Molecular Biology of the Cell
Vol. 13, 158–168, January 2002
Mammalian Homolog of Drosophila Tumor
Suppressor Lethal (2) Giant Larvae Interacts with
Basolateral Exocytic Machinery in Madin-Darby
Canine Kidney Cells
Anne Mu ?sch,*†David Cohen,* Charles Yeaman,‡§W. James Nelson,§
Enrique Rodriguez-Boulan,* and Patrick J. Brennwald?†
*M. Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, New
York 10021;‡Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242;
§Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford,
California 94305; and?Department of Cell and Developmental Biology, University of North Carolina,
Chapel Hill, North Carolina 27599
Submitted August 20, 2001; Revised October 8, 2001; Accepted October 20, 2001
Monitoring Editor: Vivek Malhotra
The Drosophila tumor suppressor protein lethal (2) giant larvae [l(2)gl] is involved in the
establishment of epithelial cell polarity during development. Recently, a yeast homolog of the
protein has been shown to interact with components of the post-Golgi exocytic machinery and to
regulate a late step in protein secretion. Herein, we characterize a mammalian homolog of l(2)gl,
called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role
in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after
contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphor-
ylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral
domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-
bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of
the exocytic machinery at the basolateral membrane, but not with other plasma membrane soluble
N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent
from or not restricted to the basolateral membrane domain. These data suggest that Mlgl
contributes to apico-basolateral polarity by regulating basolateral exocytosis.
The gene product of Drosophila L(2)GL is essential for de-
velopment of polarized epithelia (Manfruelli et al., 1996;
Bilder et al., 2000) and for cell polarity associated with asym-
metric cell divisions of neuroblasts during fly development
(Ohshiro et al., 2000; Peng et al., 2000). In concert with the
PDZ-proteins scribble and dlg, l(2)gl contributes to the cor-
rect targeting of apical determinants for epithelial cell po-
larity and mutations in l(2)gl lead to a loss of monolayer
organization and the formation of epithelial-derived tumors
(Gateff, 1978; Bilder et al., 2000). In dividing neuroblasts,
l(2)gl mediates the targeting of cell fate determinants to the
basal cortex, a prerequisite for generation of different neu-
ronal cell types (Ohshiro et al., 2000; Peng et al., 2000). The
failure of this differentiation event in l(2)gl mutants results
in brain tumors (Gateff, 1978). L(2)gl’s role as a tumor sup-
pressor, therefore, appears to be tightly associated with a
function in cell polarity. The molecular details of this role,
however, remain obscure.
Recently, l(2)gl homologs in yeast and mammalian neu-
ronal cells have been discovered to regulate a late step in
protein secretion by their ability to interact with the core
machinery that mediates the fusion of post-Golgi transport
vesicles with the plasma membrane (Fujita et al., 1998; Leh-
man et al., 1999). This core machinery for vesicle fusion is
comprised of soluble N-ethylmaleimide-sensitive factor at-
tachment receptor (SNARE) proteins that are associated
with transport vesicles (v-SNAREs) and the target mem-
brane (t-SNAREs), respectively (Sollner et al., 1993). The
complex of SNARE proteins is comprised of four parallel
helical bundles that are thought to position both membranes
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and provide the energy for the formation of a fusion pore
(Katz et al., 1998; Sutton et al., 1998; Weber et al., 1998). In
yeast, the l(2)gl homologs Sro7/Sro77 interact directly with
Sec9, a t-SNARE for vesicle fusion at the plasma membrane,
and loss of both gene products with homology to l(2)gl
results in a cold-sensitive growth defect with an accumula-
tion of post-Golgi transport vesicles (Lehman et al., 1999).
Likewise, the l(2)gl-related protein tomosyn was found in a
complex with the plasma membrane t-SNARE syntaxin 1 in
neuronal cells and antibodies to tomosyn inhibit the exocy-
tosis of dense core vesicles from PC12 cells in vitro (Fujita et
These data raise the interesting possibility that l(2)gl con-
tributes to cell polarity by regulating polarized exocytosis.
To test this hypothesis, we analyzed the subcellular distri-
bution of a ubiquitously expressed homolog of l(2)gl and its
interaction with post-Golgi t-SNAREs in a model epithelial
cell line, Madin-Darby canine kidney (MDCK) cells. MDCK
cells achieve cell polarity by directly targeting apical and
basolateral proteins in separate exocytic carriers to their
respective surface domains (Mostov et al., 2000). The speci-
ficity of membrane fusion events is predicted to result from
the presence of distinct plasma membrane t-SNAREs at the
apical and basolateral membrane domains. MDCK cells ex-
press the post-Golgi SNAREs syntaxin 2, 3, and 4 (Low et al.,
1996) and soluble N-ethylmaleimide-sensitive factor attach-
ment protein-23 (SNAP-23) (Low et al., 1998). Although en-
dogenous syntaxins are expressed at levels too low for im-
munodetection, overexpressed syntaxin 3 is restricted to the
apical membrane, whereas syntaxin 4 is specific for the
basolateral domain. Syntaxin 2 and SNAP-23 are uniformly
distributed on both surface domains (Low et al., 1996, 1998).
We demonstrate in this study that the MDCK cell ho-
molog of l(2)gl, called Mlgl, becomes associated with the
lateral membrane as MDCK cells establish a polarized phe-
notype and that this membrane-associated population spe-
cifically binds to the basolateral t-SNARE syntaxin 4, sug-
gesting a role for Mlgl in regulating basolateral exocytosis in
epithelial cells. We have identified a highly conserved phos-
phorylated peptide in Mlgl that plays a role in restricting the
protein to the lateral surface and preventing it from binding
to the apical surface domain.
MATERIALS AND METHODS
Cell Culture and Transfections
MDCK strain II cells were grown in DMEM ? 10% fetal calf serum.
Stable MDCK cell lines expressing syntaxin 2-hemagglutinin (HA),
syntaxin 3-Flag, or syntaxin 4-HA were generated by cotransfecting
the cDNAs in pCMV1 (syn2 and 4) or in pCNA3 (syn3) with
pSVNeoC600, which carries a selection marker for neomycin. Trans-
fection occurred with LipofectAMINE Plus (Invitrogen, Carlsbad,
CA) and clones were selected with G418. The syntaxin-expressing
plasmids were obtained from Dr. M. Bennett (University of Califor-
nia, Berkeley, Berkeley, CA). Mlgl/Mlgl-SA expressing clones were
generated from an MDCKII-TET OFF cell line (provided by K.
Mostov, University of California, San Francisco, San Francisco, CA)
that allows inducible expression of the recombinant proteins in the
absence of tetracycline. For maintenance, cell lines were cultured in
the presence of 20 ng/ml doxycycline. To induce expression of
m-l(2)gl proteins, cells were plated at confluency in the absence of
tetracycline for 3 d. For Ca-switch experiments, cells were split before
they reached confluency and plated at confluency in minimal essential
serum that was dialyzed against phosphate-buffered saline (PBS).
Three hours after plating, cells were either maintained in Ca-free
medium for up to 24 h or switched to regular growth medium.
Generation of Mlgl Clones and l(2)gl Antibodies
The full-length mouse clone of Mgl-1 (Tomotsune et al., 1993; Gen-
Bank accession no. NM008502) was generated by fusion polymerase
chain reaction (PCR) from 1500-bp fragments encoding the N- and
the C-terminal half of the gene product. They were obtained sepa-
rately by reverse transcription-PCR from mouse kidney total RNA.
The 450-base pair fragment encoding the C-terminal 153 residues of
Mlgl was cloned in frame into the pGEX4T-1 vector (Amersham
Biosciences, Piscataway, NJ) to prepare Mlgl-GST-fusion protein
that was used to generate polyclonal antibodies in rabbits. An
IgG-fraction of Mlgl-serum was prepared by chromatography on
DEAE-Affigel Blue (Bio-Rad, Hercules, CA) before affinity purifica-
tion of Mlgl antibodies on immobilized Mlgl-GST-fusion proteins.
Antibodies to glutathione S-transferase (GST) were subsequently
removed by passing the affinity-purified IgG fraction over a column
of immobilized GST. The full-length Mlgl cDNA was subcloned into
Bluescript SKII and the point mutations outlined in Figure 5A for
mMlglSA were introduced using the QuickChange mutagenesis kit
(Stratagene, La Jolla, CA) and verified by sequencing. Both, the Mlgl
and Mlgl-SA cDNAs were subcloned into the pTRE2-vector (CLON-
TECH, Palo Alto, CA) that allowed tetracycline-dependent expres-
sion in Tet-OFF cell lines. The Mlgl cDNA was also cloned into
pCDN3 under the T7-promotor for in vitro translation.
In Vitro Binding Assay
The GST-vectors GST-syntaxin 3 and GST-syntaxin 4 encode the
cytoplasmic domains of both syntaxins coupled to GST in the
pGEX-kg vector (Guan and Dixon, 1991). The plasmids were pro-
vided by M. Bennett (University of California, Berkeley). The GST-
SNAP-23 construct was provided by P. Roche (National Institutes of
Health, Bethesda, MD). The DNA was transformed into Escherichia
coli BL21 cells and the recombinant proteins produced and purified
on gluthathione-sepharose (Amersham Biosciences) according to
the manufacturer’s instructions.
The full-length mouse Mlgl was in vitro translated in the TNT-
coupled Reticulocyte Lysate System (Promega, Madison, WI) in the
presence of [35S]methionine. The translation product (4 ?l) was
diluted into 100 ?l of binding buffer (10 mM HEPES/KOH pH 7.4,
150 KCl, 1 mM EDTA, 0.5% Triton-X 100, 2 mM 4-(2-aminoethyl-
)benzenesulfonyl fluoride (AEBSF), 10 ?g/ml each leupeptin, pep-
statin, and antipain) and preadsorbed on 10 ?l of gluthatione-
sepharose for 2 h at 4°C. The supernatant was then incubated with
3 ?mol of fusion proteins (GST, GST-syntaxin 3, GST-syntaxin 4, or
GST-SNAP-23) immobilized on 10 ?l of gluthatione-sepharose for
2 h at 4°C. After the incubation, the unbound material was collected
and trichloroacetic acid-precipitated, whereas the sepharose beads
were washed 4? with 1 ml of binding buffer. Bound and unbound
Mlgl was solubilized in SDS-PAGE buffer and analyzed by electro-
phoreses. Quantitation of35S-labeled Mlgl in both fractions occurred
with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Cells were kept at confluency for 3 d on 15-cm culture dishes,
washed with Hanks’ balanced salt solution, and scraped from the
dish in 1 ml of homogenization buffer (20 mM HEPES/KOH pH 7.4,
0.25 M sucrose, 5 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol,
protease inhibitor cocktail [10 ?g/ml leupeptin, pepstatin A, and
antipaine], 2 mM AEBSF.
Homogenization occurred with a Ball homogenizer as described
in Musch et al. (1997). The postnuclear supernatant (PNS) was
mixed with 50% Nycodenz (Accudenz) in homogenization buffer to
LGL Homologs in Polarized Epithelia
Vol. 13, January 2002 159
give rise to a 25% solution and underlaid a step gradient of 20 and
5% Nycodenz. Fractions were collected from top to bottom after a
centrifugation at 100,000 ? g for 2 h (Figure 1C). The membrane
fraction between the 5 and 20% Nycodenz layers was pelleted for
1 h at 150,000 ? g and resuspended in either SDS sample buffer
(Figure 6D) or in Tris-buffered saline (TBS) for the extraction exper-
iments in Figure 3B
Glycerol Gradient Analysis
Contact-naı ¨ve MDCK cells were homogenized in isotonic sucrose
buffer [20 mM HEPES/KOH pH 8.0, 90 mM KOAc, 2 mM
Mg(OAc)2, 0.25 M sucrose, 1 mM pefabloc, and 10 ?g/ml each
antipain, aprotinin, bestatin, chymostatin, leupeptin, and pepstatin
A] by 10 passages through a ball bearing homogenizer (Varian
Physics, Stanford University, Palo Alto, CA). The postnuclear su-
pernatant was centrifuged at 15,000 ? g for 10 min to remove large
membrane fragments. The resulting supernatant (100 ?l) was lay-
ered onto a 1.2-ml 10-step 22.5–36% (vol/vol) glycerol gradient in 20
mM HEPES/KOH pH 8.0, 90 mM KOAc, 2 mM Mg(OAc)2. The
gradient was centrifuged at 91,000 ? g for 16 h at 4°C. Fractions (100
?l) were collected and analyzed for the presence of Mlgl by Western
blot analysis. In parallel, glycerol gradients were centrifuged con-
taining globular protein standards with known sedimentation coef-
ficients: bovine serum albumin (4.3S), ?-amylase (11.2S), and thyro-
Immunofluorescence was performed on filter-grown cells that were
fixed with 2% paraformaldehyde (PFA) in PBS for 30 min, perme-
abilized with 0.1% Triton X-100, blocked with 1% bovine serum
albumin in PBS and processed for indirect immunofluorescence. For
E-cadherin labeling, cells were fixed and extracted in methanol at
?20°C for 5 min. The following antibodies were used besides the
affinity-purified antibody against Mlgl: anti-HA, clone 12CA5
(Roche Molecular Biochemicals, Indianapolis, IN); anti-dog E-cad-
herin, monoclonal (provided by Dr. R. Kemmler, University of
Freiburg, Freiburg, Germany); anti-ZO-1, rat polyclonal (Chemicon
International, Temecula, CA); anti-gp135, monoclonal provided by
Dr. G.K. Ojakian (State University of New York, Downstate Medical
Center, Brooklyn, NY); and anti-h-dlg (Santa Cruz Biotechnology,
Santa Cruz, CA). Serial x-y or z-sections (0.5 ?m) were taken from
top to bottom on a Zeiss inverted confocal microscope with a 63?
lens and analyzed with LSM software (Carl Zeiss, Thornwood, NY).
The images were further processed in Adobe Photoshop (Adobe
Systems, Mountain View, CA).
For the analysis of the phosphorylation status of Mlgl, monolay-
ers were starved in phosphate-free medium for 90 min and labeled
in the same medium for 90 min with 100 ?Ci/ml [32P]orthophos-
phate, before being lysed in immunoprecipitation (IP) buffer (see
below) supplemented with 1? phophatase inhibitor cocktail I (P-
2850; Sigma). Immunoprecipitation of proteins from the microsomal
fraction or unfractionated homogenate of MDCK cells occurred
after extraction of the proteins in 1 ml of IP buffer (20 mM Tris/HCl
pH 8, 5 mM EDTA, 150 mM NaCl, 0.2% bovine serum albumin, 1%
Triton-X 100, 2 mM AEBSF, protease inhibitor cocktail). The lysates
were preadsorbed with 100 ?l of Pansorbin (Calbiochem) and di-
vided into equal aliquots for IP. The volume was adjusted to 500
?l/IP reaction. In pilot experiments, the amount of antibody that
precipitated the maximal amount of protein was determined for
anti-SNAP-23 (polyclonal serum, provided by P. Roche), anti-Mlgl,
anti-Flag (monoclonal M2; International Biotechnologies, New Ha-
ven, CT), anti-HA (clone 12CA5; Roche Molecular Biochemicals),
and anti-p200 (a monoclonal antibody that recognizes the head-
group of various myosin II isoforms; Narula et al., 1992) The
amounts that resulted in maximal immunoisolation were used for
the coIP experiments. The monoclonal antibodies were incubated
together with an equal amount of rabbit anti-mouse IgG (Rockland,
Gilbertsville, PA). Control IgG was normal rabbit IgG in the same
amount as the highest amount of specific IgG used. Immune com-
plexes were collected on protein A-Sepharose and washed 3? 10
min in IP buffer. Immunoblots were developed with125I-protein A
and images analyzed by PhosphorImager. When monoclonal anti-
bodies were used, the blot was incubated with 1 ?g/ml rabbit
anti-mouse IgG, between incubations with the first antibody and
We isolated the cDNA for a ubiquitously expressed ho-
molog of l(2)gl by reverse transcription-PCR from mouse
kidney total RNA with primers designed from the mouse
Mgl-1 sequence (Tomotsune et al., 1993). The resulting
cDNA was sequenced to confirm its identity and then used
to construct and purify a recombinant GST fusion protein
with the C-terminal 153 residues of Mgl-1, which was then
used as an immunogen for production of polyclonal sera in
rabbits. The resulting antiserum was then subjected to a
three-step affinity purification to isolate IgG specific to the
Mgl-1 portion of the immunogen (see MATERIALS AND
METHODS). This antibody detected a protein of ?120 kDa
in both mouse fibroblasts (3T3 cells) and MDCK cells (Figure
1A), which we will refer to as Mlgl for mammalian l(2)gl. To
determine the localization of Mlgl in polarized MDCK cells,
monolayers were grown to confluency on polycarbonate
filters. When PFA-fixed cells were labeled by indirect immu-
nofluorescence with affinity-purified Mlgl antibody and an-
alyzed by confocal microscopy, a prominent staining of the
lateral membrane was observed, whereas the apical surface
domain was devoid of Mlgl. Labeling was not restricted to
the plasma membrane but was also seen in the cytoplasm of
the cells (Figure 1B). This was confirmed by cell fraction-
ation. Approximately 30% of total Mlgl in homogenates
from confluent MDCK cells floated with the membrane frac-
tion to the interphase between 5 and 20% Nycodenz in a step
gradient (Figure 1C). As in Drosophila, MDCK Mlgl forms
high molecular weight complexes. When cell homogenates
were analyzed by velocity gradient centrifugation, most
Mlgl appeared in a complex of about 17S, with a smaller
amount recovered in a complex of about 12S (Figure 1D). In
Drosophila, large l(2)gl complexes have been shown to con-
tain homo-oligomers of the protein that bind additional
polypeptides (Strand et al., 1994a). Our initial characteriza-
tion therefore suggests that the molecular organization and
subcellular localization of l(2)gl in Drosophila and mamma-
lian epithelia are well conserved.
We next asked whether the association of Mlgl with the
lateral membrane correlates with the establishment of
MDCK cell polarity. Early events during the development of
an epithelial phenotype are associated with the redistribu-
tion of cell adhesion and tight junction markers from intra-
cellular locales in contact-naı ¨ve MDCK cells to restricted
domains of the cell surface when cells make contact with
each other (Rodriguez-Boulan and Nelson, 1989). Likewise,
the exocyst, a protein complex that regulates polarized exo-
cytic events at the plasma membrane, achieves its membrane
(Grindstaff et al., 1998). Because l(2)gl is a candidate for being
a cell polarity determinant in Drosophila and a regulator of
post-Golgi exocytosis in yeast, we tested whether Mlgl un-
dergoes a similar change in its intracellular localization dur-
ing the development of a polarized MDCK phenotype.
A. Mu ¨sch et al.
Molecular Biology of the Cell160
antibodies detect a protein of ?120 kDa in the mouse fibroblastic cell line 3T3 and in MDCK cells. Whole cell lysates from 3T3 and MDCK cells
were separated by 5–15% PAGE, and probed in Western blot with affinity-purified antibodies against mouse Mlgl . (B) Mlgl is localized in the
cytoplasm and at the lateral membrane. Confocal z-sections or x-y sections through the apical plane (1), the plane of the tight junction (2), a
midplane (3), and the basal plane (4) of confluent MDCK cells labeled for Mlgl (green) and ZO-1 (red). (C) Approximately 30% of total Mlgl is
membrane associated. A PNS from confluent MDCK cells was floated on a step gradient composed of 5, 20, and 25% Nycodenz. Fractions were
collected from top to bottom and probed for Mlgl in Western blot analysis. (D) Soluble Mlgl assembles in a ?17S complex. PNS from
“contact-naı ¨ve” MDCK cells was fractionated in a linear 22.5–36% (vol/vol) glycerol gradient. The distribution of MGL was determined by
SDS-PAGE followed by immunoblotting with the Mlgl antibody. Protein levels were quantified using a Molecular Dynamics PhosphorImager. Size
markers (indicated by the arrows) are bovine serum albumin (4.3S), ?-amylase (11.2S), and thyroglobulin (19.2S).
Mlgl associates with the lateral membrane in polarized MDCK cells and assembles into high molecular weight complexes. (A) Mlgl
LGL Homologs in Polarized Epithelia
Vol. 13, January 2002161
during the development of epithelial cell polarity. Con-
tact-naive MDCK cells were plated at confluency on poly-
carbonate filters in low Ca2?medium. On attachment,
cells were incubated in high Ca2?-medium for 0, 3, 6, or
24 h and fixed in methanol (E-cadherin labeling, left) or
PFA (Mlgl labeling, right). Indirect immunofluorescence
occurred with antibodies against E-cadherin (green) and
ZO-1 (red) or Mlgl (green) and ZO-1 (red). Presented are
reconstructed confocal sections along the x-y-axis and con-
Mlgl associates with the basolateral membrane
A. Mu ¨sch et al.
Molecular Biology of the Cell162
The signaling events that lead to development of epithe-
lial cell polarity are dependent on Ca2?in the extracellular
medium of cultured cells (Gonzalez-Mariscal et al., 1990;
Rajasekaran et al., 1996). When contact-naive MDCK cells
were plated at confluency in the absence of Ca2?, cell-cell
adhesion and tight junction formation were prevented, and
E-cadherin and ZO-1 were contained in cytoplasmic struc-
tures (Figure 2, left). Three hours after the addition of Ca2?,
E-cadherin began to appear at the lateral surface and cell-cell
contacts were established. After 6 h, tight junction staining
of ZO-1 was apparent. Mlgl distribution showed a similar
change in response to Ca2?. (Figure 2, right). In low Ca2?
medium, the protein did not exhibit any cell surface local-
ization. Instead, it accumulated in punctate cytoplasmic
structures. After the addition of Ca2?to the growth me-
dium, the association of Mlgl with the lateral surface fol-
lowed that of E-cadherin. Maximal membrane association
was achieved between 6 and 24 h after the Ca2?switch,
indicating that recruitment of Mlgl to the lateral membrane
is temporally correlated with the development of a polarized
phenotype in MDCK cells. The development of polarized
membrane domains in MDCK cells also involves a drastic
reorganization of the cortical actin cytoskeleton that results
in a tight association of cell adhesion molecules with micro-
filaments in polarized cells (Nelson et al., 1990a,b). E-Cad-
herin and the homolog of dlg, SAP97, are resistant to the
extraction by 1% Triton X-100 due to their association with
the actin cytoskeleton at the lateral membrane (Nelson et al.,
1990b; Wu et al., 1998; Figure 3A). In contrast, Mlgl was
readily extracted from the membrane when cells were incu-
bated with Triton X-100 before fixation (Figure 3A) and only
a residual amount of Mlgl resisted Triton X-100 extraction
from isolated MDCK membrane preparations (Figure 3B).
Mlgl does not appear, therefore, to be part of the cortical
cytoskeleton in MDCK cells. This contrasts with studies in
Drosophila, where the bulk fraction of the protein exhibits
resistance to Triton X-100 extraction (Strand et al., 1994b).
Our data suggest that the association with the actin cytoskel-
eton is not a conserved feature of l(2)gl and therefore un-
likely to be pivotal for its role in epithelial cell polarity.
Because homologs of l(2)gl in yeast and neurons were
found to associate with post-Golgi t-SNAREs, we tested
whether Mlgl associates with a specific SNARE at the
plasma membrane of MDCK cells. Epithelial cells possess at
least three plasma membrane t-SNAREs with different do-
main-specific localization. Whereas syntaxin 3 and 4 are
highly enriched at the apical and basolateral domain, respec-
tively, two splice variants of syntaxin 2 are distributed uni-
formly along both surface domains (Low et al., 1996). In
addition, a soluble SNARE, SNAP-23, participates as a t-
SNARE with syntaxins in vesicle fusion at the plasma mem-
brane and can associate with both surface domains in
MDCK cells (Low et al., 1996; St-Denis et al., 1999; Kawanishi
et al., 2000). Because the expression levels of endogenous
syntaxins are below the level of immunodetection, we used
MDCK cell lines that overexpress HA- or Flag-tagged syn-
taxin 2, 3, or 4 as previously characterized (Low et al., 1996).
When confluent MDCK cells were homogenized and the
microsomal fraction solubilized with 1% Triton X-100, we
could efficiently immunoprecipitate syntaxin 2, 3, and 4,
extracted for 10 min on ice with 1% Triton X-100 in Hanks; balanced salt solution before fixation and labeled for E-cadherin, dlg/SAP97, or
Mlgl in green and ZO-1 in red. (B) MDCK membrane fractions were extracted with either TBS alone or TBS supplemented with 1% Triton
X-100, 1% NP-40, 1% SDS, 6 M urea, or 500 mM NaCl and pelleted at 22,000 ? g for 10 min. The pellets were washed in TBS and analyzed
after SDS-PAGE in Mlgl immunoblots.
Membrane-associated Mlgl is sensitive to extraction with 1% Triton X-100. (A) Filter-grown confluent MDCK monolayers were
LGL Homologs in Polarized Epithelia
Vol. 13, January 2002163
SNAP-23 and Mlgl from the membrane extracts (Figure 4A,
top). When SNAP-23 immunoprecipitates from each cell line
were probed for the presence of syntaxins, SNAP-23 was
found to coprecipitate with syntaxin 4 and to a much lesser
extent with syntaxin 2, whereas coprecipitation with syn-
taxin 3 was not detectable. The coisolation of syntaxin 2 and
4 with SNAP-23 also occurred when the HA antibodies were
used to immuno-isolate syntaxins. Interestingly, Mlgl anti-
bodies coimmunoprecipitated syntaxin 4-HA and SNAP-23
from the microsomal fraction of syntaxin 4-expressing cells
with an efficiency that was comparable to the coisolation of
both t-SNAREs. The amount of SNAP-23 or syntaxin 4 that
interacted with Mlgl was ?5%, whereas ?10% of syntaxin 4
and SNAP-23 could be coprecipitated. The interaction of
Mlgl was specific for syntaxin 4 because the Mlgl antibodies
did not precipitate syntaxin 2, 3, nor SNAP-23 from syntaxin
2- or 3-expressing expressing cells. The fraction of syntaxin
4 and SNAP-23 that we found complexed with Mlgl was
comparable to the amounts of Sec9p, Sso1/2p, and Snc1/2p
that were coisolated with Sro7 in yeast (Lehman et al., 1999)
and to the amount of syntaxin 1 that coprecipitated with
tomosyn in neurons (Fujita et al., 1998).
When immunoprecipitates of syntaxin 4-HA or SNAP-23
were analyzed for the presence of Mlgl, no coisolation was
detected, most likely because the epitopes in both t-SNAREs
were buried within the complex and inaccessible to the HA
and SNAP-23 antibodies. Interestingly, coprecipitation of
Mlgl with the SNAREs was detected when lysates were first
depleted of 80% of Mlgl during an immunoprecipitation
with the Mlgl antibody. When the remaining 20% of Mlgl
was subjected to a second round of immunoprecipitation
with the SNAP-23 or HA antibodies, ?25% was coimmuno-
precipitated with these t-SNAREs (Figure 4A, bottom).
Again, this coisolation was specific for syntaxin 4-expressing
cells and did not occur with the same antibodies in syntaxin
2-expressing cells or with the SNAP-23 or Flag antibodies in
syntaxin 3-expressing cells. Analysis by velocity gradient
centrifugation of the Triton X-100–solubilized lysate showed
Mlgl in several distinct peaks. After immunodepletion, in
contrast, the distribution of the remaining 20% of Mlgl was
diffuse over the whole gradient (our unpublished data). It is
possible, therefore, that the depletion of certain Mlgl species
in the first IP induce a shift in the equilibrium between
different Mlgl complexes and the monomer that lead to the
generation of Syn4/SNAP-23–containing Mlgl complexes
where the epitopes to the HA and SNAP-23 antibodies are
accessible. We encountered a similar situation when we
tested the interaction of Mlgl with nonmuscle myosin II that
had been reported for Drosophila l(2)gl (Strand et al., 1994a).
No myosin was precipitated by the Mlgl antibody in the first
immunoprecipitation, but when the remaining 20% of Mlgl
were immunoprecipitated in a second immuno-isolation
cipitates with syntaxin 4 and
SNAP-23 and interacts with my-
osin II. (A) Top (first IP): Triton
fractions prepared from conflu-
ent syntaxin 4-HA (left)–, syn-
taxin 2-HA (middle)–, or syn-
taxin 3-Flag (right)–expressing
cells were immunoprecipitated
with normal rabbit IgG (lane 2),
anti-SNAP-23 (lane 3), anti-HA/
Flag (lane 4), or anti-Mlgl (lane
5). The immunoprecipitates were
probed by immunoblotting with
antibodies to Mlgl (top), HA/
Flag (middle), or SNAP-23 (bot-
tom). Lane 1 represents half of
the lysate used for IPs. Bottom
(2nd IP): lysate that was immu-
nodepleted of 80% of Mlgl was
reabsorbed on protein A-Sepha-
rose to deplete all IgG and sub-
sequently reprecipitated with the
same antibodies that were listed
in A; Western blot was probed
with Mlgl antibodies. (B) Triton
from MDCK cells was immuno-
precipitated with control IgG
(left, lane 2), anti-myosin II (lane
3), or anti-Mlgl (lane 4); with
control IgG (right, lane 2), anti-
Mlgl (lane 3), or anti-myosin II
(lane 4). Lysate that was de-
pleted of ?80% of Mlgl was reabsorbed on protein A-Sepharose and subsequently reprecipitated in the left panel with anti-myosin II (lane
5), control IgG (lane 6), or anti-Mlgl (lane 7); and in the right panel with anti-Mlgl (lane 5), anti-myosin II (lane 6), or control IgG (lane 7). Lane 1
in both panels represents one-fifth of lysate used for the IPs. Samples were analyzed for myosin II (left) or Mlgl (right) by Western blot.
A. Mu ¨sch et al.
Molecular Biology of the Cell164
from the same lysate, the Mlgl antibody coprecipitated my-
osin II. Likewise, myosin II antibodies coprecipitated Mlgl
only when the lysate was previously cleared of ?80% of
Mlgl in the first immunoprecipitation (Figure 4B).
Our data suggest that Mlgl interacts with the t-SNAREs
syntaxin 4 and SNAP-23 at the basolateral membrane. A
direct interaction between Mlgl and syntaxin 4 was estab-
lished for the isolated proteins in vitro (Figure 5). The full-
length mouse clone of Mgl-1 was in vitro translated in the
presence of [35S]methionine and incubated with immobi-
lized GST-fusion proteins of SNAP-23 or the cytoplasmic
domains of syntaxin 3 and syntaxin 4. Thirty-six percent of
syntaxin 4-GST bound to M-lgl, whereas other plasma mem-
brane SNARE proteins showed significantly lower affinity
for M-lgl (9% for syntaxin 3-GST and 3% for SNAP-23-GST).
The association of Mlgl with basolateral SNARE proteins
in polarized MDCK cells makes it a good candidate to
participate in basolateral secretion. Our attempts to interfere
with Mlgl function, however, have failed so far, preventing
us from testing this hypothesis directly. The introduction of
Mlgl antibodies into SLO-permeabilized cells failed to effect
exocytosis of several basolateral markers. Antisense ap-
proaches did not significantly reduce the amount of Mlgl in
confluent MDCK cells, probably due the long half-life of the
protein (Mu ¨sch, unpublished data) and/or the potential
presence of a second isoform of the protein that can be
predicted from entries in the EST database. A 10-fold over-
expression of Mlgl did not affect either the polarity or the
kinetics of apical or basolateral exocytosis (our unpublished
data). This is in agreement with data in yeast were even a
100-fold overexpression of pSro7 does not lead to any
growth defect (Brennwald, unpublished data).
In an attempt to generate mutations in Mlgl that could
yield a dominant negative phenotype, we altered the se-
quence encoding a highly conserved stretch of 25 amino
acids in the mouse cDNA by exchanging four serine resi-
dues for alanines (Figure 6A). A peptide comprised of the
corresponding sequence in Drosophila l(2)gl has previously
been shown in vitro to inhibit phosphorylation of l(2)gl by a
kinase associated with the l(2)gl complex (Kalmes et al.,
1996). We expressed the recombinant protein (mMlgl-SA) or
the wild-type mouse clone (mMlgl) under an inducible pro-
motor in MDCK cells (Figure 6B). Mouse Mgl when ex-
pressed in MDCK cells had a slightly lower apparent mo-
lecular weight than the MDCK protein. It should be noted,
however, that mouse Mlgl from 3T3 cells and MDCK Mlgl
showed similar electrophoretic behavior when resolved on
the same gel (Figure 1A) and that endogenous MDCK l(2)gl,
on the other hand, occasionally appeared as a double band
(Figure 1D). The reason for this diversity is presently un-
clear. Despite slightly higher expression levels of mMlgl-SA
compared with the wild type, the extent of32P incorporation
into the serine mutant in vivo is 3.5-fold lower than for the
wild-type recombinant protein, but still higher than
incorporation into endogenous Mlgl. These data indicate
that serine residues within the peptide are indeed phosphor-
ylated and that Mlgl possesses additional phosphorylation
sites. When the subcellular distribution of Mlgl and Mlgl-SA
in MDCK cells was compared by wide-field and confocal
microscopy, striking differences appeared (Figure 6C). Al-
though overexpression of Mlgl reveals a distribution similar
to that of endogenous Mlgl, induction of mMlgl-SA at the
same expression level showed an accumulation of the mu-
tant protein at the apical surface. This is particularly obvious
in confocal z-sections through the cells that were colabeled
for the apical membrane protein gp135. Although Mlgl is
present in the subapical cytoplasm, it does not colocalize
with the apical membrane marker. Mlgl-SA, in contrast,
overlaps with gp135 at the apical surface. Hence, we have
identified a phoshorylated peptide in the C-terminal portion
of Mlgl that plays a role in restricting Mlgl to the lateral
membrane domain and preventing it from associating with
the apical surface domain.
Biochemical analysis revealed that overexpressed Mlgl or
Mlgl-SA distributed with the same ratio between mem-
branes and the cytosol as endogenous Mlgl (Figure 5D) and
showed the same sedimentation behavior in velocity gradi-
ents as the endogenous protein (our unpublished data). It
had been suggested that Mlgl phosphorylation negatively
regulates its association with myosin II in Drosophila
(Kalmes et al., 1996). We have no evidence, however, that the
amount of myosin II that could be coprecipitated by Mlgl in
Mlgl-SA–overexpressing cells is different from that in Mlgl-
expressing cells (our unpublished data). It is likely, there-
fore, that other proteins are responsible for the specificity of
the association of Mlgl with the lateral membrane. Overex-
pression of Mlgl-SA did not appear to interfere with the
function of endogenous Mlgl. Despite the localization defect
of the recombinant protein, no effect on cell polarity or the
kinetics of protein secretion could be detected in Mlgl-SA–
expressing cells (our unpublished data).
translated in the presence of [35S]methionine and incubated with 3
?M GST, GST-SNAP-23, GST-syntaxin 3, or GST-syntaxin 4 immo-
bilized on gluthatione-sepharose. (A) Bound (B) and unbound (UB)
factions were analyzed for35S-labeled Mlgl. T represents half of
Mlgl input. The percentage of bound Mlgl was determined from
two independent experiments. (B) Image of the Coomassie-stained
gel of A.
Mlgl binds syntaxin 4-GST in vitro. Mlgl was in vitro
LGL Homologs in Polarized Epithelia
Vol. 13, January 2002165
in mMlgl-SA. Gray-boxed serine residues were changed to alanine; the homologous region of mouse Mlgl in Drosophila is outlined [D-l(2)gl].
(B) Phosphorylation of Mlgl and Mlgl-SA in vivo. Mlgl was quantitatively immunoprecipitated from detergent lysates of cells expressing
wild-type Mlgl (WT) or the phosphorylation mutant (SA). Left, Western blot of Mlgl; right, autoradiogram of phosphorylated Mlgl from cells
labeled with [32P]orthophosphate. (C) Mlgl-SA localizes at the apical membrane. x-y: wild-field immunofluorescence image of Mlgl in control
cells (left), Mlgl-expressing cells (middle), or Mlgl-SA–expressing cells (right). z: confocal z-sections through control (top), Mlgl- (middle),
and Mlgl-SA (bottom)–expressing cells; Mlgl labeling is shown on the left and in green on the right; ZO-1 in blue, gp135 in red on the right
panels. (D) Membrane association of Mlgl and Mlgl-SA. PNSs were prepared from confluent cells that either expressed recombinant Mlgl
(WT) or Mlgl-SA (SA) in the absence of tetracycline (?tet) or expressed only endogenous Mlgl in the presence of tetracycline (?tet). The
homogenate was floated on a Nydodenz gradient (see MATERIALS AND METHODS). One-third of the PNS input was compared with the
membrane fraction. Mlgl in both fractions was analyzed by immunoblot analysis.
A phosphorylation-deficient mutant of Mlgl is localized at the apical membrane in MDCK cells. (A) Scheme of serine mutations
A. Mu ¨sch et al.
Molecular Biology of the Cell166
Our characterization of a homolog of Drosophila l(2)gl in
MDCK cells revealed that the mammalian protein, like its
Drosophila counterpart, assembles into high molecular
weight complexes and associates with the lateral membrane
of polarized epithelial cells. The epithelial cell culture model
enabled us, moreover, to identify novel features of l(2)gl that
suggest that the protein contributes to cell polarity by its
ability to interact with the basolateral exocytic machinery.
Similar to homologs in yeast and a l(2)gl-related protein in
neurons, MDCK Mlgl interacts with a plasma membrane
t-SNARE. The interaction is specific for syntaxin 4, the t-
SNARE that is restricted to the basolateral membrane and
has been implicated in basolateral exocytosis (Lafont et al.,
1999; Mostov et al., 2000). Mlgl does not interact with syn-
taxin 2 or syntaxin 3, which are distributed in a nonpolar
manner or at the apical surface. SNAP-23, a t-SNARE at both
surface domains of MDCK cells, coimmunoprecipitated
with Mlgl only in cells that overexpressed syntaxin 4, sug-
gesting that a complex of syntaxin 4 and SNAP-23 associates
with Mlgl. An interaction between SNAP-23 and syntaxin 4
had been previously reported and was verified in our ex-
periments (St-Denis et al., 1999). In the absence of other
proteins, mouse Mlgl binds to syntaxin 4-GST but not to
SNAP-23-GST or syntaxin 3-GST. It remains to be estab-
lished whether the interaction of Mlgl with SNAREs in vivo
requires a syntaxin 4/SNAP-23 complex or occurs with syn-
taxin 4 independently of SNAP-23. It had been proposed
that tomosyn acts as a SNARE surrogate for syntaxin 1
because the protein sequence contains a vesicle-associated
membrane protein-like motif (Masuda et al., 1998). As with
the yeast homologs Sro7/77, Mlgl does not possess this
domain, but is nevertheless able to bind to SNARE com-
plexes, demonstrating that this is neither an essential nor
well-conserved component of this interaction. Rather, this
may reflect a need in neuronal cells to keep t-SNAREs in a
primed conformation to ensure that this is not rate limiting
during rapid or prolonged rounds of exocytosis.
Similar to proteins with a role in cell polarity and/or
polarized exocytosis, Mlgl is not membrane associated in
contact-naive MDCK cells and binds to the plasma mem-
brane only after cell polarity determinants such as E-cad-
herin have defined the lateral membrane of contacting cells.
This phenomenon has been observed for the exocyst, a sol-
uble protein complex that participates in basolateral secre-
tion and for dlg/SAP97, a PDZ-domain containing protein
at the lateral membrane (Grindstaff et al., 1998; Reuver and
Garner, 1998). Genetic studies in Drosophila have suggested
that l(2)gl and dlg are dependent on each other for function
and localization (Bilder et al., 2000). Although we have not
been able to demonstrate any physical interaction between
either Mlgl and dlg or exocyst proteins and Mlgl (Yeaman
and Mu ¨sch, unpublished data), functional interactions be-
tween the protein complexes remain to be analyzed.
The association of l(2)gl with the lateral surface domain of
epithelia is pivotal for its tumor suppressor function in
Drosophila (Manfruelli et al., 1996; Bilder et al., 2000). It is of
importance, therefore, to identify the determinants in l(2)gl
that are responsible for its domain-specific membrane asso-
ciation. Studies with membrane extracts from Drosophila
have indicated that l(2)gl phosphorylation negatively regu-
lates the association of the protein with both the membrane
and with myosin II (Kalmes et al., 1996), which led to the
suggestion that membrane association of l(2)gl occurs via
myosin and is regulated by phosphorylation. This study
identified a 25 amino acid peptide in the l(2)gl sequence that
inhibited l(2)gl phosphorylation in vitro and is highly con-
served among species. That prompted us to examine the
possibility that a related phosphorylation event might reg-
ulate Mlgl distribution in MDCK cells. We expressed a re-
combinant mouse Mlgl protein in MDCK cells that lacked
the potential phosphorylation sites within this sequence.
The recombinant protein exhibited reduced phoshorylation
levels and had indeed an altered subcellular distribution
compared with the wild type. Different from the prediction,
however, the phosphorylation mutant did not show a higher
degree of total membrane association, but instead an altered
distribution between the two membrane domains of polar-
ized MDCK cells. A significant percentage of the membrane-
associated pool was at the apical rather than the basolateral
membrane, indicating that the phosphorylated residues pre-
vent Mlgl from associating with the apical membrane.
Although it remains to be demonstrated, the interaction of
Mlgl with syntaxins at the basolateral surface together with
the established function of Mlgl in protein secretion in yeast
makes a strong case for a role of Mlgl in basolateral exocy-
tosis. Mlgl might, similarly to the function of the exocyst,
link the establishment of epithelial cell polarity to the devel-
opment of a basolateral exocytic pathway. This hypothesis
contrasts with the prevailing assumption that the role of
l(2)gl in epithelial cell polarity is related to its association
with the actin cytoskeleton (Strand et al., 1994b). The latter
hypothesis is based on the interaction of Drosophila l(2)gl
with myosin II and the resistance of its membrane pool to
extraction with nonionic detergents (Strand et al., 1994b).
Although a fraction of MDCK Mlgl was found to interact
with myosin II, the bulk of the mammalian protein does not
appear to be part of the cortical actin cytoskeleton. Rather
than being an anchor for Mlgl at the membrane, myosin II
could be subject to regulation by Mlgl in a process that leads to
vesicle fusion. Myosin II has been implicated in exocytic events
at the plasma membrane in several systems (Howell and Ty-
hurst, 1986; Mochida et al., 1994; Wilson et al., 1999; Torgerson
and McNiven, 2000). Mlgl could thus couple the steps involv-
ing myosin and the SNAREs to coordinate vesicle fusion.
We thank Drs. Paul Roche for SNAP-23 antisera, Mark Bennett for
the syntaxin-GST constructs, Keith Mostov for the MDCK-TET OFF
cells, and Guendalina Rossi for critical reading of the manuscript.
This work was supported by grants from the Mathers Charitable
Foundation; the Pew Scholars in Biomedical Sciences Program (to
P.J.B); the National Institutes of Health GM-54712 (to P.J.B.), GM-
34107 (to E.R.B.), GM35527 (to W.J.N.), and a Jules and Doris Stein
Professorship of the Research to Prevent Blindness Foundation (to
E.R.B.). C.Y. was supported by a Walter V. and Idun Y. Berry
Bilder, D., Li, M., and Perrimon, N. (2000). Cooperative regulation of
cell polarity, and growth by Drosophila tumor suppressors. Science
LGL Homologs in Polarized Epithelia
Vol. 13, January 2002 167
Fujita, Y., et al. (1998). Tomosyn: a syntaxin-1-binding protein that Download full-text
forms a novel complex in the neurotransmitter release process.
Neuron 20, 905–915.
Gateff, E. (1978). Malignant neoplasms of genetic origin in Drosophila
melanogaster. Science 200, 1448–1459.
Gonzalez-Mariscal, L., Contreras, R.G., Bolivar, J.J., Ponce, A.,
Chavez De Ramirez, B., and Cereijido, M. (1990). Role of calcium in
tight junction formation between epithelial cells. Am. J. Physiol. 259,
Grindstaff, K.K., Yeaman, C., Anandasabapathy, N., Hsu, S.C., Ro-
driguez-Boulan, E., Scheller, R.H., and Nelson, W.J. (1998). Sec6/8
complex is recruited to cell-cell contacts and specifies transport
vesicle delivery to the basal-lateral membrane in epithelial cells. Cell
Guan, K.L., and Dixon, J.E. (1991). Eukaryotic proteins expressed in
Escherichia coli: an improved thrombin cleavage and purification
procedure of fusion proteins with glutathione S-transferase. Anal.
Biochem. 192, 262–267.
Howell, S.L., and Tyhurst, M. (1986). The cytoskeleton and insulin
secretion. Diabetes Metab. Rev. 2, 107–123.
Kalmes, A., Merdes, G., Neumann, B., Strand, D., and Mechler, B.M.
(1996). A serine-kinase associated with the p127-l(2)gl tumor sup-
pressor of Drosophila may regulate the binding of p127 to non-
muscle myosin II heavy chain and the attachment of p127 to the
plasma membrane. J. Cell Sci. 109, 1359–1368.
Katz, L., Hanson, P.I., Heuser, J.E., and Brennwald, P. (1998). Genetic
and morphological analyses reveal a critical interaction between the
C-termini of two SNARE proteins and a parallel four helical arrange-
ment for the exocytic SNARE complex. EMBO J. 17, 6200–6209.
Kawanishi, M., Tamori, Y., Okazawa, H., Araki, S., Shinoda, H., and
Kasuga, M. (2000). Role of SNAP23 in insulin-induced translocation
of GLUT4 in 3T3–L1 adipocytes. Mediation of complex formation
between syntaxin4 and VAMP2. J. Biol. Chem. 275, 8240–8247.
Lafont, F., Verkade, P., Galli, T., Wimmer, C., Louvard, D., and
Simons, K. (1999). Raft association of SNAP receptors acting in
apical trafficking in Madin-Darby canine kidney cells. Proc. Natl.
Acad. Sci. USA 96, 3734–3738.
Lehman, K., Rossi, G., Adamo, J.E., and Brennwald, P. (1999). Yeast
homologues of tomosyn and lethal giant larvae function in exocy-
tosis and are associated with the plasma membrane SNARE, Sec9.
J. Cell Biol. 146, 125–140.
Low, S.H., Chapin, S.J., Weimbs, T., Komuves, L.G., Bennett, M.K.,
and Mostov, K.E. (1996). Differential localization of syntaxin iso-
forms in polarized Madin-Darby canine kidney cells. Mol. Biol. Cell
Low, S.H., Roche, P.A., Anderson, H.A., van Ijzendoorn, S.C.,
Zhang, M., Mostov, K.E., and Weimbs, T. (1998). Targeting of
SNAP-23 and SNAP-25 in polarized epithelial cells. J. Biol. Chem.
Manfruelli, P., Arquier, N., Hanratty, W.P., and Semeriva, M. (1996).
The tumor suppressor gene, lethal(2)giant larvae (1(2)g1), is re-
quired for cell shape change of epithelial cells during Drosophila
development. Development 122, 2283–2294.
Masuda, E.S., Huang, B.C., Fisher, J.M., Luo, Y., and Scheller, R.H.
(1998). Tomosyn binds t-SNARE proteins via a VAMP-like coiled
coil. Neuron 21, 479–480.
Mochida, S., Kobayashi, H., Matsuda, Y., Yuda, Y., Muramoto, K.,
and Nonomura, Y. (1994). Myosin II is involved in transmitter
release at synapses formed between rat sympathetic neurons in
culture. Neuron 13, 1131–1142.
Mostov, K.E., Verges, M., and Altschuler, Y. (2000). Membrane
traffic in polarized epithelial cells. Curr. Opin. Cell Biol. 12, 483–490.
Musch, A., Cohen, D., and Rodriguez-Boulan, E. (1997). Myosin II is
involved in the production of constitutive transport vesicles from
the trans-Golgi Network. J. Cell Biol. 138, 291–306.
Nelson, W.J., Hammerton, R.W., Wang, A.Z., and Shore, E.M.
(1990a). Involvement of the membrane-cytoskeleton in development
of epithelial cell polarity. Semin. Cell Biol. 1, 359–371.
Nelson, W.J., Shore, E.M., Wang, A.Z., and Hammerton, R.W.
(1990b). Identification of a membrane-cytoskeletal complex contain-
ing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin,
and fodrin in Madin-Darby canine kidney epithelial cells. J. Cell
Biol. 110, 349–357.
Ohshiro, T., Yagami, T., Zhang, C., and Matsuzaki, F. (2000). Role of
cortical tumor-suppressor proteins in asymmetric division of Dro-
sophila neuroblast. Nature 408, 593–596.
Peng, C.Y., Manning, L., Albertson, R., and Doe, C.Q. (2000). The
tumor-suppressor genes lgl, and dlg regulate basal protein targeting
in Drosophila neuroblasts. Nature 408, 596–600.
Rajasekaran, A.K., Hojo, M., Huima, T., and Rodriguez-Boulan, E.
(1996). Catenins and zonula occludens-1 form a complex during early
stages in the assembly of tight junctions. J. Cell Biol. 132, 451–463.
Reuver, S.M., and Garner, C.C. (1998). E-Cadherin mediated cell
adhesion recruits SAP97 into the cortical cytoskeleton. J. Cell Sci.
Rodriguez-Boulan, E., and Nelson, W.J. (1989). Morphogenesis of
the polarized epithelial cell phenotype. Science 245, 718–725.
Sollner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H.,
Geromanos, S., Tempst, P., and Rothman, J.E. (1993). SNAP recep-
tors implicated in vesicle targeting and fusion. Nature 362, 318–324.
St-Denis, J.F., Cabaniols, J.P., Cushman, S.W., and Roche, P.A.
(1999). SNAP-23 participates in SNARE complex assembly in rat
adipose cells. Biochem. J. 338, 709–715.
Strand, D., Jakobs, R., Merdes, G., Neumann, B., Kalmes, A., Heid,
H.W., Husmann, I., and Mechler, B.M. (1994a). The Drosophila
lethal(2)giant larvae tumor suppressor protein forms homo-oli-
gomers and is associated with nonmuscle myosin II heavy chain.
J. Cell Biol. 127, 1361–1373.
Strand, D., Raska, I., and Mechler, B.M. (1994b). The Drosophila
lethal(2)giant larvae tumor suppressor protein is a component of the
cytoskeleton. J. Cell Biol. 127, 1345–1360.
Tomotsune, D., Shoji, H., Wakamatsu, Y., Kondoh, H., and Takahashi,
N. (1993). A mouse homologue of the Drosophila tumor-suppressor
gene l(2)gl controlled by Hox-C8 in vivo. Nature 365, 69–72.
Sutton, R.B., Fasshauer, D., Jahn, R., and Brunger, A.T. (1998).
Crystal structure of a SNARE complex involved in synaptic exocy-
tosis at 2.4 A resolution. Nature 395, 347–353.
Torgerson, R.R., and McNiven, M.A. (2000). Agonist-induced
changes in cell shape during regulated secretion in rat pancreatic
acini. J. Cell. Physiol. 182, 438–447.
Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl,
M., Parlati, F., Sollner, T.H., and Rothman, J.E. (1998). SNAREpins:
minimal machinery for membrane fusion. Cell 92, 759–772.
Wilson, J.R., Biden, T.J., and Ludowyke, R.I. (1999). Increases in
phosphorylation of the myosin II heavy chain, but not regulatory
light chains, correlate with insulin secretion in rat pancreatic islets
and RINm5F cells. Diabetes 48, 2383–2389.
Wu, H., Reuver, S.M., Kuhlendahl, S., Chung, W.J., and Garner, C.C.
(1998). Subcellular targeting and cytoskeletal attachment of SAP97
to the epithelial lateral membrane. J. Cell Sci. 111, 2365–2376.
A. Mu ¨sch et al.
Molecular Biology of the Cell 168