Download Direct interaction of two polarity complexes implicated in epithelial

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Direct interaction of two polarity
complexes implicated in epithelial
tight junction assembly
Toby W. Hurd*||, Lin Gao†||, Michael H. Roh‡, Ian G. Macara†# and Ben Margolis*‡§
*Howard Hughes Medical Institute, ‡Departments of Biological Chemistry and §Internal Medicine, University of Michigan Medical School, Ann Arbor,
MI 48109-0650, USA
†Center for Cell Signalling, University of Virginia Health Sciences Center, Charlottesville, VA 22908-0577, USA
||These authors contributed equally to this work
e-mail: ¶[email protected] or #[email protected]
Published online: 27 January 2003; DOI: 10.1038/ncb923
Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and
basolateral membranes. Two evolutionarily conserved multiprotein complexes, Crumbs (Crb)–PALS1 (Stardust)–PATJ
(DiscsLost)1–4 and Cdc42–Par6–Par3–atypical protein
kinase C (aPKC), have been implicated in the assembly of
tight junctions and in polarization of Drosophila
melanogaster epithelia5–8. Here we identify a biochemical
and functional link between these two complexes that is
mediated by Par6 and PALS1 (proteins associated with
Lin7). The interaction between Par6 and PALS1 is direct,
requires the amino terminus of PALS1 and the PDZ domain
of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6
with a mutated PDZ domain, to the cell surface. Expression
of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of
PALS1, members of the Par3–Par6–aPKC complex and the
tight junction marker, ZO-1. Similarly, overexpression of
Par6 in MDCK cells inhibits localization of PALS1 to the tight
junction. Our data highlight a previously unrecognized link
between protein complexes that are essential for epithelial
polarity and formation of tight junctions.
or Sdt cause polarity defects in D. melanogaster epithelia12. The
mammalian homologue of Sdt is PALS14. Similar to Sdt, PALS1
interacts with the tail of mammalian Crb isoforms through its PDZ
domain. PALS1 utilizes an L27 domain to interact with a multi-PDZ
domain protein called PATJ4,13. Similarly, Discs Lost1, a D.
melanogaster orthologue of PATJ, can interact with an L27 domain
from Sdt4. Thus, analogous complexes containing Crb–PALS1–PATJ
a
L
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IP
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IP
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IP
HA–Par3
HA–Par6
Myc–PALS1
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HA–Par6
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Myc–PALS1 –
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pithelial cell lines provide an excellent model system for
exploring the molecular basis of how polarity is established
and maintained. Polarized epithelial cells contain distinct apical and basolateral membranes, each with their own complement of
cell-surface proteins9 and a tight junctional seal at the superior
aspect of the lateral surface to prevent admixture of membranes.
Genetic and biochemical evidence has implicated multiple protein
complexes in the assembly and maintenance of tight junctions.
Many components of these complexes possess post-synaptic density 95/discs large/zona occludens 1 (PDZ) domains. PDZ domains
commonly, but not exclusively, bind to the extreme carboxyl terminus of proteins and are often found in membrane-associated
guanylate kinase (MAGUK) proteins, where they are paired with
Src-homology 3 (SH3) and guanylate kinase domains10. MAGUK
proteins are crucial for several aspects of cell polarity11.
One MAGUK protein required for cell polarity in D.
melanogaster epithelia is Stardust (Sdt)2,3. Sdt interacts with the
transmembrane protein Crb through its PDZ domain. The two
proteins colocalize in a subapical complex at the top of the lateral
membrane, corresponding to the site in the epithelia of higher
organisms where the tight junction forms. Mutations in either Crb
IP
Pre
Pre
IP
IP
Par3
PALS1
E
PATJ
Par3
Myc–PALS1
IP: Par6
IP: Myc–PALS1
Figure 1 Par6 binds PALS1. a, Myc–PALS1 immunoprecipitates HA–Par6 , but not
HA–Par3, in the absence of HA–Par6 . Myc–PALS1 was immunoprecipitated from
HEK293 cells transiently transfected with Myc–PALS1, HA–Par6wt and/or HA–Par3,
and subsequently immunoblotted for HA–Par3, HA–Par6 and Myc–PALS1. Protein
expression levels in cell lysates (L) are shown adjacent to the Myc–PALS1 immunoprecipitations (IP). In the control, anti-Myc immunoprecipitations were performed on
untransfected cells. b, Immunoprecipitation of endogenous Par6 coprecipitates
endogenous Par3 and PALS1. Par6 was immunoprecipitated from cell lysates of
wild-type MDCK I cells and immunoblotted for endogenous Par3 and PALS1. c,
Myc–PALS1 immunoprecipitates endogenous Par3 from MDCK cell extracts.
Myc–PALS1 was immunoprecipitated from cell lysates of MDCK cells stably
expressing Myc–PALS1and immunoblotted for endogenous Par3 and PATJ. In the
control lane (Pre), rabbit pre-immune serum was used to perform the immunoprecipitations. Protein expression levels in cell lysates are also shown.
NATURE CELL BIOLOGY VOL 5 FEBRUARY 2003 www.nature.com/naturecellbiology
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137
L
L
Myc–PALS1(∆GUK)
Myc–PALS1(∆SH3)
Myc–PALS1(∆PDZ)
Myc–PALS1wt
HA–Par6
HA–Par6
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mut.PDZ
Myc–PALS1
102–371
GST–PALS1(1–181)
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GUK
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lysate IP: HA–Par6
PDZ
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CRIB
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letters
HA–Par3
Myc–PALS1
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lysate
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Bound to
glutathione beads
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HA–Par6
GST–PALS1(1–181)
Blot: anti-HA
Cell lysate
50% input
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HA–Par6(102–371)
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g
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HA–Par6
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Myc–Cdc42
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Cell lysate
HA–Par6
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Myc–PALS1
+
+
+
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HA–Par6
–
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+
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–
Q61L
T17N
Myc–Cdc42
Figure 2 Characterization of Par6–PALS1 interaction. a, The domain structure
of PALS1 and Par6. b, Deletion of the N-terminal 181 residues of PALS1 prevents
binding of Par6. HA–Par6 was immunoprecipitated from HEK293 cells transiently
transfected with HA–Par6 Myc–PALS1wt or PALS1 lacking the U1 and L27N, PDZ,
SH3 or GUK domains. Subsequent immunoprecipitates were blotted for
Myc–PALS1and HA–Par6 . Myc–PALS1 expression levels were determined by
immunoblotting cell lysates (bottom). c, Par6 binds to the N terminus of PALS1.
A recombinant GST fusion protein consisting of the U1 and L27N domains of
PALS1 (GST–PALS1(1–181)) or GST alone were immobilized on agarose beads and
incubated with cell extract from HEK293 cells transiently transfected with HA–Par6,
HA–Par3 or Myc–PATJ. Precipitating proteins were detected by immunoblotting for
HA–Par6 , HA–Par3 or Myc–PATJ. Cell lysates were also immunoblotted to monitor
protein expression levels. d, Coprecipitation of Myc–PALS1and HA–Par6 . Cos7
cells were cotransfected with Myc–PALS1 and different HA-tagged Par6 constructs.
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HA–Par6 was immunoprecipitated and bound Myc–PALS1 was detected by
immunoblotting. The Par6 PDZ mutant (mut.PDZ) harbours KPLG167–170AAAA
mutations. e, The Par6 PDZ domain is required for efficient interaction with the
PALS1 N terminus. GST–PALS1(1–181) was immobilized on agarose beads and
incubated with cell lysates from HEK293 cells transiently transfected with either full
length HA–Par6(1–371), HA–Par6(102–371), HA–Par6(1–271) or HA–Par6
(mut.PDZ). Expression levels of the HA–Par6 mutants are also shown (bottom).
f, Direct interaction of PALS1 with Par6. Purified GST or GST–PALS1(1–181) was
bound to glutathione beads and incubated with purified S–Par6(1–271). GST proteins were detected using anti-GST antibody and S–Par6(1–181) was detected
using HRP-tagged S protein. g, The PALS1–Par6 interaction is regulated by Cdc42.
Cos7 cells were cotransfected with Myc–PALS1and HA–Par6 in the absence or
presence Cdc42Q61L or Cdc42T17N. HA–Par6 was immunoprecipitated and bound
Myc–PALS1 was detected by immunoblotting.
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letters
and Crb–Sdt–DiscsLost (Dlt) are found in mammalian and
D. melanogaster epithelia, respectively14.
A different complex, which colocalizes with PALS1 and Sdt in
D. melanogaster and mammalian epithelia, is composed of Par3,
Par6, aPKC and Cdc42-GTP5–8. The Par3–Par6–aPKC complex is
important for determining polarity in many cell types, including
D. melanogaster neuroblasts, the Caenorhabditis elegans zygote and
mammalian epithelial cells11,15,16. Par6 contains a PDZ domain and
an adjacent Cdc42/Rac-interactive binding domain (CRIB)-like
motif, both of which are required for binding to the small GTPases
Cdc42 and Rac7,8,17. The N terminus of Par6 binds aPKC-λ and ζ7,8,16. In turn, both aPKCs and Par6 can interact with a multi-PDZ
domain protein called Par3, also known as atypical PKC isotypespecific interacting protein (ASIP) and Bazooka18,19.
The spatial and functional similarities of Crb–PALS1–PATJ and
Par3–Par6–aPKC led us to examine whether these complexes
interact. First, we transiently expressed Myc–PALS1 with haemagglutinin (HA)–Par6 or HA–Par3 in HEK 293 cells and found that
Par6, but not Par3, co-immunoprecipitated with PALS1 (Fig. 1a).
However, when all three proteins were expressed together, we
detected a complex of PALS1 with both Par6 and Par3.
Importantly, we could detect endogenous PALS1 in an immunoprecipitation of endogenous Par6 from MDCK cells (Fig. 1b).
Although Fig. 1b demonstrates that the antibodies we raised detect
PALS1 in MDCK cells by immunoblotting, they are not useful for
immunoprecipitation. Accordingly, we used a MDCK cell line
expressing Myc–PALS1 and found that endogenous Par3 and PATJ
were both co-immunoprecipitated with Myc–PALS1 (Fig. 1c).
Collectively, these data indicate that the Crb–PALS1–PATJ and
Par3–Par6–aPKC complexes can associate with one another
through a PALS1–Par6 interaction.
PALS1 is a multi-domain protein (Fig. 2a). To identify the
region of PALS1 that interacts with Par6, we tested the ability of
various Myc–PALS1 deletion mutants to co-immunoprecipitate
with HA–Par6. We found that removal of the N terminus of PALS1,
which contains the U1 and L27N domains, reduced binding to Par6
(Fig. 2b). Furthermore, a glutathione S-transferase (GST) fusion
protein containing the U1 and L27N domains (PALS1(1–181))
could bind to overexpressed Par6, but not to Par3 (Fig. 2c).
However, GST–L27N alone could not bind Par6 (data not shown),
suggesting that the binding site for Par6 exists in the PALS1 U1
domain.
Neither the N-terminal aPKC-binding site nor the C terminus
of Par6 were required for PALS1 association, but mutation of the
Par6 PDZ domain reproducibly reduced binding to PALS1 (Fig. 2d,
e). Importantly, bacterially expressed GST–PALS1(1–181) bound
specifically to S-tagged Par6(1–271), demonstrating that the interaction is direct (Fig. 2f). Next, we co-expressed PALS1 and Par6
with the constitutively active Cdc42 mutant, Cdc42Q61L, which
binds to Par6, or with inactive Cdc42 mutant, Cdc42T17N, which
does not. Interestingly, Cdc42Q61L (which is constitutively GTPbound) markedly enhanced the association of PALS1 with Par6,
suggesting the interaction might be controlled by cell signalling
events (Fig. 2g).
We then examined the functional implications of interactions
between Par6 and the Crb–PALS1–PATJ complex. We demonstrated that Crb3 is endogenously expressed in MDCK cells and exists
in a complex with PATJ and PALS1 (ref. 20). When co-expressed
with Myc–Crb3 in HeLa cells, Par6 was recruited to the plasma
membrane (Fig. 3a). However, Par6 containing the mutant PDZ
domain was not recruited (Fig. 3b). Conversely, Crb3-∆ERLI, a
Crb3 mutant that lacks four amino acid residues at its C terminus
and cannot bind PALS1, was unable to recruit Par6 to the membrane (Fig. 3c). Therefore, we suggest that Crb3 promotes recruitment of Par6 to the membrane through PALS1.
Finally, we examined if components of the Crb–PALS1–PATJ or
Par3–Par6–aPKC complexes were mislocalized in MDCK cells
expressing dominant-negative mutants that disrupt these polarity
a
Myc–CRB3
HA–Par6
Anti-Myc
Anti–HA
Myc–CRB3/
HA–Par6
Myc–CRB3/
HA–Par6
Merge
b
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Anti-Myc
Anti–HA
Myc–CRB3/
HA–Par6(mut.PDZ)
Myc–CRB3/
HA–Par6(mut.PDZ)
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Myc–CRB3-∆ERLI
Anti-Myc
Anti–HA
Myc–CRB3-∆ERLI/
HA–Par6
Myc–CRB3-∆ERLI/
HA–Par6
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Figure 3 CRB3 recruits Par6 to the plasma membrane. a, Myc–Crb3 recruits
HA–Par6 to the plasma membrane. HeLa cells were transiently transfected with
Myc–Crb3 and/or HA–Par6wt. Cells were fixed 48 h after transfection and stained
with mouse monoclonal anti-Myc (green) rabbit polyclonal anti-HA (red) antibodies.
Nuclei were stained with DAPI (blue). b, Deletion of the four C-terminal residues of
Myc–Crb3 prevents recruitment of HA–Par6 to the plasma membrane. HeLa cells
were transiently transfected with HA–Par6wt and/or Crb3 lacking the C-terminal E,
R, L and I residues (Myc–Crb3-∆ERLI). Cells were stained as in a. c, Mutation of the
Par6 PDZ domain prevents its recruitment to the plasma membrane by Myc–Crb3.
HeLa cells were transiently transfected with Myc–Crb3 and/or a Par6 containing
the KPLG167–170AAAA mutation in the PDZ-encoding region (HA–Par6 mut.PDZ).
Cells were stained as in a.
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letters
a
b
Myc–PALS1
Myc–PALS1
ZO-1
ZO-1
PALS1
aPKCζ
Myc–PATJ1(1–238)
ZO-1
PALS1
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ZO-1
aPKCζ
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ZO-1
PATJ
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d
Myc–PATJ1(1–238)
wt
Par6(wt)
PALS1
CRB3
E-cadherrin
Par6(102–371)
Par6(mut.PDZ)
PALS1
Figure 4 PATJ(1–238) disrupts aPKCζζ localization and tight junction biogenesis. a, An MDCK cell line stably expressing Myc–PATJ(1–238) was grown in lowcalcium (5 µM) media and subsequently incubated in normal-calcium (1.8 mM)
media for 24 h. Cells were then fixed, permeabilized, and immunostained with antibodies against the indicated proteins. b, MDCK cells stably expressing Myc–PALS1
or Myc–PATJ(1–238) were grown as in a. MDCK cells expressing Myc–PALS1 were
immunostained with mouse monoclonal anti-Myc (red) and rabbit polyclonal antiaPKCζ (green). Cells expressing Myc–PATJ(1–238) were stained with mouse monoclonal anti-ZO-1 (red) and rabbit polyclonal anti-aPKCζ (green). Square panels represent x-y sections, whereas rectangular panels represent x-z sections (apical to the
top, basolateral to the bottom). c, MDCK cells expressing Myc–PATJ(1–238) were
treated as in a. Subsequently, cells were immunostained with anti-E-cadherin and
anti-Crb3 antibodies (green and red, respectively). Crb3 localizes to the apical surface, whereas E-cadherin is present at the lateral membrane. d, Overexpression of
Par6 inhibits relocalization of PALS1 to cell–cell contacts during tight junction reassembly. MDCK cell lines stably expressing different Myc-tagged Par6 constructs
were subjected to calcium withdrawal after reaching confluence (as described previously). Cells were fixed 6 h after re-addition of calcium to the medium and stained
for endogenous PALS1. Untransfected cells are shown as a control.
complexes. In initial studies, we found no effects of dominantnegative PATJ on the formation of stable tight junctions4.
However, we were able to detect defects in tight junction assembly
using a calcium switch protocol. Expression of dominant-negative
PATJ(1–238) caused mis-localization of PALS1 and tight junctions
were fragmented 24 h after calcium switch, as detected by ZO-1
staining (Fig. 4a). Importantly, aPKC also mislocalized away from
the fragmented tight junctions (Fig. 4b). However, PATJ(1–238)
did not affect the global polarity of cells grown on filters, as localization of Crb3 and E-cadherin remained unchanged (Fig. 4c).
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Overexpression of wild-type Par6 or Par6-∆N, both of which disrupt tight junction assembly7,15, resulted in mis-localization of
PALS1 (Fig. 4d). In contrast, overexpression of Par6 containing a
mutated PDZ domain that binds poorly to PALS1 (Fig. 2) did not
result in the mis-localization of PALS1.
In summary, our results identify an unexpected direct interaction between PALS1 and Par6 that links two protein complexes
important for epithelial cell polarity. Crb is localized to the apical
surface of mammalian and fly epithelia and colocalizes at the tight
junction with PALS1 and PATJ2–4,21. Through PALS1, Crb may
recruit Par6 and associated proteins to the apical side of the tight
junction. Par3 also binds to the junctional adhesion molecule
(JAM-1), which may contribute to this localization22,23.
Furthermore, our results suggest that the localization of these protein complexes is a codependent process, as the disruption of one
complex affects the localization of proteins present in the other.
Consistently, components of the Par3–Par6–aPKC complex are
mislocalized in the epithelia of flies with Sdt mutations2,3. Further
studies will be necessary to determine if the absence of PALS1
affects targeting of Par3–Par6–aPKC in mammalian epithelia. Our
attempts to reduce levels of PALS1 by inhibitory RNA (siRNA)
approaches have been complicated by the fact that MDCK cells are
canine in origin and that siRNA strategies in polarized epithelial
cells are still at an early stage.
The Par3–Par6–aPKC complex is involved in polarity determination in a wide variety of tissues, including epithelia, neuroblasts,
migrating astrocytes and in Danio rerio organogenesis11,24–26. In
contrast, the Crb–PALS1–PATJ complex seems to be more specific
for epithelia, at least in D. melanogaster, suggesting it functions as a
specific adaptor assisting in the localization of the Par3–Par6–aPKC
complex in this cell type2,3. However, it remains unclear whether all
these proteins are simultaneously present in a single complex, or
whether the interactions are dynamic. For example, the PDZ
domain of Par6 binds to the N terminus of PALS1, but can also
bind to Par3 and is required for binding to Cdc42 (refs 7, 8).
Nonetheless, the interaction between PALS1 and Par3 is dependent
on Par6, indicating that Par3 and PALS1 do not compete for the
PDZ domain of Par6. The finding that a small GTPase stimulates
the interaction of PALS1 with Par6 suggests the interactions are
probably dynamic. Thus, the binding of Cdc42-GTP to the CRIB
motif may induce a conformational change in Par6 that increases
binding to PALS1. These results may also explain how small G protein signalling can contribute to the localization of the
Par3–Par6–aPKC complex, as described in other systems26–28.
Whether interaction of PALS1 with Par6 also controls downstream
signalling events, such as the modulation of aPKC activity, remains
to be determined. Nonetheless, the identification of a direct interaction between these two protein complexes is an important step in
understanding the mechanisms that promote epithelial polarity
and formation of tight junctions.
Methods
Cell culture
MDCKI and HeLa cells were grown in DMEM (Invitrogen; Carlsbad, CA) containing 100U penicillin,
100 µg ml−1 streptomycin sulphate, 2 mM L-glutamine and 10% foetal bovine serum. MDCK
Myc–Par6B, Myc–PALS1 and Myc–PATJ(1–238; refs 4,7) clones were propagated in media containing
175 µg ml−1 hygromycin B, 300 µg ml−1 G418 and 100 µg ml−1 zeocin, respectively.
Immunofluorescence microscopy and imaging
For recruitment assays, HeLa cells grown on glass coverslips were transfected with Fugene6 transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s instructions, with 0.5 µg per
well Myc–Crb3/Myc–Crb3-∆ERLI (ref. 20) and/or 0.5 µg per well HA–Par6wt/HA–Par6mut.PDZ (ref. 7)
and left for 48 h. For MDCK immunostaining, MDCK cells were seeded at high density onto 12-mm
Transwell membrane filters (0.4-µm pore size, Corning Costar, New York, NY). Coverslips and filters
were fixed in 4% formaldehyde/PBS for 20 min, permeabilized in 0.1% Triton X-100/PBS for 10 min
and blocked in 2% goat serum/PBS for 60 min. Coverslips/filters were incubated with primary antibodies in blocking solution (monoclonal anti-Myc 9E10 (1:1000), polyclonal anti-HA Y11 (1:1000),
polyclonal anti-PALS1 (1:200; ref. 4), polyclonal anti-PATJ (1:200; ref. 4), polyclonal anti-Crb3 (1:250),
monoclonal anti-E-Cadherin (Sigma, St Louis, MO; 1:1600), polyclonal anti-aPKCζ (Upstate
Biotechnology, Lake Placid, NY; 1:500) and monoclonal anti-ZO-1 (Zymed, San Francisco, CA;
1:1000)) overnight in a humidified chamber at 30 °C. After extensive washes with 2% goat serum/PBS,
filters were incubated with fluorochrome-conjugated secondary antibody (1:1,000 in 2% goat
serum/PBS) for 1 h at 30 °C. Subsequently, filters were washed several times with 2% goat serum/PBS
and mounted on glass slides with ProLong antifade reagent (Molecular Probes, Inc, Eugene, OR).
Immunofluorescence microscopy was performed at the University of Michigan Morphology and Image
Analysis Laboratory with a Zeiss LSM510 Axiovert 100M inverted confocal microscope (Carl Zeiss,
Inc., Thornwood, NY).
Immunoprecipitation and immunodetection
Plasmids (2 µg) pRK5-Myc-PALS1(wt, ∆U1 + L27N, ∆PDZ, ∆SH3 and ∆GUK), pRK5-Myc-PATJ,
pKH3-Par6B(wt, 102–371, 1–271 and mut.PDZ) and pHA3–Par3 were transfected using Fugene6 into
HEK293 cells grown to 50% confluency on 10-cm dishes. After 48 h, cells were collected in 0.5 ml lysis
buffer (50 mM Tris-HC at pH 7.4, 150 mM sodium chloride, 10% glycerol, 1% Triton X-100, 1.5 mM
magnesium chloride, 1 mM EGTA, 10 mM sodium fluoride, 10 mM Na4P2O7, 1 mM Na3VO5, 1 mM
phenyl methylsulphonyl fluoride, 10 µg ml−1 leupeptin and 20 µg ml−1 aprotinin). For MDCK
immunoprecipitation and immunodetection experiments, cells were grown to confluence on 15-cm
dishes and extracted in 1 ml of lysis buffer. Lysates were cleared by centrifugation (12,000g for 15 min
at 4 °C), with a fraction kept for immunoblotting. The remainder was used for immunoprecipitation
with monoclonal anti-Myc 9E10, polyclonal anti-HA Y11 or anti-Par6 T20 (Santa Cruz Biotechnology,
Santa Cruz, CA), as described previously. Precipitated proteins were washed three times with cold PBS
supplemented with 10% glycerol, eluted with sample buffer and resolved on 4–12% gradient bis-Tris
gels using the NuPAGE electrophoresis system (Invitrogen). Proteins were electrophoretically transferred from the gels onto nitrocellulose membranes. Membranes were blocked in 5% bovine serum
albumin/Tris-buffered saline (TBS) for 30 min and incubated with primary antibody in 5% bovine
serum albumin/TBS for 2 h at room temperature. After extensive washing with 0.1% Triton X100/TBS, membranes were soaked in TBS supplemented with 5% skimmed milk powder and secondary antibody conjugated to horseradish peroxidase. Incubation with secondary antibody was performed for 1 h at room temperature or for 4 h at 4 °C. Membranes were washed with 0.1% Triton X100/TBS and bands were visualized using ECL reagent (Amersham Biosciences, Piscataway, NJ).
For pull-down experiments, HEK293 lysates were incubated with GST or GST–PALS1(1–181)
bound to Sepharose beads overnight at 4 °C. Sepharose beads were washed and immunoblotted as for
immunoprecipitations.
RECEIVED 10 SEPTEMBER 2002; REVISED 6 NOVEMBER 2002; ACCEPTED 25 NOVEMBER 2002;
PUBLISHED 27 JANUARY 2003.
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ACKNOWLEDGMENTS
We thank S. Laurinec for antibody generation and A. Liu for assistance with tissue culture and confocal microscopy. We thank A. Saltiel for helpful discussions. We acknowledge the University of Michigan
Microscopy and Image Analysis Core for allowing us to use the confocal microscope. This work was
partially supported by grant CA40042 from the National Institutes of Health, DHHS (to I.G.M). B.M.
is an investigator of the Howard Hughes Medical Institute.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
NATURE CELL BIOLOGY VOL 5 FEBRUARY 2003 www.nature.com/naturecellbiology
©2003 Nature Publishing Group