ARTICLES
A molecular network for de novo generation of the
apical surface and lumen
David M. Bryant1,5, Anirban Datta1,5, Alejo E. Rodríguez-Fraticelli3, Johan Peränen4, Fernando Martín-Belmonte3
and Keith E. Mostov1,2,6
To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is regulated
by polarity complexes that are hypothesized to direct downstream events, such as polarized membrane traffic, although this
interconnection is not well understood. We have found that Rab11a regulates apical traffic and lumen formation through the Rab
guanine nucleotide exchange factor (GEF), Rabin8, and its target, Rab8a. Rab8a and Rab11a function through the exocyst to
target Par3 to the apical surface, and control apical Cdc42 activation through the Cdc42 GEF, Tuba. These components assemble
at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical
exocytosis during lumen formation, revealing an interaction between the machineries of vesicular transport and polarization.
Most internal epithelial organs consist of a monolayer of polarized epithelial cells surrounding a central lumen. Polarization requires the interaction of the signalling complexes and scaffolds that define cortical domains
with membrane-sorting machinery 1. In yeast, traffic from the trans-Golgi
network to the cell surface is regulated by Ypt32p and Sec4p (ref. 2), homologues of mammalian Rab11 and Rab8, respectively. Ypt32p recruits Sec2p
(homologue of mammalian Rabin8), a GEF for Sec4p. Sec2p and Sec4p in
turn interact with the exocyst, which docks vesicles to the cell surface3.
Definition of cortical domains in metazoa involves a complex of Par3,
Par6, atypical protein kinase C (aPKC) and the GTPase, Cdc42 (ref. 4).
This complex is a master regulator of polarity, conventionally depicted
upstream of membrane-trafficking machinery. How this complex interfaces with membrane transport is poorly understood.
Here, we show a molecular mechanism for lumen and apical surface
formation, linking Rab8a and Rab11a, the exocyst, annexin2, Cdc42
and its GEF Tuba, and the Par3–aPKC complex. This pathway shows
how the membrane traffic and cortical polarity machineries cooperate
to generate the apical surface and lumen de novo.
RESULTS
Apical polarization during lumen formation
On plating into 3D culture, individual MDCK (Madin-Darby canine kidney) cells proliferate and assemble into cyst structures—a polarized spherical
monolayer surrounding a central lumen. Lumenogenesis requires the apical membrane determinant gp135/podocalyxin5 (PCX in figures). Initially,
podocalyxin is localized at the extracellular matrix-contacting surface in
MDCK aggregates (Fig. 1a, 12 h and Supplementary Information, Fig. S1a),
before polarity inversion occurs, with β-catenin and Na+/K+-ATPase at cell–
cell junctions and podocalyxin now at the lumen (Fig. 1a, 24–48 h; arrows
and Supplementary Information, Fig. S1d)6,7. Lumens start to form at a site
termed the ‘pre-apical patch’ (PAP), where opposing plasma membranes are
separated, but the podocalyxin signal does not give an optically resolvable
lumen by confocal microscopy7. The lumen can only be visualized after
expansion and further separation of apical membranes (Fig. 1b). Conversely,
apical proteins syntaxin-3 and GFP–CNT1 (green fluorescent protein fused
to concentrative nucleoside transporter-1) label the entire surface before
concentrating at the lumen (Supplementary Information, Fig. S1b, c, e, f)5.
Binding of antibodies to GFP–VSVG–podocalyxin (VSVG; vesicular
stomatis viral G protein) at the periphery of cysts, followed by incubation
to allow lumenogenesis, revealed that podocalyxin at the PAP (arrowheads) and in vesicles (arrows) is at least partially derived from transcytosed peripheral podocalyxin (Supplementary Information, Fig. S1g).
Transcytosis of podocalyxin to the luminal surface to establish the PAP
represents formation of apical–basal polarization. We therefore examined localization of select polarity (Par3–aPKC), trafficking (exocyst
complex; Sec8–Sec10–Sec15A), and junctional (occludin) proteins during lumen initiation. Strikingly, although these proteins showed differing
localizations before lumen formation, all converged transiently during
lumen initiation (Fig. 1c–k and Table 1). When GFP–podocalyxin was
peripheral, Par3 and Sec8 co-localized in puncta at the edge of cell–cell
contacts (Fig. 1c; arrowheads). When GFP–podocalyxin was internalized
and transcytosed, some Par3 and Sec8 concentrated at the first detectable
site of GFP–podocalyxin delivery to the nascent apical surface (Fig. 1d;
arrow). We term this the apical membrane initiation site (AMIS). Later,
1
Department of Anatomy, University of California, San Francisco, CA 94143‑2140, USA. 2Department of Biochemistry and Biophysics, University of California, San
Francisco, CA 94143‑2140, USA. 3Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain. 4Institute of
Biotechnology, Viikinkaari 9, University of Helsinki, FIN‑00014 Helsinki, Finland. 5These authors contributed equally to this work.
6
Correspondence should be addressed to K.E.M. ([email protected]).
Received 16 February 2010; accepted 30 July 2010; published online 03 October 2010; DOI: 10.1038/ncb2106
nature cell biology VOLUME 12 | NUMBER 11 | NOVEMBER 2010
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A RT I C L E S
a
24 h
48 h
AMIS
PAP
Open Lumen
f
g
h
i
j
k
PCX/Occludin/nuclei
`-cat/PCX/nuclei
12 h
b
Early aggregate
PAP
Early aggregate
AMIS
d
Open lumen
e
Par3 /Sec8/GFP–PCX
c
Open lumen
PCX/aPKC/nuclei
L
Figure 1 Characterization of lumen initiation in MDCK cysts. (a)
Representative immunofluorescence confocal microscopy images showing
development of polarity in MDCK cysts incubated with antibodies against
the indicated proteins, and Hoescht (to visualize nuclei). Arrow at 24 h after
plating indicates polarity inversion, with podocalyxin now at the cyst interior
and formation of a PAP. Arrow at 48 h after plating indicates the opening of
the luminal space. (b) Schematic representation of cyst development from
a. Black lines, plasma membrane; red lines, apical surface; blue, nuclei.
(c–k) Transient localization of polarity and trafficking machinery to an apical
membrane initiation site (AMIS) in representative MDCK cysts at different
stages during lumen initiation. Bottom: higher‑magnification images of the
indicated regions showing localization of individual proteins (c–e), or in f–k
individual proteins (left, middle) and a merge of these two images (right).
Arrowheads in c–e indicate Sec8 and Par3 localization. Arrow in d indicates
co‑localization of Sec8, Par3 and GFP–podocalyxin (green) at the AMIS.
Arrows in f indicate co‑localization of podocalyxin and occludin at the AMIS;
arrowheads in g and h indicate localization of occludin at the edges of the
lumen (g) and at the tight junctions (h). In i arrowheads indicate peripheral
podocalyxin and aPKC. Arrows in i indicate localization of aPKC at the AMIS.
In j–k arrowheads indicate aPKC at tight junction regions. Scale bars, 20 μm.
Par3 and Sec8 enriched at the tight junction (Fig. 1e; arrowheads). We
define early apical structures where several tight-junction markers have
become distinctly localized from podocalyxin as the PAP7, compared
with the AMIS that forms earlier, where tight-junction markers and
podocalyxin cannot be resolved by confocal microscopy.
In contrast, Sec10 and occludin initially localized along the entire
cell–cell contact in early aggregates, which had peripheral podocalyxin
(Supplementary Information, Fig. S2a and data not shown). Podocalyxin
delivered to the AMIS partially overlapped with Sec10 and occludin
(Fig. 1f and Supplementary Information, Fig. S2b; arrows). Although
occludin remained along the entire contact, Sec10 condensed toward
the AMIS. As the lumen expanded, Sec10 and occludin enriched at the
tight junction (Fig. 1g, h and Supplementary Information, Fig. S2c–f),
although some occludin remained along cell–cell contacts (Fig. 1h).
aPKC follows yet a different pattern, with distinct pools initially localized
with peripheral podocalyxin (arrowheads) and the AMIS (Fig. 1i; arrows),
before enriching at PAP edges and finally the tight junction and lumen
(Fig. 1j, k; arrowheads). These data show the complex movement of trafficking and cortical polarity proteins that converge transiently at the AMIS.
and Supplementary Information, Fig. S3f–l). In contrast to control cysts
with a single lumen, apical podocalyxin and basolateral β-catenin, knockdown of Rab8 (Rab8a/Rab8b) and Rab11 (Rab11a/ Rab25, but not Rab11b)
family members significantly decreased single lumenogenesis (Fig. 2f) so
that cysts had multiple lumens and accumulated podocalyxin in vesicles
(Fig. 2b–e; arrowheads and data not shown) close to the cell surface (marked
by β-catenin). For single lumen-perturbing knockdowns, phenotypes
were confirmed using additional shRNAs (Supplementary Information,
Fig. S3l), and additional cargoes (Supplementary Information, Fig. S3a–c).
Perturbation of Rab10, Rab11b, Rab13 and Rab14 did not markedly perturb
lumenogenesis, and were not investigated further (Fig. 2f).
The Rab11 family regulates transcytosis8 and lumenogenesis in diverse
systems9–11. GFP–Rab11a localized to vesicles underlying the AMIS (marked
by Par3; arrow in Fig. 2g), then remained on subapical vesicles once lumens
expanded (Fig. 2h and Table 1). Podocalyxin transcytosed to the AMIS
through Rab11a-positive vesicles. When podocalyxin was peripheral (Fig.
2i; arrow), GFP–Rab11a localized to juxtanuclear and peripheral vesicles
(Fig. 2i; white and yellow arrowheads, respectively). On internalization,
podocalyxin localized to GFP–Rab11a-positive vesicles (Fig. 2j; arrowheads), then both were delivered to the cyst interior (Fig. 2k; arrowheads).
Here, regions of podocalyxin devoid of GFP–Rab11a began to emerge
(Fig. 2k–m; arrows), representing podocalyxin surface delivery. Similarly,
immunoglobulin A (IgA) transcytosed to the PAP (Supplementary
The Rab8 and Rab11 GTPase families direct lumen initiation
We examined AMIS and lumen formation on perturbation of select Rab
GTPases involved in apical, basolateral or junctional trafficking (Fig. 2a–f
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Table 1 Distribution of trafficking and polarity proteins during lumen initiation and expansion.
Lumen Stage
Protein
AMIS
PAP
Open Lumen
Occludin
Cell–cell contact
Cell–cell contact
Cell–cell contact/tight junction
Par3
AMIS
PAP
Tight junction
Tight junction/luminal
aPKC
AMIS/peripheral
PAP
Podocalyxin
Vesicles/peripheral
PAP
Lumen
Rab8a
Vesicles
Subapical vesicles
Subapical vesicles
Rab11a
Vesicles
Subapical vesicles
Subapical vesicles
Tight junction
Sec8
AMIS
PAP
Sec10
Cell–cell contact
PAP
Tight junction
Sec15A
Vesicles
Subapical vesicles
Subapical vesicles
Information, Fig. S1h). As the lumen expanded, GFP–Rab11a clustered
underneath the apical surface (Fig. 2l, m). Notably, overexpression of GFP–
Rab11a (wild-type or an activated Q70L substitution mutant) increased
single lumenogenesis, whereas dominant negative GFP–Rab11aS25N attenuated single lumenogenesis and accumulated podocalyxin intracellularly
(Supplementary Information, Fig. S4a; data not shown). Thus, Rab11a
promotes transcytosis to the AMIS and single lumenogenesis.
Rab8 family GTPases were also required for single lumen formation (Fig. 2b, c, f), and Rab8a localized to transcytosing podocalyxin
vesicles and the AMIS7 (Table 1 and data not shown). Knockdown of
Rab11a resulted in upregulation of Rab8a, and vice versa (Supplementary
Information, Fig. S3f, g), suggesting compensation or cooperation
between Rab11 and Rab8 families. Therefore, we knocked down Rab8
and Rab11 family members, alone or in combination (Supplementary
Information, Fig. S4b). Of tested combinations, co-knockdown of Rab8a
and Rab8b, with or without Rab11a knockdown, most markedly reduced
single lumenogenesis. This suggests that the Rab8 family may function
downstream of Rab11a. Accordingly, Rab8a knockdown blocked the
increase in single lumenogenesis induced by GFP–Rab11aQ70L expression
(Supplementary Information, Fig. S4c). These data are consistent with the
hypothesis that the Rab8 family functions, at least in part, downstream of
Rab11a during apical transport and lumenogenesis, although the precise
interaction between these Rab proteins may be more complex.
Regulation of Rab8 during lumenogenesis
Rab11 binds to the Rab GEF Rabin8 and stimulates its activity towards Rab8
(ref. 12). We reasoned that Rab11a may control subapical Rabin8–Rab8
targeting. In control cysts, a small pool of Rabin8, and to a lesser extent
Rab8a, localized to dispersed puncta, with some clustered subapically
(Fig. 3a; arrows). Expression of GFP–Rab11aWT, but not GFP–Rab11aS25N,
strongly enhanced recruitment of Rabin8 and Rab8a to Rab11a-positive
subapical vesicles (Fig. 3a; arrowheads). Similarly to endogenous Rab8a
(Fig. 3a), GFP–Rab8aWT was cytoplasmic and in subapical vesicles, the
latter of which was enhanced on activated GFP–Rab8aQ67L expression
(Supplementary Information, Fig. S4f). Thus, active Rab11a recruits active
Rab8a to subapical vesicles, probably through Rabin8.
In western blots of MDCK lysates, Rabin8 was identified in two bands corresponding to its α and β isoforms: both possess the Rab11-binding region12
(Fig. 3b and Supplementary Information, Fig. S5a). On Rabin8α knockdown
some podocalyxin accumulated in vesicles (Fig. 3c; arrowheads), and there
was also a small (but statistically significant) decrease in the proportion of
cysts with single lumens (Fig. 3e). This effect is only modest probably because
of compensatory upregulation of Rabin8β observed on Rabin8α knockdown
(Fig. 3b). Dual Rabin8α and Rabin8β knockdown caused cell death, precluding further analysis (data not shown). In cysts with endogenous Rabin8α
knockdown, expression of RNAi-resistant GFP–hRabin8αWT (GFP-tagged
human Rabin8α), which localized to the luminal region (arrows), restored
single lumenogenesis and podocalyxin localization (Fig. 3d, e). Conversely,
Rabin8α GEF-domain mutants (Supplementary Information, Fig. S5a–c)
further decreased single lumenogenesis and co-accumulated with podocalyxin on vesicles (Fig. 3d, e; arrowheads) beneath the surface marked by
F-actin. Similarly, overexpression of GFP–TBC1D30WT, a GAP (GTPaseactivating protein) specific to the Rab8 family13, but not GAP-deficient
GFP–TBC1D30R140A, perturbed single lumenogenesis (Supplementary
Information, Fig. S5d, e). These data suggest that a Rab11a–Rabin8α–Rab8a
cascade, inhibited by TBC1D30, is part of a regulatory module that governs
apical transport and lumenogenesis.
The exocyst and Par3–aPKC complexes regulate apical
polarization
Rab8a and Rab11a interact with the Sec15 exocyst subunit 14, in turn
linking to Sec10 and other subunits as part of a chain tethering vesicles to
the basolateral3 and apical membranes15. The exocyst also interacts with
the Par3–aPKC complex 16,17. As these factors converge at the AMIS, we
examined their requirement in apical traffic and single lumenogenesis.
In contrast to control cysts with apical podocalyxin and basolateral
β-catenin (Fig. 4a), Sec15A knockdown (Supplementary Information,
Fig. S3m) resulted in accumulation of podocalyxin in prominent GFP–
Rab11a-positive vesicles close to the apical surface (Fig. 4b; arrows, 4c;
arrowheads). This Rab11a compartment seemed expanded relative to
control cysts (compare Fig. 2m). Additionally, cysts were defective in
apical polarization, mistargeting apical cargo to regions of cell–cell contact (Fig. 4b and Supplementary Information, Fig. S3d). Accordingly,
Sec15A knockdown caused almost complete loss of single lumenogenesis
(Fig. 4d). Similarly, Sec10 knockdown decreased single lumenogenesis
and caused vesicular accumulation of podocalyxin (Supplementary
Information, Fig. S2g–i). Thus, the exocyst regulates podocalyxin transport from Rab11a-positive vesicles to the forming apical surface.
We also examined the role of the exocyst on Par3 transport and AMIS
formation. In control cysts, Par3 localized to tight junctions (Fig. 4e; arrows).
In Sec15A knockdown cysts, Par3 showed varying, though always abnormal,
localization. In regions where a PAP formed, Par3 was recruited to the surface (Fig. 4f). However, in regions of vesicular podocalyxin accumulation,
Par3 failed to be recruited to the surface and an AMIS was undetectable
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A RT I C L E S
Knockdown
Control
Rab8a
a
Rab8b
Rab11a
c
Rab25
e
d
PCX/β-catenin/nuclei
b
1.4
AMIS
*
1.0
*
*
*
0.8
0.6
***
0.4
***
***
***
0.2
0.0
Rab
KD/DN:
8a
KD
8b
KD
10
KD
11a
KD
11b
KD
13
DN
14
DN
Open Lumen
g
h
l
m
Par3/GFP–Rab11a/nuclei
1.2
Control
Single lumen formation (c.f. control)
f
25
KD
Early aggregate
Expanding lumen
j
k
GFP–Rab11a
PCX/nuclei
i
Figure 2 The Rab8 and Rab11 GTPase families direct lumen initiation.
(a–e) Representative confocal microscopy images of an MDCK cyst at
48 h incubated with antibodies against the indicated proteins on stable
expression of shRNA to knockdown Rab8a (b), Rab8b (c), Rab11a (d) and
Rab25 (e). Control indicates stable expression of scrambled‑sequence
shRNA (a). Bottom: higher‑magnification images of the indicated regions,
showing podocalyxin localization (left), β‑catenin localization (middle),
and a merge of these two images (right). Arrowheads indicate vesicular
podocalyxin accumulation. (f) Proportion of single lumens in cysts with
either stable knockdown or overexpression of dominant‑negative alleles
(DN) of indicated Rab GTPases at 48 h, normalized with respect to control
cells. Line represents 0.75‑fold single lumenogenesis. Values represent the
mean ± s.d. of three or more experiments. Asterisk indicates P < 0.05 and
triple asterisks indicate P < 0.0001. Control, n = 868; Rab8a, n = 302;
Rab8b, n = 312; Rab10, n = 336; Rab11a, n = 307; Rab11b, n = 1,528;
Rab13, n = 315; Rab14, n = 331; Rab25, n = 310. (g, h) Representative
images of a cyst expressing GFP–Rab11a (green) and incubated with
Hoescht (blue) and with antibodies against Par3 (red) during lumen
initiation. Bottom: higher‑magnification images of the indicated regions
showing GFP–Rab11a (left), Par3 (middle) and a merge of these two
images (right). Arrow (g) and arrowheads (h) indicate localization of Par3.
(i–m) Experiments performed as in g and h except cells were incubated
with antibodies against podocalyxin (red). In i; arrow indicates podocalyxin,
and yellow and white arrowheads indicate localization of GFP–Rab11a
to peripheral and juxtanuclear vesicles, respectively. In j, k; arrowhead
indicates co‑localization of podocalyxin and GFP–Rab11a. In k–m; arrows
indicate areas devoid of GFP–Rab11a, and arrowheads indicate clustering
of GFP–Rab11a underneath the lumen. Scale bars, 20 μm.
(Fig. 4f; arrowhead). In cysts with endogenous Sec15A knockdown, expression of RNAi-resistant GFP–Sec15AWT, which localized to subapical vesicles
(Fig. 4g), rescued single lumenogenesis, surface delivery of podocalyxin and
Par3 localization (that is, at tight junctions once lumens had formed).
To test the role of exocyst coupling to Rabs, we used GFP–Sec15AN691A,
a mutant that does not bind to Rab11a15. This mutant was completely
unable to rescue the trafficking and single lumenogenesis defects caused
by knockdown of endogenous Sec15A (Fig. 4h). Thus, coupling of exocyst to Rab8a or Rab11a is required for surface targeting of podocalyxin
and Par3 to the AMIS.
Similar to exocyst knockdown, Par3 knockdown also resulted in intracellular podocalyxin accumulation close to the surface marked by β-catenin
(Fig. 4i; arrows), in vesicles co-labelled for GFP–Rab11a (Fig. 4j; arrows),
and a strong disruption of single lumenogenesis (Fig. 4l). Par3 knockdown
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A RT I C L E S
b
Knockdown
Rabin8_5
GFP–Rab11aS25N
Rabin8_4
GFP–Rab11aWT
Control
MDCK
Rabin8/nuclei
a
_
`
Rabin8
GAPDH
e
1.5
*
+
+
–
–
+
+
A
–
GFP–hRabin8_:
**
01
0.0
Rabin8_ knockdown:
**
A
0.5
L1
F201A
1.0
WT
Single lumen formation (c.f. control)
Rabin8_ KD
WT
F2
GFP–hRabin8_/PCX/F-actin
96
d
PCX/nuclei
Rab8a/nuclei
c
Figure 3 A Rab11–Rabin8–Rab8 complex governs apical transport and single
lumenogenesis. (a) Rab11a recruits Rabin8 and Rab8a to subapical vesicles.
Control MDCK cysts and cysts expressing GFP–Rab11a (wild‑type Rab11a;
WT or an S25N substitution mutant; both green) were grown for 48 h and
immunostained for either endogenous Rabin8 or Rab8a (both in red). There is
diffuse vesicular labelling, with low‑level subapical accumulation of Rabin8 and
Rab8a (arrows) in control cysts. Arrowheads indicate marked co‑recruitment
and clustering of Rabin8 and Rab8a vesicles to the subapical region, which
was absent in the GFP–Rab11aS25N mutant. (b) Knockdown of Rabin8 by two
different shRNA were assessed by western blot analysis. Control cells stably
expressed a scrambled‑sequence shRNA. GAPDH was used as a loading control.
(c) Representative confocal microscopy image of an MDCK cyst at 48 h, stably
expressing shRNA to knockdown Rabin8α, and incubated with anti‑podocalyxin
antibodies, and Hoescht. Arrowheads indicate intracellular accumulation
of podocalyxin. (d) Representative confocal images of MDCK cysts, stably
expressing shRNA to knockdown Rabin8α and expressing either RNAi‑resistant
GFP–hRabin8α or a GFP– hRabin8αF201A mutant, were incubated with antibodies
against podocalyxin and F‑actin. Arrows indicate luminal GFP–hRabin8αWT
localization, and arrowheads indicate co‑localization of GFP– hRabin8αF201A
and podocalyxin on vesicles. (e) Proportion of cysts with a single lumen in
MDCK cysts stably expressing shRNA to knockdown Rabin8α, and expressing
GFP–Rabin8αWT, GFP–Rabin8αL196A, or GFP–Rabin8αF201A, as indicated. Values
represent the means ± s.d. of three or more experiments. Asterisk indicates
P < 0.05, double asterisk indicates P < 0.001. Control, n = 312; Rabin8α
KD, n = 340; Rabin8α KD + GFP–hRabin8αWT, n = 316; Rabin8α KD + GFP–
hRabin8αL196A, n = 317; Rabin8α KD + GFP–hRabin8αF201A, n = 328. At the
bottom of a and c and right of d are higher‑magnification images of the indicated
regions showing localization of individual proteins (left, middle) and a merge
of these two images (right). Scale bars, 20 μm. Uncropped images of blots are
shown in Supplementary Information, Fig. S7a.
also mistargeted some GFP–CNT1 to cell–cell contacts (Supplementary
Information, Fig. S3e). Moreover, on Par3 knockdown, Sec8 was not
recruited to surface regions adjacent to vesicular podocalyxin (Fig. 4k),
representing a failure to form the AMIS.
Inhibition of aPKC, using its pseudosubstrate inhibitor (aPKCPS; Fig. 4m, n) similarly perturbed AMIS and single lumenogenesis,
causing accumulation of podocalyxin in Rab11a-positive vesicles, close
to the surface marked by β-catenin (Fig. 4n; arrowheads). Additionally,
aPKC inhibition caused lack of podocalyxin internalization from the
periphery in some cells (Fig. 4n; arrows), probably representing an
additional function of aPKC at this locale (see Fig. 1i). Together, these
data demonstrate a crucial role for the exocyst–Par3–aPKC complex
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A RT I C L E S
d
1.5
1.0
0.5
g
f
Sec15A knockdown
GFP–Sec15A
5
2
A_
GFP–Sec15AN691A
h
Par3 /PCX/nuclei
e
Control
*
*
Co
0.0
KD:
A_
GFP–Rab11a
Se
c1
5
β-cat c
ntr
ol
β-cat b
Se
c1
5
Sec15A knockdown
Single lumen formation
(c.f. control)
Control
PCX/nuclei
a
1.5
1.0
0.5
Control
4
3
r3_
Pa
r3_
*
aPKC-PS
n
Apple–Rab11a
PCX/β-catenin
m
Pa
Co
0.0
KD:
*
ol
Sec8
ntr
GFP–Rab11a k
PCX/nuclei
β-cat j
Single lumen formation
(c.f. control)
l
Par3 knockdown
i
Figure 4 The exocyst and Par3–aPKC regulate lumenogenesis. (a–c) Sec15A is
required for AMIS formation. Representative images of a cyst at 48 h treated
with Hoescht and with antibodies against podocalyxin, and β‑catenin (a, b) or
expressing GFP–Rab11a (c). Cells were stably expressing either scrambled‑
sequence shRNA (control) or shRNA specific to Sec15A. Bottom: higher‑
magnification images of the indicated regions showing localization of individual
proteins (left, middle) and a merge of these two images (right). Arrows (b)
indicate localization of podocalyxin and arrowheads (c) indicate podocalyxin in
vesicles labelled with GFP–Rab11a. (d) Proportion of cysts with a single lumen
on stable expression of a control scrambled sequence shRNA or two different
types of Sec15A shRNA, as indicated. Values represent the mean ± s.d. of three
or more different experiments. Asterisk indicates P < 0.001. Control, n = 334;
Sec15A_2, n = 355; Sec15A_5, n = 345. (e–h) Representative images of cysts
at 48 h treated with Hoescht and antibodies against podocalyxin and Par3.
Cells were stably expressing either scrambled sequence shRNA (control; e) or
shRNA specific to Sec15A. In g, h the cysts are expressing an RNAi‑resistant
GFP–Sec15A or a GFP–Sec15AN691A Rab‑uncoupled mutant. Par3 targeting to
1040
the AMIS requires the exocyst. (e–h) Arrow indicates localization of Par3. (f,
h) Arrowhead indicates podocalyxin vesicle coalescence. (i–k) The Par3–aPKC
complex is required for lumen initiation. MDCK cells stably expressing shRNA
specific to Par3 were imaged 48 h after plating. Arrows indicate podocalyxin
localization. (l) Proportion of cysts with single lumens after knockdown of
Par3 using two different shRNA, as indicated. Scrambled‑sequence shRNA
was used as a control. Values represent the mean ± s.d. of three or more
experiments. Asterisk indicates P <0.001. Control, n = 326; Par3_3, n = 348;
Par3_4, n = 369. (m) Images of control cysts expressing Apple–Rab11a, and
immunostained for podocalyxin and β‑catenin. (n) Images of cysts expressing
Apple–Rab11a, treated with aPKC‑pseudosubstrate inhibitor (aPKC‑PS) and
immunostained for podocalyxin and β‑catenin. In n, arrowheads indicate
podocalyxin accumulation in Apple–Rab11a vesicles (beneath the surface
marked by β‑catenin) and arrows indicate podocalyxin localization at the
periphery of cells. At the bottom of a–c, e–k, and to the right of m and n are
higher‑magnification images of the indicated regions showing localization of
individual proteins and a merge of these images. Scale bars, 20 μm.
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A RT I C L E S
in podocalyxin delivery from Rab11a-positive vesicles to form the
lumen.
Annexin2–Cdc42 associates with Rab11a-positive vesicles
during lumenogenesis
Luminal targeting of aPKC in MDCK cysts requires interaction of
GTP–Cdc42 with the phosphatidylinositol (4,5)-bisphosphate-binding
protein, Annexin2 (Anx2)6. Anx2 both transits to the surface through, and
regulates the function of, Rab11a recycling vesicles18,19. We thus examined
the interaction between Anx2, Cdc42 and the Rab11a–Rab8a module.
In early cysts with peripheral podocalyxin (arrow), and subperipheral Apple–Rab11a, GFP–Anx2 localized to the surface (Supplementary
Information, Fig. S6a). When podocalyxin was in condensed Rab11apositive vesicles beneath the AMIS, some GFP–Anx2 now also localized
to these vesicles (Supplementary Information, Fig. S6b; arrowheads). Once
podocalyxin was at the open lumen (Supplementary Information, Fig. S6c;
arrow), GFP–Anx2 localized to both apical and basolateral surfaces, but no
longer to subapical Rab11a-positive vesicles. Thus, GFP–Anx2 transiently
associates with Rab11a-positive vesicles during lumen initiation.
In contrast to controls expressing wild-type Anx2, which had luminal
podocalyxin, subapical Apple–Rab11a, and apical and basolateral GFP–
Anx2 (Supplementary Information, Fig. S6d, f), expression of dominant
negative Anx2 (Anx2 XM) perturbed lumenogenesis and caused the
accumulation of GFP–Anx2 and podocalyxin in Rab11a-positive vesicles (Supplementary Information, Fig. S6e; arrowheads). Conversely,
knockdown of Rab8a or Rab11a caused intracellular accumulation of
podocalyxin in structures co-labelled with GFP–Anx2 (Supplementary
Information, Fig. S6g, h; arrowheads). Thus, Anx2 and Rab8a–Rab11a
cooperate in the delivery of podocalyxin to the apical surface.
We next examined whether Cdc42 associated with Rab11a-positive
vesicles. Unlike Anx2, GFP–Cdc42, although possessing a large cytoplasmic pool, co-localized with subapical Apple–Rab11a in cysts with
open lumens (Fig. 5a; arrowheads). Activated Cdc42 (GFP–Cdc42Q61L)
localized to cell–cell contacts and the luminal region, marked by podocalyxin (Fig. 5b; arrowheads). As GFP–Cdc42Q61L removed cytoplasmic
background labelling, and expression did not perturb single lumenogenesis (Fig. 6i), we used this allele to further examine Cdc42 localization. In
early cysts with peripheral podocalyxin (arrows), GFP–Cdc42Q61L localized to the surface (Fig. 5c). When podocalyxin was internalized into
Rab11a-positive vesicles and subsequently concentrated at the AMIS,
GFP–Cdc42Q61L now extensively overlapped with these vesicles (Fig. 5d,
e; arrowheads). As the PAP (Fig. 5f) and open lumen (Fig. 5g) formed,
podocalyxin and Rab11a were no longer co-localized, whereas GFP–
Cdc42Q61L maintained some overlap with both (arrows). Thus, active
Cdc42 associates with Rab11a-positive vesicles during lumenogenesis.
Tuba–Cdc42 function in apical transport from Rab8a/Rab11apositive vesicles
We next determined whether Cdc42 is required for transport from
Rab11a-positive vesicles. As shown previously 6, Cdc42 knockdown
perturbed lumenogenesis (Fig. 5n), and resulted in accumulation
of podocalyxin in vesicular apical compartments (VACS; arrows)
or vesicles (arrowheads) close to the surface marked by β-catenin
(Fig. 5i). Notably, on Cdc42 knockdown, intracellular podocalyxin
was localized to Rab8a/Rab11a-positive vesicles, suggesting Cdc42
regulates transport from these vesicles (Fig. 5l; arrowheads).
Intersectin 2 and Tuba have been identified as the only Cdc42-specific
GEFs essential for MDCK lumenogenesis20,21. As intersectin-2 knockdown did not disrupt transport of podocalyxin in cysts20, we examined
if Tuba regulates Cdc42-dependent podocalyxin transport. Tuba knockdown phenocopied Cdc42 knockdown, disrupted single lumenogenesis
and accumulated podocalyxin in Rab8a/Rab11a-positive vesicles (Fig. 5j,
m, n; arrowheads). Knockdown of Tuba or Cdc42 blocked the increase
in single lumenogenesis induced by expression of GFP–Rab11a (Cdc42
had a larger effect; Fig. 5n), suggesting that Rab11a operates upstream
of both Tuba and Cdc42. Thus, Tuba-dependent Cdc42 activation is
required for podocalyxin apical transport.
Tuba is required for Cdc42 apical targeting 21. We examined whether
Rab8a or Rab11a also influenced Cdc42 activation. Knockdown of
Rab8a, but not Rab11a, markedly decreased global GTP–Cdc42 levels
(Fig. 6a). Similarly, overexpression of GFP–Rab8aQ67L, but not GFP–
Rab11aQ70L, activated Cdc42 (Fig. 6b), suggesting that Rab8a influences
global Cdc42 activation.
We examined whether Rab8a and Rab11a regulate apical Cdc42 targeting. A YFP-tagged p21-binding domain (PBD–YFP) probe of activated Cdc42 (ref. 6) labelled the luminal surface, along with podocalyxin
(Fig. 6c; arrowheads), and to a lesser extent cell–cell contacts, mirroring
the localization of activated Cdc42 (Fig. 5b). Rab8a knockdown abrogated PBD–YFP membrane association, despite retaining luminal podocalyxin labelling (Fig. 6d; arrowhead). Strikingly, Rab11a knockdown
resulted in a loss of apical (arrowhead), but not basolateral, PBD–YFP
(arrows) (Fig. 6e). Thus, Rab8a is required for global activation and
surface targeting of Cdc42, whereas Rab11a controls apical Rab8a, and
consequently, active Cdc42 targeting.
We reasoned that as Rab8a and Rab11a influence apical targeting of
active Cdc42, overexpression of active Cdc42 may rescue single lumenogenesis on knockdown of Rab8a or Rab11a. Indeed, expression of active
Cdc42 (GFP–Cdc42Q61L) rescued apical targeting of podocalyxin (Fig. 6g,
h), and single lumenogenesis, in cysts with Rab8a or Rab11a knockdown
(Fig. 6i). These data support the conclusion that Cdc42, regulated by Rab8a
and Rab11a, is required for apical transport of podocalyxin. Therefore,
Rab11a regulates a molecular network directing the apical polarity and
trafficking machineries to initiate de novo lumen formation.
DISCUSSION
How membrane trafficking and polarity-complex machineries work
together to form the apical surface and lumen is a fundamental issue22.
We describe a molecular chain linking membrane-trafficking machinery with delivery of the Par3–aPKC–Cdc42 complex to the nascent
apical surface during lumen formation (Fig. 7a). This emphasizes
the complex spatiotemporal orchestration needed to construct a new
membrane (see Table 1).
Podocalyxin is initially localized to the extracellular matrix-contacting
periphery (Fig. 7a), before internalization into Rab11a-positive vesicles,
to which Rab8a is recruited through the GEF Rabin8 (which is inhibited
by the GAP, TBC1D30; Fig. 7b). Apical vesicle delivery and lumenogenesis is regulated by both Rab proteins. The exocyst, a Rab effector, docks
vesicles with the apical surface to create the AMIS, in cooperation with
Par3–aPKC (Fig. 7b). Anx2 and Cdc42 associate with Rab8a/Rab11apositive vesicles, regulating apical transport and single lumenogenesis,
dependent on the Cdc42 GEF, Tuba. Par6 probably bridges Cdc42 to
the aPKC–Par3–exocyst complex at the AMIS. Thus, apical polarity and
nature cell biology VOLUME 12 | NUMBER 11 | NOVEMBER 2010
© 2010 Macmillan Publishers Limited. All rights reserved
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A RT I C L E S
Apple–Rab11a
GFP–Cdc42/nuc
a
GFP–Cdc42Q61L
PCX/nuc
Peripheral
c
PAP
g
Lumen
PCX/GFP–Cdc42Q61L/Rab11a
f
Vesicular
AMIS
e
d
PCX/ GFP–Cdc42Q61L/Rab11a
b
Control
Cdc42 KD
Tuba KD
i
j
PCX/β-cat/nuclei
h
Control
k
Cdc42 knockdown
l
m
Tuba knockdown
n
1.4
MDCK
GFP–Rab11a
PCX/GFP–Rab11a/Rab8a
Single lumen formation
(c.f. control)
1.2
1.0
**
0.8
***
0.6
0.4
***
***
***
***
0.2
Figure 5 Tuba and Cdc42 regulate transport from Rab8a/Rab11a‑positive
vesicles. (a–b) Representative images of 48 h cysts either co‑expressing
Apple–Rab11a and GFP–Cdc42, and stained for nuclei (a), or expressing
GFP–Cdc42Q61L and labelled for podocalyxin and nuclei (b). Arrowheads
indicate co‑localization of Cdc42 and Rab11a for a, or localization of
GFP–Cdc42Q61L and podocalyxin at the luminal surface for b. (c–g) Images
of cysts expressing GFP–Cdc42Q61L at indicated stages of development. (c)
Arrows indicate peripheral localization of podocalyxin. (d, e) Arrowheads
indicate co‑localization of GFP–Cdc42Q61L and podocalyxin in Rab11a‑positive
vesicles. (f, g) Arrows indicate localization of GFP–Cdc42Q61L and podocalyxin
at the luminal surface. (h–j) Images of cysts at 48 h stably expressing shRNA
specific to Cdc42 or Tuba. Cells were treated with Hoescht and with antibodies
specific to podocalyxin and β‑catenin. (i, j) Arrow indicates accumulation
of podocalyxin in VACS; arrowheads indicate podocalyxin localization in
1042
Co
n
Cd trol
c4
2_
2
Cd
c4
2_
3
Tu
ba
_1
Tu
ba
_2
Co
ntr
o
Cd
c4 l
2_
2
Tu
ba
_1
0.0
KD:
vesicles. (k–m) Images of cysts at 48 h stably expressing GFP–Rab11a and
shRNA specific to Cdc42 and Tuba, and treated with antibodies specific to
podocalyxin and Rab8a. (k) Arrowheads indicate subapical GFP–Rab11a and
Rab8a localization. (l, m) Arrowheads indicate co‑localization of podocalyxin,
GFP–Rab11a and Rab8a in vesicles.Bottom (a–m): higher‑magnification
images of the indicated regions showing localization of individual proteins
and a merge of these images. Scale bars, 20 μm. (n) Proportion of MDCK
cysts, and cysts expressing GFP–Rab11a, with a single lumen at 48 h after
plating on stable expression of either shRNA specific to Cdc42 or Tuba, or a
control scrambled‑sequence shRNA. Values represent the mean ± s.d. of three
or more experiments. Double asterisks indicate P < 0.001, triple asterisks
indicate P < 0.0001. Control, n = 311; Cdc42_2, n = 321; Cdc42_3,
n = 307; Tuba_1, n = 319; Tuba_2, n = 319; GFP–Rab11a control, n = 600;
GFP–Rab11a + Cdc42_2, n = 316; GFP–Rab11a + Tuba_1, n = 308.
nature cell biology VOLUME 12 | NUMBER 11 | NOVEMBER 2010
© 2010 Macmillan Publishers Limited. All rights reserved
Control
Rab8a knockdown
d
4
3
2
0
Total
Rab11a knockdown
GFP–Rab
e
i
1.2
MDCK
GFP–Cdc42Q61L
*
Single lumen formation
(c.f. control)
1.0
g
*
0.8
0.6
0.4
0.2
0.0
h
KD:
Control
Rab8a
Rab11a
GFP–Cdc42Q61L
PCX/nuclei
f
11aQ70L
1
8aQ67L
11aQ70L
GTP
*
5
MDCK
1a
a
0
KD:
Anti-Cdc42
20
PCX/PBD–YFP/nuclei
c
GFP
6
GTP–Cdc42 levels
(c.f. control)
*
40
8aQ67L
MDCK
60
Ra
b1
Total
GFP–Rab
80
Ra
b8
Rab11a
Rab8a
GTP
b
100
Co
Anti-Cdc42
Control
Knockdown
ntr
ol
a
GTP-Cdc42 levels
c.f. control (percentage)
A RT I C L E S
Figure 6 Rab8a/Rab11a regulate Cdc42 during apical transport. (a, b)
Rab8a–Rab11a control Cdc42 activation and targeting. GTP–Cdc42 levels
were assessed in MDCK cells stably expressing shRNA specific to Rab8a
or Rab11a (a), or MDCK cells expressing activated Rab8a and Rab11a
mutants (b), by pulldown and western blotting. Cell lysate was used to
determine total Cdc42 levels. In a, graph represents GTP–Cdc42 levels
normalized to total Cdc42 expression, and presented as a percentage of
control cell levels of GTP–Cdc42. In b, these values are presented as a
fold‑change, compared with control GTP–Cdc42 levels. Values represent
the mean ± s.d. of three different experiments. Asterisk indicates
P < 0.05. (c–e) Localization of a PBD–YFP probe to detect activated
Cdc42 (and possibly also Rac) in MDCK cysts 48 h after plating. (c)
Arrowheads indicate podocalyxin and PBD–YFP co‑localization. (d)
Arrowheads indicate podocalyxin localization (which is not associated
with PBD–YFP when there is knockdown of Rab8a). (e) Arrowhead
indicates loss of apical PBD–YFP and arrow indicates localization of
PBD–YFP to the basolateral membrane. (f–h) Rescue of Rab8a/Rab11a
knockdown by active Cdc42 overexpression in MDCK cysts at 48 h after
plating. Arrowheads indicate apical targeting of GFP–Cdc42 Q61L and
podocalyxin. Bottom (c–h): higher‑magnification images of the indicated
regions showing localization of individual proteins (left, middle) and a
merge of these two images (right). Scale bar, 20 μm. (i) Effect of Rab8a
and Rab11a knockdown on proportion of cysts with single lumens at
48 h after plating. Values represent means ± s.d. Asterisk indicates
P < 0.05. Control, n = 868; Rab8a KD, n = 302; Rab11a KD, n = 307;
GFP–Cdc42Q61L Control, n = 343; GFP–Cdc42Q61L + Rab8a KD, n = 319;
GFP–Cdc42Q61L + Rab11a KD, n = 308. Uncropped images of blots are
shown in Supplementary Information, Fig. S7a.
membrane domain identity is initiated de novo by membrane delivery from Rab8a/Rab11a-positive vesicles. Finally, the luminal space is
expanded by pumps and channels (Fig. 7b)7,23.
Podocalyxin is the earliest marker of apical polarization that we have
studied, and its initial appearance at the nascent apical surface marks the
AMIS. Notably, the Rab11a–Rabin8α–Rab8a cascade also regulates mammalian ciliogenesis, and the homologous yeast pathway regulates budding 24, suggesting it is an ancient polarity-generating module.
The exocyst is a Rab effector required for transport of podocalyxin from
Rab11a endosomes. This transport also requires the Par3–aPKC polarity complex, revealing a mutual interdependence of these complexes for
localization to the AMIS. Similarly, in developing neurites, association of
the exocyst and Par3–aPKC is required for Par3 localization to growing
neurite tips17. Interaction of the exocyst with Par3–aPKC may focus exocystdependent vesicle docking events transiently to the AMIS, allowing initiation of apical polarity, before both complexes relocalize to the tight junction
for subsequent transport events. That the exocyst was also required for Par3
localization demonstrates that vesicle trafficking operates both upstream and
downstream of cortical polarity proteins, suggesting a feedback loop.
Rab11a-positive vesicles also deliver the Crumbs3–Pals1–PatJ complex to early lumens25. In Drosophila, Crumbs functions to dissociate
Par3 from Par6 and aPKC at the apical surface, restricting Par3 to apicolateral borders26,27. Similarly, Crumbs3 delivered to the AMIS may
exclude a pool of Par3 from the nascent apical surface, allowing it to
concentrate to the sides of the developing lumens, such as in the transition from the AMIS to the PAP reported here.
nature cell biology VOLUME 12 | NUMBER 11 | NOVEMBER 2010
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1043
A RT I C L E S
a
PCX
aPKC
Early aggregate
Apical membrane
initiation
Par3
Sec8
Pre-apical Patch
Open lumen
PCX
PCX
Sec10
L
aPKC
Par3
Sec8
Sec10
b
Anx2
Cdc42
Par
6
Tuba
aPKC
TBC1D30
Rab8a
Par3
AMIS
Apical vesicle
(podocalyxin)
Sec8
Sec10
Rabin8α
Rab11a
Sec15A
Figure 7 A molecular network for de novo lumen generation. (a) Schematic
representation of the different stages of lumenogenesis and apical
polarization in MDCK cysts. Initially, podocalyxin is localized to the periphery
of cysts (early aggregate), before internalization into Rab8a and Rab11a‑
positive vesicles, and delivery to the AMIS (apical membrane initiation).
As podocalyxin at the apical domain and tight junctions become separately
localized, the AMIS progresses to a PAP, representing the early stages of
apical–basal polarization. Expansion then allows opening of the luminal
space. Note the co‑accumulation of polarization and trafficking machinery at
the AMIS, despite varying localization during other stages of lumenogenesis.
Red lines, podocalyxin; black lines, plasma membrane; grey ovals, nuclei;
brown ovals, tight junctions; brown rectangle, AMIS; L, lumen. (b) A
schematic representation of the molecular network involved in delivery of
apical vesicles (podocalyxin) to the AMIS during apical membrane initiation.
Par6 and aPKC form a complex with Cdc42 (ref. 28). Cdc42, in conjunction with Anx2 (ref. 6), regulates vesicular transport to the apical
surface from Rab8a/Rab11a-positive vesicles. However, Cdc42 also regulates the orientation of cell division in conjunction with Par6–aPKC20,21,29.
Indeed, the Cdc42 GEFs, intersectin 2 and Tuba, regulate Cdc42 activation and orientation of division during MDCK lumenogenesis20,21.
Intersectin-2 knockdown results in multiple lumens without vesicular
podocalyxin accumulation20, suggesting it functions in cell division
but not vesicle transport. In contrast, Tuba regulates apical targeting of
Cdc42 (ref. 21) and, as demonstrated here, podocalyxin transport from
Rab8a/Rab11a-positive vesicles.
Active Cdc42 localized to Rab11a-positive vesicles. In yeast, Cdc42 is
also delivered to the bud site30, suggesting that vesicular transport of Cdc42
to membrane being generated de novo is a conserved polarity-generating
event. Notably, Rab8a and Rab11a were required for Cdc42 activation
at the lumen. Rab8a regulated global Cdc42 activation; Rab11a regulated apical Cdc42 targeting. That global Cdc42 activation was markedly
decreased on Rab8a, but not Rab11a, knockdown suggests that, whereas
Tuba functions downstream of Rab11a and regulates a pool of Cdc42
activation in Rab11a-positive vesicles, Rab8a may influence additional
GEF proteins, such as intersectin 2. How Rab8a and Rab11a influence
Cdc42 GEFs remains to be elucidated. Similarly, we demonstrate a role
for aPKC in apical transport from Rab11a-positive vesicles, suggesting
that the Cdc42–Par6–aPKC complex may function in membrane transport, in addition to cell division31,32. These data reveal a novel role for
Anx2–Cdc42–aPKC–Par3 in conjunction with the exocyst and Tuba in
apical transport to the AMIS.
Our knockdown and overexpression experiments resulted in multiple lumens and accumulation of apical proteins in subapical vesicles.
Perturbation of apical traffic could cause multiple lumen formation
through several overlapping mechanisms. Reduced apical delivery may
prevent initially small lumens from enlarging and consolidating into one
central lumen, which occurs in several mammalian organs33. Multiple small
lumens also require ion pumping to hydrostatically enlarge and coalesce
lumens. Apical trafficking defects could cause defective junctions and/or
mislocalization of pumps and channels, preventing enlargement23.
Orientation of mitosis also regulates lumen formation. In Caco-2 cysts,
Cdc42 knockdown correlates with disrupted spindle orientation, without
notable apical transport defects29. Caco-2 lumen formation requires artificially increasing cyclic AMP, which strongly promotes apical exocytosis34, potentially masking detection of apical transport defects. As Rab8a,
Rab11a, exocyst and Par3–aPKC also have roles in spindle orientation
and/or cytokinesis35–37, the extent to which their function in cell division is separate from apical trafficking is unknown. Apical membrane
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© 2010 Macmillan Publishers Limited. All rights reserved
A RT I C L E S
traffic might be needed to localize proteins that orient mitosis, such as
Cdc42, aPKC and LGN (leucine-glycine-asparagine repeat-containing
protein)20,21,38,39. Notably, multiple lumens can occur without disruption
to apical transport 20,39, suggesting that the two processes can be uncoupled. For example, in MDCK and other systems, lumens can form in
the absence of cell division or apoptosis7,40–43, instead requiring vesicular
transport of podocalyxin to the cyst interior. Thus, coordination of division orientation and apical transport mechanisms are probably central
in the generation of a single lumen.
Recently, the Par proteins, as well as aPKC and Cdc42, have been demonstrated as regulators of polarity through endocytosis32. We demonstrate that the membrane traffic, especially exocytosis, is both upstream
and downstream of the Par complex. Our data support an emerging view
of the Par complex as a multifunctional platform modulating membrane
traffic31,32, and suggest Cdc42–aPKC–Par3 as a convergence between the
machineries of cortical polarization and vesicular transport.
METHODS
Methods and any associated references are available in the online version
of the paper at http://www.nature.com/naturecellbiology/
Note: Supplementary Information is available on the Nature Cell Biology website
ACKNOWLEDGEMENTS
We thank F. Barr, E. Brown, J. Stow, W. Guo, I. Macara, K. Simons, J. Wilson, T.
Weimbs, and A. Zahraoui for gifts of reagents and unpublished data, and the
Mostov lab for kind assistance. Supported by a Susan G Komen Foundation
Fellowship (D.M.B.), a DOD Breast Cancer Concept Award (A.D.), NIH grants
R01DK074398, R01AI25144 and P01AI53194 (K.E.M.), grants of the Human
Frontiers Science Program (HFSP-CDA00011/2009), Marie Curie (IRG-209382),
MICINN (BFU2008-01916) and (CONSOLIDER CSD2009-00016) to F.M.-B.; and
a JAE fellowship (MICINN) to A.E.R.F.
AUTHOR CONTRIBUTIONS
D.M.B., A.D., A.E.R.F, F.M.-B. and J.P. designed the experiments. D.M.B., A.D.,
A.E.R.F. and J.P. did the experimental work. D.M.B. and K.E.M. analysed the
experiments. D.M.B. and K.E.M wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturecellbiology
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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tion the apical surface during epithelial morphogenesis. J. Cell. Biol. 183, 625–633
(2008).
30. Harris, K. P. & Tepass, U. Cdc42 and vesicle trafficking in polarized cells. Traffic 11,
1272–1279 (2010).
31. Balklava, Z., Pant, S., Fares, H. & Grant, B. D. Genome‑wide analysis identifies a gen‑
eral requirement for polarity proteins in endocytic traffic. Nat. Cell Biol. 9, 1066–1073
(2007).
32. Shivas, J. M., Morrison, H. A., Bilder, D. & Skop, A. R. Polarity and endocytosis: recipro‑
cal regulation. Trends Cell Biol. 20, 445–452 (2010).
33. Hogan, B. L. & Kolodziej, P. A. Organogenesis: molecular mechanisms of tubulogenesis.
Nat. Rev. Genet. 3, 513–523 (2002).
34. Brignoni, M. et al. Exocytosis of vacuolar apical compartment (VAC) in Madin‑Darby
canine kidney epithelial cells: cAMP is involved as second messenger. Exp. Cell. Res.
205, 171–178 (1993).
35. Matheson, J., Yu, X., Fielding, A. B. & Gould, G. W. Membrane traffic in cytokinesis.
Biochem. Soc. Trans. 33, 1290–1294 (2005).
36. Zhang, H., Squirrell, J. M. & White, J. G. RAB‑11 permissively regulates spindle align‑
ment by modulating metaphase microtubule dynamics in Caenorhabditis elegans early
embryos. Mol. Biol. Cell 19, 2553–2565 (2008).
37. Pohl, C. & Jentsch, S. Final stages of cytokinesis and midbody ring formation are
controlled by BRUCE. Cell 132, 832–845 (2008).
38. Horne‑Badovinac, S. et al. Positional cloning of heart and soul reveals multiple roles
for PKC lambda in zebrafish organogenesis. Curr. Biol. 11, 1492–1502 (2001).
39. Zheng, Z. et al. LGN regulates mitotic spindle orientation during epithelial morphogen‑
esis. J. Cell Biol. 189, 275–288 (2010).
40. Yu, W. et al. Formation of cysts by alveolar type II cells in three‑dimensional culture
reveals a novel mechanism for epithelial morphogenesis. Mol. Biol. Cell 18, 1693–
1700 (2007).
41. Liu, K. D. et al. Rac1 is required for reorientation of polarity and lumen formation
through a PI 3‑kinase‑dependent pathway. Am. J. Physiol. Renal Physiol. 293,
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42. Tanimizu, N., Miyajima, A. & Mostov, K. E. Liver progenitor cells fold up a cell mon‑
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nature cell biology VOLUME 12 | NUMBER 11 | NOVEMBER 2010
© 2010 Macmillan Publishers Limited. All rights reserved
1045
METHODS
DOI: 10.1038/ncb2106
METHODS
Cyst culture. MDCK cysts were subcultured in 5% fetal bovine serum (FBS; Gibco),
minimum essential medium (MEM) or grown in 3D Matrigel cultures (BD), as
described6. Cells were trypsinized to a single cell suspension at 1.5 × 104 cells ml–1
in complete medium containing 2% Matrigel. Suspensions (250 μl) were plated
into 8-well coverglass chambers (Nunc), pre-coated with 5 μl of 100% Matrigel.
Cells were grown for 24–48 h before fixation in 4% paraformaldehyde (PFA). In
some experiments, cells were treated with aPKC-PS (40 μM; Invitrogen) from the
time of plating to inhibit aPKCs6. In most instances, exogenous proteins were from
stably expressing cell lines. For Anx2 XM experiments, cells in 2D were transiently
transfected with Anx2 XM (Lipofectamine 2000) 24 h before plating into 3D.
RNAi. Stable RNAi was achieved by viral shRNA. For Sec10 knockdown, cells were
transfected with siRNA oligonucleotides, as previously described6. In all instances,
knockdown was verified by western blot or quantitative real-time PCR (Q-PCR)
procedures, normalized to GAPDH expression (Brilliant-II SYBR Green Kit,
Agilent). Q-PCR primers are presented in Supplementary Information, Table S1.
RNAi target sequences are presented in Supplementary Information, Table S2.
Rab8a_1, Rab10, Rab11a_1 and Rab11b, in pRVH1-puro or –hygro retroviruses,
were previously published44. All other shRNAs were generated in pLKO.1-puro45,
or pLKO.1-blast, which was constructed by exchanging the puromycin resistance
gene for blasticidin. Cdc42, Par3 and Tuba shRNAs were adapted for pLKO.1 from
published sequences21,46. pLKO.1 lentiviruses were constructed according to the
Addgene pLKO.1 protocol (www.addgene.org) using iRNAi (www.mekentosj.
com), and target sequences were based on an (AA)N19 algorithm. RNAi sequences
were submitted to BLAST (NCBI) to verify target specificity. For isoform-specific
RNAi to Rabin8, shRNAs predicted to target the α isoform (Rabin8_4 or Rabin8_5)
or to both α and β (Rabin8_2) isoforms of canine Rabin8 were extrapolated from
sequence alignment with human Rabin8 splice forms (mined from NCBI). For
knockdown and rescue experiments, GFP–tagged plasmids of transcripts from
human or rat, which are not targeted by anti-canine shRNAs, were used.
Virus production and transduction. Retrovirus production was performed
essentially as previously described44, except pRVH1 plasmids were transfected
into 293-GPG cells (O. Weiner, UCSF)47. Post transfection (48 h), viral supernatants were collected daily for 7 days. For lentivirus production, pLKO.1 plasmids
were co-transfected with ViraPower packaging mix into 293-FT cells according
to manufacturer’s instructions (Invitrogen). All supernatants were centrifuged to
remove cell debris and frozen in liquid nitrogen for further use.
For retrovirus transduction (pRVH1), subconfluent cultures of MDCK cells,
16 h after plating, were incubated with virus-containing supernatants supplemented with 10 μg ml–1 Polybrene (Millipore) for 24 h at 32 °C. On changing
to fresh medium, cells were incubated for a further 24 h at 37 °C, before passage into appropriate antibiotic-containing medium. For lentivirus transduction
(pLKO.1), subconfluent MDCK cultures, 1–4 h after plating, were infected with
virus-containing supernatants for 12–16 h at 37 °C. Viral supernatants were then
diluted 1:1 with growth medium, cultured for a further 48 h, then passaged into
appropriate antibiotic-containing medium. Hygromycin (0.5 mg ml–1), puromycin
(5 μg ml–1), and blasticidin (12.5 μg ml–1) were used.
Plasmids and cell lines. Plasmids, the genes cloned into them and their source,
and cell lines expressing the indicated genes and their source, were as follows: GFP–
Cdc42 (used also for expression of the Q61L mutant; Addgene); GFP–Rab11a (used
for expression of wild type and the S22N mutant; E. Brown, UCSF, USA); GFP–
Rab8a (used for expression of wild type and the Q67L mutant) and GFP–Sec15A (J.
Stow, University of Queensland, Australia); GFP–VSVG–podocalyxin and pRVH1puro/hygro (used for expression of Rab8a, Rab10, Rab11a and Rab11b; K. Simons,
EMBL, Germany); GFP–TBC1D30 (used for expression of wild type TBC1D30 and
the R140A mutant; F. Barr, University of Liverpool, UK); GFP–Cdc42, YFP–PBD,
GFP–Annexin2 (used for expression of wild type and XM)6 and GFP–Rab13 (used
for expression of the T22N mutant; A. Zharaoui, CEA CNRS, France); GFP–Rab14
(used for the expression of the S25N mutant; J. Wilson, University of Arizona,
USA); GFP–Rabin8α48 and Syntaxin3-2×myc (T. Weimbs, UCSD, USA)49 and
GFP–CNT150. All additional plasmids were constructed through site-directed
mutagenesis (Quikchange) or standard subcloning. For generation of stable lines,
transfected cells underwent fluorescence activated cell sorting (FACS) after selection to obtain appropriate expression levels.
Antibodies and immunolabelling. Primary mouse antibodies used were: antiCdc42 (1:1,000; BD Biosciences); anti-GAPDH (1:10,000; Millipore, Billerica,
MA); anti-gp135/podocalyxin (1:1,000; G. Ojakian, SUNY, USA); anti-myc
(1:200; 9E10, Santa Cruz Biotechnology); anti-p58 and anti-Na+/K+-ATPase
(1:200; K. Matlin); anti-Tuba (1:500; Abnova) and anti-VSVG (1:1,000; P5D4)51.
Primary rabbit antibodies used were: anti-aPKCζ (1:200; C-20) and antiβ-catenin (1:200; Santa Cruz Biotechnology); anti-GFP (1:1,000; Invitrogen);
anti-occludin (1:200; Invitrogen); anti-Par3 (Immunofluorescence, 1:100, western blot, 1:1,000; Millipore); anti-Rabin8 (immunofluorescence, 1:50; western
blot, 1:500; ProteinTech); anti-Rab8a (immunofluorescence, 1:50, western blot,
1:500)48; anti-Rab8b (1:500; ProteinTech); anti-Rab10 (1:500; Sigma); anti-Rab11a
(immunofluorescence, 1:100, western blot, 1:1,000; Millipore); anti-Rab25 (1:500;
Cell Signaling Technology); anti-Sec8 (1:100; Enzo Life Sciences); anti-Sec10
(immunofluorescence, 1:250 in methanol:acetic acid, western blot, 1:250; W. Guo,
University of Pennsylvania, USA). Rat anti-ZO-1 (1:200; R40.76, B. Stevenson)
was also used. Alexa fluorophore-conjugated secondary antibodies (1:250 for
all secondary antibodies) or Phalloidin (1:400; Invitrogen) and Hoescht to label
nuclei (10 μg ml–1), were utilized. Cysts were stained as previously described6 .
Statistics. Single lumen formation was quantified as previously described6. The
percentage of cysts with a single lumen was determined, and normalized to control
cysts. Values are mean ± s.d. from three replicate experiments, with n ≥100 cysts per
replicate. Significance was calculated using a paired, two-tailed Student’s t-test.
Transcytosis and IgA uptake. For podocalyxin transcytosis, cells stably expressing GFP–VSVG–podocalyxin were plated into 8-well chamber slides and grown
for 12 h at 37 °C. Slides were placed on ice and washed twice with serum-free
medium at 4 °C before incubation at 4 °C for 30 min with serum-free medium
containing the indicated antibodies. Cysts were then washed twice with cold
serum-free medium, before being grown for 24 h at 37 °C in fresh serum-containing medium supplemented with 2% Matrigel.
Polymeric immunoglobulin A (pIgA), provided by J. P. Vaerman (Catholic
University of Louvain, Belgium), was biotinylated using sulfo-NHS-LC-biotin
(Pierce). For IgA uptake, MDCK PTR-9 cells stably expressing human TfR and
rabbit polymeric immunoglobulin receptor (pIgR)52 were grown for 36 h in 3D
to induce cyst formation, then incubated with 100 μg ml–1 biotinylated IgA in
complete medium for 40 min. Biotinylated IgA was detected using fluorescently
labelled streptavidin (Invitrogen).
GTPase activation. GTP loading of Cdc42 was by GST-PAK pull down
(Cytoskeleton, Denver), according to manufacturer’s instructions, with modifications. Cells were lysed in Mg Lysis Buffer (Millipore) containing 1 mM PMSF
(phenylmethanesulfonylfluoride) and protease inhibitor cocktail, and lysates were
passed through a 27.5 gauge needle (BD), and cleared of debris by centrifugation.
A sample of lysate was taken for protein concentration determination (BCA),
and lysates were snap-frozen. Protein (3 μg) from the appropriate condition was
incubated with 20 μg of GST-PAK bead for 30 min at 4 °C. Beads were collected by
centrifugation and washed three times in buffer before SDS–polyacrylamide gel
electrophoresis (SDS–PAGE). Total Cdc42 and GTP–Cdc42 levels were detected
by western blotting using an anti-Cdc42 antibody (BD Biosciences).
Rabin8 mutagenesis. GEF mutants of Rabin8 were modelled on critical, conserved residues in the Rab GEF domain governing Sec2p–Sec4p interactions53.
L196A and F201A mutants were generated from pEGFP-C1-Rabin8α and pGEX2T-Rabin8α48 (Quikchange). Decreased, direct association of Rab8a with Rabin8
mutants was verified by GST pulldown. Briefly, GST–Rabin8, GST–Rabin8L196A,
GST–Rabin8F201A (15 °C overnight; 200 μM IPTG) or GST protein expression (37 °C
for 3 h) was induced in bacteria. Lysates containing GST fusion proteins were incubated with glutathione–agarose beads (Sigma) at 4 °C for 2 h, then washed three
times during 30 min with binding-buffer (50 mM Tris at pH 7.5, 150 mM NaCl,
2 mM MgCl2 and 1% Triton X-100). Rab8AT22N and Rab8AQ67L were translated
in vitro using a TNT Quick kit (Promega) according to manufacturer’s instructions. The in vitro translation products were incubated with GST proteins coupled
to glutathione–agarose beads in binding buffer (50 mM Tris at pH 7.5, 150 mM
NaCl, 2 mM MgCl2 and 1% Triton X-100) on a rotating wheel at 4 °C for 1 h. The
beads were washed four times with binding buffer over 30 min. Bound material
was eluted from the beads with Laemmli sample buffer and loaded onto a 12%
nature cell biology
© 2010 Macmillan Publishers Limited. All rights reserved
METHODS
DOI: 10.1038/ncb2106
SDS–PAGE gel. As a control, 1/10 of the in vitro translation reactions were used.
Bands were visualized by autoradiography of the dried gels. Densitometry revealed
that the L196A and F201A mutants decreased association with Rab8a by 39% and
52%, respectively, confirming their reduction-of-function characteristics.
44. Schuck, S., Manninen, A., Honsho, M., Fullekrug, J. & Simons, K. Generation of single and
double knockdowns in polarized epithelial cells by retrovirus‑mediated RNA interference.
Proc. Natl Acad. Sci. USA 101, 4912–4917 (2004).
45. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed
viral high‑content screen. Cell 124, 1283–1298 (2006).
46. Sfakianos, J. et al. Par3 functions in the biogenesis of the primary cilium in polarized
epithelial cells. J. Cell Biol. 179, 1133–1140 (2007).
47. Ory, D. S., Neugeboren, B. A. & Mulligan, R. C. A stable human‑derived packaging cell
line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc.
Natl Acad. Sci. USA 93, 11400–11406 (1996).
48. Hattula, K., Furuhjelm, J., Arffman, A. & Peranen, J. A Rab8‑specific GDP/GTP exchange
factor is involved in actin remodeling and polarized membrane transport. Mol. Biol. Cell
13, 3268–3280 (2002).
49. Kreitzer, G. et al. Three‑dimensional analysis of post‑Golgi carrier exocytosis in epithelial
cells. Nat. Cell Biol. 5, 126–136 (2003).
50. Mangravite, L. M., Lipschutz, J. H., Mostov, K. E. & Giacomini, K. M. Localization of
GFP‑tagged concentrative nucleoside transporters in a renal polarized epithelial cell line.
Am. J. Physiol. Renal. Physiol. 280, F879–F885 (2001).
51. Kreis, T. E. Microinjected antibodies against the cytoplasmic domain of vesicular sto‑
matitis virus glycoprotein block its transport to the cell surface. Embo J. 5, 931–941
(1986).
52. Brown, P. S. et al. Definition of distinct compartments in polarized Madin‑Darby canine
kidney (MDCK) cells for membrane‑volume sorting, polarized sorting and apical recycling.
Traffic 1, 124–140 (2000).
53. Sato, Y. et al. Asymmetric coiled‑coil structure with guanine nucleotide exchange activity.
Structure 15, 245–252 (2007).
nature cell biology
© 2010 Macmillan Publishers Limited. All rights reserved
s u p p l e m e n ta r y i n f o r m at i o n
DOI: 10.1038/ncb2106
GFP-PCX
Na/K-ATPase / nuc
Syntaxin-3 / nuclei
b
c
d
e
f
Open Lumen
Inverted
a
GFP-CNT1
PCX / nuclei
g
GFP-VSVG-PCX / nuc
α-VSVG
α-GFP
uptake
surface bind
α-myc
Rab11a / IgA / Nuclei
40 min IgA uptake
h
Figure S1 Localization of apical and basolateral proteins during cystogenesis.
(a-f) Early apical polarization is a feature of podocalyxin in cysts. MDCK
cysts stably expressing GFP-podocalyxin (a,d), Syntaxin-3-2xmyc (b,e),
or GFP-CNT1 (c,f) (all green) were co-stained for nuclei (nuc, blue), and
either Na/K-ATPase (red, a,d), or podocalyxin (red, c,f) 12 h after plating
(a-c) or once lumens had formed (48 h, d-f). Note that GFP-podocalyxin,
but not Syntaxin-3 or GFP-CNT1 are excluded from cell-cell contacts in
early aggregates. Smaller panels (c,f,
right) depict
higher magnification
Figure
S1 - Mostov
images of regions indicated. Arrowheads, GFP-CNT1 at cell-cell contacts.
(g) Podocalyxin transcytoses to the lumen. Binding of antibodies (α-myc,
α-VSVG, α-GFP; red) to cysts expressing GFP-VSVG-podocalyxin (green) at
4°C, followed by allowing lumen development to occur revealed transcytosis
of α-VSVG and α-GFP, but not non-specific α-myc antibodies, with
podocalyxin from the periphery to the PAP (arrowheads), and to intracellular
vesicles (arrows). Yellow, colocalization of podocalyxin and bound antibodies.
(h) Lumens are accessible to transcytosed IgA. MDCK cysts stably expressing
pIgR were grown for 36 h, and incubated with 100 µg/ml biotinylated
IgA (IgA) for 40 min to allow transcytosis. Cells were stained for Rab11a
(green), nuclei (blue), and IgA using fluorescent streptavidin (red). Note IgA
transcytosed to the PAP (arrow). Bar, 20 µm.
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1
© 2010 Macmillan Publishers Limited. All rights reserved.
s u p p l e m e n ta r y i n f o r m at i o n
e
b
f
β-cat
Sec10 / ZO-1
g
DIC
a
PCX
Sec10 / ZO-1
Control
PCX / Sec10 / nuc
1
Sec10 KD
2 3 pool
Sec10
Sec10 KD
1.5
1.0
*
0.5
0.0
(p
C
on
tro
l
i
0
oo K
l) D
Control
c1
h
Se
d
Sec10 / PCX / nuc
c
Single lumen formation
(c.f. control)
Actin
Figure S2 Sec10 analysis during lumen formation. (a-d) Sec10 labeling
condenses towards the AMIS and tight junctions. During lumenogenesis,
Sec10 (red) initially localizes along the cell-cell contact (a) when
podocalyxin (green) is peripheral, then condenses towards the AMIS,
overlapping with podocalyxin (b), before redistributing to sub-luminal
puncta (c,d). (e-f) Sec10 localizes to the tight junctions once lumens form.
72h MDCK cysts were labeled for Sec10 (red), ZO-1 (blue), and either
podocalyxin (e) or β-catenin (f) (both in green). Arrowheads, Sec10 and
ZO-1 at tight junction regions. (g-i) Knockdown of Sec10 disrupts single
lumenogenesis. Lysates of MDCK transfected with siRNAs against Sec10
2
alone (1, 2, 3), in combination (pool), or with control siRNAs were blotted for
Sec10 and actin as a loading control (g). In contrast to control cysts (h) with
luminal podocalyxin (red, arrowhead) and subapical Sec10 puncta (green),
Sec10 knockdown cysts lost Sec10 labeling, did not form a single lumen,
and showed vesicular accumulation of a pool of podocalyxin (arrows). Sec10
knockdown significantly reduced single lumenogenesis (i). Lumenogenesis
quantitation values represent the average of ≥ three different experiments ±
S.D., where *p <0.05 and n ≥ 100 cysts/replicate. Control, n = 445; Sec10
KD, n = 363. In a-f cysts were co-imaged for DIC to show cell outlines.
Smaller panels depict higher magnification of regions indicated. Bar, 20 µm.
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© 2010 Macmillan Publishers Limited. All rights reserved.
Rab11a KD
b
Rab8a KD
c
Rab25_4
Rab25_2
Control
KD
***
0.4
***
***
***
***
Rab10 KD
***
***
0.2
0.0
Rab
KD:
Rab25
**
0.6
_4
k
Rab11b
0.8
25
Rab11a
KD
_2
Control
11
a
KD
_3
Control
8b
0%
1.0
_1
*
20%
1.2
8b
*
40%
_2
60%
1.4
8a
80%
Control
8a
_1
% mRNA expression
c.f. control
l
100%
Control
GAPDH
GAPDH
_2
GAPDH
25
GAPDH
Rab10
Rab8b
_1
Rab11a
11
a
Rab11a
i
Rab8b_3
Rab8a
j
Par3 KD
e
KD
Rab8b_1
Rab8a
h
Control
g
Rab8a_2
Control
Rab8a/11a
Rab11a_1
Rab8a_1
Control
f
Sec15A KD
d
KD
KD
Rab11a_2
Control
a
Single lumen formation (c.f. control)
GFP-CNT1
Na/K-ATPase / nuc
s u p p l e m e n ta r y i n f o r m at i o n
GAPDH
*
*
40%
Par3
Cdc42
Tuba
Tuba_2
KD
Tuba_1
Cdc42_3
60%
Cdc42_2
80%
p
KD
Control
o
Control
Par3_4
Par3_3
n
100%
L
S
20%
GAPDH
_5
5A
c1
Se
c1
Sc
ra
m
5A
bl
e
_2
0%
KD:
Se
% mRNA expression
c.f. control
m
Control
KD
GAPDH
GAPDH
Figure
S3 - Mostov
Figure S3 Characterization
of MDCK
RNAi. (a-e) Apical exocytosis machinery
kncockdown disrupts lumen formation in cysts. MDCK GFP-CNT1 (green)
cysts stably expressing control (a), Rab11a (b; Rab11a_2), Rab8a (g;
Rab8a_2), Sec15A (d; Sec15A_2), or Par3 (e; Par3_4) shRNAs were grown
for 48 h and labeled for Na/K-ATPase (red) and nuclei (blue). Note apical
GFP-CNT1, and basolateral Na/K-ATPase, localization in control cysts, and
disruption to GFP-CNT1 localization, but not Na/K-ATPase, localization in
apical exocytosis/polarity machinery. Arrowheads, GFP-CNT1 at cell-cell
contacts. (f-k, m-p) Validation of shRNAs. Knockdown with (f) primary Rab8a
and Rab11a shRNAs alone or in combination, (g) secondary Rab8a and
Rab11a shRNAs, (h) both Rab8b shRNAs, (i) Rab10 shRNA, (j) primary
Rab11a and Rab11b shRNA, (k) both Rab25 shRNA, (m) both Sec15A
shRNA, (n) both Par3 shRNA, (o) both Cdc42 shRNA, and (p) both Tuba
shRNA was verified by blotting with appropriate antibodies (f-i, k, n-p), or
by qRT-PCR (j, m), with GAPDH used as a control in all instances. For qRTPCR, values are mean ± SD from three replicates. *p < 0.05. (l) Validation
of multiple Rab8/11 family member shRNAs on single lumen formation.
Single lumen formation in 48 h cysts expressing indicated shRNA to Rab8
and Rab11 family members was quantified, confirming that multiple
shRNAs targeting Rab8a, Rab8b, Rab11a, and Rab25 all disrupt single
lumenogenesis. Line represents 0.75-fold single lumenogenesis, normalized
to control levels. Values represent the average of ≥ three different experiments
± S.D., where **p <0.001, ***p<0.0001. Control, n = 868; Rab8a_1, n =
409; Rab8a_2, n = 302; Rab8b_1, n = 303; Rab8b_3, n = 312; Rab11a_1,
n = 1,463; Rab11a_2, n = 307; Rab25_2, n = 315; Rab25_4, n = 310.
Smaller panels depict higher magnification of regions indicated. Bar, 20 µm.
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3
© 2010 Macmillan Publishers Limited. All rights reserved.
***
**
1.0
***
0.5
0.0
C
on
tro
l
Single lumen formation
(c.f. control)
1.5
Single lumen formation
(c.f. control)
c
WT S25N Q70L
GFP-Rab11a
**
*
1.2
1.0
0.8
0.6
***
0.4
***
***
***
0.2
0.0
Rab
KD:
MDCK
GFPRab11aQ70L
2.0
1.4
d
Control
b
a
Single lumen formation (c.f. control)
s u p p l e m e n ta r y i n f o r m at i o n
***
***
***
***
***
***
8a 8b 8a 11a 11b 25 8a 8b 8a 11a 11a
8b
11a 11a 8b 11b 25
11a
GFP-Rab8a / PCX / nuclei
WT
Q67L
1.5
1.0
**
0.5
0.0
Scramble
Rab8a KD
Figure S4 Additional Rab GTPase analysis during lumen formation. (a)
Rab11a promotes lumen formation. Quantitation of single lumenogenesis
at 48 h in control cysts or cysts stably expressing GFP-Rab11a (WT, S25N,
or Q70L) revealed that GFP-Rab11a WT or Q70L expression significantly
increased single lumenogenesis compared to control (MDCK alone), while the
S25N mutant strongly suppressed single lumenogenesis. Values represent
the average of ≥ three different experiments ± S.D., where **p <0.001,
***p<0.0001. Control, n = 868; GFP-Rab11a WT, n = 600; GFP-Rab11a
S25N, n = 614; GFP-Rab11a Q70L, n = 355. (b) Quantiation of single
lumenogenesis at 48 h in cysts expressing indicated combinations of stable
RNAi to Rab8 and Rab11 family members revealed that co-knockdown of
Rab8 family members, with or without Rab11a co-knockdown, strongly
perturbed single lumenogenesis, suggesting Rab8a/b function downstream of
Rab11a in lumenogenesis. Line represents 0.75-fold single lumenogenesis,
normalized to control levels. Values represent the average of ≥ three different
experiments ± S.D., where **p <0.001, ***p<0.0001. Control, n = 868;
Rab8a, n = 409; Rab8b, n = 312; Rab8a+b, n = 316; Rab11a, n = 307;
Rab11b, n = 1,528; Rab25, n = 310; Rab8a+11a, n = 304; Rab8b+11a,
n = 316; Rab8a+8b+11a, n = 313; Rab11a+11b, n = 295; Rab11a+25, n
= 305. (c) Rab8a functions with Rab11a in lumen formation. Examination
of single lumenogenesis in 48 h MDCK or MDCK GFP-Rab11aQ70L cysts
stably expressing control or Rab8a RNAi revealed that Rab11aQ70L expression
increased single lumenogenesis, dependent on Rab8a expression, suggesting
that Rab8a functions downstream of Rab11a activation. Values represent the
average of ≥ three different experiments ± S.D., where **p <0.001. Scramble,
n = 331; Scramble + GFP-Rab11aQ70L, n = 355; Rab8a KD, n = 320; Rab8a
KD + GFP-Rab11aQ70L, n = 306. (d) Rab8a localizes to subapical endosomes.
MDCK GFP-Rab8a WT or Q67L (both green) cysts grown for 48 h were costained for podocalyxin (red) and nuclei (blue). Arrows, subapical GFP-Rab8a.
Smaller panels indicate higher magnification of regions indicated. Bar, 20 µm.
Figure S4 - Mostov
4
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© 2010 Macmillan Publishers Limited. All rights reserved.
s u p p l e m e n ta r y i n f o r m at i o n
hsRabin8α
b
RBR
hsRabin8β
+
+
-
-
+
+
-
Q67L
-
-
-
+
+
-
-
+
01
A
Rab8a
hsRabin8
cfRabin8
rnRabin8
scSec2p
aa: 189
211
A
ST
G
A
T
L1
96
W
01
W
L1
A
R140A
GFP-TBC1D30
PCX / nuclei
WT
Coomassie
Stain
F2
d
Input
+
F2
L1
9
6A
cfRabin8α
Pull-down
Rab8a:
T22N
96
Sec2
T
a
GST-Rabin8α:
1.5
1.0
1.20
Rab8a T22N
Rab8a Q67L
1.00
0.80
0.60
0.40
0.20
0.00
*
0.5
WT
L196A F201A
GST-Rabin8α:
1D
C
FP
G
D
TB
C
M
FP
G
30
R TBC
14 1
0A D
30
0.0
K
Single lumen formation
(c.f. control)
e
Binding of Rab8a to Rabin8α
(c.f. control)
c
Figure S5 Characterization of Rab8 regulatory proteins. (a) Cartoon diagram of
Rabin8 isoforms, and conserved, critical guanine nucleotide exchange activity
residues. Amino acid positions refer to human Rabin8α. RBR, Rab11-binding
region; Cf, canis familiaris; hs, homo sapiens; rn, rattus norvegicus; sc,
saccharomyces cerevisiae. Boxed residues indicate crucial residues for GEF
activity. (b-c) Characterization of Rab8a binding to Rabin8α. In vitro binding
of Rab8a-T22N to recombinant GST-Rabin8α WT, GST-Rabin8α-L196A, GSTRabin8α-F201A and GST, and Rab8a-Q67L to GST-Rabin8α WT and GSTRabin8α-L196A (b), as described in Methods. Lanes at right indicate 1/10
of input of in vitro translated Rab8a-T22N and Rab8a-Q67L. Corresponding
GST-proteins from beads used for the binding assays were run on SDS-PAGE
and stained with Coomassie blue. Densitometry revealed that the L196A
and F201A mutants decreased association with Rab8a-T22N by 39% and
52%, respectively, confirming their reduction-of-function characteristics (c).
Note negligible association of any Rabin8α allele with Rab8a-Q67L. (d-e)
TBC1D30 GAP activity inhibits single lumen formation. MDCK cysts stably
expressing WT or GAP-deficient (R140A) GFP-TBC1D30 (green) grown for
48 h were costained for podocalyxin (red) and nuclei (blue). Quantitation (e)
revealed that GFP-TBC1D30 strongly suppressed single lumenogenesis, and
that this required its GAP activity (i.e. R140A mutant did not perturb single
lumenogenesis). Lumenogenesis quantitation values represent the average of
≥ three different experiments ± S.D., where *p <0.05. Total cysts: MDCK, n =
329; GFP-TBC1D30, n = 322; GFP-TBC1D30R140A, n = 305. Smaller panels
indicate higher magnification of regions indicated. Bar, 20 µm.
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Figure S5 - Mostov
5
© 2010 Macmillan Publishers Limited. All rights reserved.
PCX / GFP-Anx2 / nuc
a
f
Peripheral
Control
PCX localization:
AMIS
b
g
Rab8a KD
c
h
Luminal
PCX / GFP-Anx2 / Apple-Rab11a
Apple-Rab11a / GFP-Anx2 / PCX
s u p p l e m e n ta r y i n f o r m at i o n
Anx2 WT
e
Anx2 XM
Rab11a KD
Figure S6 Annexin2 associates with Rab11a vesicles. (a-c) Anx2
transiently associates with Rab11a vesicles. Examination of cysts
expressing GFP-Anx2 (green), Apple-Rab11a (red), and labelled for
podocalyxin (blue), revealed that Anx2 localized to Rab11a vesicles
(arrowheads) during lumen initiation (b, AMIS), but not when podocalyxin
was peripheral (a), or once lumens formed (c, luminal podocalyxin, arrows).
(d-e) Dominant negative Anx2 (Anx2 XM) disrupts single lumenogenesis.
In control cysts (d) GFP-Anx2 (green) was at the apical and basolateral
surface, Apple-Rab11a (pseudo-coloured blue) was in subapical vesicles,
6
d
and podocalyxin (red) was at the lumen. In contrast, expression of Anx2
XM disrupted single lumenogenesis and caused the accumulation of a
pool of podocalyxin and Anx2 in Apple-Rab11a vesicles (e, arrowheads).
(f-h) Rab8a/11a knockdown accumulates a pool of intracellular Anx2.
In contrast to control cysts (f) with luminal podocalyxin (red) and apical
and basolateral GFP-Anx2 (green), Rab8a (g) or Rab11a (h) knockdown
perturbed lumenogenesis and co-accumulated Anx2 and podocalyxin in
intracellular vesicles (arrowheads). Blue, nuclei. Smaller panels indicate
higher magnification of regions indicated. Bar, 20 µm.
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© 2010 Macmillan Publishers Limited. All rights reserved.
Fig 6b
11a Q70L
WB: Cdc42
8a Q67L
GFP-Rab
MDCK
11a Q70L
8a Q67L
MDCK
11a Q70L
8a Q67L
MDCK
Rabin8_4
GFP-Rab
GFP-Rab
Control
Rabin8_5
Fig. 3b
Control
Rabin8_4
Rabin8_5
s u p p l e m e n ta r y i n f o r m at i o n
WB: GFP
Total lysate
Control
Rab11a KD
Control
Fig 6a
Rab8a KD
Rab11a KD
WB: GAPDH
Rab8a KD
Control
WB: Rabin8
GST-PAK
pulldown
Fig S2g
Sec10 KD
1 2 3 pool
Sec10
Actin
GST-PAK
pulldown
Control
Rab8a KD
Rab11a KD
Rab8a_2
Rab11a_2
Fig S3g
8a/11a KD
8a/11a KD
Rab11a KD
Rab8a KD
Control
Control
Rab8a KD
Rab11a KD
8a/11a KD
Fig S3f
Control
KD
WB: Rab8a
Total lysate
WB: Rab11a
WB: Rab8a
WB: GAPDH
WB: GAPDH
WB: Rab11a
Rab8a
Figure S7a - Mostov
Figure S7 Full scans of Western blots.
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7
© 2010 Macmillan Publishers Limited. All rights reserved.
s u p p l e m e n ta r y i n f o r m at i o n
Control
KD
Rab25_4
Fig S3k
Rab25_4
Rab25_2
Control
Rab8b_3
Rab8b_1
Control
Rab8b_3
Rab8b_1
Control
KD
KD
Rab25_2
Fig S3h
KD
Rab25
Fig S3o
KD
Cdc42_3
Cdc42_3
Par3_4
Par3_3
Control
Cdc42_2
k
KD
Control
KD
Cdc42_2
Fig S3n
Par3_4
Par3_3
j
Control
KD
Rab10 KD
Control
Rab10 KD
Control
Fig S3i
WB: Rab25
Control
WB: GAPDH
WB: Rab8b
WB: GAPDH
WB: GAPDH
WB: GAPDH
WB: Rab10
WB: GAPDH
WB: Par3
WB: Cdc42
m
Fig S5b
Pull-down
Rab8a:
T22N
+
+
+
-
-
Q67L
-
-
-
+
+
Rab8a:
T22N
Q67L
Pull-down
Tuba_2
Tuba_1
WB: Tuba
l
KD
Control
Fig S3p
Input
+
+
-
-
-
+
G
ST
A
T
96
L1
W
A
A
01
F2
T
96
W
L1
WB: GAPDH
Rab8a
GST-Rabin8α:
Figure S7 continued
Figure S7b - Mostov
8
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© 2010 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 1: Q-RT PCR Primer sequences.
Target
GAPDH
Rab11a
Rab11b
Sec15A
Primer
Fwd
Rev
Fwd
Rev
Fwd
Rev
Fwd
Rev
Sequence (5’ – 3’)
AGTCAAGGCTGAGAACGGGAAACT
CATGGTTCACGCCCATCACAAACA
GCATCCAGGTTGATGGGAAA
AGGCACCTACAGCTCCACGA
GCTGGTGGGCAACAAGAGTG
GGTGGAATCCAAGGCTGAGG
GTCAGCCTGCCAGCATCTGT
CTGCTGAACAGCTCCCATGC
© 2010 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 2: RNAi target sequences
Target
Control
Cdc42_2
Cdc42_3
Par3_3
Par3_4
Rab8a_1
Rab8a_2
Rab8b_1
Rab8b_3
Rab10
Rab11a_1
Rab11a_2
Rab11b
Rab25_2
Rab25_4
Rabin8_4
Rabin8_5
Sec10_1
Sec10_2
Sec10_3
Sec15A_2
Sec15A_5
Tuba_1
Tuba_2
Vector
pLKO.1-puro
pLKO.1-blast
pLKO.1-blast
pLKO.1-puro
pLKO.1-puro
pRVH1-puro/hygro
pLKO.1-puro
pLKO.1-blast
pLKO.1-blast
pRVH1-puro
pRVH1-puro/hygro
pLKO.1-puro
pRVH1-puro
pLKO.1-blast
pLKO.1-blast
pLKO.1-puro
pLKO.1-puro
siRNA
siRNA
siRNA
pLKO.1-puro
pLKO.1-puro
pLKO.1-blast
pLKO.1-blast
Sequence (5’ – 3’)
CCGCAGGTATGCACGCGT
GATTACGACCGCTGAGTTA
GCGATGGTGCCGTTGGTAA
GACATCATGAAAGCTAGAA
GAGTATGGAGAGGCACATC
(AA)GACAAGTTTCCAAGGAACG
GGGCCCTCCCCCTCCAATACT
(AA)GCGGGCAGAGCCAGGAATT
(AA)GAGGAGAGAAGTTAGCAAT
(AA)GCTGAAGATATCCTTCGAAAG
(AA)GGCACAGATATGGGACACA
(AA)GGTTTGTCATTCATTGAG
(AA)GAACATCCTCACAGAGATC
(AA)AGAGATCTTCACCAAAGTG
(AA)CCAGGCGCTGGTCAGAAGA
(AA)GCCCTGTAAACACAGAATT
(AA)GCTGGGATATTTCAAAGAG
GCACATCAGCTATGTAGCAACCAA
CAGATCCTCCTGATTTACCAAGGAT
CCAAAGACTTCAAGATTCCACTGGTA
(AA)GAGGATGAGAATGAAGAGG
(AA)GCACGGGTCATGATAGTTT
GGAAATATGCAGATGGTGA
GCAGAGAAGTTAAAGGACA
© 2010 Macmillan Publishers Limited. All rights reserved.