Resource

Proteomics of Primary Cilia by Proximity Labeling

Graphical Abstract Authors
David U. Mick, Rachel B. Rodrigues,
Ryan D. Leib, Christopher M. Adams,
Allis S. Chien, Steven P. Gygi,
Maxence V. Nachury

Correspondence
nachury@stanford.edu

In Brief
Primary cilia organize cellular signaling
events in a specialized
microenvironment. Mick et al. apply
proximity labeling using cilia-APEX to
study the ciliary proteome. They uncover
unexpected signaling molecules,
including kinases PKA and AMPK, inside
cilia and further use a proteomic profiling
approach to unravel molecular defects of
Ift27/Bbs19 mutant cilia.

Highlights
d APEX labeling enables proteomic analyses of a non-
membrane-enclosed compartment

d Cilia-APEX identifies the kinases PKA, AMPK, and LKB1 in

primary cilia

d PKA functions inside cilia to phosphorylate GLI3 and regulate

Hedgehog signaling

d Proteomic profiling of Ift27/Bbs19 cilia detects ciliary

accumulation of BBSome

Mick et al., 2015, Developmental Cell 35, 497512

November 23, 2015 2015 Elsevier Inc.
http://dx.doi.org/10.1016/j.devcel.2015.10.015
Developmental Cell
Resource
Proteomics of Primary Cilia by Proximity Labeling
David U. Mick,1 Rachel B. Rodrigues,2 Ryan D. Leib,3 Christopher M. Adams,3 Allis S. Chien,3 Steven P. Gygi,2
and Maxence V. Nachury1,*
1Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA
2Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
3Stanford University Mass Spectrometry, Stanford University, Stanford, CA 94305, USA
*Correspondence: nachury@stanford.edu
http://dx.doi.org/10.1016/j.devcel.2015.10.015
SUMMARY
While cilia are recognized as important signaling or-
ganelles, the extent of ciliary functions remains un-
known because of difficulties in cataloguing proteins
from mammalian primary cilia. We present a method
that readily captures rapid snapshots of the ciliary
proteome by selectively biotinylating ciliary proteins
using a cilia-targeted proximity labeling enzyme
(cilia-APEX). Besides identifying known ciliary pro-
teins, cilia-APEX uncovered several ciliary signaling
molecules. The kinases PKA, AMPK, and LKB1
were validated as bona fide ciliary proteins and
PKA was found to regulate Hedgehog signaling in
primary cilia. Furthermore, proteomics profiling of
Ift27/Bbs19 mutant cilia correctly detected BBSome
accumulation inside Ift27 / cilia and revealed that
b-arrestin 2 and the viral receptor CAR are candidate
cargoes of the BBSome. This work demonstrates
that proximity labeling can be applied to proteomics
of non-membrane-enclosed organelles and sug-
gests that proteomics profiling of cilia will enable a
rapid and powerful characterization of ciliopathies.
INTRODUCTION
Cilia organize and tune signal transduction cascades by dynam-
ically concentrating signaling molecules in a specialized environ-
ment (Goetz and Anderson, 2010; Nachury, 2014). This dynamic
compartmentalization of signaling by cilia is best exemplified by
the Sonic Hedgehog (Hh) pathway where signal activation elicits
the ciliary accumulation of the GLI transcription factors and the
seven transmembrane protein Smoothened (SMO), and the
ciliary removal of the pathway inhibitors Patched1 (PTC1) and
GPR161, a G protein coupled receptor (Corbit et al., 2005; Mu-
khopadhyay et al., 2013; Rohatgi et al., 2007). Remarkably, in un-
stimulated cells, the cilium is required for the conversion of GLI2
and GLI3 into transcriptional repressors even though GLI2 and
GLI3 are hardly detectable inside cilia (Tukachinsky et al.,
2010; Wen et al., 2010). Proteins that rapidly traverse the cilium
can thus be altered within cilia. Yet tools to identify such transient
visitors of the cilium are currently lacking.
Primary cilium dysfunction causes a variety of hereditary disor-
ders collectively named ciliopathies (Bettencourt-Dias et al.,
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 497
2011; Fliegauf et al., 2007). The characterization of Bardet-Biedl
syndrome (BBS) gene products led to the discovery of the
BBSome, a coat complex that removes signaling molecules
from the cilium (Jin et al., 2010; Lechtreck et al., 2013; Liew
et al., 2014). In a landmark study, the biochemical purification
of flagella from the single-cell organism Chlamydomonas rein-
hardtii was leveraged to identify proteins that accumulate in
bbs4 mutant flagella (Lechtreck et al., 2009). Similarly, the finding
that the translation factor EF1a abnormally enters cilia of cep290/
mks4/nphp6 mutants was the first indication that MKS and NPHP
proteins are part of a diffusion barrier at the base of cilia (Craige
et al., 2010). In contrast, the comprehensive profiling of alter-
ations of the ciliary proteome in mammalian cells is currently
not feasible since isolation of primary cilia is not sufficiently repro-
ducible and preparations are heavily contaminated by microvilli
(Mayer et al., 2008). In one study, protein correlation profiling
(PCP) was used to identify novel ciliary proteins (Ishikawa et al.,
2012). However, the required mass spectrometric (MS) analyses
of 25 sucrose gradient fractions make PCP impractical as a
routine technique. Furthermore, PCP suffers from limited sensi-
tivity, and the number of signaling factors and membrane pro-
teins discovered in the PCP ciliary proteome was limited.
Proximity labeling is an emerging technology that relies on
enzymatic activities capable of generating free biotinyl radicals
to enable the rapid and spatially restricted labeling of proteins
in the vicinity of the enzyme. While proximity labeling was initially
used to uncover protein-protein interactions (Firat-Karalar et al.,
2014; Lambert et al., 2015; Roux et al., 2012), recent studies
leveraged proximity labeling to identify the protein contents of
the membrane-enclosed mitochondrial matrix and intermem-
brane space (Lam et al., 2015; Rhee et al., 2013). Here, we
show that proximity labeling can be applied to capture minute-
scale snapshots of ciliary protein contents. The described
method is rapid, simple to implement, and uncovered several
ciliary protein kinases. Most remarkably, proximity labeling is
capable of identifying signaling proteins that survey the ciliary
interior and whose presence inside cilia had thus far escaped
detection. Finally, this method can be used to profile the prote-
ome of ciliopathy mutant cells and rapidly uncover the underlying
molecular defects.
RESULTS
Selective Biotinylation of Ciliary Proteins by Cilia-APEX
To globally biotinylate ciliary proteins (Figure 1A), we fused the
ascorbate peroxidase APEX to a number of ciliary targeting
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498 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
signals. In the presence of H2O2, APEX catalyzes the conversion
of biotin-phenol into biotin-phenoxyl radicals. Such radicals are
short lived (<5 ms), have a small labeling radius (<20 nm), are
membrane-impermeable, and can covalently react with elec-
tron-rich amino acids such as Tyr, Trp, His, and Cys (Rhee
et al., 2013). The greatest ciliary enrichment of APEX was
observed when fused to NPHP3[1203] (Nakata et al., 2012),
and the NPHP3[1203]-GFP-APEX fusion (hereafter named
cilia-APEX) was highly enriched in primary cilia of inner medullary
collecting duct epithelial cells (IMCD3) (Figure 1B), mouse em-
bryonic fibroblasts (MEFs), and retinal pigmented epithelial cells
(RPE1-hTERT) (Figures 1C and S1). Staining cells with Alexa
Fluor 647-labeled streptavidin (SA647) revealed that addition of
biotin-phenol and hydrogen peroxide (H2O2) led to cilia-specific
biotinylation by cilia-APEX (Figures 1B and 1C). No ciliary biotin
signal was detected in the absence of H2O2, biotin-phenol, or
with a mislocalized control-APEX fusion in which the myristoyla-
tion site that mediates ciliary localization of NPHP3[1203] was
mutated (Figure 1B). Western blot analyses showed similar
expression of cilia-APEX and control-APEX in stable IMCD3
cell lines (Figure 1D, lanes 11 and 12), and the biotinylated pro-
teins could be efficiently isolated by streptavidin chromatog-
raphy of cell lysates (Figure 1D). Probing the isolated biotinylated
proteins for ciliary markers revealed that acetylated tubulin and
the IFT-B components IFT88 and CLUAP1 were specifically
recovered from cilia-APEX cells subjected to labeling, but not
in the absence of labeling or from control-APEX cells treated
with biotin-phenol and H2O2 (Figure 1D, lanes 1315). Mean-
while, highly abundant proteins, such as tubulin, actin, GAPDH,
or Annexin V were either absent or greatly depleted from the
streptavidin eluates, thus demonstrating the high specificity re-
sulting from the stringent streptavidin-based purifications under
denaturing conditions (Figure 1D). We conclude that cilia-APEX
selectively labels ciliary proteins.
Cilia-APEX Identifies a Variety of Ciliary Proteins
We next conducted liquid chromatography (LC)/MS-MS ana-
lyses of cilia-APEX-labeled samples. To control for the re-
spective contributions of endogenous biotinylated proteins
and of non-ciliary proteins biotinylated by APEX, we also sub-
jected unlabeled cilia-APEX and labeled control-APEX samples
to LC/MS-MS analyses (Figure 2A). By selecting proteins that
were enriched at least 5-fold (estimated by spectral counts,
see Supplemental Information) in the cilia-APEX sample
compared to either control, we identified 622 candidate ciliary
proteins (Figure 2A). To identify reproducible hits, three inde-
pendent replicates were carried out. We grouped the final
cilia-APEX proteome in a high confidence Tier 1 and a low
confidence Tier 2. Tier 1 consists of 162 candidate ciliary pro-
teins that were identified by at least six spectral counts and
in at least two experiments (Figure 2B; Table S1). Tier 2 con-
sists of 208 additional proteins identified by at least six spectral
counts in one experiment. Among the candidate ciliary
proteins, we identified a large number of known ciliary proteins,
such as subunits of the ciliary trafficking complexes IFT-A,
IFT-B, kinesin 2, and dynein 1b; the small GTPases ARL3,
ARL6, and ARL13B, as well as Septins 2 and 7 (Ghossoub
et al., 2013; Hu et al., 2010) and EvC complex subunits (Pusa-
pati et al., 2014) (Figure 2C). Several microtubule-associated
proteins previously localized to cilia (Ghossoub et al., 2013;
Patzke et al., 2010; Schrder et al., 2007), such as CSPP1,
MAP4, or EB1 were also identified in Tier 1. Centrosomal
proteins and transition zone proteins were conspicuously
absent from the cilia-APEX proteome, consistent with the
highly specific localization of cilia-APEX inside cilia and the
estimated 20 nm labeling radius of biotinyl radicals generated
by APEX.
Unexpected candidate ciliary proteins were identified by
cilia-APEX. These include all subunits of the spindle and kinet-
ochore associated (SKA) complex, four tetraspanins, ubiquity-
lation enzymes, as well as proteins associated with ciliary
functions, but not known to localize to cilia (e.g., AZI1 and
Dishevelled 3) (Figure 2C). To assess the cilia-APEX proteome,
we transfected epitope-tagged constructs into IMCD3 cells.
SKA1 was indeed found inside cilia (Figures 2D and S2A).
Given that the SKA complex enables the kinetochore to track
depolymerizing microtubules in mitosis (Schmidt et al., 2012),
SKA might function during cilium shortening to track the
axonemal tip. Similarly, the tetraspanins CD81 and CD82
were found in cilia (Figure 2D). Tetraspanins are palmitoylated
4-pass membrane proteins that shape membrane structure
by influencing lipid bilayer geometry. Intriguingly, all four
tetraspanins identified by cilia-APEX have been reproducibly
found in extracellular vesicles (Mathivanan et al., 2010; Simp-
son et al., 2008), pointing to a possible role of these tetraspa-
nins in ciliary ectosome formation, a biologically important
phenomenon in C. reinhardtii (Cao et al., 2015; Wood et al.,
2013) and nematodes (Wang et al., 2014). Finally, the clathrin

Figure 1. Cilia-APEX Specifically Biotinylates Ciliary Proteins

(A) Diagram of the cilia-APEX labeling strategy. The cells expressing cilia-APEX (or control-APEX) were seeded at high density and ciliation was induced by
serum-starvation. The cells were pre-incubated with 0.5 mM biotin-phenol for 45 min, labeled for 1 min by the addition of 1 mM H2O2, and the reaction was
terminated by washing the cells in quenching buffer. The cells were either immediately lysed or fixed for processing for immunofluorescence microscopy.
(B) Immunofluorescence of stable IMCD3 cell lines expressing control-APEX or cilia-APEX in the presence or the absence of labeling reagents. The APEX fusion
proteins were directly detected by GFP fluorescence (ARL13B is a cilium marker detected by antibody and the biotinylated proteins were revealed by SA647). The
merged insets show primary cilia with channels shifted to aid visualization. The scale bars in (B) and (C) represent 5 mm (main) and 1 mm (insets).
(C) Immunofluorescence microscopy of MEF and human RPE cells after transient transfection with cilia-APEX (see Figure S1 for individual channels).
(D) Biotinylated proteins from cell lysates generated from control-APEX or cilia-APEX after APEX-labeling (+) or from cilia-APEX after mock-labeling (biotin-phenol
was omitted; ) were isolated by streptavidin chromatography and samples analyzed by SDS-PAGE and western blotting. The biotinylated proteins were de-
tected by streptavidin-HRP, and the indicated proteins were detected by specific antibodies. The asterisks indicate endogenous biotinylated proteins. Note the
slower migration of the control-APEX enzyme is caused by the absence of the myristoyl moiety (lanes 11 versus 12). Also note that the cilia-APEX enzyme is
efficiently biotinylated, whereas control-APEX is not, suggesting that APEX does not undergo self-biotinylation. The Load and Unbound represent 0.1% of the
total lysate and Elution 10% of the total eluate.
See also Figure S1.

Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 499
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D E

Figure 2. Ciliary Proteins from APEX-Labeling in IMCD3 Cells

(A) Venn diagram showing identified proteins after streptavidin chromatography from the indicated APEX-labeling reactions. The number in the yellow segment
represents cilia-specific proteins enriched at least 5-fold in the experimental sample compared to both controls.
(B) Venn diagram showing the overlap of cilia-specific proteins between the three independent replicates. The yellow segment represents candidate ciliary
proteins identified in at least two out of three experiments and with a total spectral count R6 (Tier 1).
(C) Candidate ciliary proteins from (B) grouped into functional categories. The asterisks denote cilia-specific proteins from Tier 2 that are known interaction partners
of Tier 1 proteins. The previously known ciliary proteins are shown in gray and the novel candidate ciliary proteins are shown in black. In blue are novel ciliary proteins
confirmed by independent methods in this study (see Figures 3, 5, and S2). MT, microtubule. The complete list of identified proteins is shown in Table S1.
(D) IMCD3 cells were transiently transfected with the indicated fusion proteins for 24 hr, after which ciliation was induced for another 24 hr before cells were fixed
in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and proteins detected by GFP fluorescence (LAP-SKA1), anti-FLAG, or anti-HA antibodies. The
cilia were counterstained using anti-ARL13B or anti-acTub antibodies as indicated. Only the merged images are shown (see Figure S2 for individual channels).
The merged insets show primary cilia with channels shifted to aid visualization. The scale bars represent 5 mm (main) and 1 mm (insets).
(E) Table comparing prominent protein categories identified in primary cilia by PCP (Ishikawa et al., 2012) versus cilia-APEX. Note the absence of ribosomal
subunits and chaperones, frequent contaminants of proteomic studies, in the cilia-APEX proteome.
See also Figure S2.

adaptor TOM1L2 (TOM1-like 2) was found in cilia (Figure 2D).
TOM1 and TOM1L2 (both Tier 1 hits) are evolutionarily
conserved ESCRT-0 proteins that carry out early endocytic
sorting of ubiquitinated cargoes by linking them to Myosin-VI
(Tumbarello et al., 2012). The findings that the canonical
ESCRT-0 complex Hrs/STAM mediates the ciliary exit and lyso-
somal degradation of polycystin-1 and -2 (Hu et al., 2007) and
that ubiquitination enhances ciliary exit (Xu et al., 2015) suggest
a possible role of TOM1/TOM1L2 in these processes.
Taking into account all Tier 1 proteins that we detected in
cilia in the course of this study, the immunofluorescence valida-
tion rate of candidate ciliary proteins in the cilia-APEX prote-
ome is 56% (Figure S2B). However, as outlined below, we
believe that the sensitivity of cilia-APEX for certain proteins is
greater than that of immunofluorescence, and that some pro-
teins scored as false positive by microscopy may be present
in cilia at very low steady-state levels. Furthermore, the data set
we selected for validation by immunofluorescence did not
include Tier 1 proteins that were previously known to localize
to cilia. Thus, the 56% validation rate we calculate is likely
underestimated. Compared to the PCP cilium proteome (Ishi-
kawa et al., 2012), cilia-APEX identified many more

500 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
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Figure 3. AC6 and PKA Are Present in IMCD3 Cilia

(A) Cultured mouse cortical neurons (DIV 15) and serum-starved IMCD3 cells were fixed in 4% paraformaldehyde (PFA) and analyzed by immunostaining for AC3
and glutamylated tubulin (GluTub). The AC3 channels were normalized to one another.
(B) IMCD3 cells were starved for 16 hr to induce ciliation and fixed in 4% paraformaldehyde (PFA). After permeabilization with 0.1% saponin, the cells were
incubated overnight with antibodies against AC5/6 and acetylated tubulin. The images showing AC5/6 localization were deconvolved.
(C) IMCD3 cells stably expressing NG-PKA RIa were processed and imaged as in (B).
All scale bars represent 5 mm (main) and 1 mm (insets). All insets show primary cilia, and the merged insets are offset to aid visualization.

transmembrane and microtubule-associated proteins, and in
particular signaling-relevant proteins, such as protein kinases
and predicted calcium-binding proteins (Figure 2E). Cilia-
APEX thus represents a significant advance given the ease of
the approach and the broad coverage of structural, trafficking,
and signaling components.
Discovery of a Ciliary AC6/Cyclic AMP/PKA Signaling
Axis that Regulates Hh Signaling
Adenylate cyclase 3 (AC3) serves as a specific ciliary marker of
neurons and glial cells and is generally considered to be the pre-
dominant cyclic AMP (cAMP)-generating enzyme in primary cilia
(Bishop et al., 2007) (Figure 3A). Yet, consistent with previous

Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 501
findings (Kwon et al., 2010; Masyuk et al., 2008) and the absence
of AC3 from the cilia-APEX proteome, we failed to detect AC3 in
primary cilia of IMCD3 cells (Figure 3A). Meanwhile, AC6 was a
strong Tier 1 hit in the cilia-APEX proteome (Figure 2C; Table S1),
and an antibody recognizing AC5 and AC6 revealed a clear
ciliary signal in IMCD3 cells (Figure 3B). In the absence of AC5-
and AC6-specific antibodies, we were unable to determine
which of these two proteins is present in cilia of IMCD3 cells.
However, out of 36 spectral counts that could be assigned to
either AC5 or AC6 in cilia-APEX, 32 were unique to AC6, four
were common to AC5 and AC6, and none were specific to
AC5. These results suggest that AC6 is the main ciliary adenylate
cyclase in IMCD3 cells. The presence of three distinct adenylate
cyclases in cilia poses the question of their respective functional
significance. While AC3 has been detected in cilia of neurons
(Bishop et al., 2007), kidney cells (Nikonova et al., 2014), and fi-
broblasts (Halbritter et al., 2013; Ou et al., 2009), a recent publi-
cation found AC5 and 6 to be the major regulators of Hh signaling
in the chicken neural tube and in mouse cerebellar granular
neuron precursors (Vuolo et al., 2015). Interestingly, while
AC3, 5, and 6 are all activated by Gas, only AC5 and 6 are in-
hibited by Gai or free Ca2+ (Sadana and Dessauer, 2009). Given
that most ciliary G protein coupled receptors (GPCRs) are
coupled to either Gas or Gai, modulating the respective levels
of AC3, 5, and 6 in cilia would provide a means for different
cell types to tune their response to ciliary GPCR activity.
A prominent signaling target of cAMP is the cAMP-dependent
protein kinase PKA. At rest, PKA exists as an auto-inhibited com-
plex of regulatory (R) and catalytic (C) subunits and binding of
cAMP to R subunits triggers the release of active C subunits.
PKA exerts a powerful inhibition on Hh signaling through phos-
phorylation of the GLI2 and GLI3 transcription factors, and the
phenotype of a PKA-C mutant mouse is as severe as that of a
Ptc1 / mouse (Tuson et al., 2011). Paradoxically, while the
localization of adenylate cyclases predicts that cAMP produc-
tion takes place within cilia, immunocytochemistry found some
concentration of PKA-C at the base of cilia (Barzi et al., 2010; Tu-
son et al., 2011). It has therefore been proposed that cAMP
diffusing out of cilia activates PKA at the base and that PKA de-
posits inhibitory phosphorylations on GLI2/3 before or after
GLI2/3 have passed through cilia (Mukhopadhyay and Rohatgi,
2014). However, it is conceivable that the pools of ciliary
PKA-C have remained undetectable because they are too labile
for conventional fixation methods or because of low sensitivity of
the immunological reagents. The presence of the PKA R subunit
Ia (PKA-RIa) in Tier 1 and of PKA-Ca and RIIb in Tier 2 of the cilia-
APEX proteome suggested that sensing of cAMP by PKA might
take place inside cilia (Figure 2C). Indeed, stably expressed
NeonGreen(NG)-tagged PKA-RIa can be detected in primary
cilia of IMCD3 cells (Figure 3C). Congruently, Jacob et al.
(2011) have reported that genetic ablation of PKA-RIa leads to
Hh signaling defects.
The detection of an R subunit of PKA in cilia suggested that the
PKA holoenzyme may enter and become activated within cilia by
sensing intraciliary cAMP levels. To directly test the functional
significance of ciliary localization of PKA, we turned our attention
to Hh signaling and, in particular, GLI3 processing. Upon phos-
phorylation by PKA, GLI3 undergoes partial proteolysis and be-
comes converted from a 190 kDa polypeptide (GLI3FL) into a
502 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
83 kDa transcriptional repressor (GLI3R). We reasoned that if
PKA phosphorylates GLI3 within cilia, then inhibiting PKA inside
cilia should block GLI3 processing. To test this prediction, we
directed the specific PKA inhibitor peptide PKI (Knighton et al.,
1991) to cilia by expressing NPHP3[1203]-GFP-PKI (cilia-PKI)
stably and at low levels in IMCD3 cells (Figure 4A). While high
level expression of GFP-PKI has been reported to inhibit Hh
signaling (Iglesias-Bartolome et al., 2015), the site of PKI action
has not been investigated. To control for the specificity of the
PKI inhibitory peptide and the importance of ciliary targeting,
we expressed cilia-PKI-4A, a 4-residue mutant that fails to block
PKA activity and NPHP3[1203;G2A]-GFP-PKI (control-PKI), a
mutant that fails to target to cilia (Figures 4A and S3). All con-
structs were expressed at similarly low levels as evidenced by
western blot (Figure 4D) and the extremely weak fluorescent
signal of the diffusely distributed control-PKI (Figure S3). Consis-
tent with the weak localization of GFP at the pericentriolar matrix
(Breslow et al., 2013), control-PKI was slightly enriched at the
ciliary base. Importantly, cilia-PKI clearly localized to the ciliary
shaft, distal to the basal body (marked by ninein) and the transi-
tion zone (marked by CEP290) (Figure 4B). Moreover, all IMCD3
cell lines appropriately responded to the SMO agonist (SAG) by
redistributing SMO to cilia and removing GPR161 from cilia (Fig-
ures 4C and S3). In contrast, while control-PKI and cilia-PKI-4A
cells produced high levels of GLI3R at rest, cilia-PKI expression
resulted in a clear reduction in GLI3R (Figure 4D), similar to the
effect of genetically removing PKA C activity (Tuson et al.,
2011). Furthermore, Hh pathway stimulation results in decreased
processing of GLI3FL into GLI3R and the GLI3R/GLI3FL ratio was
appropriately decreased in control-PKI and cilia-PKA-4A cells
treated with SAG (Figures 4E and 4F). In contrast, GLI3R levels
and the GLI3R/GLI3FL ratio were barely altered by SAG addition
to cilia-PKI cells. This demonstrates that inhibiting PKA activity
within cilia leads to defective processing of GLI3 and points to-
ward a direct function of PKA in cilia.
Hence, consistent with the well-established functions of cAMP
inside olfactory cilia (Anholt, 1989), we propose that cAMP is
sensed inside cilia by the PKA holoenzyme to transmit Hh signals
to the rest of the cell. In this model, cilia harbor high levels of
cAMP owing to the activity of AC3, 5, and 6 and the tonic stimu-
lation of Gas by GPR161 (Mukhopadhyay et al., 2013). PKA thus
becomes activated inside cilia where it phosphorylates GLI3FL
and primes it for proteolytic processing into GLI3R. Once the
Hh pathway becomes activated, GPR161 exits cilia and SMO
enters cilia where it may inhibit AC5/6 either through Gai (Riobo
et al., 2006; Barzi et al., 2011) or elevated ciliary Ca2+ (DeCaen
et al., 2013). These alterations result in lower levels of ciliary
cAMP, decreased phosphorylation of GLI3FL by PKA inside cilia,
and reduced production of GLI3R (Figure 4G).
LKB1-AMPK Signaling in Primary Cilia
Liver kinase B1 (LKB1) is a master regulator of cell growth and
epithelial polarity that is frequently altered in cancer (Jansen
et al., 2009). Instead of relying on phosphorylation for its activa-
tion, LKB1 is activated through formation of a heterotrimer with
the pseudokinase STRAD and the scaffolding protein MO25 (Ze-
qiraj et al., 2009). Since vertebrates have two paralogues of
STRAD (a and b) and two paralogues of MO25 (a and b), the
functional diversification of the four STRAD-MO25-LKB1
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C D E

F G

Figure 4. Cilium-Specific Inhibition of PKA Perturbs GLI3 Processing during Hh Signaling

(A) Diagrams of cell lines expressing cilia-PKI to inhibit PKA activity within primary cilia, the non-inhibiting mutant cilia-PKA-4A, and control-PKI expressed in the
cytosol.
(B) Ciliated IMCD3-[cilia-PKI] cells stained for ninein and CEP290. The cilia-PKI was detected by GFP fluorescence. The scale bar represents 1 mm.
(CF) Ciliated cells were treated with 200 mM SAG or vehicle control for 16 hr.
(C) IMCD3-[cilia-PKI] cells were stained for acetylated tubulin, GPR161, or SMO. The channels were shifted to aid visualization (see Figure S3 for individual
channels and control cell lines).
(D and E) Indicated cell lines were analyzed by immunoblotting for GLI3, GFP, and actin (D), the GLI3R and GLI3FL signals were quantified by densitometry
(ImageJ) and the GLI3R/GLI3FL ratios were plotted in (E).
(F) The effect of SAG addition on the GLI3R/GLI3FL ratios was measured. The average from three independent experiments is shown. The error bars depict SD
(n = 3).
(G) Model depicting the ciliary AC/cAMP/PKA signaling axis during Hh signaling (see text for details and also Figure S3).

complexes remains an intriguing yet unsolved question. Interest-
ingly, STRADb, but not STRADa, was found in the cilia-APEX
proteome (Figure 2C) and transfection of epitope-tagged
STRADb confirmed a significant ciliary enrichment of STRADb
(Figure 5A). Since LKB1 has been localized to primary cilia of
Madin-Darby canine kidney (MDCK) cells (Boehlke et al.,
2010), and since LKB1 was a Tier 2 hit in the cilia-APEX
proteome, we tested the contribution of STRADb to LKB1
localization. In our experimental setup, endogenous LKB1 was
undetectable in IMCD3 cilia unless exogenous STRADb was

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504 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
introduced into the cell (Figure 5A), thus suggesting that STRADb
functions as a ciliary targeting subunit for the STRAD-MO25-
LKB1 complex. To identify the ciliary targeting determinants of
STRADb, we compared the sequences of STRADa and STRADb
proteins from five different organisms. Whereas the N-terminal
extensions of STRADa orthologs displayed significant differ-
ences in length and composition, the STRADb N termini were
highly conserved among species and contained two conserved
cysteine residues near the amino terminus, suggestive of poten-
tial palmitoylation sites (Figure 5B). Congruent with prior evi-
dence of acylation mediating ciliary targeting (Constantine
et al., 2012; Follit et al., 2010), STRADb[C6S,C8S] no longer
localized to the primary cilium, but was instead found dispersed
throughout the cell, similar to the distribution of STRADa
(Figure 5C). When LKB1-NG was co-expressed with the STRAD
variants, STRADb directed LKB1 to the primary cilium, while
STRADb[C6S,C8S] and STRADa directed LKB1 to the cytosol
and nucleus. Hence, palmitoylation of STRADb is likely
to direct the LKB1/STRADb/MO25 holoenzyme to the primary
cilium, opening the possibility of a cilia-localized LKB1
signaling cascade regulated by STRADb palmitoylation. Alterna-
tively, it is conceivable that the two cysteine residues at positions
6 and 8 may be directly recognized by the ciliary import
machinery.
The major downstream targets of LKB1 are AMP kinase
(AMPK) family members. AMPK itself is activated when ATP is
depleted and the cellular levels of AMP are increased, and the
LKB1-AMPK axis integrates the metabolic state of the cell to
regulate energy production (Jansen et al., 2009). Examination
of the cilia-APEX data set revealed the presence of AMPK sub-
units b2 in Tier 1 and a1 in Tier 2. Surprisingly, NG-AMPKb2 sta-
bly expressed in IMCD3 cells was not detectable in primary cilia
(Figure 5D). Several proteins such as SMO, GLI2, and GLI3 have
been proposed to rapidly cycle in and out of cilia and are only de-
tected in cilia under signaling conditions (Corbit et al., 2005; Kim
et al., 2009; Tukachinsky et al., 2010; Wen et al., 2010), or when
ciliary exit is compromised by inhibition of Dynein 1b (Kim et al.,
2009; Ocbina and Anderson, 2008), or by removal of the
BBSome export factor IFT27 (Eguether et al., 2014; Liew et al.,
2014). Remarkably, AMPKb2 was strongly enriched in primary
cilia of Ift27 / cells (Figure 5D), indicating that AMPK is likely
to rapidly cycle into and out of cilia. Collectively, these findings
suggest that IMCD3 cilia may serve as signaling hubs for energy
metabolism through a STRADb-LKB1-AMPK pathway. In this
context, it is worth noting that SMO ligands that translocate
SMO to cilia increase AMPK activity and alter glucose meta-
bolism (Teperino et al., 2012). Since the effects of SMO on
AMPK and metabolism occur on the scale of minutes, do not
require a transcriptional response or SMO activation and depend
on intact cilia, a tantalizing conclusion is that SMO translocation
leads to activation of AMPK within cilia. In addition, AMPK has
recently been shown to regulate Hh signaling through destabiliz-
ing phosphorylation of GLI1 (Li et al., 2015). Finally, LKB1 dele-
tion results in increased stability of GLI3R (Jacob et al., 2011).
AMPK may thus respond to Hh inputs and fine-tune the Hh
response within cilia.
Comparative Cilia-APEX Analyses Rapidly Identify
Alterations of the Ciliary Proteome
Ift27 / cells accumulate specific signaling proteins in their cilia
(Figure 5D) (Eguether et al., 2014; Liew et al., 2014) and, unlike
dynein 1b or IFT-A mutants, Ift27 mutant cilia do not suffer
from structural abnormalities. We therefore sought to apply the
cilia-APEX technology to globally compare the protein content
of Ift27 / cilia to that of wild-type cilia. Cilia-APEX was stably
expressed in Ift27 / cells and displayed the same ciliary local-
ization and labeling efficiency as in wild-type (WT) cells (Fig-
ure S4). We conducted large-scale APEX labeling experiments
in triplicate in each cell line, followed by streptavidin affinity-
chromatography and MS analyses, and calculated ratios of the
average spectral counts for each identified protein. To correct
for the variance in protein recovery between the different sam-
ples, we calculated the mean ratio of spectral counts between
WT and Ift27 / for non-ciliary contaminants (118 mitochondrial
proteins). Assuming that the variability in the control group is
representative of the whole data set, we set a significance
threshold at 2.75 SD around the mean value of the mitochondrial
control group to ensure a false positive rate of less than 1%.
Out of 2,070 proteins identified in WT or Ift27 / samples, only
57 showed a significant change between the two genotypes (Fig-
ure 6A; Table S2). Congruent with the recent finding that the
BBSome accumulates in Ift27 / cilia (Eguether et al., 2014;
Liew et al., 2014), five BBSome subunits scored among the top
21 enriched proteins and all five were enriched at least 4.4-fold
in the Ift27 / cilia-APEX samples when compared to WT cilia-
APEX samples. Moreover, LZTFL1, a BBSome interactor known
to accumulate in Ift27 / mutant cilia (Eguether et al., 2014), was
also near the top of the enriched list (Figure 6A, top inset).
Together, these results demonstrate the proof-of-concept of
the comparative cilia-APEX approach.
Analysis of major functional groups revealed that ciliary kine-
sins, most EvC zone components, and most IFT components
only showed non-significant changes, although a general trend
of depletion was observed for IFT subunits (Figure 6B).
Among the significantly depleted proteins were the microtu-
bule-binding proteins EB2 and MAP1B, several proteins involved

Figure 5. LKB1 Localization to IMCD3 Cilia Is Mediated by STRADb Palmitoylation

(A) IMCD3 cells were transiently transfected with STRADb33FLAG, serum-starved for 16 hr, and fixed in 4% paraformaldehyde (PFA) and stained with anti-FLAG,
anti-LKB1, and anti-acTub antibodies to visualize the primary cilium.
(B) Multiple sequence alignment of STRADa and STRADb from five different organisms each using ClustalW 2.0.11. The black boxes indicate identical residues in
at least four out of five species, and the gray boxes indicate similar amino acids. The black underlining indicates the STRAD core domain used for crystallization in
Zeqiraj et al. (2009). Only the N-terminal part of alignment is shown in the image (Homo sapiens, Hs; Mus musculus, Mm; Bos Taurus, Bt; Danio rerio, Dr; Xenopus
laevis, Xl; and Xenopus tropicalis, Xt).
(C) IMCD3 cells were co-transfected with LKB1-NG and indicated STRAD isoforms and mutants and analyzed as in (A).
(D) Ift27 / and WT IMCD3 cells stably expressing NG-AMPKb2 were analyzed as in Figure 3C.
All scale bars represent 5 mm (main) and 1 mm (insets). In all merged insets, channels were offset to aid visualization.

Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 505
 A

Figure 6. Comparative Cilia-APEX Proteomics Identifies Proteins Accumulating in or Depleted from Ift27/ Cilia
(A) Cilia-APEX labeling, streptavidin chromatography, and LC-MS/MS were performed in three independent experiments in WT and Ift27 / cells. The y axis
represents the log2-transformed ratios of average spectral counts between Ift27 / and WT samples. Each vertical line represents one protein. The mean (m) and
SD (s) of log2[SpC(Ift27 / )/SpC(WT)] calculated for a control group of 118 mitochondrial proteins are 0.29 and 0.62, respectively, leading to significance
thresholds of 1.98 (m + 2.75s) and 1.40 (m 2.75s) indicated by dashed red lines. The insets show magnified views of the significantly enriched (top) and depleted
(bottom) proteins. The BBSome subunits and LZTFL1 are shown in green, and the proteins whose abundance is altered by IFT27 deletion and was confirmed by
fluorescence microscopy are shown in orange.
(B) Proteins from selected functional groups are displayed.
See also Figure S4.

in clathrin-independent endocytosis (flotillin and endophilin-A2), teins (Figures 6A and 6B). Consistent with previous reports (Bo-
as well as Aurora Kinase A. Most unexpected was the presence tilde et al., 2013; Pasek et al., 2012), CLUAP1 was detected at
of the IFT-B subunit CLUAP1 among the top 30 depleted pro- the basal body as well as in foci along the length of WT cilia,

506 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
similar to other IFT-B components (Figure 7A). While the amount
of CLUAP1 at the basal body appeared unchanged in Ift27 /
cells, the levels of CLUAP1 along the length of the cilium were
significantly reduced (Figures 7A and 7B). Meanwhile, the total
cellular abundance of CLUAP1 was unaffected by Ift27 deletion
(Figure 7C). Thus, while Ift27 deletion has been reported to not
significantly affect IFT88 localization to cilia (Eguether et al.,
2014; Liew et al., 2014) or the ciliary abundance of other IFT sub-
units (Figures 6A and 6B), CLUAP1 appears to be reduced in
Ift27 / cilia. Whether this is due to general reduction of IFT-B
within Ift27 / cilia, as suggested by the general trend of IFT
depletion (see Figure 6A), or whether this defect is specific for
CLUAP1 remains to be investigated. Similar to the IFT27/25
module, CLUAP1 may be considered a peripheral IFT-B subunit,
since it has not been identified in initial preparations of mamma-
lian IFT. Therefore, it is conceivable that CLUAP1 requires IFT27
for proper association with the IFT-B complex.
The top enriched protein in Ift27 / cilia is the Coxsackie and
Adenovirus Receptor (CAR), an integral component of the
epithelial tight junction complex (Cohen et al., 2001). Expression
of epitope-tagged proteins revealed very few cilia positive for
CAR in WT cells. Similarly, CAR-positive cilia were rarely found
when staining for endogenous CAR (Figures 7D and 7E). How-
ever, when analyzing Ift27 / mutants, the majority of cilia
were clearly positive for CAR, even though the total levels of
CAR were unchanged (Figures 7C7E). The possible role of the
BBSome in trafficking the viral receptor CAR out of cilia remains
enigmatic. CAR is normally found on the basolateral side of tight
junctions where it associates with cytoskeletal and signaling pro-
teins (Cohen et al., 2001; Coyne et al., 2004; Sollerbrant et al.,
2003). One first possibility is that the presence of CAR inside cilia
is detrimental to the cell and that the BBSome functions as a
clearing device as previously suggested for phospholipase D in
Chlamydomonas (Lechtreck et al., 2013). Since CAR is typically
not present on apical membranes, the entry of Coxsackie virus
and Adenovirus into the organism occurs after breaching of tight
junctions on respiratory and intestinal cells. However, in the
absence of the clathrin adaptor AP1B, as naturally occurring in
retinal pigmented epithelium cells, CAR becomes misrouted to
the apical membrane and adenoviruses can efficiently enter cells
from the apical side (Diaz et al., 2009). Similarly, when BBSome
function is compromised, CAR may accumulate within cilia and
permit entry of Adenovirus and Coxsackie virus from the apical
side of the cells. More research will be required to test whether
BBS patients and animal models are more susceptible to viral in-
fections by Adenovirus and Coxsackie virus. Alternatively, it is
conceivable that CAR performs a thus far unsuspected function
inside cilia. Similar to several ciliopathy mouse models, CAR
knockout mice die between embryonic day (E)11 and E13 from
insufficient heart function and display cardiac pericardial edema
(Dorner et al., 2005).
Another top hit in our comparative proteomics was b-arrestin 2
(Figure 6A), and we observed increased levels of b-arrestin 2 in
Ift27 / cilia by fluorescence microscopy (Figures 7F and S5).
b-arrestins mediate clathrin-dependent internalization of acti-
vated GPCRs by binding to the phosphorylated cytoplasmic tails
of GPCRs (Reiter and Lefkowitz, 2006). b-arrestin 2 has been
previously detected inside cilia (Molla-Herman et al., 2008) and
was suggested to participate in SMO ciliary trafficking (Kovacs
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 507
et al., 2008). Since the BBSome also functions in the trafficking
of SMO (Zhang et al., 2011, 2012), b-arrestin 2 may cooperate
with the BBSome to remove SMO from cilia.
Thus, the comparative cilia-APEX analyses have uncovered an
unexpected candidate cargo of the BBSome (CAR) and a puta-
tive adaptor for BBSome-mediated export (b-arrestin 2), further
highlighting the power of the comparative cilia-APEX method.
DISCUSSION
Work in the past decade has established the importance of the
primary cilium in signaling and in human diseases characterized
by obesity, retinal degeneration, and kidney cysts. However, our
understanding of the physico-chemical environment of cilia and
how it endows cilia with signaling properties remains fragmen-
tary. A major technological limitation to the understanding of
events that take place inside cilia lies in our inability to obtain
reproducible and pure fractions of primary cilia for sensitive anal-
ysis of the ciliary proteome by MS. Here, we show that the APEX
labeling technology is a versatile, readily applicable, and highly
effective approach to study the protein content of primary cilia.
The cilia-APEX fusion functions in a variety of cell types and en-
ables a quantitative analysis of the alterations in ciliary protein
contents resulting from a specific mutation. This technology
promises a comprehensive and quantitative assessment of dif-
ferences in the ciliary proteomes between healthy and disease
states, between different cell types, between different geno-
types, and upon exposure to specific drugs and other small mol-
ecules. The highly specific information provided by the compar-
ative cilia-APEX approach allows for the generation of precise
molecular hypotheses as exemplified by the identification of
most BBSome subunits, as well as the BBSome associated pro-
tein LZTFL1 as proteins that accumulate in Ift27 / cilia. Within a
few weeks, the comparative cilia-APEX study of Ift27 / versus
WT cells produced a definitive molecular hypothesis that had
taken over 1 year to be developed by traditional methods of tan-
dem affinity purification (TAP) tagging and searching for the
physiologically relevant interactors of IFT27 (Liew et al., 2014).
Furthermore, the comparative cilia-APEX study uncovered
entirely unexpected candidate BBSome cargoes and adaptors
that had escaped detection by traditional biochemical associa-
tion methods. Our proof-of-concept study of Ift27 / versus
WT cells thus suggests that cilia-APEX profiling may constitute
a rapid and powerful method for the characterization of ciliop-
athy mutants.
Interestingly, AMPKb2, which was clearly identified by cilia-
APEX, was initially not detected inside cilia by immunohisto-
chemistry. Since AMPKb2 becomes clearly detectable inside
cilia once exit is impaired, this suggests that AMPKb2 rapidly
traverses the cilium. Considering that the APEX labeling reaction
is performed for 1 min, the extent of biotinylation of a given pro-
tein integrates the number of molecules that visited the cilium
either transiently or stablywithin this time frame. In theory, a
cilium-resident protein present at 600 copies in the cilium at
steady state will be biotinylated by cilia-APEX as efficiently as
a protein present at 10 copies in the cilium at steady state, but
whose ciliary turn-over rate is 1 s. This interpretation is congruent
with the extremely low number (150 copies) of known ciliary
signaling molecules such as the ciliary Ca2+ channel PKD1L1/
 A B

Figure 7. Validation of CLUAP1 Depletion and CAR and b-Arrestin 2 Enrichment in Ift27/ Cilia
(A) WT and Ift27 / cells expressing cilia-APEX were starved for 16 hr to induce ciliation, fixed in 4% paraformaldehyde (PFA), permeabilized using 0.1% saponin,
and stained for endogenous CLUAP1. The yellow arrows point at the basal body.
(B) Ciliary CLUAP1 signals in cilia were quantified in WT (n = 52) and Ift27 / (n = 42). The error bars represent SEM.
(C) Western blots of WT and Ift27 / cell lysates.
(D) Immunofluorescence microscopy as in (A) using anti-CAR antibody to stain native CAR.
(E) Percentages of cilia with detectable CAR staining inside cilia in WT and Ift27 / cell lines. The error bars represent SD between three independent experiments
(60 or more cilia were counted in each experiment).
(F) WT and Ift27 / cells stably expressing b-arrestin2-GFP were analyzed by fluorescent microscopy as in (A).
All scale bars represent 5 mm (main) and 1 mm (insets). All merged insets show primary cilia with the channels shifted to aid visualization. See also Figure S5.

508 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
PKD1L2 in cilia (DeCaen et al., 2013) and suggests that other
proteins with enzymatic activities that function inside cilia
may have escaped detection by traditional methods. The accu-
mulation of signaling proteins in Ift27 / cilia highlights the
Ift27 / mutant as a promising tool to identify ciliary proteins
whose steady-state levels have remained below the detection
limit of immunological reagents. Other mutants defective in
exit from cilia such as IFT-A and dynein-1b may prove similarly
useful.
In light of the ability of cilia-APEX to detect proteins transiently
visiting cilia, it is likely that several of the false positives that we
have been unable to detect in cilia by immunofluorescence (Fig-
ure S2B) may only become detectable by standard imaging
methods once studied in the appropriate export mutant. The
absence of common contaminants such as chaperones and ri-
bosomal proteins from the cilia-APEX proteome suggests a
very low rate of true false positives. Meanwhile, the absence
of several known IFT polypeptides from the cilia-APEX data set
is likely to represent true negatives and may reflect the selectivity
of the proximity biotinylation method toward electron-rich amino
acids exposed on the surface of proteins. Consistently, most of
the missing IFT subunits in the cilia-APEX data set are small pro-
teins that likely bear no surface-exposed amino acids reactive
with biotin-phenoxyl radicals.
A unique feature of the APEX technology is its ability to provide
temporal snapshots of the ciliary proteome with minute-scale
resolution, and cilia-APEX has the potential to reveal dynamic
changes in the ciliary protein content after exposure to signaling
ligands such as Hh. Moreover, the success of cilia-APEX gener-
alizes the applicability of proximity labeling for proteomics of
cellular subcompartments or domains beyond membrane-en-
closed organelles such as mitochondria (Lam et al., 2015;
Rhee et al., 2013). In particular, the absence of transition zone
and basal body proteins from the cilia-APEX proteome suggests
that the rapid and highly localized labeling provided by the APEX
enzyme may be leveraged to capture snapshots of the proteome
of other protrusions such as lamellipodia, filopodia, and
microvilli.
EXPERIMENTAL PROCEDURES
Detailed information about the generation of cell lines and plasmids, as well as
antibodies and reagents used in this study, can be found in the Supplemental
Information together with detailed descriptions of instrumentation and soft-
ware used for immunofluorescence microscopy, image analyses, as well as
MS analyses.
APEX-Labeling Experiments
Serum-starved cells were incubated in the presence of 0.5 mM biotin-phenol
for 45 min, after which hydrogen peroxide (H2O2) was added to a final concen-
tration of 1 mM and the plates immediately swirled to ensure equal distribution.
After 1 min, the biotin-phenol- and H2O2-containing medium was aspirated
and cells were washed three times with quenching buffer (PBS supplemented
with 10 mM sodium ascorbate, 10 mM sodium azide, and 5 mM Trolox). For
microscopic analysis, cells were immediately fixed. For proteomics and west-
ern blot analyses, lysis buffer (6 M urea, 0.3 M NaCl, 1 mM EDTA, 1 mM EGTA,
10 mM sodium ascorbate, 10 mM sodium azide, 5 mM Trolox, 10% glycerol,
and 25 mM Tris/HCl [pH 7.5]) was immediately added and cells lyzed by
scraping them off the culture dish. The cell lysate was cleared by centrifugation
(16,000 3 g for 20 min) and the supernatant used immediately for further ana-
lyses or stored at 80 C.
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 509
Image Analysis
Analysis of imaging data was performed using ImageJ software (NIH). For
measurement of the amounts of CLUAP1 in cilia, three planes from z stacks
acquired at 0.3 mm interval were projected and the cilia defined by segmented
lines (width 3 pixels) based on cilia-APEX signal. The mean signal was sub-
tracted by the mean background and multiplied by cilium length. The average
of at least 20 cilia was calculated for three independent experiments and SEMs
were calculated. For CAR, at least 60 cilia were counted in three independent
experiments and percentages of positive cilia and SEs calculated.
Streptavidin Chromatography
Protein concentrations of lysates prepared from APEX-labeling experiments
were determined by Bradford assay and lysates adjusted to equal protein con-
centrations. Lysates were diluted 10-fold with wash buffer (lysis buffer without
urea supplemented with 0.5% Triton X-100, and 0.1% SDS), from which a
sample was taken as loading control. Diluted lysates were added onto Strep-
tavidin-Sepharose beads (Thermo Scientific) and biotinylated proteins were
captured for 1 hr at room temperature. Beads were washed extensively first
with wash buffer, then with 4 M urea 10 mM Tris/HCl (pH 7.5), and finally
with 4 M urea, 10 mM Tris/HCl (pH 7.5), and 50 mM biotin. For SDS-PAGE an-
alyses, bound material was eluted by boiling beads in SDS sample buffer sup-
plemented with 2 mM biotin. For proteomics analyses, bound proteins were
eluted by boiling beads in filter aided sample preparation (FASP) buffer
(4% SDS, 0.1 mM DTT, and 0.1M Tris/HCl [pH 7.5]), and SDS was replaced
with 2 M urea by FASP before reduction, alkylation, and trypsin digestion as
described (Wisniewski et al., 2009). For comparative proteomics of WT and
Ift27 / cells, beads were directly subjected to alkylation and elution by trypsin
digest.
Hh Signaling Experiments
To visualize trafficking events during Hh signaling, 50,000 cells were seeded
onto glass coverslips in 24-well plates, grown for 24 hr, and serum starved
for 16 hr in the presence of 200 mM SAG or DMSO as vehicle control before fix-
ation in 4% paraformaldehyde. For western blot analyses, 300,000 cells were
seeded into 6-well plates before treatments as above. Cells were washed in
13 PBS and lysed in buffer (0.3 M NaCl, 1 mM EDTA, 1 mM EGTA, 10% glyc-
erol, 25 mM Tris/HCl [pH 7.5], 0.5% Triton X-100, 0.1% SDS, and protease
inhibitors) for 15 min. Lysates were cleared by centrifugation and protein
concentrations determined by Bradford assay. There was 8 mg of total protein
separated by SDS-PAGE and analyzed by western blot using indicated
antibodies.
MS Analyses
Tryptic peptides were desalted and separated by LC before MS-MS analyses.
Mass spectra were processed and analyzed to identify proteins according to
the mouse Uniprot database. Data were validated at a 2% or 1% false discov-
ery rate. See the Supplemental Information for details.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and two tables and can be found with this article online at http://
dx.doi.org/10.1016/j.devcel.2015.10.015.
AUTHOR CONTRIBUTIONS
D.U.M. and M.V.N. conceived the project and wrote the paper with input from
the other authors. D.U.M. established the cilia-APEX workflow, designed and
performed the experiments, and analyzed the data. R.D.L., C.M.A., and A.S.C.
helped to establish the MS workflow and performed and analyzed the MS ex-
periments to define the cilia-APEX proteome. R.B.R., R.D.L., and S.P.G.
performed the MS experiments and analyzed the data for the comparative
cilia-APEX proteomics.
ACKNOWLEDGMENTS
We thank Chris Jacobs, Hiroshi Hamada, Jeff Bergelson, Carsten Janke, Tam-
ara Caspary, Kathryn Anderson, Saikat Mukhopadhyay, Suzie Scales, Michel
Bornens, and Sophie Saunier for the gifts of antibodies; Iain Cheeseman and
Mark Scott for the gifts of cDNAs; the DuBois lab for help with synthesis of
biotin-phenol; David Martinelli for the gift of mouse cortical neurons; Anna
Okumu, Krysta D. Wyatt, and Jaclyn S. Lee for technical assistance; and
Bianca Schrul and the Nachury lab for helpful comments, careful reading of
the manuscript, and discussions. This work was supported in part by NIH
grants to M.V.N. (GM089933) and S.P.G. (GM67945) and by a Stanford
Dean of Research-Stanford University Mass Spectrometry (SUMS) Seed
Grant to M.V.N. D.U.M. was supported by an EMBO long-term fellowship
and an A.P. Giannini Foundation fellowship. SUMS acknowledges support
from NCRR (S10RR027425).
Received: May 27, 2015
Revised: September 26, 2015
Accepted: October 19, 2015
Published: November 12, 2015
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