Beruflich Dokumente
Kultur Dokumente
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
Resource
*Correspondence: nachury@stanford.edu
http://dx.doi.org/10.1016/j.devcel.2015.10.015
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 497
A
C D
498 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
signals. In the presence of H2O2, APEX catalyzes the conversion compared to either control, we identified 622 candidate ciliary
of biotin-phenol into biotin-phenoxyl radicals. Such radicals are proteins (Figure 2A). To identify reproducible hits, three inde-
short lived (<5 ms), have a small labeling radius (<20 nm), are pendent replicates were carried out. We grouped the final
membrane-impermeable, and can covalently react with elec- cilia-APEX proteome in a high confidence Tier 1 and a low
tron-rich amino acids such as Tyr, Trp, His, and Cys (Rhee confidence Tier 2. Tier 1 consists of 162 candidate ciliary pro-
et al., 2013). The greatest ciliary enrichment of APEX was teins that were identified by at least six spectral counts and
observed when fused to NPHP3[1203] (Nakata et al., 2012), in at least two experiments (Figure 2B; Table S1). Tier 2 con-
and the NPHP3[1203]-GFP-APEX fusion (hereafter named sists of 208 additional proteins identified by at least six spectral
cilia-APEX) was highly enriched in primary cilia of inner medullary counts in one experiment. Among the candidate ciliary
collecting duct epithelial cells (IMCD3) (Figure 1B), mouse em- proteins, we identified a large number of known ciliary proteins,
bryonic fibroblasts (MEFs), and retinal pigmented epithelial cells such as subunits of the ciliary trafficking complexes IFT-A,
(RPE1-hTERT) (Figures 1C and S1). Staining cells with Alexa IFT-B, kinesin 2, and dynein 1b; the small GTPases ARL3,
Fluor 647-labeled streptavidin (SA647) revealed that addition of ARL6, and ARL13B, as well as Septins 2 and 7 (Ghossoub
biotin-phenol and hydrogen peroxide (H2O2) led to cilia-specific et al., 2013; Hu et al., 2010) and EvC complex subunits (Pusa-
biotinylation by cilia-APEX (Figures 1B and 1C). No ciliary biotin pati et al., 2014) (Figure 2C). Several microtubule-associated
signal was detected in the absence of H2O2, biotin-phenol, or proteins previously localized to cilia (Ghossoub et al., 2013;
with a mislocalized control-APEX fusion in which the myristoyla- Patzke et al., 2010; Schrder et al., 2007), such as CSPP1,
tion site that mediates ciliary localization of NPHP3[1203] was MAP4, or EB1 were also identified in Tier 1. Centrosomal
mutated (Figure 1B). Western blot analyses showed similar proteins and transition zone proteins were conspicuously
expression of cilia-APEX and control-APEX in stable IMCD3 absent from the cilia-APEX proteome, consistent with the
cell lines (Figure 1D, lanes 11 and 12), and the biotinylated pro- highly specific localization of cilia-APEX inside cilia and the
teins could be efficiently isolated by streptavidin chromatog- estimated 20 nm labeling radius of biotinyl radicals generated
raphy of cell lysates (Figure 1D). Probing the isolated biotinylated by APEX.
proteins for ciliary markers revealed that acetylated tubulin and Unexpected candidate ciliary proteins were identified by
the IFT-B components IFT88 and CLUAP1 were specifically cilia-APEX. These include all subunits of the spindle and kinet-
recovered from cilia-APEX cells subjected to labeling, but not ochore associated (SKA) complex, four tetraspanins, ubiquity-
in the absence of labeling or from control-APEX cells treated lation enzymes, as well as proteins associated with ciliary
with biotin-phenol and H2O2 (Figure 1D, lanes 1315). Mean- functions, but not known to localize to cilia (e.g., AZI1 and
while, highly abundant proteins, such as tubulin, actin, GAPDH, Dishevelled 3) (Figure 2C). To assess the cilia-APEX proteome,
or Annexin V were either absent or greatly depleted from the we transfected epitope-tagged constructs into IMCD3 cells.
streptavidin eluates, thus demonstrating the high specificity re- SKA1 was indeed found inside cilia (Figures 2D and S2A).
sulting from the stringent streptavidin-based purifications under Given that the SKA complex enables the kinetochore to track
denaturing conditions (Figure 1D). We conclude that cilia-APEX depolymerizing microtubules in mitosis (Schmidt et al., 2012),
selectively labels ciliary proteins. SKA might function during cilium shortening to track the
axonemal tip. Similarly, the tetraspanins CD81 and CD82
Cilia-APEX Identifies a Variety of Ciliary Proteins were found in cilia (Figure 2D). Tetraspanins are palmitoylated
We next conducted liquid chromatography (LC)/MS-MS ana- 4-pass membrane proteins that shape membrane structure
lyses of cilia-APEX-labeled samples. To control for the re- by influencing lipid bilayer geometry. Intriguingly, all four
spective contributions of endogenous biotinylated proteins tetraspanins identified by cilia-APEX have been reproducibly
and of non-ciliary proteins biotinylated by APEX, we also sub- found in extracellular vesicles (Mathivanan et al., 2010; Simp-
jected unlabeled cilia-APEX and labeled control-APEX samples son et al., 2008), pointing to a possible role of these tetraspa-
to LC/MS-MS analyses (Figure 2A). By selecting proteins that nins in ciliary ectosome formation, a biologically important
were enriched at least 5-fold (estimated by spectral counts, phenomenon in C. reinhardtii (Cao et al., 2015; Wood et al.,
see Supplemental Information) in the cilia-APEX sample 2013) and nematodes (Wang et al., 2014). Finally, the clathrin
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 499
A C
D E
adaptor TOM1L2 (TOM1-like 2) was found in cilia (Figure 2D). tion rate of candidate ciliary proteins in the cilia-APEX prote-
TOM1 and TOM1L2 (both Tier 1 hits) are evolutionarily ome is 56% (Figure S2B). However, as outlined below, we
conserved ESCRT-0 proteins that carry out early endocytic believe that the sensitivity of cilia-APEX for certain proteins is
sorting of ubiquitinated cargoes by linking them to Myosin-VI greater than that of immunofluorescence, and that some pro-
(Tumbarello et al., 2012). The findings that the canonical teins scored as false positive by microscopy may be present
ESCRT-0 complex Hrs/STAM mediates the ciliary exit and lyso- in cilia at very low steady-state levels. Furthermore, the data set
somal degradation of polycystin-1 and -2 (Hu et al., 2007) and we selected for validation by immunofluorescence did not
that ubiquitination enhances ciliary exit (Xu et al., 2015) suggest include Tier 1 proteins that were previously known to localize
a possible role of TOM1/TOM1L2 in these processes. to cilia. Thus, the 56% validation rate we calculate is likely
Taking into account all Tier 1 proteins that we detected in underestimated. Compared to the PCP cilium proteome (Ishi-
cilia in the course of this study, the immunofluorescence valida- kawa et al., 2012), cilia-APEX identified many more
500 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
A
transmembrane and microtubule-associated proteins, and in Discovery of a Ciliary AC6/Cyclic AMP/PKA Signaling
particular signaling-relevant proteins, such as protein kinases Axis that Regulates Hh Signaling
and predicted calcium-binding proteins (Figure 2E). Cilia- Adenylate cyclase 3 (AC3) serves as a specific ciliary marker of
APEX thus represents a significant advance given the ease of neurons and glial cells and is generally considered to be the pre-
the approach and the broad coverage of structural, trafficking, dominant cyclic AMP (cAMP)-generating enzyme in primary cilia
and signaling components. (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 83 kDa transcriptional repressor (GLI3R). We reasoned that if
of AC3 from the cilia-APEX proteome, we failed to detect AC3 in PKA phosphorylates GLI3 within cilia, then inhibiting PKA inside
primary cilia of IMCD3 cells (Figure 3A). Meanwhile, AC6 was a cilia should block GLI3 processing. To test this prediction, we
strong Tier 1 hit in the cilia-APEX proteome (Figure 2C; Table S1), directed the specific PKA inhibitor peptide PKI (Knighton et al.,
and an antibody recognizing AC5 and AC6 revealed a clear 1991) to cilia by expressing NPHP3[1203]-GFP-PKI (cilia-PKI)
ciliary signal in IMCD3 cells (Figure 3B). In the absence of AC5- stably and at low levels in IMCD3 cells (Figure 4A). While high
and AC6-specific antibodies, we were unable to determine level expression of GFP-PKI has been reported to inhibit Hh
which of these two proteins is present in cilia of IMCD3 cells. signaling (Iglesias-Bartolome et al., 2015), the site of PKI action
However, out of 36 spectral counts that could be assigned to has not been investigated. To control for the specificity of the
either AC5 or AC6 in cilia-APEX, 32 were unique to AC6, four PKI inhibitory peptide and the importance of ciliary targeting,
were common to AC5 and AC6, and none were specific to we expressed cilia-PKI-4A, a 4-residue mutant that fails to block
AC5. These results suggest that AC6 is the main ciliary adenylate PKA activity and NPHP3[1203;G2A]-GFP-PKI (control-PKI), a
cyclase in IMCD3 cells. The presence of three distinct adenylate mutant that fails to target to cilia (Figures 4A and S3). All con-
cyclases in cilia poses the question of their respective functional structs were expressed at similarly low levels as evidenced by
significance. While AC3 has been detected in cilia of neurons western blot (Figure 4D) and the extremely weak fluorescent
(Bishop et al., 2007), kidney cells (Nikonova et al., 2014), and fi- signal of the diffusely distributed control-PKI (Figure S3). Consis-
broblasts (Halbritter et al., 2013; Ou et al., 2009), a recent publi- tent with the weak localization of GFP at the pericentriolar matrix
cation found AC5 and 6 to be the major regulators of Hh signaling (Breslow et al., 2013), control-PKI was slightly enriched at the
in the chicken neural tube and in mouse cerebellar granular ciliary base. Importantly, cilia-PKI clearly localized to the ciliary
neuron precursors (Vuolo et al., 2015). Interestingly, while shaft, distal to the basal body (marked by ninein) and the transi-
AC3, 5, and 6 are all activated by Gas, only AC5 and 6 are in- tion zone (marked by CEP290) (Figure 4B). Moreover, all IMCD3
hibited by Gai or free Ca2+ (Sadana and Dessauer, 2009). Given cell lines appropriately responded to the SMO agonist (SAG) by
that most ciliary G protein coupled receptors (GPCRs) are redistributing SMO to cilia and removing GPR161 from cilia (Fig-
coupled to either Gas or Gai, modulating the respective levels ures 4C and S3). In contrast, while control-PKI and cilia-PKI-4A
of AC3, 5, and 6 in cilia would provide a means for different cells produced high levels of GLI3R at rest, cilia-PKI expression
cell types to tune their response to ciliary GPCR activity. resulted in a clear reduction in GLI3R (Figure 4D), similar to the
A prominent signaling target of cAMP is the cAMP-dependent effect of genetically removing PKA C activity (Tuson et al.,
protein kinase PKA. At rest, PKA exists as an auto-inhibited com- 2011). Furthermore, Hh pathway stimulation results in decreased
plex of regulatory (R) and catalytic (C) subunits and binding of processing of GLI3FL into GLI3R and the GLI3R/GLI3FL ratio was
cAMP to R subunits triggers the release of active C subunits. appropriately decreased in control-PKI and cilia-PKA-4A cells
PKA exerts a powerful inhibition on Hh signaling through phos- treated with SAG (Figures 4E and 4F). In contrast, GLI3R levels
phorylation of the GLI2 and GLI3 transcription factors, and the and the GLI3R/GLI3FL ratio were barely altered by SAG addition
phenotype of a PKA-C mutant mouse is as severe as that of a to cilia-PKI cells. This demonstrates that inhibiting PKA activity
Ptc1 / mouse (Tuson et al., 2011). Paradoxically, while the within cilia leads to defective processing of GLI3 and points to-
localization of adenylate cyclases predicts that cAMP produc- ward a direct function of PKA in cilia.
tion takes place within cilia, immunocytochemistry found some Hence, consistent with the well-established functions of cAMP
concentration of PKA-C at the base of cilia (Barzi et al., 2010; Tu- inside olfactory cilia (Anholt, 1989), we propose that cAMP is
son et al., 2011). It has therefore been proposed that cAMP sensed inside cilia by the PKA holoenzyme to transmit Hh signals
diffusing out of cilia activates PKA at the base and that PKA de- to the rest of the cell. In this model, cilia harbor high levels of
posits inhibitory phosphorylations on GLI2/3 before or after cAMP owing to the activity of AC3, 5, and 6 and the tonic stimu-
GLI2/3 have passed through cilia (Mukhopadhyay and Rohatgi, lation of Gas by GPR161 (Mukhopadhyay et al., 2013). PKA thus
2014). However, it is conceivable that the pools of ciliary becomes activated inside cilia where it phosphorylates GLI3FL
PKA-C have remained undetectable because they are too labile and primes it for proteolytic processing into GLI3R. Once the
for conventional fixation methods or because of low sensitivity of Hh pathway becomes activated, GPR161 exits cilia and SMO
the immunological reagents. The presence of the PKA R subunit enters cilia where it may inhibit AC5/6 either through Gai (Riobo
Ia (PKA-RIa) in Tier 1 and of PKA-Ca and RIIb in Tier 2 of the cilia- et al., 2006; Barzi et al., 2011) or elevated ciliary Ca2+ (DeCaen
APEX proteome suggested that sensing of cAMP by PKA might et al., 2013). These alterations result in lower levels of ciliary
take place inside cilia (Figure 2C). Indeed, stably expressed cAMP, decreased phosphorylation of GLI3FL by PKA inside cilia,
NeonGreen(NG)-tagged PKA-RIa can be detected in primary and reduced production of GLI3R (Figure 4G).
cilia of IMCD3 cells (Figure 3C). Congruently, Jacob et al.
(2011) have reported that genetic ablation of PKA-RIa leads to LKB1-AMPK Signaling in Primary Cilia
Hh signaling defects. Liver kinase B1 (LKB1) is a master regulator of cell growth and
The detection of an R subunit of PKA in cilia suggested that the epithelial polarity that is frequently altered in cancer (Jansen
PKA holoenzyme may enter and become activated within cilia by et al., 2009). Instead of relying on phosphorylation for its activa-
sensing intraciliary cAMP levels. To directly test the functional tion, LKB1 is activated through formation of a heterotrimer with
significance of ciliary localization of PKA, we turned our attention the pseudokinase STRAD and the scaffolding protein MO25 (Ze-
to Hh signaling and, in particular, GLI3 processing. Upon phos- qiraj et al., 2009). Since vertebrates have two paralogues of
phorylation by PKA, GLI3 undergoes partial proteolysis and be- STRAD (a and b) and two paralogues of MO25 (a and b), the
comes converted from a 190 kDa polypeptide (GLI3FL) into a functional diversification of the four STRAD-MO25-LKB1
502 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
A B
C D E
F G
complexes remains an intriguing yet unsolved question. Interest- Madin-Darby canine kidney (MDCK) cells (Boehlke et al.,
ingly, STRADb, but not STRADa, was found in the cilia-APEX 2010), and since LKB1 was a Tier 2 hit in the cilia-APEX
proteome (Figure 2C) and transfection of epitope-tagged proteome, we tested the contribution of STRADb to LKB1
STRADb confirmed a significant ciliary enrichment of STRADb localization. In our experimental setup, endogenous LKB1 was
(Figure 5A). Since LKB1 has been localized to primary cilia of undetectable in IMCD3 cilia unless exogenous STRADb was
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 503
A
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 et al., 2008). Since the BBSome also functions in the trafficking
of CLUAP1 at the basal body appeared unchanged in Ift27 / of SMO (Zhang et al., 2011, 2012), b-arrestin 2 may cooperate
cells, the levels of CLUAP1 along the length of the cilium were with the BBSome to remove SMO from cilia.
significantly reduced (Figures 7A and 7B). Meanwhile, the total Thus, the comparative cilia-APEX analyses have uncovered an
cellular abundance of CLUAP1 was unaffected by Ift27 deletion unexpected candidate cargo of the BBSome (CAR) and a puta-
(Figure 7C). Thus, while Ift27 deletion has been reported to not tive adaptor for BBSome-mediated export (b-arrestin 2), further
significantly affect IFT88 localization to cilia (Eguether et al., highlighting the power of the comparative cilia-APEX method.
2014; Liew et al., 2014) or the ciliary abundance of other IFT sub-
units (Figures 6A and 6B), CLUAP1 appears to be reduced in DISCUSSION
Ift27 / cilia. Whether this is due to general reduction of IFT-B
within Ift27 / cilia, as suggested by the general trend of IFT Work in the past decade has established the importance of the
depletion (see Figure 6A), or whether this defect is specific for primary cilium in signaling and in human diseases characterized
CLUAP1 remains to be investigated. Similar to the IFT27/25 by obesity, retinal degeneration, and kidney cysts. However, our
module, CLUAP1 may be considered a peripheral IFT-B subunit, understanding of the physico-chemical environment of cilia and
since it has not been identified in initial preparations of mamma- how it endows cilia with signaling properties remains fragmen-
lian IFT. Therefore, it is conceivable that CLUAP1 requires IFT27 tary. A major technological limitation to the understanding of
for proper association with the IFT-B complex. events that take place inside cilia lies in our inability to obtain
The top enriched protein in Ift27 / cilia is the Coxsackie and reproducible and pure fractions of primary cilia for sensitive anal-
Adenovirus Receptor (CAR), an integral component of the ysis of the ciliary proteome by MS. Here, we show that the APEX
epithelial tight junction complex (Cohen et al., 2001). Expression labeling technology is a versatile, readily applicable, and highly
of epitope-tagged proteins revealed very few cilia positive for effective approach to study the protein content of primary cilia.
CAR in WT cells. Similarly, CAR-positive cilia were rarely found The cilia-APEX fusion functions in a variety of cell types and en-
when staining for endogenous CAR (Figures 7D and 7E). How- ables a quantitative analysis of the alterations in ciliary protein
ever, when analyzing Ift27 / mutants, the majority of cilia contents resulting from a specific mutation. This technology
were clearly positive for CAR, even though the total levels of promises a comprehensive and quantitative assessment of dif-
CAR were unchanged (Figures 7C7E). The possible role of the ferences in the ciliary proteomes between healthy and disease
BBSome in trafficking the viral receptor CAR out of cilia remains states, between different cell types, between different geno-
enigmatic. CAR is normally found on the basolateral side of tight types, and upon exposure to specific drugs and other small mol-
junctions where it associates with cytoskeletal and signaling pro- ecules. The highly specific information provided by the compar-
teins (Cohen et al., 2001; Coyne et al., 2004; Sollerbrant et al., ative cilia-APEX approach allows for the generation of precise
2003). One first possibility is that the presence of CAR inside cilia molecular hypotheses as exemplified by the identification of
is detrimental to the cell and that the BBSome functions as a most BBSome subunits, as well as the BBSome associated pro-
clearing device as previously suggested for phospholipase D in tein LZTFL1 as proteins that accumulate in Ift27 / cilia. Within a
Chlamydomonas (Lechtreck et al., 2013). Since CAR is typically few weeks, the comparative cilia-APEX study of Ift27 / versus
not present on apical membranes, the entry of Coxsackie virus WT cells produced a definitive molecular hypothesis that had
and Adenovirus into the organism occurs after breaching of tight taken over 1 year to be developed by traditional methods of tan-
junctions on respiratory and intestinal cells. However, in the dem affinity purification (TAP) tagging and searching for the
absence of the clathrin adaptor AP1B, as naturally occurring in physiologically relevant interactors of IFT27 (Liew et al., 2014).
retinal pigmented epithelium cells, CAR becomes misrouted to Furthermore, the comparative cilia-APEX study uncovered
the apical membrane and adenoviruses can efficiently enter cells entirely unexpected candidate BBSome cargoes and adaptors
from the apical side (Diaz et al., 2009). Similarly, when BBSome that had escaped detection by traditional biochemical associa-
function is compromised, CAR may accumulate within cilia and tion methods. Our proof-of-concept study of Ift27 / versus
permit entry of Adenovirus and Coxsackie virus from the apical WT cells thus suggests that cilia-APEX profiling may constitute
side of the cells. More research will be required to test whether a rapid and powerful method for the characterization of ciliop-
BBS patients and animal models are more susceptible to viral in- athy mutants.
fections by Adenovirus and Coxsackie virus. Alternatively, it is Interestingly, AMPKb2, which was clearly identified by cilia-
conceivable that CAR performs a thus far unsuspected function APEX, was initially not detected inside cilia by immunohisto-
inside cilia. Similar to several ciliopathy mouse models, CAR chemistry. Since AMPKb2 becomes clearly detectable inside
knockout mice die between embryonic day (E)11 and E13 from cilia once exit is impaired, this suggests that AMPKb2 rapidly
insufficient heart function and display cardiac pericardial edema traverses the cilium. Considering that the APEX labeling reaction
(Dorner et al., 2005). is performed for 1 min, the extent of biotinylation of a given pro-
Another top hit in our comparative proteomics was b-arrestin 2 tein integrates the number of molecules that visited the cilium
(Figure 6A), and we observed increased levels of b-arrestin 2 in either transiently or stablywithin this time frame. In theory, a
Ift27 / cilia by fluorescence microscopy (Figures 7F and S5). cilium-resident protein present at 600 copies in the cilium at
b-arrestins mediate clathrin-dependent internalization of acti- steady state will be biotinylated by cilia-APEX as efficiently as
vated GPCRs by binding to the phosphorylated cytoplasmic tails a protein present at 10 copies in the cilium at steady state, but
of GPCRs (Reiter and Lefkowitz, 2006). b-arrestin 2 has been whose ciliary turn-over rate is 1 s. This interpretation is congruent
previously detected inside cilia (Molla-Herman et al., 2008) and with the extremely low number (150 copies) of known ciliary
was suggested to participate in SMO ciliary trafficking (Kovacs signaling molecules such as the ciliary Ca2+ channel PKD1L1/
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 507
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 Image Analysis
proteins with enzymatic activities that function inside cilia Analysis of imaging data was performed using ImageJ software (NIH). For
measurement of the amounts of CLUAP1 in cilia, three planes from z stacks
may have escaped detection by traditional methods. The accu-
acquired at 0.3 mm interval were projected and the cilia defined by segmented
mulation of signaling proteins in Ift27 / cilia highlights the lines (width 3 pixels) based on cilia-APEX signal. The mean signal was sub-
Ift27 / mutant as a promising tool to identify ciliary proteins tracted by the mean background and multiplied by cilium length. The average
whose steady-state levels have remained below the detection of at least 20 cilia was calculated for three independent experiments and SEMs
limit of immunological reagents. Other mutants defective in were calculated. For CAR, at least 60 cilia were counted in three independent
exit from cilia such as IFT-A and dynein-1b may prove similarly experiments and percentages of positive cilia and SEs calculated.
useful.
Streptavidin Chromatography
In light of the ability of cilia-APEX to detect proteins transiently
Protein concentrations of lysates prepared from APEX-labeling experiments
visiting cilia, it is likely that several of the false positives that we were determined by Bradford assay and lysates adjusted to equal protein con-
have been unable to detect in cilia by immunofluorescence (Fig- centrations. Lysates were diluted 10-fold with wash buffer (lysis buffer without
ure S2B) may only become detectable by standard imaging urea supplemented with 0.5% Triton X-100, and 0.1% SDS), from which a
methods once studied in the appropriate export mutant. The sample was taken as loading control. Diluted lysates were added onto Strep-
absence of common contaminants such as chaperones and ri- tavidin-Sepharose beads (Thermo Scientific) and biotinylated proteins were
captured for 1 hr at room temperature. Beads were washed extensively first
bosomal proteins from the cilia-APEX proteome suggests a
with wash buffer, then with 4 M urea 10 mM Tris/HCl (pH 7.5), and finally
very low rate of true false positives. Meanwhile, the absence with 4 M urea, 10 mM Tris/HCl (pH 7.5), and 50 mM biotin. For SDS-PAGE an-
of several known IFT polypeptides from the cilia-APEX data set alyses, bound material was eluted by boiling beads in SDS sample buffer sup-
is likely to represent true negatives and may reflect the selectivity plemented with 2 mM biotin. For proteomics analyses, bound proteins were
of the proximity biotinylation method toward electron-rich amino eluted by boiling beads in filter aided sample preparation (FASP) buffer
acids exposed on the surface of proteins. Consistently, most of (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
the missing IFT subunits in the cilia-APEX data set are small pro-
described (Wisniewski et al., 2009). For comparative proteomics of WT and
teins that likely bear no surface-exposed amino acids reactive Ift27 / cells, beads were directly subjected to alkylation and elution by trypsin
with biotin-phenoxyl radicals. digest.
A unique feature of the APEX technology is its ability to provide
temporal snapshots of the ciliary proteome with minute-scale Hh Signaling Experiments
resolution, and cilia-APEX has the potential to reveal dynamic To visualize trafficking events during Hh signaling, 50,000 cells were seeded
changes in the ciliary protein content after exposure to signaling 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-
ligands such as Hh. Moreover, the success of cilia-APEX gener-
ation in 4% paraformaldehyde. For western blot analyses, 300,000 cells were
alizes the applicability of proximity labeling for proteomics of seeded into 6-well plates before treatments as above. Cells were washed in
cellular subcompartments or domains beyond membrane-en- 13 PBS and lysed in buffer (0.3 M NaCl, 1 mM EDTA, 1 mM EGTA, 10% glyc-
closed organelles such as mitochondria (Lam et al., 2015; erol, 25 mM Tris/HCl [pH 7.5], 0.5% Triton X-100, 0.1% SDS, and protease
Rhee et al., 2013). In particular, the absence of transition zone inhibitors) for 15 min. Lysates were cleared by centrifugation and protein
and basal body proteins from the cilia-APEX proteome suggests concentrations determined by Bradford assay. There was 8 mg of total protein
separated by SDS-PAGE and analyzed by western blot using indicated
that the rapid and highly localized labeling provided by the APEX
antibodies.
enzyme may be leveraged to capture snapshots of the proteome
of other protrusions such as lamellipodia, filopodia, and MS Analyses
microvilli. 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-
EXPERIMENTAL PROCEDURES ery rate. See the Supplemental Information for details.
Detailed information about the generation of cell lines and plasmids, as well as
SUPPLEMENTAL INFORMATION
antibodies and reagents used in this study, can be found in the Supplemental
Information together with detailed descriptions of instrumentation and soft-
Supplemental Information includes Supplemental Experimental Procedures,
ware used for immunofluorescence microscopy, image analyses, as well as
five figures, and two tables and can be found with this article online at http://
MS analyses.
dx.doi.org/10.1016/j.devcel.2015.10.015.
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 509
Bornens, and Sophie Saunier for the gifts of antibodies; Iain Cheeseman and DeCaen, P.G., Delling, M., Vien, T.N., and Clapham, D.E. (2013). Direct
Mark Scott for the gifts of cDNAs; the DuBois lab for help with synthesis of recording and molecular identification of the calcium channel of primary cilia.
biotin-phenol; David Martinelli for the gift of mouse cortical neurons; Anna Nature 504, 315318.
Okumu, Krysta D. Wyatt, and Jaclyn S. Lee for technical assistance; and Diaz, F., Gravotta, D., Deora, A., Schreiner, R., Schoggins, J., Falck-Pedersen,
Bianca Schrul and the Nachury lab for helpful comments, careful reading of E., and Rodriguez-Boulan, E. (2009). Clathrin adaptor AP1B controls adeno-
the manuscript, and discussions. This work was supported in part by NIH virus infectivity of epithelial cells. Proc. Natl. Acad. Sci. USA 106, 11143
grants to M.V.N. (GM089933) and S.P.G. (GM67945) and by a Stanford 11148.
Dean of Research-Stanford University Mass Spectrometry (SUMS) Seed
Dorner, A.A., Wegmann, F., Butz, S., Wolburg-Buchholz, K., Wolburg, H.,
Grant to M.V.N. D.U.M. was supported by an EMBO long-term fellowship
Mack, A., Nasdala, I., August, B., Westermann, J., Rathjen, F.G., and
and an A.P. Giannini Foundation fellowship. SUMS acknowledges support
Vestweber, D. (2005). Coxsackievirus-adenovirus receptor (CAR) is essential
from NCRR (S10RR027425).
for early embryonic cardiac development. J. Cell Sci. 118, 35093521.
Received: May 27, 2015 Eguether, T., San Agustin, J.T., Keady, B.T., Jonassen, J.A., Liang, Y., Francis,
Revised: September 26, 2015 R., Tobita, K., Johnson, C.A., Abdelhamed, Z.A., Lo, C.W., and Pazour, G.J.
Accepted: October 19, 2015 (2014). IFT27 links the BBSome to IFT for maintenance of the ciliary signaling
Published: November 12, 2015 compartment. Dev. Cell 31, 279290.
Firat-Karalar, E.N., Rauniyar, N., Yates, J.R., 3rd, and Stearns, T. (2014).
REFERENCES Proximity interactions among centrosome components identify regulators of
centriole duplication. Curr. Biol. 24, 664670.
Anholt, R.R. (1989). Molecular physiology of olfaction. Am. J. Physiol. 257, Fliegauf, M., Benzing, T., and Omran, H. (2007). When cilia go bad: cilia defects
C1043C1054. and ciliopathies. Nat. Rev. Mol. Cell Biol. 8, 880893.
Barzi, M., Berenguer, J., Menendez, A., Alvarez-Rodriguez, R., and Pons, S. Follit, J.A., Li, L., Vucica, Y., and Pazour, G.J. (2010). The cytoplasmic tail of
(2010). Sonic-hedgehog-mediated proliferation requires the localization of fibrocystin contains a ciliary targeting sequence. J. Cell Biol. 188, 2128.
PKA to the cilium base. J. Cell Sci. 123, 6269. Ghossoub, R., Hu, Q., Failler, M., Rouyez, M.-C., Spitzbarth, B., Mostowy, S.,
Barzi, M., Kostrz, D., Menendez, A., and Pons, S. (2011). Sonic Hedgehog- Wolfrum, U., Saunier, S., Cossart, P., Nelson, W.J., and Benmerah, A. (2013).
induced proliferation requires specific Ga inhibitory proteins. J. Biol. Chem. Septins 2, 7 and 9 and MAP4 colocalize along the axoneme in the primary
286, 80678074. cilium and control ciliary length. J. Cell Sci. 126, 25832594.
Bettencourt-Dias, M., Hildebrandt, F., Pellman, D., Woods, G., and Godinho, Goetz, S.C., and Anderson, K.V. (2010). The primary cilium: a signalling centre
S.A. (2011). Centrosomes and cilia in human disease. Trends Genet. 27, during vertebrate development. Nat. Rev. Genet. 11, 331344.
307315. Halbritter, J., Bizet, A.A., Schmidts, M., Porath, J.D., Braun, D.A., Gee, H.Y.,
Bishop, G.A., Berbari, N.F., Lewis, J., and Mykytyn, K. (2007). Type III adenylyl McInerney-Leo, A.M., Krug, P., Filhol, E., Davis, E.E., et al.; UK10K
cyclase localizes to primary cilia throughout the adult mouse brain. J. Comp. Consortium (2013). Defects in the IFT-B component IFT172 cause Jeune
Neurol. 505, 562571. and Mainzer-Saldino syndromes in humans. Am. J. Hum. Genet. 93, 915925.
Boehlke, C., Kotsis, F., Patel, V., Braeg, S., Voelker, H., Bredt, S., Beyer, T., Hu, J., Wittekind, S.G., and Barr, M.M. (2007). STAM and Hrs down-regulate
Janusch, H., Hamann, C., Godel, M., et al. (2010). Primary cilia regulate ciliary TRP receptors. Mol. Biol. Cell 18, 32773289.
mTORC1 activity and cell size through Lkb1. Nat. Cell Biol. 12, 11151122. Hu, Q., Milenkovic, L., Jin, H., Scott, M.P., Nachury, M.V., Spiliotis, E.T., and
Botilde, Y., Yoshiba, S., Shinohara, K., Hasegawa, T., Nishimura, H., Shiratori, Nelson, W.J. (2010). A septin diffusion barrier at the base of the primary cilium
H., and Hamada, H. (2013). Cluap1 localizes preferentially to the base and tip maintains ciliary membrane protein distribution. Science 329, 436439.
of cilia and is required for ciliogenesis in the mouse embryo. Dev. Biol. 381, Iglesias-Bartolome, R., Torres, D., Marone, R., Feng, X., Martin, D., Simaan,
203212. M., Chen, M., Weinstein, L.S., Taylor, S.S., Molinolo, A.A., and Gutkind, J.S.
Breslow, D.K., Koslover, E.F., Seydel, F., Spakowitz, A.J., and Nachury, M.V. (2015). Inactivation of a Ga(s)-PKA tumour suppressor pathway in skin stem
(2013). An in vitro assay for entry into cilia reveals unique properties of the sol- cells initiates basal-cell carcinogenesis. Nat. Cell Biol. 17, 793803.
uble diffusion barrier. J. Cell Biol. 203, 129147. Ishikawa, H., Thompson, J., Yates, J.R., 3rd, and Marshall, W.F. (2012).
Cao, M., Ning, J., Hernandez-Lara, C.I., Belzile, O., Wang, Q., Dutcher, S.K., Proteomic analysis of mammalian primary cilia. Curr. Biol. 22, 414419.
Liu, Y., and Snell, W.J. (2015). Uni-directional ciliary membrane protein traf- Jacob, L.S., Wu, X., Dodge, M.E., Fan, C.-W., Kulak, O., Chen, B., Tang, W.,
ficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding. Wang, B., Amatruda, J.F., and Lum, L. (2011). Genome-wide RNAi screen re-
eLife 4, 4. veals disease-associated genes that are common to Hedgehog and Wnt
Cohen, C.J., Shieh, J.T., Pickles, R.J., Okegawa, T., Hsieh, J.T., and signaling. Sci. Signal. 4, ra4.
Bergelson, J.M. (2001). The coxsackievirus and adenovirus receptor is a trans- Jansen, M., Ten Klooster, J.P., Offerhaus, G.J., and Clevers, H. (2009). LKB1
membrane component of the tight junction. Proc. Natl. Acad. Sci. USA 98, and AMPK family signaling: the intimate link between cell polarity and energy
1519115196. metabolism. Physiol. Rev. 89, 777798.
Constantine, R., Zhang, H., Gerstner, C.D., Frederick, J.M., and Baehr, W. Jin, H., White, S.R., Shida, T., Schulz, S., Aguiar, M., Gygi, S.P., Bazan, J.F.,
(2012). Uncoordinated (UNC)119: coordinating the trafficking of myristoylated and Nachury, M.V. (2010). The conserved Bardet-Biedl syndrome proteins
proteins. Vision Res. 75, 2632. assemble a coat that traffics membrane proteins to cilia. Cell 141, 12081219.
Corbit, K.C., Aanstad, P., Singla, V., Norman, A.R., Stainier, D.Y.R., and Reiter, Kim, J., Kato, M., and Beachy, P.A. (2009). Gli2 trafficking links Hedgehog-
J.F. (2005). Vertebrate Smoothened functions at the primary cilium. Nature dependent activation of Smoothened in the primary cilium to transcriptional
437, 10181021. activation in the nucleus. Proc. Natl. Acad. Sci. USA 106, 2166621671.
Coyne, C.B., Voelker, T., Pichla, S.L., and Bergelson, J.M. (2004). The cox- Knighton, D.R., Zheng, J.H., Ten Eyck, L.F., Xuong, N.H., Taylor, S.S., and
sackievirus and adenovirus receptor interacts with the multi-PDZ domain pro- Sowadski, J.M. (1991). Structure of a peptide inhibitor bound to the catalytic
tein-1 (MUPP-1) within the tight junction. J. Biol. Chem. 279, 4807948084. subunit of cyclic adenosine monophosphate-dependent protein kinase.
Craige, B., Tsao, C.-C., Diener, D.R., Hou, Y., Lechtreck, K.F., Rosenbaum, Science 253, 414420.
J.L., and Witman, G.B. (2010). CEP290 tethers flagellar transition zone micro- Kovacs, J.J., Whalen, E.J., Liu, R., Xiao, K., Kim, J., Chen, M., Wang, J., Chen,
tubules to the membrane and regulates flagellar protein content. J. Cell Biol. W., and Lefkowitz, R.J. (2008). Beta-arrestin-mediated localization of smooth-
190, 927940. ened to the primary cilium. Science 320, 17771781.
510 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.
Kwon, R.Y., Temiyasathit, S., Tummala, P., Quah, C.C., and Jacobs, C.R. Pasek, R.C., Berbari, N.F., Lewis, W.R., Kesterson, R.A., and Yoder, B.K.
(2010). Primary cilium-dependent mechanosensing is mediated by adenylyl (2012). Mammalian Clusterin associated protein 1 is an evolutionarily
cyclase 6 and cyclic AMP in bone cells. FASEB J. 24, 28592868. conserved protein required for ciliogenesis. Cilia 1, 20.
Lam, S.S., Martell, J.D., Kamer, K.J., Deerinck, T.J., Ellisman, M.H., Mootha, Patzke, S., Redick, S., Warsame, A., Murga-Zamalloa, C.A., Khanna, H.,
V.K., and Ting, A.Y. (2015). Directed evolution of APEX2 for electron micro- Doxsey, S., and Stokke, T. (2010). CSPP is a ciliary protein interacting with
scopy and proximity labeling. Nat. Methods 12, 5154. Nephrocystin 8 and required for cilia formation. Mol. Biol. Cell 21, 25552567.
Lambert, J.-P., Tucholska, M., Go, C., Knight, J.D.R., and Gingras, A.-C. Pusapati, G.V., Hughes, C.E., Dorn, K.V., Zhang, D., Sugianto, P., Aravind, L.,
(2015). Proximity biotinylation and affinity purification are complementary and Rohatgi, R. (2014). EFCAB7 and IQCE regulate hedgehog signaling by
approaches for the interactome mapping of chromatin-associated protein tethering the EVC-EVC2 complex to the base of primary cilia. Dev. Cell 28,
complexes. J. Proteomics 118, 8194. 483496.
Lechtreck, K.F., Johnson, E.C., Sakai, T., Cochran, D., Ballif, B.A., Rush, J., Reiter, E., and Lefkowitz, R.J. (2006). GRKs and beta-arrestins: roles in recep-
Pazour, G.J., Ikebe, M., and Witman, G.B. (2009). The Chlamydomonas rein- tor silencing, trafficking and signaling. Trends Endocrinol. Metab. 17, 159165.
hardtii BBSome is an IFT cargo required for export of specific signaling pro- Rhee, H.-W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A., and
teins from flagella. J. Cell Biol. 187, 11171132. Ting, A.Y. (2013). Proteomic mapping of mitochondria in living cells via
spatially restricted enzymatic tagging. Science 339, 13281331.
Lechtreck, K.F., Brown, J.M., Sampaio, J.L., Craft, J.M., Shevchenko, A.,
Evans, J.E., and Witman, G.B. (2013). Cycling of the signaling protein phos- Riobo, N.A., Saucy, B., Dilizio, C., and Manning, D.R. (2006). Activation of het-
pholipase D through cilia requires the BBSome only for the export phase. erotrimeric G proteins by Smoothened. Proc. Natl. Acad. Sci. USA 103, 12607
J. Cell Biol. 201, 249261. 12612.
Li, Y.-H., Luo, J., Mosley, Y.-Y.C., Hedrick, V.E., Paul, L.N., Chang, J., Zhang, Rohatgi, R., Milenkovic, L., and Scott, M.P. (2007). Patched1 regulates hedge-
G., Wang, Y.-K., Banko, M.R., Brunet, A., et al. (2015). AMP-activated protein hog signaling at the primary cilium. Science 317, 372376.
kinase directly phosphorylates and destabilizes Hedgehog pathway transcrip- Roux, K.J., Kim, D.I., Raida, M., and Burke, B. (2012). A promiscuous biotin
tion factor GLI1 in medulloblastoma. Cell Rep. 12, 599609. ligase fusion protein identifies proximal and interacting proteins in mammalian
cells. J. Cell Biol. 196, 801810.
Liew, G.M., Ye, F., Nager, A.R., Murphy, J.P., Lee, J.S., Aguiar, M., Breslow,
D.K., Gygi, S.P., and Nachury, M.V. (2014). The intraflagellar transport protein Sadana, R., and Dessauer, C.W. (2009). Physiological roles for G protein-regu-
IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3. Dev. lated adenylyl cyclase isoforms: insights from knockout and overexpression
Cell 31, 265278. studies. Neurosignals 17, 522.
Masyuk, A.I., Gradilone, S.A., Banales, J.M., Huang, B.Q., Masyuk, T.V., Lee, Schmidt, J.C., Arthanari, H., Boeszoermenyi, A., Dashkevich, N.M., Wilson-
S.-O., Splinter, P.L., Stroope, A.J., and LaRusso, N.F. (2008). Cholangiocyte Kubalek, E.M., Monnier, N., Markus, M., Oberer, M., Milligan, R.A., Bathe,
primary cilia are chemosensory organelles that detect biliary nucleotides via M., et al. (2012). The kinetochore-bound Ska1 complex tracks depolymerizing
P2Y12 purinergic receptors. Am. J. Physiol. Gastrointest. Liver Physiol. 295, microtubules and binds to curved protofilaments. Dev. Cell 23, 968980.
G725G734. Schrder, J.M., Schneider, L., Christensen, S.T., and Pedersen, L.B. (2007).
Mathivanan, S., Ji, H., and Simpson, R.J. (2010). Exosomes: extracellular EB1 is required for primary cilia assembly in fibroblasts. Curr. Biol. 17, 1134
organelles important in intercellular communication. J. Proteomics 73, 1907 1139.
1920. Simpson, R.J., Jensen, S.S., and Lim, J.W.E. (2008). Proteomic profiling of
exosomes: current perspectives. Proteomics 8, 40834099.
Mayer, U., Ungerer, N., Klimmeck, D., Warnken, U., Schnolzer, M., Frings, S.,
and Mohrlen, F. (2008). Proteomic analysis of a membrane preparation from rat Sollerbrant, K., Raschperger, E., Mirza, M., Engstrom, U., Philipson, L.,
olfactory sensory cilia. Chem. Senses 33, 145162. Ljungdahl, P.O., and Pettersson, R.F. (2003). The Coxsackievirus and adeno-
virus receptor (CAR) forms a complex with the PDZ domain-containing protein
Molla-Herman, A., Boularan, C., Ghossoub, R., Scott, M.G.H., Burtey, A.,
ligand-of-numb protein-X (LNX). J. Biol. Chem. 278, 74397444.
Zarka, M., Saunier, S., Concordet, J.-P., Marullo, S., and Benmerah, A.
(2008). Targeting of beta-arrestin2 to the centrosome and primary cilium: Teperino, R., Amann, S., Bayer, M., McGee, S.L., Loipetzberger, A., Connor,
role in cell proliferation control. PLoS ONE 3, e3728. T., Jaeger, C., Kammerer, B., Winter, L., Wiche, G., et al. (2012). Hedgehog
partial agonism drives Warburg-like metabolism in muscle and brown fat.
Mukhopadhyay, S., and Rohatgi, R. (2014). G-protein-coupled receptors,
Cell 151, 414426.
Hedgehog signaling and primary cilia. Semin. Cell Dev. Biol. 33, 6372.
Tukachinsky, H., Lopez, L.V., and Salic, A. (2010). A mechanism for vertebrate
Mukhopadhyay, S., Wen, X., Ratti, N., Loktev, A., Rangell, L., Scales, S.J., and Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein
Jackson, P.K. (2013). The ciliary G-protein-coupled receptor Gpr161 nega- complexes. J. Cell Biol. 191, 415428.
tively regulates the Sonic hedgehog pathway via cAMP signaling. Cell 152,
Tumbarello, D.A., Waxse, B.J., Arden, S.D., Bright, N.A., Kendrick-Jones, J.,
210223.
and Buss, F. (2012). Autophagy receptors link myosin VI to autophagosomes
Nachury, M.V. (2014). How do cilia organize signalling cascades? Philos. to mediate Tom1-dependent autophagosome maturation and fusion with the
Trans. R. Soc. Lond. B Biol. Sci. 369, 20130465. lysosome. Nat. Cell Biol. 14, 10241035.
Nakata, K., Shiba, D., Kobayashi, D., and Yokoyama, T. (2012). Targeting of Tuson, M., He, M., and Anderson, K.V. (2011). Protein kinase A acts at the
Nphp3 to the primary cilia is controlled by an N-terminal myristoylation site basal body of the primary cilium to prevent Gli2 activation and ventralization
and coiled-coil domains. Cytoskeleton (Hoboken) 69, 221234. of the mouse neural tube. Development 138, 49214930.
Nikonova, A.S., Plotnikova, O.V., Serzhanova, V., Efimov, A., Bogush, I., Cai, Vuolo, L., Herrera, A., Torroba, B., Menendez, A., and Pons, S. (2015). Ciliary
K.Q., Hensley, H.H., Egleston, B.L., Klein-Szanto, A., Seeger-Nukpezah, T., adenylyl cyclases control the Hedgehog pathway. J. Cell Sci. 128, 29282937.
and Golemis, E.A. (2014). Nedd9 restrains renal cystogenesis in Pkd1-/- Wang, J., Silva, M., Haas, L.A., Morsci, N.S., Nguyen, K.C.Q., Hall, D.H., and
mice. Proc. Natl. Acad. Sci. USA 111, 1285912864. Barr, M.M. (2014). C. elegans ciliated sensory neurons release extracellular
Ocbina, P.J.R., and Anderson, K.V. (2008). Intraflagellar transport, cilia, and vesicles that function in animal communication. Curr. Biol. 24, 519525.
mammalian Hedgehog signaling: analysis in mouse embryonic fibroblasts. Wen, X., Lai, C.K., Evangelista, M., Hongo, J.-A., de Sauvage, F.J., and Scales,
Dev. Dyn. 237, 20302038. S.J. (2010). Kinetics of hedgehog-dependent full-length Gli3 accumulation in
Ou, Y., Ruan, Y., Cheng, M., Moser, J.J., Rattner, J.B., and van der Hoorn, F.A. primary cilia and subsequent degradation. Mol. Cell. Biol. 30, 19101922.
(2009). Adenylate cyclase regulates elongation of mammalian primary cilia. Wisniewski, J.R., Zougman, A., Nagaraj, N., and Mann, M. (2009). Universal
Exp. Cell Res. 315, 28022817. sample preparation method for proteome analysis. Nat. Methods 6, 359362.
Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc. 511
Wood, C.R., Huang, K., Diener, D.R., and Rosenbaum, J.L. (2013). The cilium Zhang, Q., Nishimura, D., Seo, S., Vogel, T., Morgan, D.A., Searby, C., Bugge,
secretes bioactive ectosomes. Curr. Biol. 23, 906911. K., Stone, E.M., Rahmouni, K., and Sheffield, V.C. (2011). Bardet-Biedl syn-
Xu, Q., Zhang, Y., Wei, Q., Huang, Y., Li, Y., Ling, K., and Hu, J. (2015). BBS4 drome 3 (Bbs3) knockout mouse model reveals common BBS-associated
and BBS5 show functional redundancy in the BBSome to regulate the degra- phenotypes and Bbs3 unique phenotypes. Proc. Natl. Acad. Sci. USA 108,
dative sorting of ciliary sensory receptors. Sci. Rep. 5, 11855. 2067820683.
Zeqiraj, E., Filippi, B.M., Deak, M., Alessi, D.R., and van Aalten, D.M.F. (2009). Zhang, Q., Seo, S., Bugge, K., Stone, E.M., and Sheffield, V.C. (2012). BBS
Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mecha- proteins interact genetically with the IFT pathway to influence SHH-related
nism of kinase activation. Science 326, 17071711. phenotypes. Hum. Mol. Genet. 21, 19451953.
512 Developmental Cell 35, 497512, November 23, 2015 2015 Elsevier Inc.