RESEARCH ARTICLE 2071
Development 139, 2071-2083 (2012) doi:10.1242/dev.077347
© 2012. Published by The Company of Biologists Ltd
Clathrin and AP-1 regulate apical polarity and lumen
formation during C. elegans tubulogenesis
Hongjie Zhang1, Ahlee Kim1, Nessy Abraham1, Liakot A. Khan1, David H. Hall2, John T. Fleming1 and
Verena Gobel1,*
SUMMARY
Clathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma
membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to
basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1
adaptor in apical polarity during de novo lumenal membrane biogenesis in the C. elegans intestine. We show that CHC-1/AP-1mediated polarized transport intersects with a sphingolipid-dependent apical sorting process. Depleting each presumed
trafficking component mislocalizes the same set of apical membrane molecules basolaterally, including the polarity regulator
PAR-6, and generates ectopic lateral lumens. GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble
asymmetrically at polarized plasma membrane domains in a co-dependent and AP-1-dependent manner. Based on these findings,
we propose a trafficking pathway for apical membrane polarity and lumen morphogenesis that implies: (1) a clathrin/AP-1
function on an apically directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical
trafficking path.
INTRODUCTION
Biological tubes are composed of polarized epithelial cells with
their apical sides generating the lumenal surface and their
basolateral sides contacting adjacent cells or the extracellular
matrix. Polarizing cues come from inside the cell (e.g. through
polarized trafficking), from the extracellular environment, or
from the plasma membrane itself (Mellman and Nelson, 2008).
Many such cues, which are highly conserved between species,
have been identified, but their integration during the complex
process of tissue morphogenesis is not well understood. It is
assumed that plasma membrane-associated polarity
determinants, such as the apical partitioning-defective (PAR)
complex PAR-3/PAR-6/aPKC, define membrane domain
identities, whereas polarized trafficking directs specific
membrane components to these domains. Although there is little
evidence for the intrinsic ability of vesicular trafficking to define
polarized membrane domains, recent analysis of tubulogenesis
has demonstrated that it may determine such domains by
recruiting the polarity complex components themselves. RAB11–RAB-8-mediated vesicular delivery of CDC-42, for instance,
was shown to be required for recruiting the apical PAR complex
to promote apical domain and lumen biogenesis in MDCK 3D
tissue culture (Bryant et al., 2010).
Membrane lipids, such as phosphoinositides, are wellcharacterized sorting molecules that have also been implicated in
the asymmetric placement of polarity complex components.
Membrane lipids assume a specific place in vesicular sorting, as
1
Department of Pediatrics, Massachusetts General Hospital and Harvard Medical
School, Boston, MA 02114, USA. 2Center for C. elegans Anatomy, Department of
Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
*Author for correspondence ([email protected])
Accepted 2 April 2012
they themselves may be asymmetrically assorted on plasma
membranes (van Meer et al., 2008). For example,
phosphoinositides determine both polarized trafficking and
polarized domain identities when inserted into the plasma
membrane (Di Paolo and De Camilli, 2006; Rodriguez-Boulan et
al., 2005). PtdIns(4,5)P2 (PIP2) enrichment at apical membranes by
the lipid phosphatase PTEN is also required for CDC-42 and PAR6 recruitment in MDCK lumen morphogenesis (Martin-Belmonte
et al., 2007). Similarly, glycosphingolipids (GSLs), which are
saturated obligate membrane sphingolipids (SLs) that are thought
to laterally assemble into membrane microdomains (lipid rafts), are
enriched on both apical plasma membranes and endomembranes,
and apically sort lipids and proteins (Simons and Gerl, 2010). In C.
elegans, GSLs define apical membrane domain identities in the
expanding intestine and are also required to recruit PAR-6 to the
lumen (Zhang et al., 2011).
Clathrin, the prototypical post-Golgi vesicle coat, is primarily
studied for its roles in endocytosis and signaling at the plasma
membrane. Recently, however, clathrin was shown to regulate
basolateral sorting through the epithelial cell-specific AP-1B
adaptor, revealing its additional role in membrane-directed
trafficking (Deborde et al., 2008; Folsch et al., 1999). Vesicle coat
formation, in turn, depends on vesicle membrane lipid composition.
PIP2, for instance, functions at several steps in clathrin coat
formation, possibly in an AP-2 adaptor-dependent manner
(Antonescu et al., 2011). Thus, vesicle lipids, coats, adaptors and
their interaction might play a crucial role in the generation of
polarized plasma membrane domains.
In a systematic screen for apicobasal polarity and tubulogenesis
defects in the C. elegans intestine, we identified clathrin and several
subunits of its AP-1 adaptor as being required for apical polarity and
lumen formation. Clathrin/AP-1 depletion caused defects similar to
those caused by the depletion of GSL-biosynthetic enzymes (also
identified in this screen). Further analysis revealed that both
trafficking components cooperate in apical sorting.
DEVELOPMENT
KEY WORDS: Caenorhabditis elegans, Polarity, Tubulogenesis, Clathrin, AP-1, Sphingolipids
2072 RESEARCH ARTICLE
Strains and culture conditions
C. elegans strains were maintained, cultured and crossed using standard
techniques (Brenner, 1974). See supplementary material Table S1 for strain
list. chc-1(tm2866)III/+ was balanced with hT2[qIs48] (Miskowski et al.,
2001). aps-1(tm935)V/+ was balanced with nT1[qIs51] (Belfiore et al.,
2002). The temperature-sensitive strain chc-1(b1025) was maintained at
16°C unless indicated otherwise.
RNAi and screens
A systematic C. elegans tubulogenesis RNAi screen was designed and
carried out as previously described, using animals carrying an erm-1::gfp
transgene, outlining the lumens of the intestine, the excretory canal and the
gonad (Zhang et al., 2011). RNAi was performed by feeding (Timmons et
al., 2001).
Standard RNAi conditions (used in the screen) were defined as dsRNA
induction by 2 mM IPTG. Mild RNAi conditions were empirically
determined for specific genes after testing serial concentrations of IPTG
and/or dilutions with mock RNAi bacteria: for chc-1, IPTG was titrated
down to 2 nM; for aps-1, RNAi bacteria were diluted 1:10 with mock
RNAi bacteria. For double RNAi, equal amounts of RNAi bacteria of two
clones were mixed. RNAi initiated after completion of embryogenesis
involved placing eggs or larvae on RNAi plates for evaluating the same
generation.
DsRed feeding
chc-1(b1025ts) animals were fed on plates containing DsRed RNAi
bacteria for at least 12 hours. The DsRed bacterial feeding strain contains
a DsRed plasmid in HT115 bacteria that constitutively produces a faint red
color.
Phenotype reversal
chc-1(b1025ts) mutant hermaphrodites were allowed to lay eggs for 1 hour
(at 16°C) and subsequently removed. The plates with eggs were transferred
to 22°C for 5 hours, then returned to 16°C. Animals were singled the next
day and phenotype development and reversal were observed for 6 days.
Lipid labeling and assessment of vesicle association
For lipid labeling, 150 l E. coli OP50 or HT115 were spiked with 2 l 5
mM labeled lipid stock solutions (NBD-C6-glucosylceramide stock was
100 M), for a feeding period of ~8 hours. The same amounts were used
for double labeling. Lipids used were: BODIPY-FL-labeled C5-ceramide
(N-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl]sphingosine), BODIPY-TR-labeled C5-ceramide (N-[(4-(4,4-difluoro-5-(2thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl]sphingosine),
BODIPY-FL-labeled C5-sphingomyelin (N-[4,4-difluoro-5,7-dimethyl-4bora-3a,4a-diaza-s-indacene-3-pentanoyl]sphingosylphosphocholine) (all
from Invitrogen) and NBD-C6-glucosylceramide (N-[6-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]hexanoyl]-D-glucosyl-1-1⬘-sphingosine)
(Avanti Polar Lipids).
Vesicle colocalization was quantified by counting the number of either
overlapping or associated (defined as overlapping or in contact) vesicles in
a 800 ⫻ 400 pixel grid area of thin confocal sections, unless otherwise
indicated.
Genetic interactions
To examine genetic interactions between clathrin, the AP-1 adaptor and
SL-biosynthetic enzymes, eggs or L4 stage larvae of wild type, chc1(b1025ts), let-767(s2716) and let-767(s2819) mutants, all carrying the
erm-1::gfp transgene, were placed on RNAi plates containing aps-1, apb1, let-767, sptl-1, apa-2 or mock RNAi bacteria. Polarity phenotypes,
lethality and arrest stages were evaluated in the same or the next
generation.
Plasmids and DNA transformation
Translational GFP fusion proteins were generated by in-frame joining of
genomic DNA of the gene of interest with GFP by PCR, using the stitching
method (Hobert, 2002). The ERM-1::mCherry plasmid was generated by
replacing the GFP with mCherry coding sequences in a plasmid expressing
an ERM-1::GFP fusion protein (Gobel et al., 2004). Briefly, mCherry DNA
was PCR amplified, digested with SmaI and KpnI and ligated to the 3⬘ end
of the erm-1 full-length genomic DNA. DNA was prepared from multiple
independent isolates, verified by restriction digestion and sequencing, and
a mixture was used for germline transformation of animals by
microinjection (Mello et al., 1991). Constructs were injected at 10-100
ng/ml, along with the dominant transgene marker rol-6(su1006).
Antibodies and immunofluorescent staining
Animals were collected in M9 on poly-L-lysine (Sigma)-coated slides,
permeabilized by freeze cracking and fixed by sequential incubation in
methanol and acetone (Miller and Shakes, 1995). Immunofluorescent
staining was performed as described (Zhang et al., 2011). Antibodies were
diluted with blocking solution at the following concentrations: MH27 (antiAJM-1), 1:20; MH33 (anti-IFB-2), 1:20; anti-DLG-1, 1:10 (all from the
Developmental Studies Hybridoma Bank, University of Iowa); ICB4,
1:500 (gift from M. de Bono, Laboratory of Molecular Biology, Cambridge
University, UK); Alexa Fluor 568 phalloidin (actin), 1:20 (Molecular
Probes); TRITC-conjugated goat anti-mouse IgG, 1:100 (Sigma); Cy5conjugated goat anti-mouse IgG, 1:250 (Jackson ImmunoResearch).
Epifluorescence and confocal microscopy
For large-scale screens, animals were directly observed on plates under an
Olympus SZX12 dissecting microscope (Olympus America Center Valley,
PA, USA) equipped with a high-power stereo fluorescence attachment
(Kramer Scientific Corporation, Amesbury, MA). For detailed
characterization, live worms were mounted in M9 buffer on glass slides,
immobilized with 10 mM sodium azide (Sigma), and visualized by confocal
and Nomarski microscopy. Confocal images were acquired on a TCS SL
laser-scanning microscope (Leica Microsystems, Bannockburn, IL, USA).
Single-plane images were taken as 6-50 sections along the z-axis at 0.2 m
intervals, and projection images generated by merging. Multi-channel images
were taken with minimal laser settings unless indicated otherwise. Laser
settings with increased sensitivity were defined as laser power at 60%, 488
nm beam path at 50%, pinhole (airy) at 1.755. Identical laser and confocal
settings were used when comparing experimental animals with controls.
To separate fluorescently labeled from autofluorescent intestinal
vesicles, empirical scanner settings were established by restricting the
wavelengths of the fluorescence filters (green filter spectrum to 500-515
nm, red filter spectrum to 630-700 nm). A considerable decrease in the
sensitivity of detecting fluorescently labeled vesicles was accepted. For all
overlay experiments, animals were sequentially scanned to exclude false
positives caused by channel bleed-through. Images were arranged using
Adobe Photoshop with occasional small adjustments for contrast and
brightness, and fluorescence intensity was quantified using ImageJ
software.
Transmission electron microscopy (TEM)
TEM procedures were carried out according to protocols previously
described (Hall, 1995; Zhang et al., 2011). chc-1(b1025ts) L1 larvae were
obtained by placing isolated eggs at 16°C for 6 hours, then at 22°C
overnight. Thin sections were cut on a Reichert Ultracut E ultramicrotome,
collected on formvar-coated gold grids, contrasted with uranyl acetate and
lead citrate and viewed in a JEOL 1011 electron microscope equipped with
a digital imaging system (Advanced Microscopy Techniques, Danvers,
MA, USA) at 80 kV.
Statistics
Data are expressed as mean ± s.d. Statistical significance was determined
at the *P<0.05, **P<0.01 and ***P<0.001 levels by Student’s two-tailed
t-test using Microsoft Excel software.
RESULTS
Clathrin (CHC-1) depletion disrupts apical
membrane polarity and generates lateral lumens
A chromosome III and genome-wide RNAi screen of all lethal
genes tracking the asymmetric distribution of apical ERM-1::GFP
in the C. elegans intestine identified several distinct classes of
DEVELOPMENT
MATERIALS AND METHODS
Development 139 (11)
Clathrin and AP-1 in apical polarity
RESEARCH ARTICLE 2073
polarity phenotypes, most involving the cytoplasmic
mislocalization of this apical membrane marker (Zhang et al.,
2011). Two classes were distinguished by basolateral membranous
ERM-1::GFP misplacement in: (1) the embryonic pre- to early
post-intercalation intestine with absent or incomplete lumen
formation (Fig. 1B,C, left and middle; 1A for anatomy); and (2) the
embryonic or early larval fully intercalated intestine, accompanied
by the formation of multiple lateral ectopic lumens (Fig. 1B,C,
right; 1D; Fig. 2A,B). Both phenotypic classes also arrested at
these respective stages. RNAi with chc-1, the C. elegans clathrin
heavy chain ortholog, generated the former phenotype, whereas
RNAi with aps-1, the clathrin AP-1 adaptor complex sigma
subunit, and apb-1, the AP-1 or AP-2 adaptor complex beta
subunit, generated the latter phenotype.
To confirm the RNAi results and examine whether the two
classes of phenotypes affected the same polarization process,
different chc-1 RNAi and mutant conditions were characterized. In
contrast to the pre-comma/comma stage embryonic arrest of chc-
DEVELOPMENT
Fig. 1. Apicobasal polarity alteration and ectopic lumen formation in CHC-1-depleted intestines. (A)Schematic of the pre- and postintercalation wild-type C. elegans intestine. (Left) E16 stage, with ten dorsal and six ventral cells (three ventral cells obscured). Arrow shows
direction of intercalation. (Right) E20 stage, cells arranged into nine intestinal (INT) rings in bilateral symmetry (four cells in first INT). (B)(Top)
Apical/lumenal ERM-1::GFP in wild-type (WT) at pre-comma (left, beginning of intercalation; image obtained with increased laser settings, see
Materials and methods), comma (middle, intercalation almost complete) and 3-fold embryo (right, fully intercalated intestine) stages. (Bottom)
Images combined with staining of actin (phalloidin) and intermediate filaments (anti-IFB2) to outline intestinal tube and lumen. (C)ERM-1::GFP
displacement and lumen defects in chc-1(RNAi) and chc-1(b1025ts) animals. (Top) Non-polarized ERM-1::GFP in embryos arrested at pre-comma
and comma stage at mid- to late intercalation (left and middle); ectopic lateral lumen formation in fully intercalated intestine in 3-fold embryo
(right, arrowhead). (Bottom) Ectopic lumens in L4 (arrowheads) and L1 (small arrows) chc-1(b1025-16°C) larvae (compare with TEM images in D).
Note the wild-type 2.5-fold embryo (thin arrow). P, pharynx. Large arrows, excretory canals. Confocal images are shown, with and without
corresponding Nomarski images; anterior is left and dorsal up. (D)TEM micrographs of L1 intestines. (Top) Wild-type, showing oval lumen (L),
tightly adjacent terminal web (arrowheads) and dense microvilli (long arrows). (Middle, bottom) chc-1(b1025-22°C), showing deformed main
lumen (L) with stunted microvilli, ectopic lateral lumens (EL) with terminal web (arrowheads) and short or almost normal microvilli (long arrows).
Intact apical junctions (short arrows) are seen in both wild type and chc-1(b1025); note excess junctions between ectopic lumens.
Fig. 2. Depletion of four different AP-1 adaptor subunits
phenocopies the polarity and ectopic lumen defect induced by
CHC-1 depletion. (A,B)aps-1 and apb-1 RNAi phenotypes (left) with
phenotypes of corresponding presumed null alleles (right) showing
basolateral ERM-1::GFP displacement (arrows) and ectopic lumen
formation (arrowheads). TEM micrographs of APB-1-depleted L1
intestines (B, bottom) show severely deformed main lumen (L) and
ectopic lumens (EL), both with sparse and stunted microvilli (long arrow),
terminal web (arrowheads) dissociated from main lumen, and intact
apical junctions (short arrows). (C,D)apg-1(RNAi) and apm-1(RNAi)
phenotypes (compare with A,B). (E)Genetic interactions between chc1(b1025), aps-1 RNAi (left) and apb-1 RNAi (right). Double mutant/RNAi
animals show enhancement, with appearance of more severe
phenotypes (compare with supplementary material Fig. S4D) and earlier
arrest. E2, E3 and L1 indicate arrest at 2-fold embryo, 3-fold embryo and
L1 larval stages. Mean ± s.d.; n3 (N>200 animals per experiment).
1(RNAi) animals (penetrance, 100%), the majority of chc1(b1025ts) animals grew into fertile adults at 16°C [with CHC-1 at
4.5% of wild-type levels (Sato et al., 2009)], but ~90% displayed
the late multiple lumen phenotype (Fig. 1C, bottom; ~20% as late
embryos). Shifting early chc-1(b1025ts) embryos to 22°C or 25°C
Development 139 (11)
[CHC-1 at 2% of wild-type levels and unstable (Sato et al., 2009)],
caused ~100% 3-fold embryonic arrest with ectopic lateral lumens
in the intercalated intestine, similar to the predominant arrest stage
of homozygous progeny of balanced chc-1(tm2866) mutants
(carrying an 847 bp chc-1 deletion; not shown). The earlier and
more severe nature of the chc-1(RNAi) phenotype as compared
with the chc-1(b1025ts and tm2866) mutant phenotype suggests a
maternal chc-1 requirement (Fig. 1C). In less severely affected
mutants, basolateral ERM-1::GFP was observed prior to ectopic
lumen development (supplementary material Fig. S1A, right).
Conversely, mild chc-1 RNAi conditions, allowing ~20% of
animals to reach the L2-L3 larval stage, developed ectopic lumens
subsequent to basolateral ERM-1::GFP displacement
(supplementary material Fig. S1A, left). Initiating chc-1 RNAi
from the L1 stage (supplementary material Fig. S1B; see below)
enhanced the mild chc-1(b1025) ectopic lumen phenotype at the
permissive temperature, inducing fully penetrant L1-L2 larval
arrest while increasing the number of ectopic lumens per animal
(not shown). Thus, maternal and zygotic chc-1 products dosedependently regulate apical polarity and lumen morphogenesis in
the C. elegans intestine, and the two classes of phenotypes appear
to disrupt the same process of membrane polarization, with the
basolateral displacement of apical membrane components
preceding ectopic lumen formation.
chc-1(RNAi) embryos displayed non-polarized, pan-membranous
ERM-1::GFP from the time of its appearance during late intestinal
intercalation, suggesting an early CHC-1 requirement. To
determine whether CHC-1 was required for the establishment of
membrane polarity, we examined ERM-1::GFP during early
intestinal polarization in chc-1(RNAi) and chc-1(b102522°C,RNAi) embryos, using increased laser settings for confocal
analysis (see Materials and methods). Under these conditions, wildtype apical ERM-1::GFP is detected at approximately the start of
intercalation, when nuclei have moved to the future apical
membrane and cytoplasmic vesicles towards the future basolateral
membrane, and apical junctions are at the spot-junction stage
(Leung et al., 1999). At this stage, chc-1(RNAi) embryos displayed
either apical ERM-1::GFP or both apical and partial basolateral
ERM-1::GFP (supplementary material Fig. S2). ERM-1::GFP
subsequently increased pan-membranously in the arrested, nonexpanding embryonic intestine, with persistently higher apical
signal intensity (supplementary material Fig. S2) (in contrast to the
decreasing apical signal intensity during larval intestinal expansion
in animals with milder phenotypes, see below). This did not
suggest the loss of an initially intact polarity, particularly given the
possibilities of basolateral ERM-1::GFP being at subdetection
levels at early stages and incomplete RNAi phenotypes. Consistent
with the latter possibility, basolateral ERM-1::GFP displacement
was stronger in chc-1(b1025-22°C,RNAi) than in chc-1(RNAi)
embryos (supplementary material Fig. S2). Moreover, the
polarization of nuclei and cytoplasmic vesicles was also impaired
in chc-1(RNAi) embryos, even in those with exclusive apical ERM1::GFP (supplementary material Fig. S2). Furthermore, from an
early stage on, excess junction material was detected on lateral
membranes, suggesting that it could be a part of the process of
apical membrane biogenesis at the lateral side (supplementary
material Fig. S3). Nevertheless, junction assembly appeared
remarkably intact and included the formation of apicolateral
junctions (compare with the analysis of junctions in larvae below).
To determine whether CHC-1 is required for membrane polarity
maintenance, ERM-1::GFP was examined in animals in which chc-1
RNAi had been initiated after completion of embryogenesis. Late
DEVELOPMENT
2074 RESEARCH ARTICLE
RESEARCH ARTICLE 2075
chc-1 RNAi was sufficient to induce ERM-1::GFP displacement,
small ectopic lumens and late larval/adult lethality (supplementary
material Fig. S1B). Adult induced chc-1 RNAi, which was capable
of reducing CHC-1::GFP, had no detectable effect (supplementary
material Fig. S1C; data not shown). To test whether the polarity
defects were reversible, chc-1(b1025ts-16°C) embryos were shifted
to 22°C for 5 hours and then returned to the permissive temperature.
This downshift decreased the number and size of ectopic lumens and
altered their character (supplementary material Fig. S1D; compare
with Fig. S6 for TEM studies of different ectopic lumens).
We conclude that CHC-1 is required to maintain and possibly
also to establish apical membrane polarity and a single lumen in
the C. elegans intestine in an apparently junction-independent
manner. CHC-1 function in polarity maintenance appears to be
restricted to embryonic and larval epithelia, where polarity defects
and even ectopic lumens remain reversible. These findings are
compatible with a CHC-1 function in apical sorting of membrane
components and/or polarity regulators during de novo membrane
biogenesis.
16°C, chc-1(b1025) grow to fertile adults with a mild ectopic
lumen phenotype (see above; Fig. 1C, bottom), whereas aps1(RNAi), apb-1(RNAi) and chc-1(b1025) at 22°C arrest as 3-fold
embryos or L1 larvae with multiple ectopic lumens (Fig. 2A,B).
chc-1(b1025);aps-1(RNAi) and chc-1(b1025);apb-1(RNAi) double
mutant/RNAi animals (at either 16°C or 22°C) revealed strong
enhancement by arresting earlier than either single mutant/RNAi
animal and resembled the severe chc-1(RNAi) phenotype with only
partially intercalated intestines, pan-membranous ERM-1::GFP,
cytoplasmic ERM-1::GFP inclusions and incomplete apical lumen
formation (Fig. 2E; supplementary material Fig. S4D). L1-initiated
aps-1 and apb-1 RNAi, which induce only very mild polarity
defects on their own (supplementary material Fig. S1B), increased
the number of ectopic lumens in chc-1(b1025-16°C) animals and
caused an earlier growth arrest (not shown).
We conclude that the similar CHC-1 and AP-1 reduction-offunction phenotypes result from different degrees of interference
with the same, or with different aspects of the same, process of
membrane polarization and lumen formation.
The AP-1 adaptor functions together with clathrin
in the regulation of apical polarity
The similarity between the phenotype of chc-1(b1025-22°C) and
that of aps-1(RNAi) and apb-1(RNAi) suggested that these genes
act in the same polarity process. To test this and to determine
whether the aps-1(RNAi) and apb-1(RNAi) phenotypes were
adaptor- and possibly subunit-specific, we characterized different
RNAi and mutant conditions. The C. elegans AP-1 adaptor
complex contains four subunits: APB-1/beta, APS-1/sigma, UNC101 or APM-1/mu, and APG-1/gamma. aps-1(RNAi) and apb1(RNAi) animals arrested as 2- to 3-fold embryos or L1 larvae with
basolateral ERM-1::GFP mislocalization and/or small ectopic
lumens at the apicolateral angle of the intercalated intestine
(penetrance, 90-95%; Fig. 2A,B; supplementary material Fig.
S4B). Homozygous progeny of heterozygous aps-1(tm935) and
apb-1(tm1369) alleles (carrying 1100 bp and 500 bp deletions,
respectively; supplementary material Fig. S4A) arrested as early
larvae and copied the RNAi polarity defects, but with a less severe
phenotype of predominantly lateral ERM-1::GFP displacement,
also suggesting a maternal effect (Fig. 2A,B, right). apg-1(RNAi)
fully recapitulated the aps-1(RNAi) and apb-1(RNAi) phenotype,
including ectopic lumen formation (penetrance, 99%; Fig. 2C), and
apm-1(RNAi) caused predominantly basolateral ERM-1::GFP
misplacement (penetrance, 90%) and L2-L3 arrest (Fig. 2D),
whereas unc-101(RNAi) did not show obvious defects in the
intestine (not shown). Likewise, interference with the endocytic
AP-2 clathrin adaptor alpha subunit apa-2 failed to show obvious
polarity defects (supplementary material Fig. S4B). Thus, the
clathrin AP-1 adaptor is required for apical polarity, whereas its
AP-2 adaptor may be dispensable. All AP-1 subunits are required
for function, with the possible exception of mu/UNC-101. APB-1
appears to be the C. elegans AP-1 beta subunit.
In further agreement with a common CHC-1/AP-1 function in
apical membrane polarity, aps-1 and apb-1, but not apa-2, RNAi
initiated after completion of embryogenesis induced a polarity
defect in the mature larval intestine that resembled the chc-1(larval
RNAi) phenotype (supplementary material Fig. S1B), and aps1(RNAi), apb-1(RNAi), apg-1(RNAi) and chc-1(b1025) mutant
animals displayed an excretory canal apical membrane and lumen
biogenesis defect (supplementary material Fig. S4C). To examine
genetic interactions, we analyzed chc-1(b1025ts);aps-1(RNAi) and
chc-1(b1025ts);apb-1(RNAi) double mutant/RNAi animals. At
Clathrin/AP-1 depletion mislocalizes multiple
apical molecules, including PAR-6, and reduces
RAB-11+ apical membrane-associated vesicles
To determine whether CHC-1/AP-1 depletion caused a general
polarity defect, additional submembranous and integral
membrane proteins were analyzed in chc-1, aps-1 and apb-1
RNAi/mutant animals. All apical molecules examined, i.e. actin
filaments (via phalloidin), intermediate filaments (via IFB-2), the
Par polarity complex component PAR-6, the oligopeptide
transporter OPT-2 and the water channel AQP-4, were displaced
laterally and/or cytoplasmically, prior to accumulating around
ectopic lateral lumens (Fig. 3B-F; 3A for anatomy). By contrast,
the basolaterally enriched molecules ICB4 (an unidentified panmembranous molecule), the ERM-1 family member NFM-1, the
Na+/H+ transporter NHX-7 and the aquaporin AQP-1, were less
affected, albeit partially cytoplasmically displaced (Fig. 3G-J).
Over time, molecules were lost from the apical, while being
gained at the basolateral, membrane, which is compatible with
an underlying apical sorting defect during membrane expansion
(Fig. 3C,E, arrows; supplementary material Fig. S5).
Transmission electron microscopy (TEM) revealed a
corresponding coincidental structural conversion of apical and
basolateral domains: microvilli were shortened and the terminal
web dissociated from the apical lumen, while both appeared de
novo at lateral circularized membranes, confirming them as ectopic
lumenal membranes (Fig. 1D; Fig. 2B; supplementary material Fig.
S6A-D). TEM and confocal analysis of several apical junction
components in animals depleted of CHC-1 or AP-1 subunits, as
well as feeding experiments with fluorescently labeled bacteria,
revealed intact junctions at their wild-type apicolateral sealing
positions, indicating that the membrane polarity defects are
unlikely to be a consequence of junction assembly defects (e.g.
membrane equilibration) (Fig. 1D; Fig. 3K-N; supplementary
material Fig. S6; compare with supplementary material Fig. S3).
Instead, excess junctions formed around ectopic lumens along
lateral membranes during their acquisition of apical membrane
characteristics (Fig. 1D; Fig. 3K-M; supplementary material Fig.
S6). However, membrane pockets that lacked apical junctions were
also observed in larvae with mild phenotypes and might represent
the reversion of milder polarity defects during epithelial expansion
(supplementary material Fig. S6E; Fig. S1D) (Zhang et al., 2011).
DEVELOPMENT
Clathrin and AP-1 in apical polarity
2076 RESEARCH ARTICLE
Development 139 (11)
Fig. 3. Placement of polarized transmembranous,
submembranous and junction molecules in AP-1-depleted
intestines. (A)Schematics of wild-type and AP-1-depleted intestines
showing apical membranes (green), basolateral membranes (blue) and
apical junctions (red). (B-J)Apicobasal membrane components in wildtype (wt, left) and APS-1- or APB-1-depleted L1 larval intestines (mt,
middle and right column in B-H, right column in I,J). aps-1, apb-1 and
chc-1 mutant/RNAi animals show similar displacement of each marker
(N>30 each). All apical markers are displaced basolaterally and/or
cytoplasmically before being displaced to ectopic lumens (EL): (B) actin
(phalloidin), ERM-1 overlay; (C) intermediate filaments (IFB-2); (D) OPT2; (E) PAR-6; (F) AQP-4. Note concomitant decrease of markers at apical
membrane (arrows in C,E, see supplementary material Fig. S5 for
quantification) and the presence of sealed ectopic lumens at the main
lumen. Pan-membranous and basolateral markers are partially
cytoplasmically displaced: (G) NFM-1 (pan-membranous punctate
expression construct shown here); (H) pan-membranous ICB4; (I,J)
basolateral NHX-7 and AQP-1, not visibly affected. (K-M)The apical
junction components AJM-1, HMP-1/alpha-catenin and DLG-1/Discs
large are contiguous (arrows) at apicolateral boundaries in both wild
type and mutant. Note additional junctions surrounding ectopic lumens
(arrowheads). (N)Intestinal section of CHC-1-depleted larva fed with
DsRed bacteria. DsRed is seen in the main lumen but not ectopic lumen
(arrow), indicating a sealed junction. Confocal images shown
throughout.
We conclude that the loss of CHC-1 and its AP-1 adaptor
complex results in a general apicobasal polarity conversion,
without apparent preceding junction assembly defects. The lateral
displacement of apical membrane components corresponds to the
structural transformation of the lateral into an apical membrane
with ectopic lateral lumen formation.
To investigate the requirement of clathrin/AP-1 for post-Golgi
trafficking, we examined RAB-5 early, RAB-7 late, RAB-10
basolateral and RAB-11 apical recycling endosomes in wild-type
and mutant/RNAi intestines (Chen et al., 2006). Most
conspicuously, GFP::RAB-11 vesicles were lost from the lumen of
aps-1(RNAi) and apb-1(RNAi) larval intestines at early stages of
polarity conversion. Furthermore, L1-specific apical GFP::RAB-7
aggregates were reduced in number and apical GFP::RAB-5 and
Clathrin/AP-1 genetically interact with
sphingolipid-biosynthetic enzymes in apical
sorting
The polarity phenotype induced by clathrin/AP-1 depletion closely
resembles that induced by interference with specific fatty acid- and
SL-biosynthetic enzymes that affect polarity through the
biosynthesis of GSLs (Zhang et al., 2011). To determine whether
clathrin/AP-1 and SLs function together in apical sorting, genetic
interactions between clathrin, its adaptors and SL-biosynthetic
enzyme genes were investigated. Since both GSLs and clathrin are
required for oocyte viability, partial loss-of-function conditions
were examined (Grant and Hirsh, 1999; Nomura et al., 2011).
Simultaneously decreasing both SL biosynthesis and clathrin or
AP-1 enhanced the polarity phenotype of either and generated
novel phenotypes.
let-767(s2819) and let-767(s2176) are moderate and severe lossof-function alleles, respectively, of the SL-biosynthetic enzyme
steroid dehydrogenase/3-ketoacyl-CoA reductase (Entchev et al.,
2008; Kuervers et al., 2003). Progeny of mutants balanced with a
duplication (sDp3) that have lost sDp3 die as larvae after
transitioning from the basolateral polarity to the ectopic lumen
phenotype (Kuervers et al., 2003; Zhang et al., 2011). Standard
chc-1, aps-1 and apb-1, but not apa-2, RNAi caused sterility in
both let-767 alleles, suggesting an interaction as early as during
oocyte development, but precluding further analysis. To bypass this
early requirement, RNAi was introduced after embryogenesis was
DEVELOPMENT
GFP::RAB-10 vesicle subfractions were also depleted (Fig. 4). In
larvae with fully developed phenotypes, only a residual string of
vesicles remained along basal membranes (supplementary material
Fig. S7; data not shown).
We conclude that CHC-1/AP-1 are required for the apical
localization of RAB-11-associated, presumably lumenal
membrane-forming endosomes, and also affect apical subfractions
of other post-Golgi vesicles in the C. elegans intestine.
Clathrin and AP-1 in apical polarity
RESEARCH ARTICLE 2077
Fig. 4. Effect of clathrin/AP-1 depletion on post-Golgi vesicles. Selective decrease of apical endosomal subfractions in aps-1(RNAi) L1 larval
intestines at early stages of polarity conversion [lower panels; similar results were obtained for apb-1- and chc-1(RNAi) animals], as compared with
wild type (upper panels; compare with supplementary material Fig. S7 for later stages of polarity conversion). Confocal images (left) and
quantification of vesicles or fluorescence intensity (right) are shown. (A)Decrease of GFP::RAB-11 vesicles from the apical membrane (arrows).
(B)Reduced subapical GFP::RAB-5 enrichment (arrows). (C)Decrease of L1-specific GFP::RAB-7 apical aggregates (arrows). (D)Decrease of subapical
GFP::RAB-10 (arrows); note the perinuclear GFP::RAB-10 assembly. L, lumen (also partially outlined); N, nucleus. Mean ± s.e.m. (n3); *P<0.05,
**P<0.01, two-tailed t-test. N>50 animals for each marker.
We conclude that CHC-1/AP-1 and SLs contribute to the same
or a parallel apical sorting function during polarized membrane
biogenesis, supporting a role of clathrin/AP-1 on an apical
trafficking route.
GFP::CHC-1 vesicles assemble underneath the
apical membrane cytoskeleton and associate with
BODIPY-Cer vesicles near Golgi membranes
CHC-1/AP-1 and SLs could act sequentially or concomitantly on
the same vesicle population or on different vesicles traveling
along the same or an associated route. To assess their potential
physical interaction in polarized trafficking, the subcellular
distribution of vesicle- and plasma membrane-associated clathrin
and SLs was examined during wild-type intestinal development.
An intestine-specific vha-6p-gfp::chc-1 transgene generates pancytoplasmic GFP puncta, which overlay a CHC-1 antibody and
a chc-1p-gfp::chc-1 transgene that partially rescues clathrin
function-defective dnj-25(RNAi) animals (Greener et al., 2001;
Sato et al., 2009). From approximately the time of intestinal
lumen formation, GFP::CHC-1 puncta asymmetrically
assembled in a linear fashion along the apical membrane,
assuming their adult intestinal expression pattern (Fig. 6A,B).
Unexpectedly, these clathrin puncta were found to collect
underneath the ERM-1-associated submembranous cytoskeleton,
suggesting that they are not coated pits and might serve other
than endocytic functions (Fig. 6B; supplementary material Fig.
S8A).
BODIPY-labeled ceramide [BODIPY-Cer; Cer is the
immediate precursor of glucosylceramide (GlcCer), the GSL
backbone] and NBD-labeled GlcCer, when fed to C. elegans,
DEVELOPMENT
complete. L1-initiated chc-1, aps-1 and apb-1, but not apa-2, RNAi
causes a mild phenotype with basolateral ERM-1::GFP
misplacement and/or ectopic lumens in ~5-40% of animals
(supplementary material Fig. S1B). This phenotype was
dominantly enhanced in let-767(s2176);sDp3 and let767(s2819);sDp3 animals carrying the duplication (themselves
wild type in appearance) (Fig. 5A).
The converse scenario of chc-1(b1025);sptl-1(RNAi) and chc1(b1025);let-767(RNAi) double mutants also demonstrated
enhancement. sptl-1 encodes serine palmitoyltransferase, which
catalyzes the first step in SL biosynthesis, and has a more severe
RNAi polarity phenotype than let-767(RNAi) (Zhang et al.,
2011). At the permissive temperature, chc-1(b1025) displays a
mild polarity phenotype with ectopic lumens in ~90% of late
embryos or larvae (see above; Fig. 5B,D, Emb-EL, L1-EL),
whereas sptl-1(RNAi) and let-767(RNAi) animals arrest as L1
larvae with ERM-1::GFP basolateral mislocalization, followed
by its enrichment at apicolateral angles where ectopic lumens
subsequently emerge (Fig. 5B,D, L1-B). In chc-1(b1025);sptl1(RNAi) and chc-1(b1025);let-767(RNAi) animals, 10-20%
embryos displayed basolateral ERM-1::GFP displacement (in
addition to ectopic lumens) and 30-50% of L1 larvae (~80 hours
after hatching) displayed enlarged ectopic lumens or multiple
small ectopic lumens with coincident basolateral ERM-1::GFP
displacement, all rarely seen in either single mutant/RNAi
condition (Fig. 5B,D, Emb-EL/B, L1-EL/B). The ability of mild
chc-1 loss to accelerate the development of the SL loss-mediated
polarity phenotype was also reflected in the earlier arrest of chc1(b1025);sptl-1(RNAi) double mutants (embryonic versus larval
lethality; Fig. 5C).
2078 RESEARCH ARTICLE
Development 139 (11)
also localize to intestinal puncta and additionally label the
lumenal membrane (Zhang et al., 2011). GSLs are enriched at,
and function on, lumenal leaflets of Golgi, vesicle and plasma
membranes (Simons and Gerl, 2010). Several lines of evidence
indicated that exogenous BODIPY-Cer at least partially reflects
the endogenous location and function of GSLs: red and green
fluorescent BODIPY-Cer colocalized at apical membranes and
formed fully overlapping puncta and ring structures with
reciprocal bleaching of fluorescence, suggesting that they share
endomembranes and plasma membranes (supplementary material
Fig. S8D, left column); BODIPY-Cer puncta overlapped NBDGlcCer
and
BODIPY-sphingomyelin
(SM)
puncta
(supplementary material Fig. S8D, middle columns) and
colocalized with endosomal markers (see below); BODIPY-Cer
and NBD-GlcCer partially rescue SL-dependent polarity defects
and are themselves displaced during polarity conversion (Zhang
et al., 2011).
The plasma membrane-associated position of BODIPY-Cer was
found to be apical of ERM-1, overlapping the integral membrane
protein AQP-4, and slightly extending on the lumenal side (where
it collected in small puncta; Fig. 6C; supplementary material Fig.
S8B). Cytoplasmic BODIPY-Cer vesicles, like GFP::CHC-1
vesicles, formed perinuclear patterns juxtaposed to MANS+ Golgi
membranes (Fig. 6D-E). BODIPY-Cer vesicle subfractions
partially or fully overlapped RAB-11 puncta and were surrounded
by RAB-7 rings, but did not colocalize with RAB-5+ or RAB-10+
endosomes or LMP-1+ lysosomes (supplementary material Fig.
S8D; Fig. S9A,B).
We assessed BODIPY-Cer and GFP::CHC-1 colocalization by
sequential confocal scanning of thin sections (to exclude falsepositive overlay) and tightening of channel spectra to reduce
autofluorescence (small vesicles are preferentially lost with this
approach; see Materials and methods). Under these conditions, more
than 50% of cytoplasmic BODIPY-Cer vesicles associated (partially
overlapped or were in contact) with GFP::CHC-1 vesicles (versus
~10% of GFP::CHC-1 vesicles associating with BODIPY-Cer
vesicles; Fig. 6F,G). Perinuclearly, the association increased to over
70% for BODIPY-Cer vesicles (and ~30% for GFP::CHC-1
vesicles).
We conclude that the subcellular localization and partial
association of BODIPY-Cer and GFP::CHC-1 are compatible with
the possibility that clathrin and vesicle SLs interact at one or
several steps on an apically directed post-Golgi vesicular
trafficking route during lumenal membrane biogenesis.
DEVELOPMENT
Fig. 5. Genetic interactions
between clathrin/AP-1 and SLbiosynthetic enzymes. (A)Dominant
enhancement of chc-1, apb-1 and
aps-1(larval RNAi) polarity phenotypes
by two let-767 alleles (s2176 and
s2819). Only let-767;sDp3 animals
carrying the duplication are shown.
Note absence of interaction with apa2. Mean ± s.d. is shown; n3 (N>200
animals per experiment); *P<0.05,
two-tailed t-test. (B,C)Enhancement
of let-767(RNAi) and sptl-1(RNAi)
polarity defects (B) and lethality (C) in
chc-1(b1025-16°C) mutants. Progeny
were evaluated prior to full phenotype
development [80 hours (B) and 104
hours (C) after egg laying]. Note the
absence of wild-type polarity and the
appearance of novel phenotypes in
double mutant/RNAi animals (B) and
the increased lethality in chc1(b1025);sptl-1(RNAi) animals (C).
Emb, embryo; L1, L1 stage larva; EL,
ectopic lumen; B, basolateral ERM-1
::GFP displacement; Emb-EL/B and L1EL/B, novel enhanced phenotypes
(indicated by color). (D)Confocal
images of representative phenotypes
shown in B. Large arrows, basolateral
ERM-1::GFP displacement; small
arrows, apicolateral ERM-1::GFP
accumulation; arrowheads, ectopic
lumens; thin arrow, large ectopic
lumen.
Clathrin and AP-1 in apical polarity
RESEARCH ARTICLE 2079
The asymmetric distribution of GFP::CHC-1 and
BODIPY-Cer vesicles to polarized membrane
domains is dependent on each other and on AP-1
To examine whether the distribution and partial association of
GFP::CHC-1 and BODIPY-Cer vesicles was specific and relevant
to polarized membrane biogenesis, we followed their subcellular
localization during polarity conversion subsequent to perturbing
clathrin/AP-1 or SL biosynthesis. Interference with SL biosynthesis
displaced GFP::CHC-1 vesicles to lateral membranes during early
stages, and to ectopic lumenal membranes during later stages of
polarity conversion, and it decreased the submembranous apical
clathrin population and the overall number of clathrin vesicles (Fig.
7A; supplementary material Fig. S10A). Conversely, clathrin
depletion basolaterally misassembled Cer vesicles and decreased
their overall number (Fig. 7B). BODIPY-Cer became displaced to
ectopic lateral lumens, lateral to the lateralized ERM-1::GFP,
during late stage polarity conversion.
We next asked whether the association and co-dependent polarized
distribution of GFP::CHC-1 and BODIPY-Cer vesicles was AP-1
dependent. aps-1 but not apa-2 RNAi displaced GFP::CHC-1 and
BODIPY-Cer to basolateral membranes and to ectopic lateral lumens
(Fig. 7C). aps-1 RNAi also reduced the apical membrane-associated
GFP::CHC-1 population and the number of GFP::CHC-1 vesicles
(Fig. 7C; supplementary material Fig. S10A), and it abolished the
perinuclear assembly of GFP::CHC-1 and BODIPY-Cer vesicles,
eliminating their Golgi-proximal association (Fig. 7C).
To determine whether GFP::CHC-1 and BODIPY-Cer vesicles
might be secondarily recruited to an apically transformed lateral
membrane domain during polarity conversion, the temporal
relationship between vesicle misrouting and the displacement of
apical membrane components was examined. The lateral
displacement of BODIPY-Cer upon aps-1 RNAi was found to occur
independently of ERM-1::GFP basolateral displacement
(supplementary material Fig. S10B,C). Moreover, laterally assembled
DEVELOPMENT
Fig. 6. Subcellular localization and
association of GFP::CHC-1 and
BODIPY-Cer in wild-type
intestines. (A)Confocal and
Nomarski images of comma (left) and
2-fold (right) embryo with GFP::CHC1 vesicles assembling at the intestinal
lumen (arrows). Intestinal width is
indicated by arrowheads. (B)Punctate
and linear GFP::CHC-1 assembles
underneath the lumenal cytoskeleton,
adjacent to, but not overlapping
with, ERM-1::Cherry (higher
magnification in inset), and assembles
perinuclearly (vha-6-gfp::chc-1
shown). N, nucleus. (C)BODIPY-Cer
colocalizes with transmembranous
AQP-4::GFP at the apical/lumenal
membrane. (Top) Isolated single
lumenal membrane sleeve (equivalent
to boxed area, lower left; compare
with supplementary material Fig.
S8B). Note that BODIPY-Cer extends
into the lumen (arrows). (Bottom)
Both lumenal membrane sleeves.
Note the irregular BODIPY-Cer puncta
and patches in the lumen, adjacent to
the apical membrane (arrows).
(D)GFP::CHC-1 assembles with
BODIPY-Cer perinuclearly (for clarity,
one nucleus is circled and one
arrowed). Note the weak perinuclear
GFP::CHC-1 rings decorated with
BODIPY-Cer vesicles (arrows, right).
(E)BODIPY-Cer vesicles close to, but
not overlapping, MANS::GFP-labeled
Golgi membranes. Note the identical
perinuclear pattern as in D (examples
of nuclei indicated as in D). (F)A
subset of BODIPY-Cer vesicles
partially overlaps GFP::CHC-1
(yellow). Boxed region is shown at
higher magnification in the inset.
(G)Quantification of cytoplasmic and
perinuclear vesicle association. Mean
± s.e.m. (n3, N>200 vesicles per
counting).
2080 RESEARCH ARTICLE
Development 139 (11)
BODIPY-Cer vesicles occasionally overlapped with transient ERM1::GFP vesicles that formed prior to the basolateral displacement of
ERM-1::GFP at the initial stage of polarity conversion
(supplementary material Fig. S10B,C) (Zhang et al., 2011).
We conclude that the polarized membrane association of
GFP::CHC-1 and BODIPY-Cer vesicles is dependent on each other
and on AP-1, raising the possibility that SL-rich vesicle membranes,
at least transiently, recruit clathrin through AP-1 at Golgi and/or postGolgi endosomal membranes to generate an apically directed vesicle
population. The displacement of these vesicles early during polarity
conversion is compatible with their initial and direct contribution to
membrane polarization and lumen biogenesis.
DISCUSSION
Clathrin/AP-1 regulate apical polarity and lumen
formation in the developing C. elegans intestine
Clathrin functions on many, particularly endocytic, trafficking
routes, but has not been implicated on biosynthetic routes towards
the apical membrane (McMahon and Boucrot, 2011). Recently,
however, clathrin was shown to regulate basolateral polarity in
mammalian epithelial cell lines, largely dependent on its epithelial
cell-specific adaptor AP-1B, characterized by its mu1B subunit
(Deborde et al., 2008; Folsch et al., 1999). Our findings now reveal
a role for clathrin/AP-1 in the regulation of apical polarity in the
expanding C. elegans intestinal epithelium. The requirement of
several AP-1 subunits for apical polarity suggests a sorting function
that cannot be exclusively attributed to subunit specificity. We and
others (Shafaq-Zadah et al., 2012) note, however, that RNAi with
APM-1/mu but not UNC-101/mu [which are both ubiquitously
expressed and equally similar to mu1A and mu1B (Shim et al.,
2000)] generates apical polarity defects [as confirmed in a
presumed unc-101 null mutant (Shafaq-Zadah et al., 2012)]. UNC101 directs polarized vesicular transport along dendrites, which,
although anterograde, might involve a basolateral component
(Dwyer et al., 2001). A different mu/UNC-101-specific sorting
function, perhaps one that is tissue specific, is thus not excluded.
Basolateral sorting appears to be also affected by loss of CHC1/AP-1, albeit to a lesser degree in our hands. Although apical mis-
DEVELOPMENT
Fig. 7. Interdependent and AP-1-dependent polarized distribution of GFP::CHC-1 and BODIPY-Cer vesicles. Confocal images of control
(left) versus early (middle) and late (right) stage mutant/RNAi L1 intestinal polarity phenotypes (except middle image in B, which shows a late stage
phenotype). (A)GFP::CHC-1 misplacement to basolateral (short arrows) and ectopic lumenal membranes (arrowheads) by sptl-1 RNAi. Note the
concomitant GFP::CHC-1 reduction at apical membrane (long arrows) and nuclei (N) (obscured by ectopic lumens in late stage phenotype).
(B)BODIPY-Cer misplacement to ectopic lateral lumens (arrowheads) in chc-1(b1025) on the lateral/lumenal side of ERM-1::GFP. Note the
concomitant apical BODIPY-Cer loss (long arrows; wild-type lumenal membrane staining is obscured by intralumenal BODIPY-Cer patches; compare
with Fig. 6C). (C)GFP::CHC-1 and BODIPY-Cer vesicle biogenesis, localization and association require APS-1. (Left column) Wild-type GFP::CHC-1
and BODIPY-Cer vesicles assemble perinuclearly (examples indicated by circles, arrows) and at the lumenal membrane. (Middle column) Basolateral
displacement of GFP::CHC-1 (long arrows; note placement on both sides of lateral membrane indicated by double arrow) and BODIPY-Cer vesicles
(short arrows) during early polarity conversion. (Right column) GFP::CHC-1 and BODIPY-Cer assemble at ectopic lumens (arrowheads) during later
stages of polarity conversion; GFP::CHC-1 is placed at the cytoplasmic side and BODIPY-Cer at the lumenal side of ectopic lumens.
sorting might secondarily affect basolateral membrane components,
a role of CHC-1/AP-1 in both apical and basolateral transport could
be envisioned as an early sorting step requiring additional signals,
or as a directional switch of apical and basolateral cargo or vesicles.
In vivo interference with CHC-1 and several AP-1 subunits,
however, primarily causes an apical polarity defect in the C.
elegans intestine, with complete transformation of lateral into
apical membrane domains. This phenotype closely resembles the
C. elegans intestinal polarity defect caused by the depletion of
GSLs, which are membrane lipids with a documented role in
apical sorting (Simons and Gerl, 2010; Zhang et al., 2011). Loss
of CHC-1/AP-1 revealed a similar, apparently junctionindependent, conversion of apicobasal membrane domain
identities, with subsequent ectopic lumen formation, likewise
suggesting a defect in apical sorting. This phenotype furthermore
resembles the microvillus inclusion disease phenotype, which
has also been linked to polarized trafficking based on its
underlying genetic mutations in the unconventional myosin
MYO5B in humans and RAB8 in mice (Cutz et al., 1989; Muller
et al., 2008; Sato et al., 2007).
Clathrin/AP-1 cooperate with SLs in the regulation
of polarity and may function on an apical route
In principle, two trafficking routes could be perturbed by a
clathrin/AP-1- and SL-dependent sorting defect: (1) an apical
biosynthetic/exocytic route (direct, transcytotic or recycling),
delivering apical cargo to (or back to) the apical membrane (and/or
its junctions); or (2) a basolateral endocytic route, removing apical
membrane (or junction) components from the basolateral
membrane (this argument disregards the possibility of defects in
the transport of polarized membrane domain inhibitors).
The following findings support a role of CHC-1/AP-1 on a
membrane-directed apical route: CHC-1/AP-1 depletion
phenocopies the polarity defect induced by the loss of GSLbiosynthetic enzymes that genetically interact; it induces apical loss
and basolateral gain of apical characteristics on expanding
membranes, compatible with apical misrouting; it removes apical
membrane-forming (such as RAB-11) vesicles from the lumen;
AP-1 loss depletes clathrin-coated vesicles and SLs from the lumen
and misplaces both to the basolateral membrane; and CHC-1
associates with Cer-rich vesicles near the Golgi and the endocytic
recycling compartment, which are documented sorting stations for
membrane-directed transport (Rodriguez-Boulan et al., 2005). We
have no evidence for a role of the clathrin endocytic AP-2 adaptor
in this process, although its function in the C. elegans intestine is
unclear and was only tested here by RNAi (of note, apa-2 RNAi
was, however, able to enhance clathrin lethality; data not shown).
Together, these findings could suggest a scenario in which SL-rich
membrane microdomains recruit AP-1/clathrin at Golgi and/or
post-Golgi endosomal membranes to regulate apical sorting. A
similar process was recently proposed for PtdIns(3,4,5)P3 (PIP3)
recruiting AP-1B to recycling endosomes to regulate basolateral
sorting (Fields et al., 2009).
This interpretation has several implications. First, it places
clathrin/AP-1 on a novel apical biosynthetic route, whereas their
role in the removal of apical membrane components from
basolateral membranes would place these molecules on previously
established endocytic and perhaps endosome-to-lysosome-directed
routes. However, the anterograde sorting function of CHC-1/AP-1
at the Golgi, although not apically directed, is well characterized
(Sachse et al., 2002). There is also evidence for the presence and
function of clathrin/AP-1 on other apically destined vesicle
RESEARCH ARTICLE 2081
populations: Drosophila AP-1 localizes to both Golgi and
endosomal membranes and colocalizes with RAB-11 (Benhra et
al., 2011); AP-1 functions in the biogenesis of apically moving
secretory granules (Burgess et al., 2011; Lui-Roberts et al., 2005);
and a CHC-1/AP-1-dependent plasma membrane-directed
secretory endo-lysosomal compartment was recently characterized
(Laulagnier et al., 2011).
Second, it implies a convergence of clathrin-dependent and
SL/lipid microdomain (raft)-dependent trafficking pathways, which
are currently thought of as distinct trafficking machineries in
endocytosis (Grant and Donaldson, 2009; Le Roy and Wrana,
2005). However, clathrin/AP-1-dependent sorting functions of SLs
have been reported on plasma, Golgi and endosomal membranes
that include apical trafficking routes (Masuyama et al., 2009; Puri
et al., 2001). Of note, clathrin-independently endocytosed
BODIPY-Cer (as used in this study) was found on clathrindependently endocytosed vesicles, and Cer-enriched microdomains
were returned to the plasma membrane via RAB-11 recycling
endosomes, demonstrating that these two components can converge
on a single apically directed vesicle (Sharma et al., 2003).
Third, it suggests that the submembranous apical string of
GFP::CHC-1 vesicles, recruited to the lumen during its biogenesis,
contains apically targeted vesicles. Plasma membrane-associated
fluorescently labeled clathrin vesicles are generally interpreted as
coated pits involved in AP-2-dependent endocytosis (Greener et al.,
2001; Sato et al., 2009). TEM, live-cell imaging and singlemolecule tracking are, however, currently redefining clathrin coats
and clathrin vesicle populations at plasma membranes, some of
which are AP-1 associated (Keyel et al., 2004; Mattheyses et al.,
2011; Saffarian et al., 2009).
Clathrin-coated and SL-rich vesicles assemble
at polarized domains of expanding plasma
membranes in a co-dependent and AP-1dependent manner
CHC-1/AP-1-mediated sorting could either directly deliver
multiple apical membrane components or could secondarily
determine apical membrane domains through the direct or indirect
recruitment of specific polarity determinants. PAR-6 is an obvious
candidate downstream effector for this apical polarity pathway,
possibly recruited through CDC-42 (see Shafaq-Zadah et al., 2012).
Another candidate effector is ERM-1/ezrin, which is proposed to
be sufficient to initiate apical membrane and microvillus biogenesis
(ten Klooster et al., 2009; Zhu et al., 2010). Lateral displacement
of such molecules could initiate the transformation of lateral into
apical domains and generate lateral lumens.
Intriguingly, however, we find that the interference with CHC1/AP-1 and/or SL biosynthesis not only misdirects polarized
membrane components, but also mislocalizes entire vesicle
populations during polarized membrane biogenesis. Moreover, the
assembly of Cer-labeled vesicles at ectopic sites of apical
membrane biogenesis during the initial phase of polarity
conversion in the C. elegans intestine raises the possibility that
these vesicles are primary effectors of membrane polarization,
rather than being secondarily recruited or generated by the apical
or apically altered lateral membrane domain. The sorting process
itself might thus directly contribute to determining polarized
plasma membrane domains, with CHC-1/AP-1 conferring
directional cues to the vesicles themselves. AP-1/CHC-1
recruitment by SL-rich vesicle membranes could, for example,
choose, set in place, or enable vesicles to use a specific actomyosin
or microtubule track for their directional movement to, or back to,
DEVELOPMENT
Clathrin and AP-1 in apical polarity
the apical membrane. For instance, both clathrin and AP-1 have
been implicated in microtubule connections: the N-terminus of
CHC binds directly to the spindle, serving the traffickingindependent role of clathrin in mitosis (Royle et al., 2005); and the
AP-1 accessory molecule Gadkin associates with the kinesin motor
KIF5, directly linking AP-1-associated vesicles and microtubules
in mammalian cells (Schmidt et al., 2009). Directional vesicle
movements during apical membrane and lumen biogenesis have
been observed early on, in vivo and in vitro, and include the shift
of the entire trans-Golgi endomembrane system during MDCK
polarity conversion towards the new apical membrane (Wang et al.,
1990; Rodriguez-Fraticelli et al., 2011).
Acknowledgements
We thank D. Baillie (Fraser University, Burnaby, BC, Canada), H. Fares
(University of Arizona, Tucson, AZ, USA), K. Kemphues (Cornell University,
Ithaca, NY, USA), T. Kurzchalia (Max Planck Institute of Molecular Cell Biology
and Genetics, Dresden, Germany), T. Lamitina (University of Pennsylvania,
Philadelphia, PA, USA), K. Nehrke (University of Rochester Medical Center,
Rochester, NY, USA), G. Ruvkun (Massachusetts General Hospital, Harvard
Medical School, Boston, MA, USA), J. Simske (Case Western Reserve University
School of Medicine, Cleveland, OH, USA), K. Strange (Vanderbilt University
Medical Center, Salisbury Cove, ME, USA), the Caenorhabditis Genetics Center
(NIH Center for Research Resources) and S. Mitani (National Bioresource
Project, Tokyo University, Japan) for strains, plasmids and antibodies and in
particular B. Grant (Rutgers University, Piscataway, NJ, USA) for kindly
providing numerous strains pertinent to this study; G. Ruvkun for the lethal
RNAi library; M. McKee (MGH Microscopy Core/partially funded by the IBD
grant DK43351 and BA DE award DK57521) for TEM work; E. Membreno for
C. elegans maintenance; and H. Weinstein and A. Walker (Massachusetts
General Hospital) for ongoing support.
Funding
This work was supported by the National Institutes of Health [grants
HD044589 and GM078653] and a Mattina R. Proctor Award to V.G.
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.077347/-/DC1
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