Making the Connection: Ciliary Adhesion Complexes Anchor Basal Bodies to the Actin Cytoskeleton

Developmental Cell

Article

Making the Connection:Ciliary Adhesion ComplexesAnchor Basal Bodies to the Actin CytoskeletonIoanna Antoniades,1,2 Panayiota Stylianou,1,2 and Paris A. Skourides1,*1Department of Biological Sciences, University of Cyprus, P.O. Box 20537, Nicosia 2109, Cyprus2These authors contributed equally to this work

*Correspondence: [email protected]://dx.doi.org/10.1016/j.devcel.2013.12.003

SUMMARY

Cilia have been associated with diverse develop-mental and physiological processes, and defects incilia underlie a number of genetic conditions. Severallines of evidence support a critical role of the actincytoskeleton in ciliogenesis and ciliary function.Here,we show thatwell-characterized focal adhesion(FA) proteins, including FAK, Paxillin, and Vinculin,associate with the basal bodies of multiciliated cellsand form complexes (CAs) that interact with theactin cytoskeleton. FAK downregulation leads tociliogenesis defects similar to those observed whenthe actin cytoskeleton is disrupted, including defectsin basal body migration, docking, and spacing,suggesting that CAs link basal bodies to the actincytoskeleton. The important role of FA proteins inciliogenesis leads us to propose that evolutionarilyFA proteins, many of which are found in primitiveflagellated unicellular eukaryotes,mayhave originallyevolved to perform functions at flagella and werelater co-opted for use in cell adhesion.

INTRODUCTION

Cilia are microtubule-based organelles extending from basal

bodies on the surface of vertebrate cells. There are two main

types of cilia; primary and motile cilia. While primary cilia can

be found on almost every cell type, motile cilia are restricted to

specialized epithelia and are essential for generating fluid flow.

Such epithelia include the airways, the ventricles of the brain,

and the oviducts (Knowles and Boucher, 2002; Sawamoto

et al., 2006; Shah et al., 2009; Worthington and Cathcart,

1963). Motile cilia can be found in single copy, as motile mono-

cilia, or multicilia (Goetz and Anderson, 2010; Pazour and

Witman, 2003; Pedersen et al., 2008; Roy, 2009), and the disrup-

tion of ciliary function has been shown to be responsible for

a variety of human diseases termed ciliopathies (Afzelius,

1976, 2004; Baker and Beales, 2009; Bisgrove and Yost, 2006;

Hildebrandt et al., 2011;Wallingford andMitchell, 2011; Zariwala

et al., 2007). Perhaps the most striking results regarding the

importance of cilia come from the fly, where DSas-4 mutant

larvae, which lack centrioles, develop into morphologically

70 Developmental Cell 28, 70–80, January 13, 2014 ª2014 Elsevier In

normal adults, which die shortly after birth because their sensory

neurons lack cilia (Basto et al., 2006).

The dependence of ciliogenesis on the actin cytoskeleton is

well documented, and both components of the actin cyto-

skeleton, as well as regulators of actin dynamics, have been

shown to play important roles in various aspects of ciliogenesis

(Bershteyn et al., 2010; Boisvieux-Ulrich et al., 1990; Dawe et al.,

2007; Ioannou et al., 2013; Klotz et al., 1986; Lemullois et al.,

1988; Pan et al., 2007; Ravanelli and Klingensmith, 2011;

Tamm and Tamm, 1988). In multiciliated cells, basal bodies

form de novo deep within the cytoplasm (Sorokin, 1968) and

are subsequently transported, via an actin-myosin-based mech-

anism, to the apical surface where they dock (Boisvieux-Ulrich

et al., 1990; Dawe et al., 2007; Klotz et al., 1986; Lemullois

et al., 1987). In addition to the proposed role of the actin cyto-

skeleton in the transport of basal bodies, the apical surface of

multiciliated cells is enriched with a dense meshwork of actin

composed of two distinct pools, the apical and the subapical

actin networks. Loss of the apical actin network leads to prob-

lems with basal body localization and polarity and appears to

be necessary for basal body docking (Boisvieux-Ulrich et al.,

1990; Park et al., 2006, 2008; Werner et al., 2011). Once basal

bodies dock at the apical surface, they must be polarized in

order for the cilia to beat in a synchronized manner and create

directional fluid flow. The subapical actin network has been

shown to be important for basal body spacing and metachronal

synchrony. This network appears to connect basal bodies

through the striated rootlets. Interestingly, disruption of the sub-

apical actin network during basal body docking results in severe

spacing issues, suggesting that spacing is determined by this

actin network, while its disruption after ciliogenesis is completed

leads to loss of metachornal synchrony (Werner et al., 2011). The

actin cytoskeleton and its regulators have also been shown to

influence the formation of primary cilia. Although the precise

role of actin on primary cilia formation is not clear, studies

suggest that modulation of the actin cytoskeleton regulates

basal body positioning and primary cilium development overall,

suggesting that primary cilia formation is promoted through the

disassembly of certain pools of highly dynamic filamentous actin

(Bershteyn et al., 2010; Kim et al., 2010; Sharma et al., 2011; Yan

and Zhu, 2013; Yin et al., 2009). However, a mutation in Talpid3

has been shown to elicit defects in primary cilia formation and

actin organization in the chicken. In these mutants, docking of

basal bodies to the apical cell membrane is defective, despite

the fact that mature basal bodies form (Yin et al., 2009). These

c.

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Figure 1. FA Proteins FAK, Paxillin, and Vin-

culin Associatewith Basal Bodies in Ciliated

Cells

(A and A0 ) Multiciliated cell expressing GFP FAK

and centrin2 RFP. Each basal body is associated

with a GFP FAK punctum at its posterior (in relation

to the tadpole’s anterior-posterior axis). GFP FAK

signal displays a gradient within the cell (in respect

to the anterior-posterior axis), with elevated signal

at the basal bodies localized at the cell’s posterior.

(B and B0) Multiciliated cell expressingGFP Paxillin

and centrin2 RFP. GFP Paxillin exhibits a similar

localization and distribution throughout the cell,

like GFP FAK.

(C and C0) Multiciliated cell expressing GFP Vin-

culin and centrin2 RFP. Localization and distribu-

tion of GFP Vinculin resembles that of GFP FAK

and GFP Paxillin.

(D) Immunofluorescence staining with a p-S732

FAK antibody in centrin2 CFP-injected embryos.

Endogenous FAK, associated with the basal

bodies, is phosphorylated on Serine 732.

(E) Immunofluorescence staining of endogenous

Vinculin in centrin2 RFP-expressing multiciliated

cells, gives a similar localization as GFP Vinculin.

(F and F0) GRP ciliated cells of a stage 17 embryo

expressing GFP FAK and centrin2 RFP, immuno-

stained for acetylated tubulin.

(G and G0) Neural tube cross section of a stage 24

embryo coexpressing GFP FAK and centrin2 RFP

and immunostained for acetylated tubulin. GFP

FAK associates with the basal bodies in both GRP

and neural tube ciliated cells.

(H and I) Immunofluorescence staining of endog-

enous Serine 732 phosphorylated FAK (H) and

Paxillin (I) in NIH 3T3 cells, costained for g tubulin

and acetylated tubulin. Endogenous FAK and

Paxillin localize at the base of primary cilia next to

the basal body in mammalian ciliated cells.

See also Figure S1.

Developmental Cell

CAs Link Basal Bodies to the Actin Network

results suggest that formation of the apical actin network affects

the attachment of basal bodies to the cell membrane and that

certain aspects of the role of the actin network may be shared

between motile and primary cilia.

In addition to the studies in animals, actin has also been

shown to be important for the control of flagellar length in

Chlamydomonas, while knockdown of actin in Giardia results

in defects in the organism’s flagella and their positioning, sug-

gesting that the requirement of actin for normal ciliogenesis

extends to flagellated unicellular eukaryotes (Dentler and

Adams, 1992; Paredez et al., 2011). Despite the critical role

of the actin cytoskeleton in ciliogenesis and ciliary function,

how the basal bodies interact with the actin cytoskeleton is

not known. Here, we show that four well-characterized focal

adhesion (FA) proteins are found associated with basal bodies

and form what we termed ‘‘ciliary adhesions’’ (CAs), connecting

basal bodies to the actin cytoskeleton of multiciliated cells.

Similar complexes are also found in monociliated cells of the

gastrocel roof plate (GRP) and primary cilia. CA protein FAK

interacts with both basal bodies and actin, while its downregu-

lation leads to defects in ciliogenesis related to actin-based

processes, suggesting that CAs functionally link basal bodies

to the actin cytoskeleton.

Deve

RESULTS

FAK, Paxillin, Vinculin, and Talin Are Associatedwith the Basal Bodies in Ciliated CellsFAs are large, dynamic protein complexes through which the

actin cytoskeleton connects to the extracellular matrix. FAK

and Paxillin are well-characterized FA proteins with critical roles

in the assembly and disassembly of FA complexes. The fact that

both FAK and Paxillin have been previously detected in associa-

tion with centrosomes raised the possibility that FA proteins also

associate with basal bodies (Herreros et al., 2000; Park et al.,

2009). Using green fluorescent protein (GFP) fusions of FAK,

Paxillin, and Vinculin in combination with live imaging, we noted

that all three proteins localize in close association with the basal

bodies, visualized using centrin2 red fluorescent protein (RFP),

in Xenopus multiciliated cells (Figures 1A–1C0). Control cells

expressing GFP alone show that GFP by itself does not localize

at the basal bodies (Figures S1A and S1A0 available online), and

western blotting showed that expression levels of GFP Paxillin

and GFP Vinculin were similar to those of the endogenous

proteins (Figures S1B and S1C). In addition, a fourth FA protein,

Talin, fused to FusionRed also localizes at the basal bodies of

multiciliated cells (Figure S1D). To further verify the GFP fusion

lopmental Cell 28, 70–80, January 13, 2014 ª2014 Elsevier Inc. 71

Figure 2. FA Proteins Present an Anterior-

Posterior Gradient in Multiciliated Cells

(A–B) Intensity color-coded maximum intensity

projections of centrin2 RFP (A) and GFP Vinculin

(A0) reveals the presence of a Vinculin gradient

in multiciliated cells. The signal of centrin2 RFP

is uniform (A), while the GFP Vinculin signal is

stronger at the cell’s posterior (A0 ). In (B), multi-

ciliated cells coexpressing GFP Vinculin and

centrin2 RFP are shown. The anterior-posterior

gradient is maintained at the tissue level. The

arrows indicate cells’ posterior where the signal

for GFP Vinculin appears elevated.

(C) Multiciliated cell coexpressing mKate2 FAK,

clamp GFP, and centrin2 CFP. FAK is localized

next to the basal body at the end of the region

marked by clamp GFP.

(D and E) Confocal optical sections of a multi-

ciliated cell showing that GFP Paxillin and mKate2

FAK colocalize. GFP Paxillin is more concentrated

at the apical-most region of the basal bodies (D),

while mKate2 FAK is more concentrated slightly

below (E).

(F–G0 ) Intensity coding of ROIs of (D) and (E).

Paxillin exhibits highest intensity at 0.00 mm, as

shown in (F) and (F0 ), whereas FAK exhibits highest

intensity at 0.38 mm, as shown in (G) and (G0).

Developmental Cell


localizations, we went on to stain endogenous FAK and Vinculin

using previously characterized antibodies against these two

proteins. Since FAK associated with centrosomes was shown

to be phosphorylated on Serine 732, we used a phospho-Serine

732-specific FAK antibody in tadpoles expressing centrin2 cyan

fluorescent protein (CFP), and, as shown, the staining confirms

that endogenous FAK is associated with basal bodies and is

phosphorylated on Serine 732 (Figure 1D). In addition, immuno-

fluorescence experiments using a monoclonal Vinculin antibody

in centrin2 RFP-expressing tadpoles gives very similar results

to the GFP fusion (Figure 1E).

We went on to express GFP FAK in neural tissues to examine

the possibility that FA proteins also associate with basal bodies

in the monociliated cells of the GRP and the primary cilia of the

neural tube. As shown in Figures 1F and 1G, GFP FAK localizes

adjacent to the basal bodies of both motile monocilia of the

GRP (Figures 1F and 1F0) and primary cilia of the neural tube

(Figures 1G and 1G0). The localization of FAK and Paxillin fusion

constructs at the basal bodies of primary cilia was confirmed

using antibodies to image the endogenous proteins in NIH 3T3

cells after serum-starvation-induced ciliogenesis (Figures 1H

and 1I). These results suggest that the aforementioned FA

proteins associate with the basal bodies in all types of cilia.

FAK, Paxillin, and Vinculin Display Polarity withinMulticiliated Cells of the Xenopus EpidermisIn addition to the strong localization at the basal bodies, FAK,

Paxillin, and Vinculin display polarity within the cell. Two types

of polarity have been identified in multiciliated cells, both

controlled by the planar cell polarity pathway: rotational polarity

and tissue-level polarity (Mitchell et al., 2007, 2009; Park et al.,

2008; Wallingford, 2010). Rotational polarity refers to the align-

ment of basal bodies within each cell, and the aforementioned

FA proteins are clearly rotationally polarized (Figures 1A–1C).


Tissue-level polarity, on the other hand, refers to the coordina-

tion of the multiciliated cells across the tissue. However, FAK,

Paxillin, and Vinculin also display a third type of polarity, with

stronger signal emanating from basal bodies at the cell posterior

and weaker signal from basal bodies at the front of the cell

(in relation to the tadpole’s anterior-to-posterior axis) (Figures

1A–1C). This gradient is sharper in the case of Vinculin, in which

the first row of basal bodies, in close proximity to the cortical

actin cytoskeleton, shows a much higher level of Vinculin

compared to the second row, which is not interacting with the

cell cortex (Figures 1C and 1C0). This gradient becomes clearer

in images where both centrin as well as Vinculin are presented

using intensity coding, revealing a strong gradient in the cell (Fig-

ures 2A and 2A0). Imaging ciliated cells at lower magnifications

shows that this polarity is retained at the tissue level (Figure 2B).

The signal for all four proteins localizes immediately adjacent to

each basal body and is rotationally polarized, but its relation to

accessory structures, like the basal foot and the rootlet, cannot

be determined without additional markers.

To better define this region, we expressed mKate2 FAK with

clamp GFP, to mark the striated rootlets, and centrin2 CFP, to

mark the basal bodies. As shown in Figure 2C, the FAK signal

is concentrated at the end of the region marked by clamp

GFP, roughly, the area where the basal foot forms and an

area where electron microscopy analysis identified interactions

between basal bodies and the actin cytoskeleton (Chailley

et al., 1989). This shows that the gradient within each cell

described earlier, is an anterior-to-posterior gradient with higher

concentrations at the basal bodies at the back of each cell

(in relation to the tadpole’s anterior-to-posterior axis).

Despite the fact that the localization of the three proteins was

nearly identical, we noticed that Paxillin and Vinculin were always

appearing simultaneously with the basal bodies in z stacks, while

FAK appeared immediately after. This suggests that Paxillin and

c.

Figure 3. FA Proteins Form CA Complexes

Connecting Basal Bodies to the Actin

Network

(A) Basal bodies (labeled with centrin2 RFP) are

docked at the apical surface of multiciliated cells,

in the same plane as apical actin (visualized with

GFP utrophin). Each basal body is found at the

center of an actin ring.

(B) mKate2 FAK is in contact with the apical actin

network.

(C) Coexpression of centrin2 RFP and GFP FAK

followed by phalloidin staining suggests that FAK

connects basal bodies to the apical actin network.

(D and D0) Multiciliated cell expressing GFP FAK,

marking CAs, and mKate2 actin, showing the

apical actin network, before (D) and after (D0)acceptor photobleaching.

(D00) mKate2 (acceptor) intensity drops after

photobleaching, while GFP (donor) intensity rises,

showing that FRET is taking place between GFP

andmKate2 and suggesting that FAK is interacting

with actin.

(E and E0) Multiciliated cell expressing centrin GFP

andmKate2 actin before (E) and after (E0) acceptorphotobleaching.

(E00) mKate2 intensity drops after photobleaching,

but the GFP intensity remains unchanged, sug-

gesting that no FRET is taking place between GFP

and mKate2, indicating the absence of a direct

interaction between centrin and actin.

See also Figure S2.

Developmental Cell


Vinculin display different distributions along the apicobasal axis

compared to FAK. In an effort to confirm this, we coexpressed

GFP Paxillin, mKate2 FAK, and centrin2 yellow fluorescent

protein (YFP). As shown in Figures 2D and 2E, FAK and Paxillin

colocalize but have a slightly different distribution along the

z axis. Paxillin appears to have the highest density at the api-

cal-most region of the basal bodies (Figures 2F and 2F0), while

FAK has a maximal density slightly below the apical-most region

of each basal body (Figures 2G and 2G0), as shown in the two

adjacent optical sections of Figures 2F–2G0.

CAs Link Basal Bodies to the Actin CytoskeletonThe presence of four FA proteins in close association with basal

bodies in a region shown to have interactions with the actin

cytoskeleton raised the possibility that these proteins are form-

ing a complex connecting basal bodies to the actin network.

As shown in Figure 3A, each basal body is in the center of a

ring of actin. On the other hand, FAK partially colocalizes with

the apical actin network, as shown in Figure 3B, suggesting

that FAK, Paxillin, Talin, and Vinculin are forming a complex

linking each basal body to the actin network. Coexpression of

GFP FAK with centrin2 RFP and staining with phalloidin shows

that FAK is in contact with both the basal bodies and the

actin network (Figure 3C). In order to confirm an interaction

between FAK and the actin network, we coexpressed GFP

FAK (donor) with mKate2 actin (acceptor) and carried out

acceptor photobleaching experiments to examine the possibility

of intermolecular fluorescence resonance energy transfer

(FRET). These experiments show that FRET is taking place

between GFP and mKate2 in ciliated cells (Figures 3D–3D00).Control experiments using centrin GFP as the donor andmKate2

Deve

actin as an acceptor fail to detect FRET, as expected, and show

that centrin does not interact with actin, in agreement with the

localization data (Figures 3E–3E00). Spot bleaching experiments

confirm that FRET is restricted to the areas containing basal

bodies (Figures S2A–S2A0 and Figure S2B). These results pro-

vide evidence that FA proteins form complexes that we termed

CAs and that CAs are connecting each basal body to the actin

network.

Given the proposed role of actin in basal body transport and

the close association of basal bodies and actin during this pro-

cess, we wanted to explore the possibility that CAs are present

during basal body migration (Boisvieux-Ulrich et al., 1990;

Ioannou et al., 2013). Imaging basal bodies (centrin2 RFP) during

ciliated cell intercalation revealed that CAs (marked by GFP Pax-

illin) are present during basal body migration, suggesting that

CAs are likely connecting basal bodies to the internal actin

network thought to be responsible for basal body transport

(Figures 4A–4A00 0 and 4B) (Ioannou et al., 2013).

Since basal bodies also connect to the subapical actin

network through the ciliary rootlets, we went on to examine

whether FA proteins are also involved in making this connection.

As shown in Figure 4C0, we found a second complex forming

below the basal bodies, in close association with the subapical

actin, suggesting that CAs are responsible for the connection

between the ciliary rootlets and the subapical actin network.

Coexpression of GFP FAK with clamp RFP and centrin2 CFP

confirms that FAK is found both in association with the basal

bodies, at the apical surface, as well as at the end of the ciliary

rootlets (Figures 4D and 4D0).In order to get a better resolution of the association of the

CAs with the actin cytoskeleton, both apical and subapical,


Figure 4. CA Complexes Link Basal Bodies

and Ciliary Rootlets to the Subapical Actin

Network

(A–A00 0) Optical sections of an intercalating multi-

ciliated cell expressing GFP Paxillin and centrin2

RFP showing that Paxillin is associated with basal

bodies during their migration to the apical surface.

(B) 3D reconstruction (y-z) of optical sections of

the intercalating cell in (A)–(A00 0).(C and C0) Multiciliated cell expressing GFP FAK

and stained with phalloidin. GFP FAK exhibits the

highest density at two focal planes. The first one

corresponds to the plane of the apical actin

network (C), while the second one appears slightly

below, at the plane of the subapical actin network

(C0). GFP FAK is in close association with both

pools of actin.

(D) Maximum intensity projection of a region of

a multiciliated cell coexpressing centrin2 RFP

(to label basal bodies), clamp RFP (to label the

striated rootlet), and GFP FAK. GFP FAK exhibits

two intensity maxima: one at the level of the cell’s

apical surface, adjacent to the basal bodies, and

a second one deeper, at the end of the ciliary

rootlets.

(D0) Confocal image from a mechanically

sectioned (along the apicobasal axis) multiciliated

cell coexpressing centrin2 CFP, clamp RFP, and

GFP FAK. The arrows mark the two maxima of

GFP FAK, one associated with the basal body and

one associated with the end of the striated rootlet.

(E) Optical section of a multiciliated cell coexpressing centrin2 CFP and mKate2 FAK and labeled with phalloidin (apical surface is at the top). The cell was initially

mechanically sectioned, approximately along its anterior-posterior axis. The subapical actin, known to connect each basal body with the end of the striated

rootlet of the cilium behind (Werner et al., 2011), appears to project from the CAs (red-labeled with mKate2 FAK) and form the characteristic discontinuous

network.

(F) Optical section of a multiciliated cell expressing mKate2 FAK and labeled with phalloidin (apical surface is at the top). The cell was initially mechanically

sectioned, approximately along its left-to-right axis. The subapical actin appears to connect CAs of neighboring basal bodies (along the left-right axis), creating

continuous loops. Again, the subapical actin appears to project from the CAs.

(G) Multiciliated cell coexpressing GFP FRNK and centrin2 RFP. GFP FRNK associates with the basal bodies in a similar way as the full-length protein. It is

localized at the posterior site of each basal body and exhibits an anterior-posterior gradient within the cell.

(H) Multiciliated cell coexpressing Paxillin C GFP and centrin2 RFP. Paxillin C GFP localizes at the posterior site of basal bodies and displays a gradient with

respect to the anterior-posterior axis of the cell.

Developmental Cell


we sectioned embryos expressing mKate2 FAK and stained

them with phalloidin. This led to an improvement of the resolu-

tion in the apicobasal axis, since lateral resolution is higher than

axial in confocal systems. We observed that the subapical

network in these cells is projecting from the CAs basally and

toward the back of the cell. We also observed that the subapi-

cal network, as described elsewhere (Werner et al., 2011),

is connecting each basal body to the one immediately behind

and in front through the rootlet, creating the characteristic

discontinuous actin network shown in Figure 4E. However,

we also noted an additional subapical network, rotated 90�

with respect to the first, creating loops between the CAs of

adjacent basal bodies (Figure 4F). This network appears to

connect basal bodies in the left-to-right direction and is

probably responsible for the spacing of basal bodies in this

direction, while the discontinuous front-to-back subapical

network is responsible for the spacing of basal bodies front

to back. Both networks appear to be originating at apical

CAs and are linked subapically at a nexus, presumably the

subapical region showing elevated FAK concentrations at the

end of the clamp-positive domain.


FAK is known to localize to FAs through its C-terminal region

and, specifically, the FAT domain. This localization is believed

to be strongly dependent on FAK’s interaction with Paxillin,

whichmaps at the C terminus of FAK (Hayashi et al., 2002; Hilde-

brand et al., 1993, 1995). We went on to explore the domains

required for FAK localization at the basal bodies. As shown in

Figure 4G, the FAK C terminus, including the two proline-rich

sequences and the FAT domain, (FRNK: FAK-Related Non-

Kinase) is sufficient for the basal body localization of the protein.

Similarly, the C terminus of Paxillin is also sufficient to drive FA

localization of the protein (Brown et al., 1996) and, as shown in

Figure 4H, is also sufficient for basal body localization, showing

that, for both proteins, the determinants for CA complex locali-

zation are similar to those for FA localization, further emphasizing

the similarity between the two complexes.

Morpholino-Based FAK Downregulation BlocksCiliogenesisAlthough the localization and FRET data are strongly sugges-

tive of the function of the CA complex, we wanted to determine

what effects the disruption of the complex would have on

c.

Figure 5. FAKKnockdown Leads to Defects

in Ciliogenesis

(A–C) Surface view (maximum intensity projection)

of the epidermis of stage 31/32 Xenopus tadpoles

stained with phalloidin and immunostained for

acetylated tubulin, which stain actin network and

cilia, respectively. In (A), cells of control embryos

appear normal, with a lot of cilia projecting from

their apical surface. In (B), morphant cells (of em-

bryos injected at one ventral blastomere, at stage

5, with 4 ng of FAK MO) have fewer cilia projecting

outward from their surface. In (C), coinjection ofHA

FAKmRNA (90 pg) with the FAKMO (4 ng) rescues

the phenotype and cells project cilia normally.

(D) Surface view (maximum intensity projection) of

the epidermis of a stage 31/32 Xenopus embryo

stained with phalloidin and immunostained for

acetylated tubulin. Coinjection of HA-FAK D375

(45 pg) mRNA with the FAK MO does not rescue

the ciliogenesis defects.

(E) Quantification of the FAK MO induced cilio-

genesis defects. Bar chart shows the percentage

of stage 31/32 embryos that present mild, mod-

erate, and severe defects or are normal with

respect to the number of cilia projecting toward

their surface. Data were collected from 130–150

cells, from ten embryos (three experiments) of

each category (control, MO injected, MO and HA

FAK injected, andMO and HA FAK D375 injected).

(F–I) 3D reconstruction (x-z) of optical sections of

ciliated cells expressing centrin2 RFP and clamp

GFP. In (F), most basal bodies have reached the

apical surface but are not docked properly in cells

injected with 4 ng of FAK MO. In (G), higher

amounts of FAKMO (8 ng) lead to amore dramatic

phenotype, as the majority of basal bodies fail to

reach the apical surface of the cell. In (H), all the

basal bodies are docked at the apical surface of

a control ciliated cell. In (I), coinjection of HA

FAK mRNA (120 pg) with the FAK MO (8 ng)

rescues the phenotype, as the basal bodies have

managed to reach the apical surface and the

majority of them have docked properly.

(J and K) Intercalating ciliated cells of stage 17

Xenopus embryos coexpressing centrin2 RFP

and GFP utrophin to label basal bodies and the

actin network, respectively. In control cells (J),

basal bodies are clustered deep in the cell,

surrounded by an actin network, and individual

basal bodies associate with actin filaments. In

cells coinjected with 8 ng of FAK MO (K), the

basal bodies are dispersed, and many of them do

not associate with actin, which fails to form an

organized network around them.

Developmental Cell


ciliogenesis. Since FAK is a well-characterized regulator of the

dynamics of FA assembly and disassembly, we postulated that

it would have an important role in CA complex function. We

thus used a previously characterized FAK morpholino (MO)

oligo (Fonar et al., 2011; Petridou et al., 2012, 2013) (after veri-

fying its efficiency at tadpole stages; Figure S3A) to block FAK

expression in multiciliated cells. As shown in Figure 5B, FAK

morphant cells have few cilia projecting from their surface

compared to control cells (Figures 5A and 5E). Coinjection of

FAK mRNA with the FAK MO effectively rescues this pheno-

type, and rescued cells project cilia normally, suggesting that

Deve

the phenotype is specific (Figures 5C and 5E). Since the

FERM domain of FAK is critical for its regulation and has

recently been shown to have an important role in the regulation

of FAK’s dynamics and function at FAs, we decided to examine

if the FERM domain was required for rescue (Cooper et al.,

2003; Petridou et al., 2013). We thus coinjected the FAK

D375 mutant, which lacks the FERM domain, with the FAK

MO. The FAK D375 mutant failed to rescue the phenotype,

showing that the FERM domain and/or proper regulation of

FAK’s kinase activity are required for FAK’s function at CAs

(Figures 5D and 5E).


Figure 6. Apical Actin Enrichment and

Rotational Polarity Are Unaffected in FAK

Morphants

(A–C) Confocal images of phalloidin-stained con-

trol (A), morphant (B), and rescued (C) ciliated cells

expressing centrin2 RFP plus enlarged close-ups

of selected regions, shown in (A0), (B0), and (C0 ),and a respective deeper optical section of each, in

(A00), (B00), and (C00), showing details of apical and

subapical actin networks. Morphants (B), unlike

controls (A) and rescued (C) cells, display reduced

numbers of basal bodies at the cell surface with

large areas completely devoid of basal bodies.

The apical actin network is present both in mor-

phants and controls; however, it appears less

organized and less dense in regions devoid of

basal bodies. The subapical actin of morphants

(B00) is completely missing in areas devoid of basal

bodies and appears disorganized in areas with

basal bodies.

(D and E) Centrin2 RFP- and clamp GFP-ex-

pressing control (D) and FAK morphant (E) multi-

ciliated cells. Spacing is disrupted, with regions

of the apical surface devoid of basal bodies in the

morphant cell. However, overall rotational polarity

is not significantly affected as most rootlets are

oriented in the same direction.

See also Figure S3.

Developmental Cell


Examination of centrin in side projections of morphant cells

(Figure 5F) reveals that, despite the fact that most basal bodies

reach the apical surface, they display variations in positioning

in the z axis, unlike the controls (Figure 5H), suggesting that

many of them do not dock. At higher doses of the FAK MO

(8 ng), basal body transport is impaired and the majority of

basal bodies remain deep in the cytosol (Figure 5G). Imaging

high-dose morphant cells during intercalation reveals that

basal bodies are often dispersed, rather than clustered like in

controls, and some clearly fail to associate with the internal actin

network, previously implicated in their transport (Boisvieux-

Ulrich et al., 1990; Ioannou et al., 2013), suggesting that the

transport defect is due to loss of association between the basal

bodies and the actin network (Figures 5J and 5K). Coinjection of

FAK mRNA also rescues the transport phenotype as shown in

Figure 5I.

In addition to the ciliogenesis and docking defects, FAK

morphants (4 ng) display spacing issues with regions of the

apical surface completely devoid of basal bodies (Figure 6B).

Despite the presence of apical actin enrichment in these

regions, the network appears less dense (Figure 6B0) and the

subapical actin is completely lost (Figure 6B00), unlike control

(Figures 6A–6A00) and rescued cells (Figures 6C–6C00), which pre-

sent uniformly organized apical and subapical actin networks.

In addition, although the apical actin of morphant cells appears

normal at regions with docked basal bodies, the subapical

actin appears less organized. Interestingly, the use of clamp

GFP with centrin2 RFP shows that rotational polarity, which is

76 Developmental Cell 28, 70–80, January 13, 2014 ª2014 Elsevier Inc.

dependent on the microtubule network,

remains relatively unaffected in FAK

morphants, especially when examining

local coordination in basal body clusters

(Figures 6D and 6E). These results suggest that FAK has an

important role in the regulation of the CA complex, and in addi-

tion, they are consistent with a role of this complex as a mecha-

nical link between basal bodies and the actin cytoskeleton.

Since the C terminus of FAK (FRNK) is sufficient for CA local-

ization, we postulated that, in a similar fashion as in FAs, it

would be able to act as a dominant negative by displacing

endogenous FAK (Gilmore and Romer, 1996; Hildebrand

et al., 1993; Schaller et al., 1992; Taylor et al., 2001). We

went on to overexpress FRNK in order to further address the

role of FAK and CAs during ciliogenesis. Expression of FRNK

partially displaces FAK from CAs but fails to induce strong

ciliogenesis defects (Figures S3B and S3C; data not shown).

In FRNK-expressing cells, most basal bodies reach the apical

surface and project cilia; however, in these cells, basal body

spacing is disrupted (Figures S3D–S3F; data not shown). Since

the subapical actin network has been shown to be responsible

for basal body spacing, this result supports a role of the CA

complex linking basal bodies to the actin network (Werner

et al., 2011). The reduced severity of the FRNK phenotype

compared to the FAK MO can be explained by the fact that

FRNK fails to displace all endogenous FAK from CAs. In

addition, FRNK only blocks kinase-dependent functions of

FAK while retaining scaffolding functions that depend on the

C-terminal FAT domain and the two proline-rich regions of

the protein (Gilmore and Romer, 1996; Hildebrand et al.,

1993; Taylor et al., 2001). This result is in agreement with a

role of CA complexes as a mechanical link between actin and

Figure 7. CA Complexes in Multiciliated Cells

(A) A diagram showing the localization of the CA complexes with respect to

the basal bodies, ciliary rootlets, and the actin cytoskeleton in multiciliated

cells. CA complexes interact with the apical and subapical pools of actin.

The upper red circle shows an x-z projection of confocal optical sections of

CA complexes and basal bodies. CAs are marked with GFP FAK (green) and

the basal bodies with centrin2 RFP (red).

(B) Proposed arrangement of the subapical actin network in multiciliated cells

in relation to the basal bodies, rootlets, and CAs.

Developmental Cell


the basal bodies and, in addition, suggests that FAK has

important kinase-independent roles at the CAs.

DISCUSSION

Overall, this work reveals a function of FA proteins in the

creation of complexes responsible for anchoring basal bodies

to the actin network of multiciliated cells. In addition, our results

show that CAs also form at the ends of ciliary rootlets and

are responsible for connecting the subapical actin network to

the ciliary rootlet. We also show that the subapical network

has two components. One discontinuous network in the front-

to-back direction connects each basal body to the one in front

and the one behind through the ciliary rootlet; and a continuous

network, at a right angle in relation to the first, connects

adjacent basal bodies in the left-to-right direction. This network

is probably responsible for spacing of basal bodies in the

left-to-right orientation while the discontinuous front-to-back

Deve

network determines spacing in the anterior-posterior axis of

the cell. The location of CAs with respect to the basal bodies,

rootlets, and the actin network is summarized in Figure 7.

The localization of the apical CA complex at the cell’s posterior

makes the most sense mechanically, since the maximum

force would be excreted by the cilium on the basal bodies

during the effective stroke rather than the recovery stroke,

pushing the basal bodies in the opposite direction of the stroke.

Linking the basal body to the actin network on this area would

ensure the immobilization of the basal body during the effective

stroke.

The similarities between CAs and FAs are striking, and the

determinants for Paxillin and FAK localization on CAs appear

to be similar to those for FA localization, although a more

detailed analysis will be required to determine if they are, in

fact, identical. FAK has been shown to be a critical regulator

of FA assembly and disassembly. Loss of FAK activity leads

to aberrant ciliogenesis and defects that are dependent on

actin-based processes, like basal body transport, docking,

and spacing, without affecting apical actin enrichment. These

results suggest that FAK, a critical regulator of FAs, is also

an important regulator of CAs, and they are consistent with

a role of this complex in the mechanical linkage of basal

bodies to the actin cytoskeleton. The proximity of the apical

CAs to the basal foot also raises the possibility that CAs are

also connecting basal bodies to the microtubule network,

shown to be responsible for establishing the rotational polarity

within multiciliated cells. However, FAK morphants show no

rotational polarity defects and, in this respect, resemble

cytochalasin-treated cells where spacing is affected but

local rotational polarity is maintained, making it unlikely that

CAs are linking basal bodies to microtubules (Werner et al.,

2011).

The presence of CA complexes in both motile and primary

cilia suggests that these proteins serve a highly conserved

function. The role of the actin cytoskeleton is well documented

both for motile cilia and primary cilia, as well as the flagella

of unicellular eukaryotes (Dentler and Adams, 1992; Engel

et al., 2011; Kim et al., 2010; Paredez et al., 2011; Sharma

et al., 2011). Interestingly, several core FA proteins have

been found in flagellated unicellular eukaryotes, including

Apusozoa and Choanoflagellates, the closest sister group

of Metazoa (Sebe-Pedros et al., 2010). Flagellar motility is

found in every major eukaryotic group (Fritz-Laylin et al.,

2010) and is believed to be an ancestral feature present

in the last common ancestor of all eukaryotic organisms

(Carvalho-Santos et al., 2011; Fritz-Laylin et al., 2010; Mitchell,

2007). This raises the possibility that the core FA proteins

originally evolved to perform functions at flagella and were

later co-opted for use in cell adhesion. Further support for

this notion comes from the fact that FA proteins were lost in

all fungi, with the exception of chytrid fungi, which are the

only members of the kingdom with flagellated gametes,

suggesting an important function for FA proteins in flagellar

function, in agreement with our results (Carvalho-Santos

et al., 2011). Future work will focus on examining the possibility

that CAs also exist in flagellated unicellular eukaryotes and will

also focus on addressing the precise role of individual proteins

within this complex.


Developmental Cell


EXPERIMENTAL PROCEDURES

Embryo Manipulations and Microinjections

Female adult Xenopus laevis were ovulated by injection of human chorionic

gonadotropin. Eggs were fertilized in vitro, dejellied in 2% cysteine (pH 7.8),

and subsequently reared in 0.1 3 Marc’s modified ringers (MMR) and staged

according to Neiuwkoop and Faber (Nieuwkoop and Faber, 1994). For micro-

injections, embryos were placed in a solution of 4% Ficoll in 0.33 3MMR and

injected using a glass capillary pulled needle, forceps, a Singer Instruments

MK1 micromanipulator, and a Harvard Apparatus pressure injector. After

injections, embryos were reared for 2 hr or until stage 8 in 4% Ficoll in

0.33 3 MMR and then washed and maintained in 0.1 3 MMR. For most

experiments, injections were made into the ventral blastomeres to target the

epidermis at the 4-cell, 8-cell, or 16-cell stage. For all experiments, we injected

morpholinos at 4 to 8 ng per blastomere and mRNAs at various amounts.

Embryos were allowed to develop to the appropriate stage and then imaged

live or fixed in MEMFA (Sive et al., 2010) for 1–2 hr at room temperature

(RT). Fixed embryos were used immediately. For live imaging, embryos were

anesthetized in 0.01% benzocaine in 0.1 3 MMR.

Immunostaining

Indirect immunofluorescence assays were carried out as described elsewhere

(Demetriou et al., 2008; Skourides et al., 1999) with modifications. NIH 3T3

cells were seeded on glass coverslips (charged with HCl), washed three

times with PBS, preextracted with 0.2% Triton X-100 for 20 s, and then

fixed for 10 min in 4% paraformaldehyde solution in PBS. Fixation was

followed by addition of 50 mM glycine solution in PBS, and then the cells

were blocked using 10% normal goat serum (Jackson Immunoresearch) for

30 min. Cells were incubated with primary antibodies diluted in 10% normal

goat serum solution in PBS for 1.5 hr. The primary antibodies used were

Paxillin mouse monoclonal (BD Transduction Laboratories) used in com-

bination with Acetyl-a-Tubulin (Lys40) rabbit polyclonal (Cell Signaling) and

g Tubulin goat polyclonal (Santa Cruz Biotechnology); and FAK [pS732]

Phosphospecific rabbit polyclonal (Invitrogen) used in combination with

Acetylated tubulin, mouse monoclonal (Santa Cruz Biotechnology) and g

Tubulin goat polyclonal (Santa Cruz Biotechnology). Cells were then washed

five times in PBS. Secondary antibodies used were Cy3 anti-mouse and

anti-rabbit (Jackson Immunoresearch), Alexa 488 anti-goat (Molecular

Probes Invitrogen), and IgG-CFL 647 anti-mouse and anti-rabbit (Santa Cruz

Biotechnology).

For whole-mount immunostaining, fixed embryos were permeabilized in

PBDT (1 3 PBS + 0.5% Triton X-100 + 1% DMSO) for several hours at

RT and blocked in PBDT + 1% normal goat serum for 1 hr at RT. Primary

antibodies were added in block solution, and embryos were incubated for

4 hr at RT or overnight at 4�C. The primary antibodies used were: Acetylated

tubulin mouse monoclonal (Santa Cruz Biotechnology), Vinculin mouse

monoclonal (Developmental Studies Hybridoma Bank), and FAK pS732 rabbit

polyclonal (Invitrogen). The next day, embryos were washed 4 3 10 min in

PBDT. Embryos were then incubated in secondary antibodies—anti-mouse

Alexa 488 (Invitrogen) or anti-mouse IgG-CFL 647 (Santa Cruz Biotech-

nology)—at RT for 1–2 hr. Actin labeling was performed using Alexa Fluor

Phalloidin 488 (Invitrogen) and Phalloidin CruzFluor 647 (Santa Cruz Biotech-

nology) at RT for 1–2 hr. Embryos were then washed 4 3 10 min in PBDT.

Embryos were postfixed in MEMFA for 10-15 min at RT, washed in 1 3 PBS

and imaged immediately.

Western Blot Analysis

Protein lysates were prepared by homogenizing embryos (by pipetting up and

down) in ice-cold MK’s modified lysis buffer (50 mM Tris, pH 8.0, 150 mM

NaCl, 0.5% NP-40, 0.5% Triton X-100, 100 mM EGTA, 5 mM NaF) supple-

mented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride and

protease cocktail; Sigma). Homogenates were cleared by centrifugation at

15,000 3 g for 30 min at 4�C (Kragtorp and Miller, 2006). Proteins (3/4–1 em-

bryo equivalent) were run on 7.5% SDS-polyacrylamide gels with the West-

ernC ladder (Bio-Rad) and blotted on to nitrocellulose membranes. Blots

were blocked in 5% milk (in TBSTw: 13 TBS buffer and 0.1% Tween 20)

and then incubated with the primary antibodies in 5% milk overnight at 4�C.The primary antibodies used were: FAK mouse monoclonal (Millipore), Paxillin


mouse monoclonal (BD Transduction Laboratories), and Vinculin mouse

monoclonal (Developmental Studies Hybridoma Bank). Visualization was

performed using horseradish peroxidase-conjugated antibodies (anti-rabbit

and anti-mouse; Santa Cruz Biotechnology), after 1 hr incubation at RT, and

signal was detected using LumiSensor (GeneScript) on a UVP BioSpectrum

Imaging System. For loading control, actin rabbit polyclonal antibody (Santa

Cruz Biotechnologies) was used.

GRP Assay

Embryos were fixed in MEMFA at stage 17. The GRP was manually dissected

and postfixed for a further 15 min. GRP tissue was then used for immunofluo-

rescence as described earlier.

Imaging and Quantification

Imaging was performed on a Zeiss LSM 710 laser scanning confocal micro-

scope with Zen 2010 software.

Quantification of the ciliogenesis defects was conducted based on the

number of cilia projecting from the apical surface of each cell. Cells have

been denoted as normal, with mild phenotype (having more than 50% of their

cilia but less than control cells), with moderate phenotype (having 50% or

lower reduction of their cilia), and with severe phenotype (having near com-

plete loss of their cilia).

Quantification of the basal body distribution was conducted by overlaying

a grid over each cell and counting the number of basal bodies included in

each cell of the grid, using Adobe Photoshop CS2 software.

Acceptor Photobleaching FRET

FRET experiments were accomplished using a laser scanning confocal micro-

scope (Zeiss LSM 710) with a Plan-Apochromat 633/1.40 Oil DIC M27 objec-

tive lens (Zeiss). Stage 33–35 embryos expressing GFP FAK and mKate2 actin

or centrin GFP and mKate2 actin were anesthetized in 0.01% benzocaine in

MMR and immobilized in silicone grease wells on glass slides. A 543 nm laser

was used for acceptor (mKate2 actin) photobleaching (100% power) within a

region of interest (ROI). A 488 nm laser (0.8% power) was used for acquisition

of GFP FAK, and emission was detected between 493 and 538 nm. A 543 nm

laser (1.2% power) was used for acquisition of mKate2 actin, and emission

was detected between 599 and 758 nm. One frame was acquired as a pre-

bleaching control, and the ROI (rectangular region) was bleached within one

frame. Zeiss Zen 2010 software was used for FRET analysis. FRET efficiency

was calculated using the following equation: FRETEfficiency = (Donor Post �Donor Pre)/(Donor Post) 3 100 (Donor Post: donor emission intensity after

photobleaching; Donor Pre: donor emission intensity before photobleaching).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures

and three figures and can be found with this article online at http://dx.doi.

org/10.1016/j.devcel.2013.12.003.

ACKNOWLEDGMENTS

We thank Drs. JohnWallingford, Brian Mitchell, and Reinhard Koster for kindly

providing plasmids. We also thank Dr. Niovi Santama for providing reagents

for the immunofluorescence experiments on NIH 3T3 cells. Finally, we thank

Charalambos Kourouklaris for generating the 3D reconstruction of the pro-

posed model. Funding was provided by the Cyprus Research Promotion

Foundation (YGEIA/BIOS/0609(BE)/14, TEXNOLOGIA/YLIKA/0311(BIE)/10).

Accepted: December 6, 2013

Published: January 13, 2014

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Documents

Making the Connection: Ciliary Adhesion Complexes Anchor Basal Bodies to the Actin Cytoskeleton