4694 | C. E. Larkins et al. Molecular Biology of the Cell
MBoC | ARTICLE
Arl13b regulates ciliogenesis and the dynamic
localization of Shh signaling proteins
Christine E. Larkinsa,b, Gladys D. Gonzalez Avilesa, Michael P. Eastb,c, Richard A. Kahnc,
and Tamara Casparya
aDepartment of Human Genetics, School of Medicine, bGraduate Program in Biochemistry, Cell and Developmental
Biology, and cDepartment of Biochemistry, School of Medicine, Emory University, Atlanta, GA 30322
ABSTRACT Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras super-
family of GTPases, is required for ciliary structure but has poorly defined ciliary functions. In
this paper, we further characterize the role of Arl13b in cilia by examining mutant cilia in vitro
and determining the localization and dynamics of Arl13b within the cilium. Previously, we
showed that mice lacking Arl13b have abnormal Sonic hedgehog (Shh) signaling; in this study,
we show the dynamics of Shh signaling component localization to the cilium are disrupted in
the absence of Arl13b. Significantly, we found Smoothened (Smo) is enriched in Arl13b-null
cilia regardless of Shh pathway stimulation, indicating Arl13b regulates the ciliary entry of
Smo. Furthermore, our analysis defines a role for Arl13b in regulating the distribution of Smo
within the cilium. These results suggest that abnormal Shh signaling in Arl13b mutant em-
bryos may result from defects in protein localization and distribution within the cilium.
The regulation of protein delivery and movement within primary
cilia is key to our understanding of how cilia are built and how their
length is controlled, as well as how cell signaling pathways are regu-
lated (Veland et al., 2009). Ciliary length and cell signaling are both
disrupted in the absence of Arl13b, a small GTPase that localizes to
cilia. Arl13bhennin (hnn) mutant mouse embryos have cilia that are one-
half the length of wild-type and a specific defect in the axoneme,
where the B-tubule of the outer doublet microtubules is not con-
nected to the A-tubule (Caspary et al., 2007). In addition, these mu-
tant embryos show a low level of expanded Shh activation in the
neural tube (Caspary et al., 2007). In mammals, Arl13b is one of ∼30
ADP-ribosylation factor (Arf) family proteins, best known for regula-
tory roles in membrane trafficking and cytoskeletal dynamics
(D’Souza-Schorey and Chavrier, 2006; Zhou et al., 2006). Therefore
defining the molecular actions of Arl13b in cilia and ciliogenesis is
expected to shed light on the links between ciliary formation and
signaling, with possible links to membrane traffic and/or cytoskele-
Almost all components of the Sonic hedgehog (Shh) signaling
pathway are localized to the cilium, and their localization shifts in
response to the Shh ligand (Corbit et al., 2005; Haycraft et al., 2005;
Rohatgi et al., 2007; Chen et al., 2009; Wen et al., 2010). In the ab-
sence of ligand, the Gli transcription factors Gli2 and Gli3 are local-
ized to the tips of cilia and are processed to form transcriptional re-
pressors (GliRs; Haycraft et al., 2005; Huangfu and Anderson, 2005;
Liu et al., 2005). This processing involves the phosphorylation and
cleavage of the full-length Glis, with the N-terminal domain acting
as the repressor and the C-terminal domain being degraded (Wang
et al., 2000). The receptor for the Shh ligand, Patched (Ptch1), is also
found in the ciliary membrane, and represses pathway activation in
the absence of ligand by inhibiting the downstream activator,
Smoothened (Smo; Rohatgi et al., 2007). When Shh ligand is pres-
ent, Shh binds Ptch1, causing it to move out of the cilium, and
this allows Smo to enter (Corbit et al., 2005; Rohatgi et al., 2007).
Smo localization to the cilium inhibits GliR formation and, via an
unknown mechanism, the full-length Glis become Gli activators
(GliAs; McMahon et al., 2003). Suppressor of Fused (Sufu), an in-
hibitor of Gli activity, is also localized to the tips of cilia, although
Sufu was found to inhibit Shh signaling independently of the cilium
University of North Carolina
Received: Dec 21, 2010
Revised: Aug 31, 2011
Accepted: Sep 26, 2011
Address correspondence to: Tamara Caspary (firstname.lastname@example.org).
Abbreviations used: Arf, ADP-ribosylation factor; EYFP, enhanced yellow fluores-
cent protein; FBS, fetal bovine serum; FRAP, fluorescence recovery after photo-
bleaching; GFP, green fluorescent protein; GliA, Gli activators; GliR, Gli transcrip-
tional repressor; IFT, intraflagellar transport; MEFs, mouse embryonic fibroblasts;
PBS, phosphate-buffered saline; PFA, paraformaldehyde; Ptch1, Patched; RFP,
red fluorescent protein; Shh, Sonic hedgehog; shRNA, short hairpin RNA; Smo,
Smoothened; Sufu, Suppressor of Fused.
“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
© 2011 Larkins et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E10-12-0994) on October 5, 2011.
Volume 22 December 1, 2011 Arl13b in ciliogenesis and Shh signaling | 4695
by binding and sequestering the Glis in the cytoplasm (Chen et al.,
2009; Jia et al., 2009; Humke et al., 2010; Tukachinsky et al., 2010).
Precisely how Shh signaling proteins are targeted and moved in
and out of the cilium is not clear, but intraflagellar transport (IFT) is
required (Huangfu et al., 2003; Haycraft et al., 2005; Liu et al., 2005).
IFT is the bidirectional movement of ciliary protein complexes and is
required to build and maintain the cilium (Kozminski et al., 1993;
Rosenbaum and Witman, 2002; Pedersen et al., 2008). Anterograde
IFT carries cargo toward the tip of the cilium, while retrograde trans-
port carries turnover products out of the cilium; deletion of antero-
grade or retrograde IFT proteins results in distinct ciliary pheno-
types, but in either case, both GliA and GliR are affected, resulting
in disrupted Shh activity (Huangfu et al., 2003; Huangfu and Ander-
son, 2005; Liu et al., 2005; May et al., 2005; Houde et al., 2006;
Ocbina and Anderson, 2008; Tran et al., 2008; Cortellino et al.,
Surprisingly, disruption of ciliary structure does not always affect
Shh signaling, as shown by Rfx3 mouse mutants, which have short
cilia and normal Shh activity (Bonnafe et al., 2004). This underscores
the ill-defined nature of the mechanisms by which a growing list of
ciliary/basal body protein mutants affect Shh signaling (Ferrante
et al., 2006; Vierkotten et al., 2007; Norman et al., 2009; Patterson
et al., 2009; Boehlke et al., 2010). Arl13bhnn mutants are unusual,
because the production and/or function of only GliA, and not GliR,
is affected in these mutants (Caspary et al., 2007). In this study,
we examine what could link all these threads: a small regulatory
GTPase, a specific anomaly in the ciliary ultrastructure, and the
unique defect in Shh signaling. We investigate the ciliary function of
Arl13b by examining its localization, dynamics, and regulation of
ciliary structure. Further, we show that many Shh signaling pathway
components are mislocalized in the absence of Arl13b, suggesting
a general role for Arl13b in protein trafficking to the cilium.
Defects in posttranslational modifications of ciliary
tubulin are consistent with defects in the architecture
of Arl13bhnn cilia
Arl13bhnn mutant mouse embryos display shortened cilia and an ul-
trastructural defect, whereby the B-tubule of the microtubule-based
outer doublets does not connect to the A-tubule (Caspary et al.,
2007). Zebrafish and Tetrahymena mutants with defects in tubulin
glutamylation display a similar phenotype in the outer doublets
(Redeker et al., 2005; Pathak et al., 2007; Dave et al., 2009). To ex-
amine such posttranslational modifications in the axoneme of
Arl13bhnn cilia, we derived primary mouse embryonic fibroblasts
(MEFs) from e12.5 Arl13bhnn and wild-type embryos. Using immuno-
fluorescence, we examined tubulin glutamylation by measuring the
average fluorescence intensity along the entire cilium relative to
background staining. We saw a significant reduction in staining of
the Arl13bhnn axoneme compared with wild-type (Figure 1, A–C).
We then extended these analyses to another tubulin posttransla-
tional modification, acetylation. Although the functional significance
of tubulin acetylation is less clear, the ciliary axoneme is highly acety-
lated and is predicted to be a mark of stable microtubules (Perdiz
et al., 2010). We found tubulin acetylation is reduced to similar lev-
els as glutamylation in Arl13bhnn MEFs (Figure 1, A–C), indicating a
more general defect in tubulin posttranslational modifications in our
Arl13b regulates ciliary length
To further investigate the role of Arl13b in ciliogenesis, we exam-
ined cilia in cultured wild-type and Arl13bhnn MEFs. Of wild-type
MEFs, 71.7% were ciliated, whereas 19.0% of Arl13bhnn MEFs had
cilia (Figure 2A). In contrast, all cells of the Arl13bhnn embryonic
node are ciliated (Caspary et al., 2007). However, reminiscent of
what we found in vivo, the Arl13bhnn MEF cilia were shorter than
wild-type cilia, suggesting Arl13b controls ciliary length both in the
embryo and in culture (Figure 2, C, D, and M).
To unravel how Arl13b might regulate ciliary length, we exam-
ined cilia in MEFs overexpressing untagged Arl13b or Arl13b-green
fluorescent protein (GFP). We found wild-type MEFs overexpressing
untagged Arl13b or Arl13b-GFP had much longer cilia than untrans-
fected cells, and both constructs rescued the ciliary length defect in
Arl13bhnn MEFs (Figure 2, B, E–H, and M). The rescued cilia were
longer than wild-type, consistent with Arl13b protein levels being
important. The cilia in wild-type or Arl13bhnn MEFs overexpressing
Arl13b-GFP were shorter than those expressing untagged Arl13b,
indicating the C-terminal GFP tag impaired Arl13b’s function, consis-
tent with previous observations with tagged versions of Arf GTPases
(Jian et al., 2010). In the few cases where Arl13b-GFP was restricted
from the cilium and instead was evident throughout the cell body
and nucleus, there was no change in ciliary length, suggesting Arl13b
needs to be in the cilium to regulate ciliary length (Figure 2M).
Of the 30 mammalian Arf family proteins, most are ∼20-kDa
proteins consisting of the Arf domain only (D’Souza-Schorey
and Chavrier, 2006; Kahn et al., 2006). Arl13b is unusual in that it
has an additional 24-kDa novel C-terminus, much of which its
nonvertebrate orthologues lack. We examined the localization
FIGURE 1: Tubulin modification defects in Arl13bhnn mutant MEFs.
(A and B) Immunofluorescence for acetylated α-tubulin (ac-tubulin;
green) and glutamylated tubulin (red) in wild-type (A) and Arl13bhnn
(B) MEFs shows reduced staining for both antibodies in Arl13bhnn
MEFs. (C) Quantification of the average fluorescence intensity of
acetylated α-tubulin and glutamylated tubulin fluorescence relative to
background staining. Error bars are ± SEM. *p < 0.0001 using
Student’s t test.
4696 | C. E. Larkins et al. Molecular Biology of the Cell
and function of the Arf domain and the
novel C-terminal domain by tagging each
with GFP and expressing each in wild-type
and Arl13bhnn MEFs. Neither half of Arl13b
could localize to cilia as the full-length ver-
sion did; we found the tagged Arf domain
in diffuse puncta within the cell body and
the tagged C-terminal domain in the nu-
cleus (Figure 2, I–L). Furthermore, neither
half of Arl13b had any effect on ciliary
length in wild-type or Arl13bhnn MEFs,
consistent with our finding that full-length
Arl13b-GFP regulated ciliary length only
when it was in cilia (Figure 2M). Thus, only
full-length Arl13b localizes to cilia to regu-
late ciliary length.
Arl13b localizes to the ciliary
We previously demonstrated that Arl13b is
expressed in cilia and does not overlap
with the basal body in fibroblasts (Caspary
et al., 2007). To define where in the cilium
Arl13b localizes, we took advantage of the
long cilia in the mouse kidney cell line
IMCD3 and used immunofluorescence. As
in the fibroblasts, Arl13b was visible along
the entire length of the cilium, but did not
colocalize with the basal body marker
g-tubulin (Figure 3, A and C). To determine
whether Arl13b within the cilium associates
with the membrane or the axoneme, we
treated cells with the detergent TritonX-100
prior to fixation. We saw a loss of Arl13b
staining in TritonX-100-treated cells, but
acetylated α-tubulin staining remained in-
tact, suggesting the majority of Arl13b is
not associated with the axoneme (Figure 3,
A–D). We also saw that the known ciliary
membrane protein SSTR3 fused with GFP
FIGURE 2: Arl13b regulates ciliary length.
(A) Quantification of the percent of ciliated
cells in wild-type and Arl13bhnn MEFs.
(B) Schematic of the Arl13b constructs that
were transfected into MEFs. (C and D)
Immunofluorescence for acetylated α-tubulin
(ac-tubulin) in wild-type and Arl13bhnn MEFs
shows shortened cilia in Arl13bhnn MEFs.
(E–L) Immunofluorescence for GFP and
acetylated α-tubulin in wild-type and
Arl13bhnn MEFs expressing GFP-tagged
Arl13b constructs. (M) Quantification of
ciliary length in MEFs with and without
transfection. The constructs are separated by
those that localize to cilia and those that do
not. Error bars are ± SD. For untransfected
wild-type vs. Arl13bhnn MEFs, p < 0.0001. For
wild-type vs. wild-type–overexpressing
Arl13b-GFP and untagged Arl13b in the
cilium, p < 0.0001; for Arl13bhnn vs. Arl13bhnn
overexpressing Arl13b-GFP and untagged
Arl13b, p < 0.0001.
Volume 22 December 1, 2011 Arl13b in ciliogenesis and Shh signaling | 4697
(SSTR3-GFP) was lost upon pretreatment with detergent, consis-
tent with Arl13b being membrane associated (Figure 3, E and F).
Curiously, we saw that Arl13b staining remained at the base and
tip of the cilium after detergent treatment prior to fixation, which
we did not observe with SSTR3-GFP. This suggests that some
Arl13b is anchored to the axoneme ends or other machinery found
at the base and tip of the cilium. It could also be an indication that
the membrane of the base and tip of the cilium is less detergent
soluble, as is the case for lipid raft domains.
We next used fluorescence recovery after photobleaching (FRAP)
to determine the dynamics of Arl13b movement within the cilium of
IMCD3 cells. To do this, we generated a lentivirus capable of driving
expression of Arl13b-GFP and infected IMCD3 cells. When we pho-
tobleached the central region of the cilium, we saw rapid recovery of
Arl13b-GFP fluorescence in the bleached region (Figure 4, A and C).
We performed the parallel analysis with SSTR3-GFP (Figure 4, B and
C) and found the same rates of recovery for Arl13b-GFP and SSTR3-
GFP (Figure 4C). To determine Arl13b turnover within the cilium, we
photobleached Arl13b-GFP as well as SSTR3-GFP in the entire cil-
ium and saw very little recovery of either protein over the course of
the experiment, which we followed for 2 min 23 s (Figure 4, D, E, and
G). In contrast, in a parallel analysis performed by photobleaching
IFT88-EYFP–expressing cells, we saw a faster rate of recovery (Figure
4, F and G), consistent with previous data showing that IFT recovery
in the cilium is faster than the recovery of ciliary membrane proteins
(Hu et al., 2010). Taken together, these data are consistent with
Arl13b associating with the membrane of the cilium.
IFT appears normal in the absence of Arl13b
As IFT is the major transport machinery that builds cilia, we wanted
to further investigate the relationship between Arl13b and IFT and
test whether Arl13b regulates IFT in mammalian cells. We took ad-
vantage of an IMCD3 cell line that stably expressed IFT88-EYFP,
which enabled us to perform FRAP and measure the rate of IFT88-
EYFP recovery. To deplete the cells of Arl13b, we used a lentivirus
coexpressing Arl13b short hairpin RNA (shRNA) and red fluorescent
protein (RFP), enabling us to specifically identify knockdown cells.
We proved the knockdown was efficient by Western blotting, as we
detected only 30% of the wild-type Arl13b levels in cells treated
with the knockdown virus (Figure 5A). Arl13b was not detected by
immunofluorescence in 50% of RFP-positive cells, and the pheno-
type was similar to what we found in the Arl13b-null MEFs: 50%
fewer cilia, and those that were present were shortened (Figure 5, B,
C, and E). When we measured the rate of recovery of IFT88-en-
hanced yellow fluorescent protein (EYFP) at the ciliary tip, we saw no
significant change between IMCD3 wild-type and Arl13b knock-
down cells (Figure 5H). Because the knockdown is sufficient to re-
flect the established Arl13b ciliary phenotype, the simplest interpre-
tation is that Arl13b is not required for nor does it provide essential
regulation of the rate of IFT. Nevertheless, we cannot rule out such
a function, as some Arl13b remains after knockdown.
Dynamic localization of Shh signaling proteins is disrupted
in Arl13bhnn MEFs
Arl13b functions in the cilium to regulate Shh signaling, because
Arl13b mutants that lack cilia (Arl13bhnn IFT172wim double mutant
embryos), like IFT172wim mutant embryos that lack cilia completely,
display no Shh response (Caspary et al., 2007). However, in contrast
to most ciliary mutants in mouse, Arl13bhnn single mutant embryos
have a low level of ligand-independent Shh pathway activation in
the neural tube, due to a specific disruption in GliA activity (Caspary
et al., 2007). Since the goal of Shh signaling is to control the balance
FIGURE 3: Ciliary Arl13b is TritonX-100 soluble. (A and C) Immunofluorescence in IMCD3 cells shows that Arl13b
colocalizes with the ciliary marker acetylated α-tubulin (A) and does not colocalize with the basal body marker g-tubulin
(C). (B and D) Triton X-100 treatment results in the loss of a majority of Arl13b staining in the cilium, although axoneme
staining with acetylated α-tubulin remains (B). (E and F) The known ciliary membrane protein SSTR3-GFP shows a similar
loss of ciliary staining with TritonX-100 treatment (F).
4698 | C. E. Larkins et al. Molecular Biology of the Cell
of GliA and GliR, and this balance requires cilia, we investigated Shh
signaling and the localization of Shh components to the cilia of
Arl13bhnn mutant MEFs with and without Shh stimulation.
First, we sought to monitor Shh activity in wild-type and Arl13bhnn
MEFs. We therefore cotransfected a firefly luciferase reporter con-
struct with eight Gli-binding sites in its promoter (8×Gli luciferase)
with a Renilla luciferase expression construct to control for transfec-
tion efficiency in wild-type and Arl13bhnn MEFs (Sasaki et al., 1997).
The levels of normalized luciferase activity in untreated wild-type
and Arl13bhnn MEFs were similar (Figure 6A). When treated with
Shh-conditioned media, wild-type MEFs showed almost a sevenfold
increase in normalized luciferase activity, whereas Arl13bhnn MEFs
displayed only a twofold increase, indicating Arl13bhnn cells have a
lowered response to the Shh ligand (Figure 6A). Because there is
also a reduction of ciliated cells in Arl13bhnn mutant cilia, it is difficult
to determine whether the reduced Shh response is caused by a re-
duction in cilia or by defective Shh signaling in the ciliated Arl13bhnn
cells or by both.
To better determine the Shh response within only the ciliated
Arl13bhnn mutant cells, we examined the dynamics of Shh compo-
nents using antibodies against the endogenous proteins. Normally,
Gli2 and Gli3 localize to the ciliary tip and are further enriched there
after Shh stimulation (Chen et al., 2009; Wen et al., 2010). Indeed,
when we measured the fluorescence intensity in the tips of cilia rela-
tive to background staining using antibodies that recognize full-
length Gli2, the N-terminal domain of Gli3 (Gli3N) or the C-terminal
domain of Gli3 (Gli3C), we found Gli2 and Gli3 were enriched
in wild-type MEFs after treatment with Shh-conditioned media
(Figure 6, B, D, F, and N; Wen et al., 2010). Interestingly, there was
no significant change in Gli2 and Gli3 enrichment in Arl13bhnn MEFs
after Shh treatment (Figure 6, C, E, G, and N). Similarly, we con-
firmed that Sufu, a mediator of Gli function, shows enrichment in the
tips of cilia after Shh treatment in wild-type, but not Arl13bhnn, MEFs
(Figure 6, H, I, and N).
Smo and Ptch1 localize along the length of the cilium in a com-
plementary manner: Ptch1 in the absence of ligand and Smo upon
pathway stimulation (Corbit et al., 2005; Rohatgi et al., 2007). As
expected, we saw Ptch1 ciliary levels decrease and Smo ciliary levels
increase after Shh stimulation in wild-type MEFs (Figure 6, J, L, and
N). We investigated how these dynamics changed in the absence of
Arl13b and found Ptch1 staining did not significantly change in re-
sponse to Shh stimulation in Arl13bhnn MEFs (Figure 6, L–N). Inter-
estingly, we found Smo localized to the cilium in Arl13bhnn MEFS
without Shh stimulation (Figure 6, J, K, and N), indicating Arl13b
FIGURE 4: Arl13b-GFP dynamics reflect those of a ciliary membrane
protein. The figure shows overexposed images for viewing purposes,
but all intensities were measured without overexposure of pixels.
(A and B) Photobleaching of a region of Arl13b-GFP (A) in the center
of the cilium shows recovery dynamics similar to SSTR3-GFP (B).
(C) Quantification of the recovery dynamics for SSTR3-GFP and
Arl13b-GFP shows no significant difference in the recovery curve
(prior to 5.9 s, p > 0.3 using Student’s t test). The relative fluorescence
intensity was quantified as the intensity in the bleached region relative
to the whole cilium. (D to G) Photobleaching Arl13b-GFP (D) and
SSTR3-GFP (E) in the whole cilium results in little fluorescence
recovery (G). IFT88-YFP has a faster recovery rate in the cilium (F and
G); p < 0.02 for both Arl13b-GFP and SSTR3-GFP, compared with
IFT88-YFP at 29.9 s. (G) Quantification of fluorescence intensity is
determined as the intensity of the bleached region (cilium) relative to
an unbleached region in the field. All experiments were corrected for
background. Error bars are ± SEM (C and G).
Volume 22 December 1, 2011 Arl13b in ciliogenesis and Shh signaling | 4699
plays a critical role in regulating the entry of Smo into cilia. We found
Smo was further enriched on Shh stimulation in Arl13bhnn MEFS
(Figure 6, K and N). However, regardless of whether or not the cells
were stimulated, Smo was found enriched in large puncta in the cilia
lacking Arl13b, in contrast to the more evenly distributed Smo stain-
ing in wild-type MEF cilia (Figure 6, J and K), arguing that Arl13b
also regulates the proper localization or targeting of Smo within the
cilium. In Arl13bhnn MEFs without Shh stimulation, Smo staining was
most often concentrated in a single puncta at either the ciliary tip
(31.8% of cells), in the proximal cilium (11.4%), in the center of the
cilium (13.6%), or in two or three puncta in multiple regions of the
cilium (22.7%). After treatment of the cells with Shh-conditioned
medium, Arl13bhnn MEFs showed a shift in the localization of ciliary
Smo such that more cells had multiple sites of concentrated Smo
(37.8%). In sum, these results demonstrate a fundamental defect in
the trafficking of Shh signaling proteins in Arl13bhnn MEFs.
Our data here point to an essential role for Arl13b in multiple as-
pects of ciliogenesis and ciliary protein localization. We saw that cell
lines with disrupted Arl13b expression have reduced numbers of
cilia, and those cilia that are present are shorter, whereas cells over-
expressing Arl13b show increased ciliary length. Taken together,
these results indicate an important role for
Arl13b in length control. Additionally, we
found abnormal tubulin modifications in
mutant cilia, consistent with the structural
defect in the axoneme that may contribute
to ciliary shortening, which supports a role
for Arl13b in regulating tubulin modifica-
tions within the cilium. Furthermore, we
found the dynamics of many Shh signaling
components were disrupted, with defective
Smo localization within the cilium, showing
the importance of Arl13b in Shh signaling
protein localization. Although these results
may imply that Arl13b has multiple inde-
pendent functions in ciliary biology, they
could also indicate a singular function of
Arl13b that affects these various aspects of
ciliary biology, such as a role in protein traf-
ficking to and within the cilium that, when
disrupted, results in defects in ciliary length,
tubulin modifications, and protein localiza-
tion to the cilium.
The role of Arl13b in ciliogenesis
We showed that loss of Arl13b led to short-
ened cilia and reduced numbers of cilia in
vitro, while overexpression of Arl13b caused
increased ciliary length. Many different cili-
ary proteins have been associated with cili-
ary length regulation, and most of these pro-
teins point to steady-state protein trafficking
to and from cilia as being the ultimate regu-
lator of ciliary length (Ishikawa and Marshall,
2011). Interestingly, the closely related
GTPase Arl6 recruits trafficking proteins of
the Bardet-Biedl syndrome complex to the
cilium and regulates ciliary length (Jin et al.,
2010; Wiens et al., 2010). We therefore think
it is likely that Arl13b also plays a similar role
in controlling protein trafficking to or from the cilium, or both.
Our findings that Arl13b is solubilized from cilia with detergent
and moves within the cilium with dynamics almost identical to the
ciliary intrinsic membrane protein SSTR3 are consistent with Arl13b
forming a quite stable association with cell membranes through N-
terminal palmitoylation, as demonstrated in 293T cells (Cevik et al.,
2010). Furthermore, these dynamics fit with evidence that ciliary
membrane proteins have very long half-lives within the cilium (Hu
et al., 2010). It is interesting that IFT proteins are tightly associated
with the ciliary membrane while being transported along the ax-
oneme and that the Arl13b Caenorhabditis elegans orthologue
ARL13 stabilizes IFT (Pigino et al., 2009; Cevik et al., 2010). How-
ever, we did not see a defect in IFT recovery using our FRAP method
to examine IFT dynamics in Arl13b knockdown cells, which could be
reflective of our method or could indicate that the vertebrate ortho-
logue of Arl13b does not regulate IFT. Other methods to examine
IFT and to determine protein interactors will be crucial for establish-
ing whether Arl13b plays a role in regulating IFT.
The ∼24-kDa C-terminal domain of Arl13b is critical to its func-
tion. The domain is vertebrate-specific and required for Arl13b lo-
calization to cilia, and we found that tagging it with GFP reduces the
ability of Arl13b to regulate ciliary length. The fact that the C-termi-
nal domain of Arl13b localizes to the nucleus when GFP-tagged is
FIGURE 5: IFT88 recovery is intact in Arl13b mutant cells. (A) Western blot showing knockdown
of Arl13b in IMCD3 cells. (B) Quantification of the percentage of cells showing cilia stained with
acetylated α-tubulin and Arl13b. (C) Quantification of ciliary length in knockdown cells compared
with no knockdown. (D and E) Immunofluorescence for (D) Arl13b and (E) acetylated α-tubulin
(ac-tubulin) costained with RFP in cells expressing the 504 knockdown construct. (F) Recovery of
IFT88-EYFP in IMCD3 cells with and without knockdown of Arl13b. Error bars are ± SEM (C and
F) and ± SD (B).
4700 | C. E. Larkins et al. Molecular Biology of the Cell
interesting, since it contains a basic sequence that
may act as a nuclear localization signal. Recent data
demonstrated that a similar sequence in the C-termi-
nus of Kif17 mediates ciliary localization and that a
truncated Kif17 lacking its N-terminus also localizes to
the nucleus (Dishinger et al., 2010). In addition,
importin-β has been localized to cilia and is required
for ciliary entry of some proteins (Fan et al., 2007;
Dishinger et al., 2010), raising the possibility that
Arl13b localizes to cilia through an importin-β interac-
tion domain in its C-terminus. As the N-terminus of
Arl13b contains palmitoylation sites (Cevik et al.,
2010), interactions with vesicles or periciliary
membrane may be required for ciliary versus nuclear
While the reduction of both acetylation and glu-
tamylation in Arl13bhnn cilia is consistent with the ar-
chitectural defect of the axoneme (Pathak et al., 2007;
Pugacheva et al., 2007), we cannot say whether the
modification defects we see are the result or the cause
of a potential defect in protein trafficking to cilia. In-
terestingly, in C. elegans, the Arl13 mutant phenotype
is rescued by loss of Arl3 function, and loss of Arl3 in-
creases tubulin acetylation in HeLa cells (Zhou et al.,
2006; Li et al., 2010), indicating these two proteins
may have inverse functions in regulating tubulin mod-
ifications. Examining a potential Arl3 and Arl13b inter-
action in mammalian cells will be important for teas-
ing apart these possibilities.
The role of Arl13b in Shh signaling
Arl13bhnn mutant embryos display defects in Shh sig-
naling in the neural tube, such that there is a ligand-
independent expansion of Shh signaling activity, while
the highest levels of activity are not reached in the
ventral neural tube (Caspary et al., 2007). To begin to
investigate this signaling defect, we examined Shh
signaling in Arl13bhnn MEFs. For our initial investiga-
tion, we determined the Shh signaling response in
FIGURE 6: Shh signaling component localization is
disrupted in Arl13bhnn MEFs. (A) Quantification of
Gli-luciferase activity relative to Renilla luciferase.
(B–M) Immunofluorescence for acetylated α-tubulin
(green) and Gli2, Gli3, Sufu, Smo, and Ptch1 (red). In
wild-type MEFs (B, D, F, H, J, and L) Gli2, Gli3, and Sufu
are enriched after Shh-conditioned media treatment,
while Ptch1 levels are reduced. Arl13bhnn MEFs (C, E, G,
I, K, and M) do not show significant enrichment of Gli2,
Gli3, Sufu, or Smo, and Ptch1 staining is not significantly
reduced after Shh-conditioned media treatment. Insets
in (J–M) are showing Ptch1 and Smo staining alone in
grayscale. (N) Quantification of average fluorescence
intensity in the tip of the cilium (Gli2, Gli3, and Sufu) or
the entire cilium (Ptch1 and Smo) relative to cell body
staining. Data from all experiments are shown. p < 10−7
for wild-type MEFs with conditioned media vs.
untreated wild-type MEFs using the Gli and Smo
antibodies; p < 0.05 using the Ptch1 and Sufu
antibodies; p < 0.04 for Smo staining in Arl13bhnn MEFS
with conditioned media vs. untreated Arl13bhnn MEFS;
p < 0.04 for Ptch1 intensities in untreated wild-type
MEFs vs. untreated Arl13bhnn MEFs.
Volume 22 December 1, 2011 Arl13b in ciliogenesis and Shh signaling | 4701
Arl13bhnn MEFs using an 8×Gli luciferase assay and saw that, without
Shh treatment, mutant MEFs had levels of Shh response similar to
wild-type MEFS; however, there was a defect in mutant cells re-
sponding to the Shh ligand. This result differs from what we found in
Arl13bhnn mutant embryos, in that there is a ligand-independent ac-
tivation of Shh signaling in the neural tube, although the highest
levels of Shh signaling cannot be reached (Caspary et al., 2007). We
predict that this discrepancy may be due to the number of ciliated
cells found in vivo versus in vitro, as all cells appear to be ciliated in
Arl13bhnn embryos, whereas less than one-half of the cells are cili-
ated in Arl13bhnn MEFs (Caspary et al., 2007). Given that loss of cilia
in Arl13bhnn mutants causes loss of Shh signaling (Caspary et al.,
2007), this could explain the lowered response to Shh in the popula-
tion of Arl13bhnn MEFs.
We next examined the localization of Shh signaling components
in the cilia of Arl13bhnn MEFs using antibodies to endogenous pro-
teins. The defects in Shh component localization we found were more
consistent with the phenotype in the Arl13bhnn mutant neural tube,
perhaps because we were able to focus on only the ciliated cells in
the population. First, we showed that Gli2 and Gli3 were not properly
enriched at the ciliary tip when Arl13bhnn MEFs were stimulated with
Shh-conditioned media. Similarly, there was a defect in shifting Sufu
localization in Arl13bhnn MEFs in response to Shh-conditioned media.
These results are reminiscent of the inability to reach the highest lev-
els of Shh signaling activity in the Arl13bhnn mutant neural tube, as it
was shown previously that Gli localization to cilia is required for path-
way activation (Tukachinsky, 2010). Next we found Smo was enriched
in cilia in the absence of Shh stimulation and, consistent with this en-
richment, we saw reduced Ptch1 staining in the cilium. The enriched
ciliary Smo was unlikely to be fully active, because the Gli proteins
were not properly enriched. As there is ligand-independent activa-
tion of Shh signaling in the neural tube, we would expect the en-
riched Smo in Arl13bhnn cilia to show a low level of activity that allows
for ligand-independent activation of the pathway.
The results presented here suggest that the relative levels of Shh
signaling proteins in cilia, as well their trafficking into and out of the
cilium, are important for activation and repression of the pathway,
and studies of other ciliary mutants support this model. For example,
the Rab23 mutant mouse has excess activation of the Shh signaling
pathway in the neural tube (Eggenschwiler et al., 2001), and Rab23
mutant cells were shown to have increased trafficking of Smo into
the cilium (Boehlke et al., 2010). However, our analysis of Arl13b ar-
gues that Smo simply being present in cilia is not sufficient for the
range of Shh response. This is supported by research examining the
retrograde ciliary dynein mutant, which also has enriched Smo in
cilia, but reduced levels of Shh pathway activity in the neural tube
(Ocbina et al., 2011). Examination of these mutants and our data
suggest there is a balance between Shh signaling protein localization
and trafficking of signaling proteins into and out of the cilium that is
important for achieving the correct levels of Shh response. Further
examination of the puncta of Smo we see in the absence of Arl13b
will likely provide the fundamental explanation for the inability of
Arl13bhnn embryos to reach high levels of Shh activation by defining
the role of Arl13b in Smo localization within the cilium. Future studies
are left to examine the mechanism of Shh signaling component in-
teractions in the cilium to better explain how relative levels of Shh
signaling proteins in cilia regulate the pathway.
MATERIALS AND METHODS
Generation of MEFs
E12.5 mouse embryos were dissected, and the head and visceral
organs were removed. The embryos were then washed in clean
phosphate-buffered saline (PBS), and single embryos were
each transferred to a 1-ml syringe. The embryos were passed
through an 18-G needle multiple times in DMEM (high glucose,
10% fetal bovine serum [FBS], penicillin, streptomycin) and trans-
ferred to a gelatinized 10-cm tissue culture dish. After the cells
reached confluency, they were split 1:5. Cells were not used after
Cells were grown on coverslips and fixed 10 min in 4% paraformal-
dehyde (PFA) in PBS, then washed in antibody wash buffer (PBS,
0.1% TritonX-100, 1% heat-inactivated goat serum) for 10 min at
room temperature. Primary antibodies were incubated overnight at
4°C, and after a series of washes, they were incubated in secondary
antibody for 1 h at room temperature. After a second series of
washes, coverslips were mounted on slides with ProLong Gold anti-
fade reagent (Invitrogen, Carlsbad, CA). Antibodies and dilutions
were as follows: Ptch1 (1:250; Raj Rahatgi, Stanford University,
Stanford, CA), Smo (1:500; Kathryn Anderson, Sloan-Kettering
Institute, New York, NY), acetylated α-tubulin (1:2500; T-6793,
Sigma-Aldrich, St. Louis, MO), g-tubulin (1:1000; T-6557, Sigma),
Gli2 (1:2000; Jonathan Eggenschwiler, Princeton University, Prince-
ton, NJ), Gli3C and Gli3N (2 μg/ml; Suzie Scales, Genentech, San
Francisco, CA; Wen et al., 2010), polyclonal GFP (1:500, AB3080,
Millipore, Billerica, MA), monoclonal GFP (1:500, MAB3580, Chemi-
con), Sufu (1:100, SC-10933, Santa Cruz Biotechnology, Santa Cruz,
CA), glutamylated tubulin (GT335, Carsten Janke, Curie Institute,
Paris), and Arl13b (1:1500).
Fluorescence intensities were measured using ImageJ software,
and were normalized to cell-body staining. All cilia in a field were
measured, and cilia were selected in the acetylated α-tubulin chan-
nel to prevent biased selection. For all antibodies except Gli2, Gli3
C-terminal, Gli3 N-terminal, and Sufu, the average intensity in the
entire cilium was measured relative to background. For the Glis and
Sufu, which only stain the tips of cilia, the fluorescence intensity in
only the tip was measured relative to background. The tip of the
cilium was determined by weakened acetylated α-tubulin staining,
as well as orientation. Three independent experiments were per-
formed for all antibodies, and a total of at least 125 cilia were mea-
sured for the Gli and Sufu antibodies, and at least 40 cilia were mea-
sured for all other antibodies. Significance was determined for all
experiments using a one-tailed Student’s t test. Smo and Ptch1
antibodies were imaged on a Zeiss LSM510 META (Carl Zeiss
Microscopy, Thornwood, NY) confocal at 63× with optical zoom. All
other antibodies were imaged on a Leica DM6000B (Leica Microsys-
tems, Buffalo Grove, IL) upright microscope at 100× with SimplePCI
software (Hamamatsu Corp., Sewickley, PA).
For removal of the ciliary membrane, cells were treated with
0.1% TritonX-100 in PBS without Ca2+ or Mg2+ for 1 min and then
fixed in 4% PFA. Antibody staining was then performed as described
in the preceding paragraphs.
IMCD3 cells were transduced with a viral construct expressing
Arl13b-GFP. All cells were grown to confluency. SSTR3-GFP cells
were from Greg Pazour and IFT88-YFP cells were from Brad Yoder.
FRAP was performed on a Zeiss LSM510 META confocal using 63×
objective with optical zoom. Briefly, cilia were photobleached using
the 488-nm laser with 25 iterations at 50% power. Images were
scanned at 3% laser power. Fluorescence intensity measurements
had background subtracted, and the bleached region was normal-
ized to the entire cilium (for determination of movement within the
4702 | C. E. Larkins et al. Molecular Biology of the Cell
We are grateful to Kathryn Anderson, Suzie Scales, Carsten Janke,
and Rajaj Rohatgi for the Smo, Gli, glutamylated tubulin (GT335),
and Ptch1 antibodies, respectively. We thank Chen-Ying Su,
Vanessa Horner, Alyssa Long, Miao Sun, Laura Mariani, Nicole
Umberger, and Karolina Piotrowska-Nitsche for helpful comments
on the manuscript. The Viral Vector Core, which generated the
Arl13b-GFP lentivirus, and the Microscopy Core of the Emory
Neuroscience NINDS Core Facilities were supported by National
Institutes of Health (NIH) grant P30-NS055077. This work was funded
by a Predoctoral Fellowship from the Greater Southeast Region
American Heart Association (C.E.L.), a Basil O’Conner Starter Scholar
Award from the March of Dimes (T.C.), and a research grant from the
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cilium) or to an unbleached region in the same field (for determina-
tion of turnover in the cilium).
Conditioned medium was generated as previously described
(Taipale et al., 2000). Briefly, 293 EcR Shh cells (ATCC; CRL-2782)
were grown to confluence, and 1 μM of MurA was added to the cells
in 2% serum DMEM. After 24 h, the conditioned medium was col-
lected and the 2% serum MurA media was replaced. A collection
was taken again after 24 h, and that medium was combined with the
For immunofluorescence of MEFs treated with Shh-conditioned
media, cells were plated at 800,000 cells per well of a six-well plate.
At confluence, cells were serum-starved in DMEM high glucose with
0% serum. After 24 h of serum-starving, conditioned medium di-
luted 1:4 was added to the cells. Untreated control cells were given
0.5% serum media at this point. After 24 h of treatment, the cells
were harvested for immunofluorescence.
The knockdown viral construct was generated using Sigma’s Mission
custom viral vector synthesis. The vector, clone ID, and targeting
sequence were pLKO.1-puro-CMV-TagRFP, TRCN0000100504, and
CCTGTCAGAAAGGTGACACTT, respectively. The targeting se-
quence targets the coding region of Arl13b starting at nucleotide
349 of the mRNA.
IMCD3 cells were transduced in a 12-well plate with the knock-
down construct at a multiplicity of infection of 5. After transduc-
tion, cells were treated with puromycin at 2 μg/ml for 3 d. After
treatment, cells were passaged to a six-well plate for immunofluo-
rescence or to be harvested for Western blot analysis.
Arl13b constructs and overexpression
Arl13b was cloned using the Gateway system (Invitrogen). Full-
length Arl13b (coding for amino acids 1–427 without stop
codon), the N-terminal domain (coding for amino acids 1–212
of Arl13b), and the C-terminal domain (coding for amino ac-
ids 210–427 of Arl13b) were cloned into pENTR/SD/D-TOPO
(K242020, Invitrogen). The inserts were recombined into the
pcDNA-DEST47 (C-terminal GFP tag; 12281010, Invitrogen) and
pcDNA-DEST40 (N-terminal GFP tag; 12274015, Invitrogen) us-
ing Gateway LR Clonase (11791-019, Invitrogen) following the
Cells (350,000 per well) were seeded in a pregelatinized six-
well plate containing coverslips in DMEM (high glucose, 10% FBS,
penicillin, streptomycin). After 24 h, the cells were transfected fol-
lowing the manufacturer’s recommendations using Lipofectamine
2000 (Invitrogen) complexed with the Arl13b expression plas-
mids. After a 5-h incubation period of cells with the complexes,
the medium was changed to serum-free DMEM High Glucose for
24 h. The cells were then fixed using 4% PFA and were subjected
to immunofluorescence. Cilia were measured using LSM Image
Examiner software (Carl Zeiss Microscopy, Thornwood, NY).
Arl13b-GFP expression virus
The L13 lentiviral mammalian expression vector was obtained from
the Emory University Viral Vector Core. The Arl13b-GFP insert was
generated by PCR from the pcDNA-DEST47 plasmid containing full-
length Arl13b and using the following primers (5′ to 3′): forward-
GCTAGCTCAAACAAGTTTGTACAAAAAAGC, reverse- GGATCCAT-
TCTAGATCGAACCACTTTG. The primers introduced a 5′ NheI site
and a 3′ BamHI site that were used to insert Arl13b-GFP into L13.
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