gene can result in the phenotypic spectrum of NPHP,
JBTS, and MKS. For example, NPHP1 mutations were reported
in patients with both NPHP and JBTS (Parisi et al., 2007),
whereas mutations in NPHP3, NPHP6/CEP290, and NPHP8/
RPGRIP1L are linked to all forms of disease along the NPHP-
JBTS-MKS spectrum (Baala et al., 2007) (Figure 1A). Because
these three diseases can be caused by mutation of the same
genes, we considered the hypothesis that these disorders may
link to defects in a specific set of cellular mechanisms and that
the associated proteins may interact and function in common
pathways. NPHP/JBTS/MKS proteins are rich in protein-protein
interaction domains (Figure S1A available online), suggesting
extensive protein-protein interactions. Studies using yeast two-
hybrid and coimmunoprecipitation (co-IP) discovered interac-
tions of NPHP1 with NPHP2, NPHP3, and NPHP4 (Mollet et al.,
2002, 2005; Olbrich et al., 2003; Otto et al., 2003). However, a
systematic connection between NPHP, JBTS, and MKS proteins
has not been explored.
The proteins encoded by genes mutated in NPHP, JBTS, and
MKS are found either within the primary cilium or at the basal
body, and several have homologs in Chlamydomonas (Hilde-
brandt et al., 2009a), suggesting that they participate in
conserved ciliary machinery. However, some of these proteins
are centrosomal even in the absence of cilia (Chang et al.,
2006), suggesting that these machines may function at the
centrosome, possibly to organize cell polarity or receptor traf-
ficking, rather than simply being a structural component of the
To better understand the molecular mechanisms underlying
NPHP-JBTS-MKS, we developed a high-confidence proteomic
strategy using the G-LAP tandem-affinity method (Torres et al.,
2009) to identify interactors associated with nine disease
proteins. Our data show that NPHP-JBTS-MKS proteins form
an interaction network that can be classified into three distinct
modules: NPHP1-4-8, NPHP5-6, and Mks. Assays for ciliogene-
sis and epithelial morphogenesis using three-dimensional (3D)
renal cultures suggest that NPHP1-4-8 links primarily to apical
organization defects seen in nephronophthisis, whereas ciliary
NPHP5-6 deficits appear central to retinal and potentially neural
deficiency, with Mks module and Hedgehog signaling defects
linked to the neural tube defects. Excitingly, our network building
strategy allowed us to propose candidates for new ciliopathy
disease genes, leading to the identification of the first human
mutations in the NPHP gene Ataxin10 (ATXN10) and JBTS
gene Tectonic2 (TCTN2).
Mapping of an NPHP-JBTS-MKS Ciliopathy
To better define physical interactions among the NPHP-JBTS-
MKS disease proteins, we used the G-LAP-Flp purification
strategy (Torres et al., 2009) to identify interacting proteins that
copurify with the nine disease gene products known at the
time that this study was initiated. The G-LAP-Flp strategy is an
optimized system for rapid generation of mammalian stable
cell lines that facilitates high-confidence and high-throughput
proteomic studies. We created clonal Flp-In cell lines stably
expressing a LAP tag (EGFP-TEV-S-peptide) fused to the amino
terminus of each individual disease protein (NPHP1, NPHP2/
inversin, NPHP3, NPHP4, NPHP5/IQCB1, NPHP6/CEP290,
NPHP8/RPGRIP1L, AHI1/Jouberin, and MKS1) in NIH 3T3 fibro-
blasts, mouse kidney inner medullary collecting duct (IMCD3)
cells, or human retinal pigment epithelial (RPE) cells. These cili-
ated cell models provide a physiologically relevant context for
studying ciliary assembly, signaling, and cystogenesis path-
ways. We then isolated complexes associated with each bait
protein by tandem affinity purification and identified interacting
proteins by mass spectrometry (MS) (Figure S1B).
To identify bona fide interactors, we optimized the LAP purifi-
cation procedure to avoid the possibility of carryover, which
would severely confound the analysis of a protein network
(Extended Experimental Procedures). For each candidate inter-
actor, we evaluated metric MS data including the total number
of peptides, the sum of ion currents contributing to a specific
(C–F) The NPHP-JBTS-MKS interaction network generated from the R script and visualized using Cytoscape. The entire network is shown in (C); individual
subgraphs that illustrate the 1-4-8 (IMCD3), 5-6 (IMCD3), and MKS (NIH 3T3) modules are shown in (D), (E), and (F). Ellipse, protein; single-headed arrows,
unreciprocated interactions (pointing to the hits); double-headed arrows (red), reciprocal interactions. Bait proteins and a subset of interactors are highlighted
using the color scheme described in Figure 1B.
See also Figure S1, Figure S2, Table S1, Table S2, Table S3, Table S5, Data S1, Data S2, and Data S3.
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 515
516 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.
mass spectrum (Sum TIC), and the percent sequence coverage.
Data from fifteen affinity purification protein mass spectrometry
(APMS) experiments were compiled into a single data set. If
any single spectrum from an identifiable peptide was found,
the corresponding protein was included (Table S1 and Data
S1). Using the method developed by Scholtens and Gentleman
mic network analysis (Extended Experimental Procedures and
Data S2), which we visualized using the Cytoscape software
package (Data S3) (Shannon et al., 2003).
This method creates a ranked list of the most probable
subgraphs, representing interactions (edges) between proteins
(nodes) in a graphical form (Figures 1D–1F and Figure S2).
Here, the nodes represent the individual NPHP, JBTS, and
MKS protein baits plus the identified hits. Hits identified in an
APMS experiment are hypothesized to interact with the bait
protein, and this connection is represented by an edge between
the bait node and the node representing the hit. If a newly
identified protein (node) shows a single interaction (edge) with
a bait, it is of lower informational value compared to a node
that shows multiple interactions with different baits; the pres-
ence of symmetric interactions, where two baits both identify
the other efficiently in the APMS experiment, is of the highest
value. Here, we began our analysis by first constructing a
network based solely on the physical interactions seen in our
APMS data set and later incorporating additional functional
data to further validate the network.
When the physical interaction data are analyzed using this
method, a striking disease protein network emerges. As high-
lighted in Figures 1B and 1C, we find that the most significant
interactors for the NPHP-JBTS-MKS disease proteins are, in
fact, other disease proteins from the same group. Our data
confirm some previously reported interactions, including the
NPHP1-NPHP2 and NPHP1-NPHP4 interactions (Mollet et al.,
2002; Otto et al., 2003). However, our analysis is more compre-
hensive and provides a quantitative view of the most abundant
interactors(TableS1). Tovalidate ournetwork, wetested several
newly identified interactions by coimmunoprecipitation and by
that NPHP-JBTS-MKS proteins do not form a single complex
but instead cluster into three biochemically distinct modules:
(1) NPHP1, NPHP4, NPHP8 (‘‘1-4-8’’), (2) NPHP5, NPHP6
(‘‘5-6’’), and (3) MKS1, MKS6 (‘‘MKS’’).
Below, we describe interactions within the specific modules.
NPHP1, NPHP4, and NPHP8 Interact and Localize to
Cell-Cell Contacts and the Ciliary Transition Zone
NPHP1, NPHP4, and NPHP8 show strong mutual interactions
via LAP tagging. LAP-NPHP1 purifications contained endoge-
nous NPHP4 and NPHP8 peptides in high abundance. Recipro-
cally, NPHP1 was identified in LAP-NPHP4 and LAP-NPHP8
purifications (Table S1 and Table S2). Visualizing purified LAP-
NPHP1 on silver-stained gels revealed a substoichiometric
band of NPHP8 and barely detectable NPHP4. LAP-NPHP4
purifications showed a band of NPHP8, NPHP1, and distinctive
breakdown products of NPHP4. LAP-NPHP8 showed high-effi-
ciency interactions with NPHP1 and NPHP4 (Figure 2A). Quite
possibly, NPHP1-4-8 form multiple or processed complexes.
We used in vitro binding to test whether these NPHP proteins
interact directly. We assayed whether in vitro-translated Myc-
tagged NPHP1, NPHP4, and NPHP8 immunoprecipitate HA-
tagged proteins produced by in vitro translation in wheat germ
extracts. We find that NPHP4 directly binds both NPHP1 and
NPHP8 in vitro and can bridge the interaction between NPHP1
and NPHP8, whereas NPHP1 and NPHP8 do not appear to
bind directly (Figures 2B, 2C, and 2H).
NPHP8, we investigated their subcellular localization. LAP-
NPHP1, LAP-NPHP4, and LAP-NPHP8 each localize diffusely
as cells approach confluence and develop into polarized epithe-
lial monolayers, these NPHP proteins accumulate to cell-cell
contacts, mostly basolateral of tight junctions (Figure 3A and
Figures S4A and S4B). The NPHP1-4-8 proteins can also be
found at a specified compartment that extends between the
basal body to the base of the axoneme, shown by costaining
with the mother centriole marker ODF2, centriole distal append-
age marker OFD1, and axonemal acetylated tubulin (Figures 3A
Figure 2. Validation of NPHP-JBTS-MKS Interactions Using Coimmunoprecipitation and In Vitro Binding Assays
(A) LAP-NPHP1, LAP-NPHP4, and LAP-NPHP8 were immunopurified from IMCD3 cells using anti-GFP antibody beads, eluted with TEV protease, and recap-
tured on S protein agarose. Eluates were separated on 4%–12% SDS-polyacrylamide gradient gels and were visualized by silver staining. NPHP1, NPHP4, and
NPHP8 species are noted by arrows.
(B and C) NPHP4 bridges the interaction between NPHP1 and NPHP8 in vitro. Myc (MT)-tagged and HA-tagged NPHP1, NPHP4, and NPHP8 were in vitro
Eluates were separated by SDS-PAGE and immunoblotted with an anti-HA antibody. HA-NPHP3 was used as a negative control.
(D) LAP-NPHP5 complexes were immunopurified from IMCD3 (left), RPE (middle), and NIH 3T3 (right) cells, and LAP-NPHP6 complexes were immunopurified
from IMCD3 cells as described in Figure 2A. Identified NPHP5 and NPHP6 species are noted by arrows.
(E) Interactions between NPHP5 and its associated proteins in vitro. Myc-tagged NPHP5 and HA-tagged interactors were in vitro translated and tested for direct
binding using the procedure described above. NPHP5 binds directly to NPHP6 via its N-terminal domain (NPHP6N).
(F) LAP-MKS1 complexes were immunopurified from NIH 3T3 cells using the procedure described in Figure 2A. Identified Mks1, B9d1, Tctn2, and Mks6 species
are noted by arrows.
(G) Validation of the interactions between Mks1 and copurified proteins Tctn2 and Ahi1. Myc-tagged Tctn2 or Ahi1 were coexpressed with Flag- or HA-tagged
Mks1 in HEK293T cells and were immunoprecipitated using anti-Myc beads or control IgG beads. Eluates were separated by SDS-PAGE and immunoblotted
with an anti-Flag or anti-HA antibody.
(H) Cartoon summarizing theinteractions among NPHP-JBTS-MKS proteins.Ellipse,protein;black line, interactionidentified by MS/MS;touching ellipses, direct
interactions validated by in vitro binding. NPHP1-4-8, NPHP5-6, and MKS modules are highlighted in blue, orange, and green, respectively.
See also Figure S3.
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 517
Inv compartment / Axoneme
518 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.
and 3B). This NPHP1-4-8 compartment is reminiscent of the
ciliary transition zone, believed to function as part of the ciliary
sensory machinery in worms (Fliegauf et al., 2006; Winkelbauer
et al., 2005). The localization of NPHP1-4-8 at transition zones
can appear even in sparse cells before the cell monolayer is fully
organized into an epithelial sheet with complete adherens and
apical junctions. This suggests that NPHP1-4-8 can be orga-
nized above the basal body independently from its organization
at cell-cell junctions, and the basal body may be sufficient to
organize the ‘‘1-4-8’’ compartment at the transition zone.
NPHP1-4-8 each contain C2 domains, which might mediate
interactions with phospholipids at cell-cell junctions or at the
Some previously reported interactors of NPHP1-4-8 were
not seen in our purifications, including NPHP3, NPHP6, and
a group of cortical regulators (Pyk2, p130Cas, PALS1, PATJ,
and Par6) (Benzing et al., 2001; Delous et al., 2009; Donaldson
et al., 2000; Olbrich et al., 2003; Murga-Zamalloa et al., 2010).
These absences likely reflect: (1) differences in the efficiency of
detecting interactions by APMS versus coimmunoprecipitation/
immunoblot, (2) the difficulty of detecting membrane-protein
interactions in detergent lysates, or (3) more interestingly, re-
wired interactions in different tissues. Notably, we did validate
that NPHP1 interacts with NPHP3 by coimmunoprecipitation,
showing that co-IP is more sensitive to show some interactions,
compared to copurification (Figure S3D).
NPHP5 and NPHP6 Form a Complex and Localize
to the Centrosome
Purifications of NPHP5 and NPHP6 consistently demonstrated
strong binding between NPHP5 (MW ?68 kD) and NPHP6/
(Figure2D,Table S1, and TableS2). Wevalidated thisinteraction
using in vitro-translated proteins and confirmed that NPHP5
binds directly to NPHP6 via an N-terminal domain spanning
amino acids 1–1207, consistent with a previously published
study (Scha ¨fer et al., 2008). In mammalian cells, NPHP6
has been shown to localize to centrosomes (Chang et al.,
2006). Consistently, LAP-NPHP5 and LAP-NPHP6 both colocal-
ize with the centrosome marker pericentrin in IMCD3 cells
(Figure 3C). LAP-NPHP5 also colocalizes with endogenous
NPHP6, but not with the centriole-distal appendage marker
OFD1 (Figure3D).Wetested whether either proteinwas required
to recruit the other to the centrosome by siRNA depletion of
NPHP5 or NPHP6. We found that NPHP5 failed to localize to
the centrosome in the absence of NPHP6 (Figure 3E). In 53%
of cells, LAP-NPHP5 is fully absent from pericentrin-positive
centrosomes, compared to only 9% of cells transfected
with a nontargeting siRNA. Conversely, depletion of NPHP5
had no effect on NPHP6 localization (data not shown). We
conclude that NPHP6 binds NPHP5 and recruits NPHP5 to the
NPHP5 also interacts with other NPHP proteins. We observed
copurification of NPHP5 with NPHP1-4-8 only in NIH 3T3 cells
and with NPHP2 only in IMCD3 cells. These interactions were
validated by co-IP in HEK293T cells but likely require additional
proteins to bridge the interactions because NPHP5 does not
bind directly to NPHP1, NPHP4, NPHP8, or NPHP2 in vitro
(Figures 2E and 2H). In contrast to the basal body localization
of NPHP5, NPHP2 was observed at the basal body but also
within the primary cilium (Figure 3F). A recent study suggested
that NPHP2 localized primarily to the proximal segment of the
axoneme (Shiba et al., 2010), which the authors termed the
‘‘inversin compartment.’’ Intriguingly, we observed NPHP2/
inversin compartment in a range of extensions along the
axonemal structure, beginning proximally but extending distally
along the axoneme (Figures 3F and 3G and Figure S4C). The
distinct localizations of NPHP1-4-8, NPHP5-6, and NPHP2
suggest a hierarchy for how these interactions are organized.
Further, the connections between these modules vary notably
among IMCD3, RPE, and NIH 3T3 cells, suggesting that these
modules may have distinct organization and functions in polar-
ized epithelial cells not seen in fibroblasts.
NPHP5 additionally copurified with proteins previously linked
to ciliogenesis. In all three cell lines, NPHP5 copurifies with
Sec3; in IMCD3 cells, NPHP5 also copurified with Sec8 and
Sec10 (Figure S2D). Sec3, Sec8, and Sec10 are components
of the exocyst complex, a protein complex that is important in
membrane trafficking and was recently shown to be required
for ciliogenesis (Zuo et al., 2009). NPHP5 at the basal body
may help to recruit the exocyst to a membrane compartment
at the cilia base. In IMCD3 cells, we also observed copurification
of NPHP5 with Ataxin 10 (Figure S2D), a protein that is linked to
Spinocerebellar Ataxia, a disease with distinctive deficiencies in
cerebellar signaling and function (Matsuura et al., 2000; dis-
cussed below). We validated the NPHP5-Ataxin10 interaction
by co-IP, but this interaction does not appear to be direct based
on in vitro binding (Figure S3A and Figures 2E and 2H).
Figure 3. Localization of NPHP 1-4-8, NPHP 5-6, and NPHP2 to the Ciliary Transition Zone, Centrosome, and the Inversin Compartment
(A) IMCD3 cells stably expressing LAP-NPHP1 (green), LAP-NPHP4 (green), or LAP-NPHP8 (green) were immunostained for pericentrin (PCNT, white) and
acetylated a-tubulin (ac-tub, red). AX, axoneme; TZ, transition zone; BB, basal body.
(C and D) NPHP5 and NPHP6 colocalize to the centrosome.
(C) IMCD3 cells stably expressing LAP-NPHP5 (green) or LAP-NPHP6 (green) were immunostained for pericentrin (PCNT, red).
(D) IMCD3 cells stably expressing LAP-NPHP5 (green) were immunostained for acetylated a-tubulin (ac-tub, red) and NPHP6 (white) or Ofd1 (white).
(E) Centrosomal localization of LAP-NPHP5 is disrupted upon depletion of NPHP6. IMCD3 LAP-NPHP5 (green) cells were transfected with siRNA against NPHP6
or control and then immunostained for pericentrin (PCNT, red). Nuclei were stained with Hoechst 33258 (blue).
(F) NPHP5-interacting protein NPHP2 localizes to the centrosome and to the cilium. IMCD3 LAP-NPHP2 cells (green) were immunostained for pericentrin (PCNT,
red) and acetylated a-tubulin (ac-tub, red). Arrows exemplify variable NPHP2/inversin compartment extensions along the axoneme.
(G) Percentage of cilia with a range of ‘‘inversin compartment/axoneme’’ ratios.
Scale bars, 10 um (A), 5 um (C and E), and 2 um (B, D, and F). See also Figure S4.
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 519
Figure 4. Functional Requirements for Ciliation and 3D Spheroid Formation Show Distinct Activities for the NPHP 1-4-8, NPHP 5-6, and MKS
(A) Depletion of NPHP5, NPHP6, and MKS1 causes ciliation defects. IMCD3 cells were transfected with siRNAs against individual disease genes, IFT88, or
control. Cells were fixed 72hr posttransfection and stained for acetylated a-tubulin (green), pericentrin (red), and DNA dye Hoechst 33528 (blue). Scale bar, 5um.
(B) Cilia were scored based on positive, adjacent staining of both pericentrin and acetylated a-tubulin. Percentage of nuclei with cilia was plotted (500–700 cells
counted). Error bars represent standard error. ***p < 0.002; **p < 0.02 (Student’s t test).
(C) Depletion of NPHP1, NPHP4, or NPHP8 cause spheroid defects in 3D kidney culture. IMCD3 cells were transferred to 3D collagen/Matrigel culture at 24 hr
posttransfection. Spheroids were fixed 72 hr later and immunostained for b-catenin (green) and ZO1 (red). Nuclei were stained with Hoechst 33528 (blue).
520 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.
Mks1 Binds to Proteins that are Important for Neural
Tube Closure, Including Mks6 and Tectonic
Extending from our core interaction network in Figure 1C, we
identified a third module consisting of Mks1 and its interacting
proteins (Figure 1F). Mks1 localizes to the base of the cilium in
vertebrates and nematodes (Bialas et al., 2009), and MKS1 is
mutated in type 1 Meckel-Gruber syndrome. Recent studies
show that Mks1 loss-of-function mouse mutant kerouac (krc)
exhibits neural tube patterning defects, polydactyly, exence-
phaly, and biliary malformations. These are similar to the known
defects in human MKS, believed to be linked to disruption of
Hh signaling (Weatherbee et al., 2009). To further characterize
this pathway, we purified proteins associated with Mks1 from
Hh-responsive NIH 3T3 cells. Mks1 copurified with all three
members of the Tectonic family of proteins (Tectonic 1–3)
(Tables S1 and S2). Tectonic is a family of three potentially
secreted or transmembrane proteins. Tectonic1 has been impli-
cated in Hh-mediated patterning of the neural tube in mouse
(Reiter and Skarnes, 2006), and our data suggest that Tectonic2
(Tctn2) is also important for Hh signaling (presented below).
Other Mks1-interacting proteins include Mks6/CC2D2A (MW
?188 kD) and B9d1 (MW ?23 kD), both of which can be visual-
ized on the silver-stained gel (Figure 2F). Mutations of MKS6
have been identified in MKS and JBTS patients and are associ-
ated with reduced ciliogenesis and neural tube defects in these
patients (Mougou-Zerelli et al., 2009; Tallila et al., 2008). B9d1,
along with B9d2 and Mks1, are the three known mammalian
proteins containing a B9 domain. C. elegans B9 proteins form
a complex that localizes to the ciliary base (Williams et al.,
2008). C. elegans B9 proteins function redundantly with nephro-
cystins to regulate sensory cilia morphology and behavior,
whereas individual mammalian B9 protein appears to function
more independently. Like Mks1, mouse B9d1 is important for
Hh signal transduction (B. Chih and A. Peterson, personal
communication). Therefore, Mks1 and its interactors may func-
tion as key regulators of the Hh signaling cascade to regulate
proper patterning of the neural tube. Mks1, Mks6, and Tectonic1
also bind to the Joubert syndrome protein Ahi1/Jouberin, which
in turn copurifies with NPHP2 (Figure 1F, Figure 2G, Figure S2B,
and Table S1), suggesting Ahi1 as a potential bridging molecule.
Functional Requirements for Ciliation, 3D Spheroid
Formation, and Hh Signaling Show Distinct Activities
for the NPHP 1-4-8, NPHP 5-6, and MKS modules
dysfunction. We therefore tested whether NPHP-JBTS-MKS
proteins are required for ciliogenesis. In IMCD3 cells, we found
that 61% of siRNA control-treated cells formed primary cilia,
as detected by staining for acetylated a-tubulin and pericentrin
(Figures 4A and 4B). As a positive control, we depleted Ift88,
an intraflagellar transport component that was previously shown
to be required for cilia formation (Pazour et al., 2000). As ex-
pected, depletion of Ift88 caused a dramatic decrease in ciliation
(Figures 4A and 4B). We then depleted Nphp5, Nphp6, Nphp2,
Nphp3, Nphp1, Nphp4, Nphp8, Ahi1, or Mks1 mRNAs by
?60%–95% using siRNAs (Figure S5B). Depletion of centroso-
esis defects. In contrast, normal numbers of cilia were observed
in cells depleted of Nphp1, Nphp4, Nphp8, Nphp2, Nphp3,
or Ahi1 (Figure 4B and Figure S5A). Our data suggest that
Nphp5, Nphp6, and Mks1 are critical for ciliogenesis, whereas
the other NPHP-JBTS-MKS proteins might not be strictly
required for ciliogenesis under standard cell culture conditions
or without much more efficient knockdown. These proteins
may instead be important for establishing tissue architecture,
regulating ciliary signaling or only affecting ciliogenesis in vivo.
To investigate the roles of NPHP-JBTS-MKS proteins in tissue
architecture, we tested the effect of depleting these proteins in
a system that reflects the cell biology of the kidney-collecting
duct and thus reports on defects seen in cystic kidney diseases.
Spheroid growth in 3D culture allows epithelial cells to organize
into polarized, ductal structures that resemble their in vivo
architecture. The spheroid systems are unique models of
epithelial cell polarity and signaling (Supplemental References).
Notably, IMCD3 cells are derived from collecting ducts at the
cortical-medullary border, thought to be the key target cells in
nephronophthisis. Using this model system, we transfected
IMCD3 cells with siRNAs for Ift88, Nphp1, Nphp4, Nphp8,
Nphp5, Nphp6, Nphp2, Nphp3, Ahi1, or Mks1 and then plated
the transfected cells in Matrigel to induce 3D spheroid growth.
After 3 days, control siRNA-treated cells formed spheroid struc-
tures with a clear lumen, apical cilia, defined tight junctions, and
clear basolateral structures. Cells depleted of Ift88 or Nphp6
developed few or grossly affected spheroids, appearing as
clumps of cells with few cilia evident, suggesting that ciliary
genes are strongly important in spheroid formation. Consis-
tently, depletion of Nphp5 or Mks1, genes that are required for
ciliation, likewise caused spheroids to form with severe defects
and few cilia evident. In striking contrast, cells depleted of
Nphp1, Nphp4, Nphp8, and Nphp2 developed spheroids with
irregular lumens, reduced sphericity, fewer ZO1-positive tight
junctions, and perturbed localization of b-catenin (Figures
4C–4E). However, no gross abnormality of ciliogenesis was
evident in these spheroids (data not shown). The apparent
partition in functional requirements between ciliation and 3D
spheroid formation suggests a distinct activity for the Nphp
1-4-8 module in organizing apical junctions versus the Nphp
5-6 and Mks1 modules in organizing cilia. We observed very
modest disorganization of spheroids in cells depleted of Ahi1
or Nphp3 (Figures 4D and 4E), suggesting these NPHP proteins
(D) Percentage of spheroids with defects. 400–700 spheroids were counted, and error bars represent standard error. NS, no spheroids formed, shown as 100%
defective. ***p < 0.001; **p < 0.01 (Student’s t test).
of variation (CV) was calculated using the formula: CV = standard deviation (R1,R2,R3) / mean (R1,R2,R3). Raw CV data from each knockdown are plotted along
with the outlier box plot. Lower quartile, 25th percentile; upper quartile, 75th percentile; top line, upper quartile + 1.5 3 interquartile range; bottom line, lower
quartile, 1.5 3 interquartile range; middle line, 50th percentile; data points outside of the lines are outliers.
See also Figure S4, Figure S5, Table S4, Table S6, and Table S7.
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 521
may participate in mechanisms that are distinct from ciliation or
1-4-8, 5-6, and Mks modules in apical organization and ciliary
function. Knockdown of nphp2, nphp5, and nphp6 leads to
body curvature defects (Otto et al., 2003; Scha ¨fer et al., 2008).
We have also found that knockdown of additional NPHP,
JBTS, and MKS proteins similarly results in body axis alterations
(Figure S5D). Kupffer’s vesicle (KV), the ciliated organ implicated
in zebrafish left-right patterning (Essner et al., 2005), is also dis-
rupted in these mutants (Figure S5C). The KV arises from dorsal
forerunner cells (DFCs). DFCs migrate attached to the overlying
surface epithelium and rearrange into rosette-like epithelial
structures and then coalesce into a single rosette that differenti-
ates into the KV with a ciliated lumen at its apical center. In
addition to cilia integrity, polarity cues and apical organization
are also crucial for KV morphogenesis and function (Oteı ´za
et al., 2008), consistent with our observation that NPHP1/4/8,
NPHP5, and MKS1 morphants all show KV defects.
Disruption of Hh signaling is thought to partly account for
the neural tube and limb phenotypes seen in MKS patients
(Weatherbee et al., 2009). MKS1 is critical for ciliogenesis,
spheroid formation, and Hh signaling. However, there is no clear
evidence demonstrating that NPHP5/6 are directly involved
in Hh signaling. To investigate the functions of NPHP5/6 in Hh
signaling, we used the standard S12 Gli-luciferase Hh signaling
assay and observed that siRNA knockdowns of Nphp5 and
Nphp6 had no effects on Hh signal transduction (Table S4).
Surprisingly, ciliogenesis was also not perturbed in these cells
(Table S4), suggesting that NPHP5/6 may function differently
in osteoblasts (S12 cells) versus in polarized epithelial cells
(IMCD3). In contrast to the requirement of Mks1 in Hh signal
transduction, the specific roles of NPHP5 and NPHP6 in Hh
signaling remain to be clarified.
Identification of Ataxin10 and Tectonic2 as New
NPHP-JBTS Disease Proteins
Our analysis of the NPHP-JBTS-MKS network reveals extensive
physical interactions among known disease proteins as well as
with proteins not currently implicated in cilia-associated dis-
orders. We reasoned that the physical interactions between
specific disease proteins lead to the phenotypic overlap of these
diseases and therefore hypothesized that some interacting
proteins from our analysis may represent unrecognized disease
loci. Remarkably, we found multiple recently reported disease
proteins identified independently from our interaction network.
These proteins include CC2D2A/MKS6 (Mougou-Zerelli et al.,
2009; Tallila et al., 2008), which interacts with AHI1 and MKS1,
and Nek8/NPHP9, which interacts with NPHP2 (Figure 1F, Fig-
ure S2E, and Figure S3C).
With the potential to use proteomic network analysis as an
unbiased means to discover new disease genes, we submitted
38 candidate genes to total genome linkage analysis to look
vals. This method is based on SNP analysis to predict candidate
intervals that are linked to causal mutations. Genes within these
intervals can then be sequenced to establish the presence of
homozygous mutation. In small families lacking pedigree infor-
mation, the number of candidate intervals can be large and
a systematic approach with complete exome sequencing pro-
vided one solution to identifying specific disease alleles
(Hildebrandt et al., 2009b). Here, we imagined that using our
high-confidence proteomic hits would provide an enriched
set to discover disease genes in NPHP/JBST/MKS patients.
to NPHP/JBTS disease: Ataxin10 (ATXN10) and Tectonic2
First, wenoted a genomic region of extensive homozygosity of
11.6 Mb on chromosome 22, with a high nonparametric linkage
score for three affected siblings in a consanguineous family
(A1197) from Turkey (Figure S6A). After genome analysis with
1M Affymetrix SNP chip, a homozygous ATXN10 mutation
(IVS8-3T > G) was identified in all three affected siblings. All
sies, consistent with nephronophthisis (Figure 5A). One of the
siblings additionally suffered from seizures and had evidence
of cerebral atrophy by imaging. This mutation was absent from
90 healthy Caucasian control samples and 86 ethnically
matched control individuals. We identified the protein encoded
by ATXN10, Ataxin10, as an NPHP5-interacting protein.
Similarly, we observed a region of extensive homozygosity in
a particular locus near the gene TCTN2 in six patients with
consanguineous background (Figure S6B). After sequencing all
exons of TCTN2, a mutation was found in one family (A1443)
from Turkey at IVS10-1G > A, affecting the obligatory splice
acceptor site, which resulted in skipping of exon 11 (Figures
5A and 5B). The mutation was also found in a heterozygous state
in the parents of a 6-year-old female who was homozygous for
the mutation. She had been previously diagnosed with Joubert
syndrome due to cerebellar vermis aplasia and hypotonia and
had no evidence for renal disease. Again, this mutation was
absent from the same control sets of Caucasian and ethnically
matched healthy individuals.
With the evidence linking TCTN2 to Joubert syndrome, we
screened additional patients and identified another two Joubert
families with frameshift or nonsense mutationsin TCTN2. Patient
UW95-3 had gross motor and communication delays and
of cardiovascular, renal, or liver disease by ultrasound and labo-
ratory testing (Figure 5A). Brain MRI revealed the molar tooth
sign (Figure 5C), consistent with Joubert syndrome. A homozy-
gous mutation was identified in TCTN2 exon 1 (c.77InsG;
p.D26GfsX51) that results in frameshift and a premature stop
codon (Figure S6C). Family MR20 has four affected siblings
with consanguineous Pakistani background. All four patients
developed childhood-onset Joubert syndrome with extremely
poor learning abilities. Brain MRI revealed the molar tooth
sign (Figure 5C). A homozygous nonsense mutation in TCTN2
exon 16 (c.C1873T; p.Q625X) was identified in all four that
were affected, but not in unaffected siblings (Figure 5A and
Thus, from a list of 38 candidates curated by our proteomic
network analysis and a modest number of patients tested, two
new human disease genes were identified.
522 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.
Tctn2 Regulates Hh Signaling and Ciliogenesis
Tctn2 was identified as an interactor of Mks1, itself shown to
regulate Hh-dependent neural tube patterning in vivo (Weather-
bee et al., 2009). The human TCTN2 mutations that we identified
areassociated with neural developmental defects. Based on this
observation, we hypothesized that Tctn2 could be a regulator of
Hh signaling. To test this hypothesis, we generated Tctn2 null
mice. On a mixed 129/Bl6 background, Tctn2 mutants have fully
penetrant neural tube closure defects, and exencephaly is
apparent at E13.5 (Figure 6A). On a Bl6 background, Tctn2?/?
embryos exhibit microphthalmia, cleft palate, and polydactyly
Tctn2 mutants also have ventricular septal defects (Figure 6D)
and can display right-sided stomach (Figure 6E) phenotypes
characteristic of ciliary defects. To determine whether Tctn2 is
requiredforcilia functionor formation, weexamined primarycilia
in mouse embryonic fibroblasts (MEFs) and neural tubes. Tctn2
was required for ciliogenesis in isolated cells and in vivo, consis-
tent with TCTN2 being a ciliopathy gene (Figures 6F and 6G).
High-level Hh signaling is required for formation of the floor
plate (Sasaki and Hogan, 1994). Tctn2 mutants lack a morpho-
logically distinct floor plate, and examination of FoxA2 expres-
sion in Tctn2?/?embryos revealed that the floor plate was not
specified. Similarly, Pax6, which is repressed by Hh signaling,
was ventrally expanded in the absence of Tctn2. Asevere reduc-
tion in Nkx2.2-expressing V3 interneuron progenitors and Islet1/
2-expressing motor neurons further suggested defects in Hh-
dependent patterning in the absence of Tctn2 (Figure 6H).
To directly determine whether Tctn2 is important for Hh
transduction, we assayed Ptc1 and Gli1, general transcriptional
targets of Hh signaling, in wild-type and Tctn2?/?MEFs.
Following pathway activation by addition of a Smoothened
agonist (SAG), both Ptc1 and Gli1 are induced ?20-fold in
wild-type MEFs, whereas Tctn2?/?MEFs display negligible
responsiveness (Figure 6I). Tctn2?/?embryos also exhibited
increased amounts of full-length, unprocessed (Gli3-190) Gli3
protein (Figure 6J), indicating that Tctn2 is important for Gli3
processing and function. Thus, Tctn2?/?neural tubes have
Gene Family # Origin
Kidney (Age at
ESRF in years)
Eye Brain Other
NPHP, Bx (2) NAD
Died at age 2,
NPHP (2) NAD NAD Liver fibrosis
NPHP (2) NAD NAD NAD
No NPHP at
age 6 yr
vermis aplasia, MTS
Nonsense N/A N/A JBTS, MTS N/A
UW95-3 East Indian
Frameshift NAD N/A JBTS, MTS NAD
Ala His Gln Lys Gly Tyr Gln Leu
Exon 10 Exon 12
Figure 5. Identification of ATXN10 and TCTN2 as New NPHP and JBTS Disease Genes
(A)Genotypeandphenotypeofpatientswithmutations inATXN10and TCTN2.Bx,biopsycompatiblewithNPHP; MTS,molartooth sign;NAD,nothingabnormal
detected; N/A, clinical data not available.
(B) RT-PCR was performed in Joubert syndrome patient A1443 using cDNA primers to exons 7 and 14 of TCTN2. Sequencing revealed an in-frame skipping of
(C) MRI images (T1) of Joubert syndrome patients MR20-3 and UW95-3 showing the molar tooth sign (MTS).
See also Figure S6.
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 523
Pax3 Pax6 Nkx2.2 Pax6 Isl1/2 Pax6
Gli1 levels (fold)
Ptc1 levels (fold)
524 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.
defects that are characteristic of altered Hh signal transduction,
and Tctn2?/?cells fail to respond to Hh agonists, suggesting
that cerebellar defects in affected individuals with TCTN2 muta-
tions may reflect defects in ciliogenesis linked to reduced Hh
The NPHP-JBTS-MKS Interactome: A Multimodule
Network Linking Ciliopathies
Using a high-confidence proteomic strategy, we have discov-
ered and begun a systematic mapping of an NPHP-JBTS-MKS
interaction network. In contrast to our earlier studies on the
Bardet-Biedl syndrome (BBS) (Nachury et al., 2007), in which
seven highly conserved BBS proteins formed a single, monodis-
perse complex, the NPHP-JBTS-MKS proteins do not form
a single complex. Instead, this large group of disease proteins
can be clustered into three biochemically and functionally
distinct modules, in which proteins within the first two modules
show notable colocalization (Figure 7). Our studies support
that genetic loss of function within each individual module drives
a unique mechanism contributing to the specific histopathologic
features of these disorders. The first module consists of NPHP1,
NPHP4, and NPHP8, localized to cell-cell contacts and to the
ciliary transition zone. They are not strongly required for ciliation,
as assayed in our in vitro models. However, when kidney epithe-
lial cells are deprived of these proteins, they form disorganized
spheroids in 3D culture characterized by irregular lumens, loss
of tight junctions, and perturbed localization of b-catenin.
NPHP1 and NPHP4 have been reported to interact with polarity
proteins PALS1, PATJ, and Par6, and depletion of NPHP1 or
NPHP4 in MDCK cells results in delayed tight junction formation
(Delous et al., 2009). These observations together with our data
support the hypothesis that NPHP1-4-8 module organizes
specialized structures at the apical surface of polarized cells
and thus may participate in pathways that are important for
epithelial morphogenesis and the establishment of tissue archi-
tecture. Given the effects of NPHP1-4-8 deficiency in pediatric
renal disease, it will be important to examine whether this
module is affected in tissues that are frequently impaired in
patients with cystic kidney disease, including liver and pancreas.
Unlike the 1-4-8 module, the centrosomal module proteins
NPHP5 and NPHP6 are indispensable for ciliation in IMCD3
cells; furthermore, cells fail to develop normal spheroids when
depleted of NPHP5 or NPHP6, underscoring the role of centro-
some/cilia integrity in tissue organization. MKS1 and its interact-
ing proteins are grouped in the third module, characterized by
their functional connection to neural tube development and Hh
Several of the NPHP proteins appear to bridge the three major
the inversin compartment, but the role of this new structure
remains mysterious. Our preliminary data suggest that NPHP3
is particularly important for localizing specific G protein-coupled
receptors (GPCRs) to the cilia (L.S. and P.K.J., unpublished
data), which provides a first clue linking NPHP proteins to
specific ciliary GPCR signaling pathways.
Collectively, the NPHP-JBTS-MKS interactome has provided
new biochemical evidence that these disorders are highly con-
nected and has suggested specific underlying mechanisms
Figure 6. Tctn2 Is Required for Ciliogenesis and Hh Signaling Transduction
(A–C) E13.5 Tctn2?/?embryos on a mixed 129/Bl6 background have fully penetrant cranial exencephaly. On a Bl6 background, E13.5 Tctn2?/?embryos display
(B) Microphthalmia (arrow) and (C) single hindlimb preaxial polydactyly, either bilaterally or unilaterally (asterisk).
(D and E) Hematoxylin and eosin staining of E14.5 Tctn2?/?embryos reveals (D) ventricular septal defects (black arrow) and (E) laterality defects as evidenced by
randomized stomach situs.
(F) Mouse embryonic fibroblasts (MEFs) derived from E12.5 Tctn2?+/?embryos are ciliated, whereas Tctn2?/?embryo-derived MEFs rarely generate cilia.
Acetylated tubulin (green) marks cilia, Ninein (red) marks basal bodies and centrosomes, DAPI (blue) marks nuclei.
(G) Immunofluorescent detection of Arl13b (red) in E9.5 transverse neural tube sections indicates that Tctn2?/?embryos display few and abnormal cilia.
(H)Tctn2 isrequiredfor patterningof theventralneuraltube. Immunofluorescence ofE9.5transverse sections between theheart andhindlimbstainedforPax6 in
red and, in green, FoxA2, Nkx2.2, Islet1/2, or Pax3.
(I)LevelsoftheHh transcriptional targetsGli1 andPtc1 wereassessedby qPCRinMEFsderivedfromE12.5Tctn2+/+and Tctn2?/?littermate embryosstimulated
with DMSO (vehicle) or smoothened agonist (SAG) for 18 hr. Tctn2+/+MEFs upregulate Gli1 and Ptc1 20- to 25-fold following SAG addition, whereas Tctn2?/?
MEFs are unresponsive. Experiments were performed three times in triplicate and values normalized to b-actin and presented as relative levels ± SEM.
***p < 0.001 (Student’s t test).
(J) Lysates from E13.5 Tctn2+/+and Tctn2?/?littermate embryos immunoblotted for Gli3. In wild-type embryos, the majority of Gli3 is processed into a truncated
repressor form (R). Tctn2 mutants have increased levels of unprocessed full-length (FL) Gli3.
See also Figure S7.
Figure 7. A Model for the NPHP-Joubert-Meckel-Gruber Network
Three interacting modules link centrosomal proteins toapicalorganizationand
to a Hedgehog regulatory network.
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 525
leading to disease progression. Our 3D culture system appears
to effectively mirror requirements for genes that suppress cystic
kidney disease, including those that function to organize apical
structures in the cell, notably NPHP1-4-8. For other genes
such as NPHP5, NPHP6, and Mks1, broader defects in centro-
some/cilia integrity and ciliary signaling may lead to tissue failure
not only in the kidney, but also in a variety of other organs,
including the neural tube and eye. Therefore, to our under-
standing, not all of the ‘‘ciliopathies’’ are simply caused by the
absence of cilia per se. They rather represent a manifestation
of defects in multiple interlinked cellular mechanisms. More
intriguingly, whereas mildrenalcysts mayarise simplyasa result
of tissue organization defect, lack of centrosome/cilia integrity
would generally predict more severe phenotypes, notably retinal
linked to cerebellar malformation and neural tube defects.
The NPHP-JBTS-MKS Network Is Distinct from BBSome
and IFT Complexes
Bardet-Biedl syndrome (BBS) shares a number of common
phenotypes with NPHP, JBTS, and MKS, including kidney cysts,
retinal degeneration, polydactyly, and mental retardation. There
is a single documented BBS family with mutations in each of
MKS1/BBS13 and NPHP6/BBS14, suggesting, at a minimum,
a genetic interaction between BBS and MKS (reviewed by Zagh-
loul and Katsanis, 2009). However, we did not observe notable
physical interactions between the NPHP-JBTS-MKS network
and components of BBSome, other BBS proteins, or more
than 14 BBS1-interacting proteins (C. Westlake and P.K.J.,
unpublished data). A reasonable hypothesis is that the shared
phenotypes are likely due to broader disruption of some key
regulatory pathways, such as the Hh signaling, Wnt signaling,
cell polarity, or centrosome control of the cytoskeleton.
We also evaluated whether the NPHP-JBTS-MKS network
proteins link to intraflagellar transport (IFT). The IFT process is
essential for the formation of cilia, and defects in IFT may lead
to cystic kidney disease and retinal degeneration (Davenport
et al., 2007; Rosenbaum and Witman, 2002). Interestingly,
NPHP-JBTS-MKS network also does not overlap with compo-
nents of IFT-A or IFT-B complex proteins (S. Mukhopadhyay
and P.K.J., unpublished data), with the notable exception of an
interaction between NPHP5 and IFT122 (Table S1). A recent
publication suggests that Chlamydomonas NPHP6/CEP290 is
a transition zone protein that is important for tethering flagellar
membrane to the transition zone, and loss of NPHP6/CEP290
results in an imbalance of IFT complexes in the flagellum (Craige
participate in a mechanism anchoring the transition zone to the
centrosome and cell cortex. It will be intriguing to determine
whether any of the mammalian transition zone proteins (such
as NPHP1, 4, and 8) or centrosomal proteins NPHP5/6 function-
ally link to IFT.
High-Confidence Proteomic Analysis Accelerates
Discovery of New Disease Genes
The identification of an NPHP-JBTS-MKS network not only
linked human genetics with underlying cellular mechanisms,
but also accelerated the discovery of new NPHP-JBTS disease
genes. The causative genes are still unknown in ?70% of
patients with NPHP (Hildebrandt et al., 2009a). Employing
protein interaction data to predict new candidate genes involved
in human genetic disorders has been explored (Lim et al., 2006;
Goh et al., 2007; Supplemental References). However, lack of
orthogonal information remains a major challenge in discovering
new disease genes (Sowa et al., 2009). Our proteomic strategy
has proved to be a highly effective approach, confirming two
new disease loci among 38 candidate genes that were sug-
gested by our network.
A pentanucleotide expansion of ATXN10 results in Spinocere-
bellar ataxia type 10, a neurodegenerative disease involving
cerebellar dysfunction leading to ataxia and seizures (Matsuura
et al., 2000). Additionally, Atxn10 has a proposed role in cere-
bellar neuron survival and neuritogenesis through an interaction
with G protein b2 subunit (Waragai et al., 2006). Depletion of
Atxn10 in 3D kidney culture leads to modest defects in both
spheroid organization and ciliogenesis (Figures S6E and S6F).
We hypothesize that these defects may directly contribute to
the kidney phenotypes observed in the NPHP patients. Further
investigation will help to clarify the various functions of Atxn10
in Nephronophthisis and in Spinocerebellar ataxia.
Tctn2 is a member of the Tectonic family proteins (Reiter and
Skarnes, 2006) and, similar to Tctn1, is important for Hh signal
transduction. Intriguingly, whereas Tctn2 is indispensible for cil-
iogenesis in the neural tube, knockdown of Tctn2 in kidney
IMCD3 cells only causes modest ciliation and spheroid defects
(Figures S6E and S6F). Such distinct requirements may reflect
differences in expression profiles as well as the complexity of
the organs involved. Indeed, Tctn2 is highly expressed in embry-
onic brain tissues but below the limit of detection in the kidney
(Figure S6G). The tissue distribution pattern of Tctn2 and the
differences in requirements for ciliogenesis are consistent with
the TCTN2 patient phenotypes, which show cerebellar vermis
aplasia but no renal disease.
Included in our NPHP-JBTS-MKS network are numerous new
interacting proteins. Many of those proteins are functioning in
fundamental cellular processes, such as cytoskeletal organiza-
tion, intracellular transport, and enzymatic reactions (Table S3).
Therefore, the NPHP-JBTS-MKS network has provided a road-
map not only for discovering new ciliopathy genes, but also for
unveiling novel disease pathways. Moreover, because cell
polarity and ciliary signaling defects have been implicated in
a number of other diseases, notably cancer, the NPHP-JBTS-
MKS network may also help to advance our understanding of
cancer and suggest potential therapeutic targets.
Proteomic Network Analysis
Data from 15 affinity purification protein mass spectrometry experiments were
compiled into a single data set. If any single spectrum from an identifiable
peptide was found, the corresponding protein was included. Of the resulting
850 unique hits, 12 proteins under current investigation were excluded from
the data set; all protein identifiers were mapped to Ensemble Gene IDs and
to common gene names. This data set is available as the tab-delimited file
Data S1 in the Supplemental Information. Further processing reduced this
data set to one comprising the results of the 3T3 and IMCD3 experiments
only and removed proteins that are either annotated to the ‘‘Keratin filament’’
526 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.
term in the GO Cellular Component Ontology or that include ‘‘keratin’’ in the
gene name.Thecompletepeptide and scoring information is available inTable
S5. An adjacency matrix was compiled for each cell line experiment, and from
these matrices, we derived two interaction networks. Each network’s maximal
bait-hit complete subgraphs were found and were merged using sensitivity
and specificity parameters of 0.7 and 0.75, respectively (Scholtens et al.,
2005). These resulting estimates of protein complex composition were plotted
from within R, and the entire networks were exported for display in Cytoscape
(Shannon et al., 2003). Enrichment of each network and its inferred subcom-
plexes for GO terms was assessed using Fisher’s exact test, with the universe
of genes defined as the set of mouse Ensemble Gene IDs having GO term
annotations. Although this test does not explicitly account for correlation of
GO term enrichment due to the ontologies’ graph structures, it serves to
provide a general view of the processes, functions, and locations of the
networks and their subgraphs. All data set manipulations and subsequent
analyses were performed using R version 2.11.1. Analysis of subgraphs
used the package ‘‘apComplex’’ version 2.14.0 (Scholtens, 2004), and gene
set enrichment analysis for GO terms used ‘‘topGO’’ version 1.16.2. The anal-
ysisscriptandsession information areincluded inDataS2intheSupplemental
figures, seven tables, and three data files and can be found with this article
online at doi:10.1016/j.cell.2011.04.019.
The authors acknowledge expert advice and contributions from Chris West-
lake, Guowei Fang, Ben Chih, Andy Peterson, Cecile Chalouni, John S. Beck,
Darryl Y. Nishimura, Charles C. Searby, Martin Griebel, John Neveu, Bogdan
Budnik, Renee Robinson, Alex Loktev, Jorge Torres, Saikat Mukhopadhyay,
Dirk Siepe, and Kevin Wright. We acknowledge the following support: J.J.M.,
NIH Medical Scientist Training Program Grant GM07365-33; R.H.G., the
Netherlands Organisation for Scientific Research VIDI grant 016.066.354 and
disciplinary Research Fellowship, University of Iowa; J.F.O., NIH grant
DK071108; D.C.S., NIH Grant CA112369; J.B.V., Canadian Institutes of Health
Research grant MOP-102758; D.A.D., KL2RR025015 and R01NS064077;
J.F.R., NIH Grant R01-AR054396, the March of Dimes, the Burroughs Well-
dation; V.C.S., NIH Grants R01-EY11298, R01-EY017168, the Roy J. Carver
Charitable Trust, Carver Endowment for Molecular Ophthalmology, and
Research to Prevent Blindness. V.C.S. is an HHMI investigator. F.H. is an
HHMI Investigator,a Doris Duke Distinguished Clinical Scientist, and aFreder-
ick G.L. Huetwell Professor. F.H. acknowledges support from the NIH grants
(DK1068306, DK1069274, and DK090917). L.S., M.J.B., X.W., S.J.S., M.K.,
Received: October 1, 2010
Revised: March 16, 2011
Accepted: April 27, 2011
Published: May 12, 2011
Baala, L., Audollent, S., Martinovic, J., Ozilou, C., Babron, M.C., Sivananda-
moorthy, S., Saunier, S., Salomon, R., Gonzales, M., Rattenberry, E., et al.
(2007). Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel
syndrome. Am. J. Hum. Genet. 81, 170–179.
Benzing, T., Gerke, P., Ho ¨pker, K., Hildebrandt, F., Kim, E., and Walz, G.
(2001). Nephrocystin interacts with Pyk2, p130(Cas), and tensin and triggers
phosphorylation of Pyk2. Proc. Natl. Acad. Sci. USA 98, 9784–9789.
Bialas, N.J., Inglis, P.N., Li, C., Robinson, J.F., Parker, J.D., Healey, M.P.,
Davis, E.E., Inglis, C.D., Toivonen, T., Cottell, D.C., et al. (2009). Functional
interactions between the ciliopathy-associated Meckel syndrome 1 (MKS1)
protein and two novel MKS1-related (MKSR) proteins. J. Cell Sci. 122,
Chang, B., Khanna, H., Hawes, N., Jimeno, D., He, S., Lillo, C., Parapuram,
S.K., Cheng, H., Scott, A., Hurd, R.E., et al. (2006). In-frame deletion in a novel
and results in early-onset retinal degeneration in the rd16 mouse. Hum. Mol.
Genet. 15, 1847–1857.
Corbit, K.C., Aanstad, P., Singla, V., Norman, A.R., Stainier, D.Y., and Reiter,
J.F. (2005). Vertebrate Smoothened functions at the primary cilium. Nature
Craige, B., Tsao, C.C., Diener, D.R., Hou, Y., Lechtreck, K.F., Rosenbaum,
J.L., and Witman, G.B. (2010). CEP290 tethers flagellar transition zone micro-
tubules to the membrane and regulates flagellar protein content. J. Cell Biol.
Davenport, J.R., Watts, A.J., Roper, V.C., Croyle, M.J., van Groen, T., Wyss,
J.M., Nagy, T.R., Kesterson, R.A., and Yoder, B.K. (2007). Disruption of intra-
flagellar transport in adult mice leads to obesity and slow-onset cystic kidney
disease. Curr. Biol. 17, 1586–1594.
Delous, M.,Hellman,N.E.,Gaude ´,H.M., Silbermann, F.,Le Bivic,A.,Salomon,
R., Antignac, C., and Saunier, S. (2009). Nephrocystin-1 and nephrocystin-4
are required for epithelial morphogenesis and associate with PALS1/PATJ
and Par6. Hum. Mol. Genet. 18, 4711–4723.
Donaldson, J.C., Dempsey, P.J., Reddy, S., Bouton, A.H., Coffey, R.J., and
cystin and both proteins localize to cell-cell contacts of polarized epithelial
cells. Exp. Cell Res. 256, 168–178.
Essner, J.J., Amack, J.D., Nyholm, M.K., Harris, E.B., and Yost, H.J. (2005).
Kupffer’s vesicle is a ciliated organ of asymmetry in the zebrafish embryo
that initiates left-right development of the brain, heart and gut. Development
Fliegauf, M., Horvath, J., von Schnakenburg, C., Olbrich, H., Mu ¨ller, D.,
Thumfart, J., Schermer, B., Pazour, G.J., Neumann, H.P., Zentgraf, H., et al.
(2006). Nephrocystin specifically localizes to the transition zone of renal and
respiratory cilia and photoreceptor connecting cilia. J. Am. Soc. Nephrol. 17,
Goh, K.I., Cusick, M.E., Valle, D., Childs, B., Vidal, M., and Baraba ´si, A.L.
(2007). The human disease network. Proc. Natl. Acad. Sci. USA 104, 8685–
mechanisms of a ciliopathy. J. Am. Soc. Nephrol. 20, 23–35.
Hildebrandt, F., Heeringa, S.F., Ru ¨schendorf, F., Attanasio, M., Nu ¨rnberg, G.,
Becker, C., Seelow, D., Huebner, N., Chernin, G., Vlangos, C.N., et al. (2009b).
A systematic approach to mapping recessive disease genes in individuals
from outbred populations. PLoS Genet. 5, e1000353.
Huangfu, D., Liu, A., Rakeman, A.S., Murcia, N.S., Niswander, L., and
Anderson,K.V.(2003).Hedgehog signalling inthemouse requiresintraflagellar
transport proteins. Nature 426, 83–87.
Lee, J.H., and Gleeson, J.G. (2010). The role of primary cilia in neuronal func-
tion. Neurobiol. Dis. 38, 167–172.
Lim, J., Hao, T., Shaw, C., Patel, A.J., Szabo ´, G., Rual, J.F., Fisk, C.J., Li, N.,
Smolyar, A., Hill, D.E., et al. (2006). A protein-protein interaction network for
human inherited ataxias and disorders of Purkinje cell degeneration. Cell
Matsuura, T., Yamagata, T., Burgess, D.L., Rasmussen, A., Grewal, R.P.,
Watase, K., Khajavi, M., McCall, A.E., Davis, C.F., Zu, L., et al. (2000). Large
expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia
type 10. Nat. Genet. 26, 191–194.
Mollet, G., Salomon, R., Gribouval, O., Silbermann, F., Bacq, D., Landthaler,
G., Milford, D., Nayir, A., Rizzoni, G., Antignac, C., and Saunier, S. (2002).
The gene mutated in juvenile nephronophthisis type 4 encodes a novel protein
that interacts with nephrocystin. Nat. Genet. 32, 300–305.
Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc. 527
Mollet, G., Silbermann, F., Delous, M., Salomon, R., Antignac, C., and Saunier, Download full-text
S. (2005). Characterization of the nephrocystin/nephrocystin-4 complex and
subcellular localization of nephrocystin-4 to primary cilia and centrosomes.
Hum. Mol. Genet. 14, 645–656.
Mougou-Zerelli, S., Thomas, S., Szenker, E., Audollent, S., Elkhartoufi, N.,
Babarit, C., Romano, S., Salomon, R., Amiel, J., Esculpavit, C., et al. (2009).
CC2D2A mutations in Meckel and Joubert syndromes indicate a genotype-
phenotype correlation. Hum. Mutat. 30, 1574–1582.
Murga-Zamalloa, C.A., Desai, N.J., Hildebrandt, F., and Khanna, H. (2010).
Interaction of ciliary disease protein retinitis pigmentosa GTPase regulator
with nephronophthisis-associated proteins in mammalian retinas. Mol. Vis.
Nachury, M.V., Loktev, A.V., Zhang, Q., Westlake, C.J., Pera ¨nen, J., Merdes,
A., Slusarski, D.C., Scheller, R.H., Bazan, J.F., Sheffield, V.C., and Jackson,
P.K. (2007). A core complex of BBS proteins cooperates with the GTPase
Rab8 to promote ciliary membrane biogenesis. Cell 129, 1201–1213.
Olbrich, H., Fliegauf, M., Hoefele, J., Kispert, A., Otto, E., Volz, A., Wolf, M.T.,
Sasmaz, G., Trauer, U., Reinhardt, R., et al. (2003). Mutations in a novel gene,
NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and
hepatic fibrosis. Nat. Genet. 34, 455–459.
Otto, E.A., Schermer, B., Obara, T., O’Toole, J.F., Hiller, K.S., Mueller, A.M.,
Ruf, R.G., Hoefele, J., Beekmann, F., Landau, D., et al. (2003). Mutations in
INVS encoding inversin cause nephronophthisis type 2, linking renal cystic
disease to the function of primary cilia and left-right axis determination. Nat.
Genet. 34, 413–420.
Oteı ´za, P., Ko ¨ppen, M., Concha, M.L., and Heisenberg, C.P. (2008). Origin and
shaping of the laterality organ in zebrafish. Development 135, 2807–2813.
Parisi, M.A., Doherty, D., Chance, P.F., and Glass, I.A. (2007). Joubert
syndrome (and related disorders) (OMIM 213300). Eur. J. Hum. Genet. 15,
Pazour, G.J., Dickert,B.L., Vucica, Y.,Seeley, E.S., Rosenbaum, J.L., Witman,
G.B., and Cole, D.G. (2000). Chlamydomonas IFT88 and its mouse homo-
logue, polycystic kidney disease gene tg737, are required for assembly of cilia
and flagella. J. Cell Biol. 151, 709–718.
Reiter, J.F., and Skarnes, W.C. (2006). Tectonic, a novel regulator of the
Hedgehog pathway required for both activation and inhibition. Genes Dev.
Rosenbaum, J.L., and Witman, G.B. (2002). Intraflagellar transport. Nat. Rev.
Mol. Cell Biol. 3, 813–825.
Salonen, R., and Paavola, P. (1998). Meckel syndrome. J. Med. Genet. 35,
Sasaki, H., and Hogan, B.L. (1994). HNF-3 beta as a regulator of floor plate
development. Cell 76, 103–115.
Scha ¨fer, T., Pu ¨tz, M., Lienkamp, S., Ganner, A., Bergbreiter, A., Ramachan-
dran, H., Gieloff, V., Gerner, M., Mattonet, C., Czarnecki, P.G., et al. (2008).
Genetic and physical interaction between the NPHP5 and NPHP6 gene prod-
ucts. Hum. Mol. Genet. 17, 3655–3662.
N., Schwikowski,B., andIdeker,T.(2003).Cytoscape:asoftware environment
for integrated models of biomolecular interaction networks. Genome Res. 13,
Scholtens, D. (2004). apComplex: Estimate protein complex membership
using AP-MS protein data. R package version 2.14.0.
Scholtens, D., Vidal, M., and Gentleman, R. (2005). Local modeling of global
interactome networks. Bioinformatics 21, 3548–3557.
Shiba, D., Manning, D.K., Koga, H., Beier, D.R., and Yokoyama, T. (2010). Inv
acts as a molecular anchor for Nphp3 and Nek8 in the proximal segment of
primary cilia. Cytoskeleton (Hoboken) 67, 112–119.
Sowa, M.E., Bennett, E.J., Gygi, S.P., and Harper, J.W. (2009). Defining the
human deubiquitinating enzyme interaction landscape. Cell 138, 389–403.
Tallila, J., Jakkula, E., Peltonen, L., Salonen, R., and Kestila ¨, M. (2008). Identi-
ciliopathy puzzle. Am. J. Hum. Genet. 82, 1361–1367.
Torres, J.Z.,Miller, J.J., and Jackson, P.K. (2009). High-throughput generation
of tagged stable cell lines for proteomic analysis. Proteomics 9, 2888–2891.
Waragai, M., Nagamitsu, S., Xu, W., Li, Y.J., Lin, X., and Ashizawa, T. (2006).
Ataxin 10 induces neuritogenesis via interaction with G-protein beta2 subunit.
J. Neurosci. Res. 83, 1170–1178.
Weatherbee, S.D., Niswander, L.A., and Anderson, K.V. (2009). A mouse
model for Meckel syndrome reveals Mks1 is required for ciliogenesis and
Hedgehog signaling. Hum. Mol. Genet. 18, 4565–4575.
Williams, C.L., Winkelbauer, M.E., Schafer, J.C., Michaud, E.J., and Yoder,
B.K. (2008). Functional redundancy of the B9 proteins and nephrocystins in
Caenorhabditis elegans ciliogenesis. Mol. Biol. Cell 19, 2154–2168.
Winkelbauer, M.E., Schafer, J.C., Haycraft, C.J., Swoboda, P., and Yoder,
B.K. (2005). The C. elegans homologs of nephrocystin-1 and nephrocystin-4
are cilia transition zone proteins involved in chemosensory perception. J.
Cell Sci. 118, 5575–5587.
Zaghloul, N.A., and Katsanis, N. (2009). Mechanistic insights into Bardet-Biedl
syndrome, a model ciliopathy. J. Clin. Invest. 119, 428–437.
Zuo, X., Guo, W., and Lipschutz, J.H. (2009). The exocyst protein Sec10 is
necessary for primary ciliogenesis and cystogenesis in vitro. Mol. Biol. Cell
528 Cell 145, 513–528, May 13, 2011 ª2011 Elsevier Inc.