Apical β1 integrin in polarized MDCK cells mediates tubulocyst formation in response to type I ...

15 Pages·1996·0.58 MB·English

Journal of Cell Science 109, 1875-1889 (1996) 1875
Printed in Great Britain ©The Company of Biologists Limited 1996
JCS9478
Apical b 1 integrin in polarized MDCK cells mediates tubulocyst formation in
response to type I collagen overlay
Anna Zuk* and Karl S. Matlin
Renal Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, 149th 13th Street,
Charlestown, MA 02129, USA
*Author for correspondence
SUMMARY
A number of epithelia form tubulocysts in vitro when inhibits the formation of these structures. To determine
overlaid with type I collagen gel. Because collagen how apical-cell collagen interactions elicit tubulocyst
receptors are generally believed to be expressed on the formation, we examined whether b 1 integrins are
basolateral domain, the mechanism by which collagen mobilized to apical plasma membranes in response to
elicits this morphogenetic response from the apical surface collagen overlay. We found that in the absence of collagen,
is unclear. To investigate the role of b 1 integrins, the major polarized monolayers of Heidelberg MDCK cells endoge-
receptor family for collagen, in this process, we overlaid nously express on apical plasma membranes a small pool
polarized monolayers of MDCK II cells grown on of the b 1 family, including a 2b 1 and a 3b 1. Collagen
permeable supports with type I collagen gel and correlated overlay does not mobilize additional b 1 integrins to apical
integrin polarity with the polarity of other apical and baso- domains. If b 1 integrins are not already apically expressed,
lateral membrane markers during tubulocyst formation. as in the C6 MDCK cell line (Schoenenberger et al. (1994)
Polarized monolayers of one clone of MDCK II cells, J. Cell Biol. 107, 527-541), b 1 integrins are not directed
referred to as Heidelberg MDCK, initially respond to apically and tubulocysts do not develop in response to
collagen overlay by stratifying; within 48 hours, lumena collagen. Thus, interaction of b 1 integrins pre-existing on
develop between the cell layers giving rise to tubulocysts. apical plasma membranes of polarized epithelia with type
Tight junctions remain intact during tubulocyst formation I collagen gel is the mechanism by which apical application
because transepithelial electrical resistance does not signif- of collagen elicits the formation of tubulocysts. Depolarized
icantly change. Major alterations are observed, however, integrins on apical plasma membranes of polarized
in the expression and localization of apical and basolateral epithelia may be relevant to the pathogenesis of disease and
membrane markers. b 1 integrins are necessary for tubulo- injury.
cyst morphogenesis because a function-blocking antibody
administered to the apical pole of the cells completely Key words: b 1 Integrin, Epithelial polarity, Tubulocyst, MDCK
INTRODUCTION integrins may also mediate cell-cell adhesion (Larjava et al.,
1990; Carter et al., 1990).
Epithelial cells form a selective permeability barrier separat- Studies on the biogenesis of epithelial polarity have focused
ing the outside world from internal compartments. To on the role of cell-cell and cell-substratum adhesion in estab-
perform this function, epithelial cells are polarized into lishing the polarized epithelial phenotype (Nelson and
discrete apical, lateral and basal domains. The apical plasma Veshnock, 1986, 1987a,b; Ekblom, 1989; Nelson and
membrane contains unique proteins and lipids not found on Hammerton, 1989; McNeil et al., 1990; Sorokin et al., 1990).
basolateral plasma membranes (Simons and Fuller, 1985; In the polarized Madin-Darby canine kidney (MDCK) cell line,
Rodriguez-Boulan and Nelson, 1989). Specialized structures in vitro studies have demonstrated that interactions with the
such as microvilli and cilia extend from the apical pole; the substratum are sufficient for establishing apical polarity
Golgi complex is positioned in the apical cytoplasm. Tight (Rodriguez-Boulan et al., 1983; Vega-Salas et al., 1987). In the
junctions, zonula adherens and desmosomes are found on absence of cell-cell contacts, MDCK cells polarize apical
lateral borders that also contain cell adhesion molecules, such plasma membrane glycoproteins in response to cell-substratum
as E-cadherin, that mediate interactions with neighboring adhesion. Basolateral membrane proteins remain randomly
cells. Basal plasma membranes contain integrin receptors distributed on plasma membranes until cell-cell contacts form
(Ruoslahti, 1991) that interact with extracellular matrix (Nelson and Veshnock, 1986, 1987a,b; Nelson and
(ECM) in the underlying basement membrane. Some Hammerton, 1989; McNeil et al., 1990). Studies on early
1876 A. Zuk and K. S. Matlin
kidney development also indicate that polarization requires the b 1, b 3, and b 4 integrin families, including the a 2b 1, a 3b 1,
cell-cell and cell-substratum adhesion. In kidney tubulogene- an unidentifieda xb 1, a 6b 4 and a vb 3(Schoenenberger et al.,
sis, the ureteric bud induces the metanephric mesenchyme to 1994; Ojakian and Schwimmer, 1994). The b 1, b 3, and b 4
condense by forming extensive cell-cell contacts. Conversion families are present on basolateral plasma membranes. Only
of the condensates into polarized tubular epithelia is later the b 1 family mediates cell attachment to collagen types I and
mediated by cell-substratum interactions with ECM (Sorokin IV and laminin (Schoenenberger et al., 1994; Ojakian and
et al., 1990). Schwimmer, 1994).
A number of studies have addressed the role of cell-cell To further study the role of b 1 integrins in epithelial polar-
contacts mediated by E-cadherin in epithelial polarization ization, we took advantage of a classic observation in which
(Nelson and Veschnock, 1986, 1987a,b; Nelson and epithelia, including thyroid, mammary gland, intestine, liver
Hammerton, 1989; McNeil et al., 1990); however, equivalent and vascular endothelia, form tubulocysts when overlaid with
studies on cell-substratum adhesion, particularly the role of the type I collagen gel (Chambard et al., 1981; Hall et al., 1982;
ECM and its receptors, have been few. Cell-ECM interactions Montesano, 1983; Montgomery, 1986; LeCluyse et al., 1994).
initiated by suspending MDCK cysts in type I collagen gel lead Because b 1 integrins are the major receptor family for
to membrane remodeling and polarity reversal (Wang et al., collagens (Hynes, 1992; Hemler et al., 1995) found on baso-
1990; Ojakian and Schwimmer, 1994). A new basal surface lateral cell surfaces (Ruoslahti, 1991), the mechanism of how
develops at the site of cell-collagen interaction and an apical collagen at apical plasma membranes elicits this morpho-
surface forms at the opposite end lining an internal lumen. genetic response was unclear. We hypothesized that apical
Further evidence that the ECM is critical in establishing the cell-collagen interactions would mobilize b 1 integrins to apical
polarized epithelial phenotype comes from studies on kidney plasma membranes to mediate the formation of tubulocysts.
development. Deposition of laminin A in the basement Using the polarized MDCK cell line, we found to our surprise
membrane surrounding the condensed mesenchyme stimulates that in the absence of collagen overlay, b 1 integrins were
the conversion of the aggregated cells into polarized epithelia endogenously expressed at low levels on apical plasma
(Sorokin et al., 1990). membranes in one clone of MDCK, even though other apical
Epithelial interactions with ECM are mediated by the and basolateral membrane markers localized exclusively to
integrins, a superfamily of cell surface receptors found on their respective plasma membrane domains. Moreover, apical
every eucaryotic cell except erythrocytes (for reviews, see application of collagen did not mobilize additional b 1 integrins
Hynes, 1992; Hemler et al., 1995). Integrins are transmem- to apical cell surfaces. Pre-existing apical b 1 integrins
brane glycoproteins that consist of unrelated a and b subunits. mediated tubulocyst formation because if receptor-ligand
Their association into noncovalent heterodimers is necessary binding was inhibited or if b 1 integrins were absent from this
for cell surface expression and ligand binding. Most epithelia surface tubulocysts did not develop in response to collagen.
express integrins belonging to the b 1, b 3, b 4 or b 5 families.
The b chains form complexes with a variety of different a
subunits, yielding receptors for collagens, laminin, fibronectin
MATERIALS AND METHODS
and vitronectin. In addition, some ab combinations recognize
more than one ECM ligand. For example, the a 1b 1 and a 2b 1
Cell culture
are receptors for collagen or laminin whereas the a 3b 1 can
MDCK II cells were originally cloned by Louvard (1980) for high
recognize collagen, laminin, or fibronectin (Hynes, 1992;
growth rate and dome formation; this clone is referred to as Heidel-
Hemler et al., 1995). Receptor-ligand binding activates trans- berg (HD) MDCK cells to identify its place of origin. HD MDCK
membrane signaling, influencing cell differentiation, behavior cells have been used extensively for studies on epithelial cell polarity
and gene expression (for reviews, see Damsky and Werb, (Matlin et al., 1981; Matlin and Simons, 1983; for review, Simons
1992; Juliano and Haskill, 1993; Clark and Brugge, 1995). and Fuller, 1985). The C6 subclone of HD MDCK (MDCK pMV7
By mediating cell-substratum interactions, the integrins 6.2.1.) was used for some experiments. C6 cells are stably transfected
have been implicated in the pathogenesis of disease and injury. with the empty pMV7 expression vector (Schoenenberger et al.,
1991). They are identical to the parental line with regard to ultra-
In carcinogenesis, novel integrin-substratum interactions influ-
structure, growth rate and transepithelial electrical resistance.
encing epithelial differentiation and polarization (Schoenen-
For routine culture, HD (passages 7-33) and C6 (passages 7-15)
berger et al., 1994) have been suggested to contribute to metas-
MDCK cells were grown in 75 cm2flasks (Falcon; Becton Dickinson
tasis (Plantefarber and Hynes, 1989; Schoenenberger and
& Co., Boston, MA) in minimal essential medium (MEM) or
Matlin, 1991; Matlin and Caplan, 1992). New integrin-ECM Dulbecco’s MEM (DME; Mediatech, Herndon, VA) containing 5%
interactions are established after epithelial injury and facilitate fetal bovine serum (FBS; Sigma Chemical Co., St Louis, MO or
wound healing and repair (for reviews, see Gailit and Clark, HyClone Laboratories, Inc., Logan, UT), 2 mM L-glutamine (Sigma)
1994; Clark, 1995). In in vitro models of acute renal failure, and 10 mM Hepes (Sigma), pH 7.4, at 37°C in 95% air, 5% CO2.
integrin-substratum interactions are altered (Gailit et al., 1993; Confluent cells were harvested with trypsin-EDTA (Gibco BRL, Life
Goligorsky et al., 1993); the a 3b 1 integrin redistributes from Technologies, Inc., Grand Island, NY) and subcultured at a 1:5
dilution twice weekly.
basal to apical cell surfaces and some integrins are lost from
For culture of cells on permeable polycarbonate supports, cells
epithelial plasma membranes (Gailit et al., 1993).
were plated at 7.8· 105cells on Transwell filters (24 mm, 0.4 m m pore
We have been investigating integrin-mediated cell-substra-
size; Corning CoStar, Cambridge, MA) pre-wetted with medium.
tum interactions in epithelial cell polarization using the MDCK
Cultures were fed every other day for 6-8 days as well as the day
cell line as a model system. We have been particularly inter- before collagen overlay. Antibiotic-antimycotic (Gibco) was included
ested in the role of the ECM in generating and maintaining in the medium at a final concentration of 100 units/ml penicillin, 100
discrete apical and basolateral domains. MDCK cells express m g/ml streptomycin and 0.25 m g/ml amphotericin B.
Apical b 1 integrin in polarized MDCK cells 1877
Antibodies for 6-8 days were incubated from the basal compartment with 100
Anti-integrin antibodies m Ci/ml [35S]methionine [35S]cysteine (EXPRE35S35S, New England
Rabbit anti-human b 1 integrin antibody was obtained from Dr E. Nuclear, Boston, MA) in labeling medium (1/10 methionine-free
MEM or DME containing FBS, glutamine, Hepes, and
Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA). This
antibiotic/antimycotic) for 16-24 hours. Apical compartments
antibody was generated against a 38mer peptide corresponding to the
contained labeling medium without isotope. After steady-state
carboxy-terminal domain (Giancotti and Ruoslahti, 1990) of the
human b 1 integrin. Under non-denaturing conditions, this antibody and labeling, filters were prepared for collagen overlay; medium was
other polyclonal b 1 integrin antibodies (Schoenenberger et al., 1994) removed only from the apical compartment and apical cell surfaces
immunoprecipitate a 2b 1, a 3b 1, an unidentified a xb 1, and precursor rinsed once with labeling medium and once with collagen solution;
b 1 from MDCK cell extracts (Schoenenberger et al., 1994). Under basal medium containing radiolabel was not removed. Apical cell
surfaces were then overlaid with 400 m l collagen solution. Filters were
denaturing conditions, this antibody and other polyclonal b 1 integrin
further incubated for up to 12 hours before processing for differential
antibodies immunoprecipitate only two polypeptides corresponding to
surface biotinylation (Schoenenberger et al., 1994) and immunopre-
the b 1 integrin subunit and its precursor (Schoenenberger et al., 1994).
cipitation of integrins.
The function-blocking rat monoclonal anti-human b 1 integrin
To examine the effects of collagen overlay on newly synthesized
antibody, AIIB2 (Hall et al., 1990), was obtained from Dr C. Damsky
integrins, cells were pulse-labeled after collagen overlay. Pulse-chase
(University of California at San Francisco, San Francisco, CA). This
experiments were done 12 hours after collagen overlay when cell
antibody immunoprecipitates only mature b 1 and its associated a
layering and lumen formation are negligible (Fig. 2B). MDCK cells
subunits in MDCK cells; it does not recognize the b 1 precursor of
were rinsed three times, 10 minutes each at 37°C with methionine-
MDCK (A. Zuk, unpublished observation).
free medium containing Hepes, glutamine, and 0.35g/l sodium bicar-
bonate (pulse-medium). MDCK cells were then pulsed for 10 minutes
Antibodies to apical and basolateral membrane markers
at 37°C from the basal cell surface with 100 m l/filter of 100 m Ci
Hybridomas secreting mouse monoclonal antibody 3F21D8 recog- [35S]methionine [35S]cysteine; 500 m l pulse-labeling medium without
nizing the apical membrane glycoprotein, gp135, were obtained from
isotope was added apically. Radiolabeled proteins were then chased
Dr G. Ojakian (SUNY, Brooklyn, NY; Ojakian and Schwimmer, for up to 90 minutes at 37°C in MEM containing 10· the normal con-
1988). Mouse monoclonal antibody 6.23.3 was generated against dog
centration of unlabeled methionine. MDCK integrins reach the cell
intestinal mucosa (Balcarova-Ständer et al., 1984). This antibody rec-
surface within 60 minutes chase (G. M. Zinkl and K. S. Matlin, unpub-
ognizes a basolateral membrane protein of 58 kDa, referred to as p58.
lished observation). After 90 minutes chase, integrins were differen-
Mouse monoclonal antibody rr1, generated against strain I MDCK
tially surface biotinylated and immunoprecipitated.
cells, recognizes the basolateral membrane protein E-cadherin
For surface biotinylation and immunoprecipitation, MDCK cells on
(Gumbiner and Simons, 1986). Culture supernatant and the antibody-
ice were rinsed three times with PBS containing 1 mM CaCl2and 0.5
secreting hybridoma were obtained from Dr B. Gumbiner (Sloan-
mM MgCl2 (PBS+) without disturbing the collagen overlay. MDCK
Kettering, New York, NY). cells were then differentially biotinylated twice, 15 minutes each, from
the apical or basal compartment or both with 0.5 mg/ml sulfo-NHS-
Collagen overlay
biotin (Pierce Chemical Co., Rockford, IL) dissolved in TEA buffer
Rat tail tendon type I collagen (Upstate Biotechnology Inc., Lake (10 mM triethanolamine, 125 mM NaCl, 2 mM CaCl2, 0.5 mM MgCl2,
Placid, NY) in 0.02 N acetic acid was dialyzed against 1/10th Ham’s pH 9.0; Gottardi and Caplan, 1992). Compartments that were not
F-12 (Gibco), pH 4.0, at 0°C. Hydrated collagen gels were cast as pre- biotinylated received only TEA buffer. Filters were then rinsed once
viously described (Zuk et al., 1989) by mixing on ice 7 ml of dialyzed in serum-free medium without mixing of apical and basal washes;
collagen (3 mg/ml), 1 ml 10· F-12 (101 mg/ml), 1 ml 10· sodium filters were then extensively rinsed with PBS+ before extraction.
bicarbonate (11.76 mg/ml; Gibco) and 1 ml FBS. Transwell filters were removed from their supports, cut in half and
In preparation for collagen overlay, MDCK cells grown to conflu- extracted in microfuge tubes (1.5 ml) containing extraction buffer (1%
ency on permeable supports were rinsed once with growth medium NP-40, 1% sodium deoxycholate (added fresh), 0.5% SDS, 10 mM Tris-
prepared as described earlier. Medium (1.5 ml) was added to the basal HCl, pH 7.5, 0.15 M NaCl, 10 mM glycine, 10 m g/ml aprotinin, 0.2
compartment; the apical compartment was overlaid with 400 m l mg/ml iodoacetamide, 1 m g/ml antipain, 17.5 m g/ml benzamidine, 1 m g
collagen solution after rinsing once with the same volume of collagen pepstatin, 10 mM glycine) with rocking at 0°C for 30 minutes. Filters
mixture. Within 20 minutes at 37°C, the collagen polymerized into a were removed by centrifugation and cell extracts in the supernatant were
hydrated gel covering MDCK apical surfaces. In order to facilitate filtered through 0.45 m m filter units (Ultrafree-MC Filters; Millipore
contact of apical cell surfaces with the polymerized type I collagen Corporation, Bedford, MA) to remove cell debris. For immunoprecipi-
gel and to avoid detachment of the collagen overlay from this cell tation, extracts were incubated overnight with rocking at 0°C with
surface, additional medium was not added to the apical compartment primary antibody; Protein A Trisacryl (Pierce) was then added and
of permeable supports overlaid with collagen. Control filters that were samples incubated further for a minimum of 2 hours. After washing three
not overlaid with collagen had 400 m l medium added to the apical times with rocking in extraction buffer without protease inhibitors and
compartment. Cultures were incubated for up to 5 days. The volume once with 10 mM Tris, pH 8.6, to remove salts, antigen-antibody
of collagen and/or medium added to the apical or basal compartments complexes were released from Protein A-Trisacryl beads by heating at
was the minimal amount needed for adequate incubation of Transwell 95°C for 3 minutes in 30 m l of 2% SDS, 20 mM Tris-HCl, pH 8.6. As
supports for the duration of the experiment. a control for 35S incorporation, 5 m l of each sample was prepared for
For some experiments, antibodies or reagents were mixed with SDS-PAGE. The remaining 25 m l were diluted tenfold with 1% Triton
collagen at the time of overlay and also added basally. Cultures were X-100, 20 mM Tris-HCl, pH 8.6, 150 mM NaCl. Streptavidin-agarose
fed from the basal compartment with fresh antibody or reagent 24 beads (Pierce) were then added and samples inverted at 4°C overnight.
hours later. Antibodies included AIIB2; reagents included mitomycin Biotinylated integrins were released from agarose beads after washing
C (Sigma), an inhibitor of cell proliferation. Heat inactivated FBS was by heating at 95°C for 3 minutes in electrophoresis sample buffer (200
used for antibody inhibition studies. mM Tris-HCl, pH 8.8, 5 mM EDTA, 15% sucrose, 0.1% bromophenol
blue, 20 mM dithiothreitol, and 2% SDS). Samples were alkylated with
Metabolic labeling and immunoprecipitation 0.5 M iodoacetamide for 15 minutes at 37°C in the dark.
Integrins were radiolabeled to steady-state prior to collagen overlay. Immunoprecipitated polypeptides were resolved on 6% SDS-
Polarized monolayers of HD MDCK cells grown on Transwell filters PAGE gels (Laemmli, 1970). Gels were fixed, impregnated with
1878 A. Zuk and K. S. Matlin
EN3HANCE (E. I. DuPont deNemours & Co., Boston, MA), dried 12 hour intervals up to 48 hours were labeled from the basal com-
and exposed to pre-flashed KODAK X-OMAT film (Eastman Kodak partment with l m Ci/ml [3H]thymidine in DME containing FBS,
Co., Rochester, NY). Hepes, glutamine and antibiotic-antimycotic. Twelve hours later,
[3H]thymidine incorporation was assayed. Filters were rinsed on ice
Light microscopy three times with PBS+, 4 minutes each, without disturbing the
Cultures were fixed in half-strength Karnovsky fixative (2.5% glu- collagen overlay. Filters were cut away from their supports with
taraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate collagen intact and proteins precipitated on ice for a minimum of 2
buffer, pH 7.4) for 1 hour, osmicated (1% OsO4), and stained en bloc hours in 1 ml 10% trichloroacetic acid (TCA). TCA was removed by
with 1% uranyl acetate, as previously described (Zuk et al., 1989). aspiration, and samples rinsed twice with 1 ml fresh ice-cold 5% TCA.
Filters were dehydrated in a graded series of ethanols, cut from the Samples were solubilized in 500 m l 0.2 M NaOH, 0.l% SDS for 1
filter holders leaving the collagen overlay intact, and infiltrated and hour at 37°C and then neutralized with an equal volume of 2 M Tris,
embedded in Epon-Araldite containing 2% DMP-30. pH 6.8. Radioactivity was measured by liquid scintillation counting
Transverse sections (0.5-1 m m) were cut using a Reichert-Jung from equal aliquots of samples. Counts per minute/sample were cal-
Ultracut microtome. Sections were stained with 1% toluidine blue (for culated by subtracting background counts (filter, with or without
light microscopy) or 0.1% toluidine blue (for immunoperoxidase collagen). Significance (P≤0.05) was determined by the one-way
stained filters) in 1% sodium borate and photographed with T-MAX analysis of variance and Student’s t-test.
ASA 100 film (light microscopy; Kodak) or Technical Pan Film ASA
100 (peroxidase immunohistochemistry; Kodak).
RESULTS
Immunohistochemistry
Apical and basolateral membrane markers were immunolocalized in
HD MDCK cells overlaid with type I collagen gel
whole-mount collagen overlaid cultures using a modification of a per-
form tubulocysts
oxidase-antiperoxidase technique previously described (Zuk and Hay,
1994). This procedure has the advantage of localizing with high res- HD MDCK cells were cultured to confluency on Transwell
olution the stable peroxidase product in plastic-embedded samples filters and then metabolically labeled to steady-state with
without the use of frozen sections (0.5-5 m m) of cells on filters and/or [35S]methionine [35S]cysteine. Apical and/or basolateral com-
confocal microscopy. partments were differentially cell surface biotinylated and cell
For peroxidase immunohistochemistry, an entire filter of MDCK cells lysates immunoprecipitated with antibody recognizing the
overlaid with collagen was rinsed in PBS+ and fixed in 2%
apical membrane glycoprotein, gp135 (mouse monoclonal
paraformaldehyde, 75 mM L-lysine, 10 mM sodium m-periodate (PLP;
antibody 3F21D8; Ojakian and Schwimmer, 1988) or the baso-
McLean and Nakane, 1974) for 1 hour. After further rinsing, endoge-
lateral membrane marker, E-cadherin (mouse monoclonal
nous peroxidase activity was quenched with 1% H2O2/PBS or 0.1%
antibody rr1; Gumbiner and Simons, 1986). Autoradiograms
phenylhydrazine/PBS for 10 minutes; cells were rinsed with PBS and
then permeabilized with 0.1% Triton X-100/PBS for 4 minutes. Non- of SDS-PAGE gels reveal that HD MDCK cells polarize apical
specific binding sites were blocked by incubating in 10% normal goat and basolateral membrane markers to their respective plasma
serum (NGS) for 45 minutes-1 hour. For incubation in antibodies, filter membrane domains. GP135 localizes exclusively to apical
holders were removed from 6-well plates and positioned basal side down plasma membranes (lane 1, Fig. 1) and is not expressed baso-
on 150 m l of antibody pipetted on Parafilm in a humidified chamber; the
apical compartment was covered with an additional 150 m l antibody.
Filters were incubated overnight at 4°C in primary antibody. Filter
holders were returned to 6-well plates, rinsed and then incubated as
described above in link antibody (goat anti-mouse; Sigma) diluted 1:20
in 10% NGS for 1 hour at room temperature. After further rinsing, filters
were incubated in peroxidase-antiperoxidase (PAP) mouse antibody
(Sigma) diluted 1:100 in 10% NGS for 1 hour at room temperature.
Filters were washed extensively and PAP complexes immunolocalized
by reacting with a freshly prepared solution of 0.05% diaminobenzidine
(Sigma) dissolved in 0.01% H2O2in 50 mM Tris-HCl, pH 7.5, for 10-
15 minutes at room temperature. The immunostained filters were then
washed and processed for light microscopy as described earlier except
that osmication and en bloc staining were omitted.
Transepithelial resistance
Transepithelial electrical resistance was measured in HD MDCK cells
grown on permeable supports with or without collagen overlay using
an Epithelial Voltohmmeter (World Precision Instruments, New
Haven, CT). Warm (37°C) medium was added to the apical (1.5 ml)
and basal (1 ml) compartments. Filters were allowed to equilibrate to
room temperature for 20 minutes. Electrical resistance was measured
according to the manufacturer’s directions. Values were calculated Fig. 1. Autoradiogram of SDS-PAGE (6%, reduced) gels analyzing
after subtracting the background contributed by blank filters plus or surface expression of gp135 and E-cadherin in HD MDCK cells labeled
minus collagen overlay, as appropriate. to steady-state with [35S]methionine [35S]cysteine. Surface biotinylated
proteins were immunoprecipitated with mouse monoclonal antibody
Thymidine incorporation directed against gp135 (A, arrow) or E-cadherin (B, arrow). HD
Cultures were labeled with [3H]thymidine (New England Nuclear) in MDCK cells express gp135 only on apical (lane 1), but not basolateral
order to measure cellular proliferation. For these experiments, HD plasma membranes (lane 2), whereas E-cadherin (B) is found only on
MDCK cells either at the time of overlay (0 hour) or at subsequent basolateral (lane 4) but not apical (lane 3) cell surfaces.
Apical b 1 integrin in polarized MDCK cells 1879
laterally (lane 2, Fig. 1), whereas E-cadherin is found only on
basolateral cell surfaces (lane 4, Fig. 1) and is not detected
apically (lane 3, Fig. 1).
To induce formation of tubulocysts, polarized monolayers
of HD MDCK cells on Transwell filters were overlaid from the
apical surface with a solution of type I collagen that polymer-
izes into a hydrated gel with incubation. At the time of overlay
(0 hours, Fig. 2A), HD MDCK cells are cuboidal shaped and
morphologically polarized along an apical to basal axis (also
Figs 4A, 5A). Monolayer morphology is uniform and
organized at this time. Collagen fibrils within the polymerized
gel contact apical epithelial surfaces (Fig. 2B). Twelve hours
later (Fig. 2B), monolayer morphology is relatively
unchanged; in a few areas, however, HD MDCK cells begin to
layer (data not shown). Cell layering is more extensive at 24
hours (Fig. 2C), frequently becoming 2-4 cells thick.
Monolayer morphology is disrupted in these areas as the
epithelium reorganizes. Changes in cell shape are noted,
ranging from cuboidal to squamous. As a second cell layer
forms that adheres to the overlaid collagen (Fig. 2C), cell-cell
contacts are retained within the original monolayer, the
forming bilayer and regions undergoing epithelial rearrange-
ment. The forming bilayer subsequently attaches to the
collagen gel and populates this surface (Fig. 2C,D,E). Lumena
then develop between cells attached to the filter and those
attached to collagen. Morphogenesis of HD MDCK cells into
a bilayer of cells separated by a lumen, i.e. tubulocyst, is
complete at 48 hours (Fig. 2D,E) and extends across the filter.
HD MDCK cells populating the gel surface and forming
tubules reacquire cuboidal cell shape (Fig. 2D).
Cell proliferation accompanies tubulocyst formation (Fig.
3). Measurement of [3H]thymidine incorporation in HD
MDCK cells at 12 hour intervals beginning at the time of
overlay and extending up to 48 hours reveals no significant
change in [3H]thymidine incorporation within the first 12 hours
of culture in the absence or presence of collagen. Proliferation
increases at subsequent time points in collagen overlaid
cultures. The most significant increase (P≤0.05) is measured
24-36 and 36-48 hours after overlay, when MDCK cells
populate the gel surface and form lumens. Mitomycin C (1-2
m g/ml), an inhibitor of cell proliferation, prevents formation of
a complete bilayer; lumena are not well developed and tubu-
locyst formation is incomplete (data not shown). This suggests
that cell division is not required for rearrangement of the
Fig. 2.Light micrographs of transverse sections (0.5 m m) of HD
MDCK cells overlaid from the apical surface with rat tail tendon
type I collagen gel. Sections were stained with 1% toluidine blue. At
0 hours (A), confluent HD MDCK cells grown on Transwell filters
are overlaid from the apical surface with type I collagen gel (see
Materials and Methods). At the time of overlay, HD MDCK cells are
cuboidal shaped and apically-basally polarized (also, Fig. 1). Twelve
hours (B) later, monolayer morphology remains relatively
unchanged. Collagen fibrils within the gel contact apical epithelial
surfaces. At 24 hours (C), cell layering is extensive and monolayer
morphology is altered in these areas. Note the heterogeneous cell
shapes found throughout the re-organizing epithelium. Cells that
appear between the monolayer and collagen gel are beginning to
attach to, and layer against, the collagen; free surfaces are developing
between the cell layers. At 48 hours (D,E), a bilayer of cells
separated by a lumen extends across the filter; lumena are well
developed in some areas. Bar, 10 m m.
1880 A. Zuk and K. S. Matlin
clonal antibodies. GP135 and p58 were thus localized in
10000
d.) collagen overlaid cultures fixed at 12 hour intervals starting at
s. the time of overlay.
m -+ 8000 Light microscopy of PAP immunostained sections reveals
E p that gp135 localizes exclusively to apical plasma membranes
MIDIN ON (c 6000 ocof ncfiornmfluinegn to HurD b MiocDhCemKi ccaell lsre astu tlhtse (tFimige. o1f, loavneer l1a)y. (HFoigw. e4vAer),,
Y TI during tubulocyst formation, gp135 expression and localiza-
H A tion changes. During early stages of cell layering, gp135 is lost
T R 4000
H- O from plasma membranes. It is frequently absent from regions
3 P of cell-collagen interaction (arrows, Fig. 4B,C) as attachments
R
2000 develop to the overlaid collagen gel. In a few areas (open
O
C arrow, Fig. 4B), however, gp135 persists at the site of cell-
N
I collagen contacts. When lumena develop (*, Fig. 4C,D) gp135
0
0-12 hrs 12-24 hrs 24-36 hrs 36-48 hrs immediately localizes to apical plasma membranes of the new
cell layer attached to collagen and to apical cell surfaces of
HOURS AFTER OVERLAY
MDCK cells attached to the filter. We did not detect internal
Fig. 3.[3H]Thymidine incorporation in HD MDCK cells with (black vacuolar staining in tubular morphogenesis at any time point
bars) or without (gray bars) collagen overlay. Filters were incubated we examined.
from the basal cell surface with 1 m Ci [3H]thymidine for 12 hour Distribution of p58, a basolateral membrane marker, was
intervals starting at the time points indicated. No significant change examined next. Initially, p58 localizes only to basolateral and
in cell proliferation is measured at 0-12 hours after collagen overlay not to apical cell surfaces of polarized HD MDCK cells (Fig.
when values are compared to non-collagen controls. The most 5A), confirming our biochemical results with the basolateral
significant increase (P≤0.05) is measured 24-36 and 36-48 hours
marker, E-cadherin (Fig. 1, lane 4). During cell layering, when
after overlay.
expression of the apical marker gp135 is reduced at points of
cell-collagen interaction, p58 is expressed over the entire
plasma membrane at regions of cell-cell, cell-substratum, and
monolayer into a bilayer but that proliferation of cells in the
cell-collagen interactions (Fig. 5B,C). As the epithelium
incipient bilayer is required for complete formation of tubulo-
populates the collagen gel surface and lumena develop between
cysts.
the cell layers, p58 reacquires a basolateral distribution and is
Transepithelial electrical resistance does not change during
lost from forming apical (i.e. free) surfaces (Fig. 5C,D).
tubulocyst formation (Table 1). Transepithelial electrical resis-
Interestingly, we found that localization of apical and baso-
tance was measured at 12 hour intervals beginning at the time
lateral membrane markers do not change during mitosis.
of overlay (0 hour) and ending 48 hours later. No loss of elec-
Whatever their distribution is prior to cell division, the same
trical resistance was detected during tubulocyst morphogen-
distribution is maintained during mitosis, confirming previ-
esis when values were compared to non-collagen overlaid
ously published results (Reinsch and Karsenti, 1994). When
controls.
gp135 expression is downregulated during cell layering, it
Apical and basolateral membrane proteins remains absent from plasma membranes during cell division
redistribute in tubulocyst formation (data not shown). When gp135 localizes to apical plasma
membranes with the formation of free lumenal surfaces, it
From the dramatic reorganization of the MDCK cell epi-
retains this apical location during subsequent mitoses that are
thelium during tubulocyst formation, it seemed likely that
responsible for populating the gel surface (data not shown).
apical and basolateral plasma membrane proteins would also
The basolateral protein, p58, which is randomly distributed on
undergo parallel rearrangements. To examine this, an
plasma membranes during cell layering, remains randomized
immunoperoxidase technique was used to follow the localiza-
during cell division. When basolaterally segregated in the
tion of gp135, an apical membrane glycoprotein (Ojakian and
presence of a free surface, it persists on basolateral plasma
Schwimmer, 1988), and p58, a basolateral membrane marker
membranes as cells within the bilayer divide (Fig. 5D).
(Balcarova-Ständer et al., 1984). Although we detected E-
Overall, these observations indicate that collagen contact
cadherin on basolateral cell surfaces both biochemically (Fig.
with the apical surface of polarized HD MDCK cells leads to
1) and by immunofluorescence microscopy (data not shown),
tubulocyst formation within 48 hours. This reorganization is
it was difficult to consistently detect this antigen with peroxi-
accomplished without significant changes in transepithelial
dase immunohistochemistry using either monoclonal or poly-
electrical resistance but with major changes in the distribution
and expression of apical and basolateral membrane proteins.
Table 1. Transepithelial electrical resistance (ohms·cm2)
of HD MDCK cells The b 1 integrin family on apical surfaces of HD
MDCK cells mediates tubulocyst formation
12 hours 24 hours 36 hours 48 hours
Because b 1 integrins are the major receptor family for
With collagen overlay 248.3±43.8 207.9±28.0 196.2±3.2 204.5±19.3
collagen (Hynes, 1992; Hemler et al., 1995), we next deter-
No overlay control 260.4±17.0 226.8±23.3 232.8±2.0 231.0±10.6
mined whether they initiate tubulocyst formation by polarized
Data represents mean ±s.e.m. of triplicate experiments, three samples HD MDCK cells overlaid with collagen gel. The function-
each. blocking anti-b 1 integrin antibody, AIIB2, which inhibits
Apical b 1 integrin in polarized MDCK cells 1881
Fig. 4.Transverse sections (0.5 m m) of HD MDCK cells cultured on Fig. 5.Transverse sections (0.5 m m) of HD MDCK cells cultured on
Transwell filters and overlaid from the apical surface with type I Transwell filters and overlaid from the apical surface with type I
collagen gel (coll). Sections were photographed after PAP collagen gel (coll). Sections were photographed after PAP
immunolocalization of gp135 with monoclonal antibody 3F21D8. immunolocalization of p58, a basolateral membrane marker. Sections
Sections were stained with 0.1% toluidine blue. At 0 hour (A), were stained with 0.1% toluidine blue. A control section (A) shows
confluent monolayers of HD MDCK cells grown on permeable that p58 localizes to basolateral cell surfaces of polarized HD
supports are overlaid from the apical surface with type I collagen gel. MDCK cells. As the epithelium layers in response to collagen
Only the apical surface exhibits strong staining of gp135 at this time. overlay and attaches to it (B,C), p58 randomizes to all cell surfaces.
After 12 hours of overlay (B) when the epithelium begins to layer, It localizes to surfaces in contact with collagen, as well to regions of
surface staining of gp135 decreases at regions of cell-collagen cell-cell and/or cell-filter interactions. In the presence of a free
interaction. In some areas, gp135 is lost (filled arrow) whereas in surface (i.e. lumen, C,D), p58 localizes basolaterally. Note the
others it persists (open arrow). Some regions of the monolayer basolateral location of p58 in the mitotic cell (D) of the bilayer
continue to localize gp135 to apical plasma membranes. As the populating the gel surface. (B) Represents 12 hours after overlay;
forming bilayer attaches to collagen (B,C), gp135 remains absent in (C,D) 36 hours after overlay. Bar, 10 m m.
regions of cell-collagen interactions (arrows). With the formation of
free lumenal surfaces (*C,D), gp135 localizes to apical plasma
membranes of HD MDCK cells attached to the filter or collagen gel.
(C,D) Represent 36 hours after overlay. Bar, 10 m m.
1882 A. Zuk and K. S. Matlin
MDCK cell attachment to collagens I and IV and laminin The redistribution of b 1 integrins from basolateral to apical
(Schoenenberger et al., 1994), was mixed with collagen prior plasma membranes in response to collagen overlay was first
to overlay of apical cell surfaces. We found that anti-b 1 examined. For these experiments, HD MDCK cells were meta-
integrin antibody significantly inhibits layering and lumen bolically labeled to steady-state with [35S]methionine
formation. Polarized HD MDCK cells do not respond to apical [35S]cysteine prior to collagen overlay. The apical compart-
collagen by reorganizing into tubulocysts. In fact, monolayer ment was then washed, overlaid with collagen and incubated
morphology does not change (Fig. 6A). We frequently further for up to 12 hours prior to differential surface biotin-
observed during processing for light microscopy that collagen ylation of apical and/or basolateral compartments and
gels are not attached to apical plasma membranes. In contrast, immunoprecipitation of b 1 integrins. Under these conditions,
the control antibody, AD7, which recognizes a Golgi-associ- any integrin detected on cell surfaces would be synthesized
ated protein (Narula et al., 1992) has no affect on tubulocyst prior to collagen overlay and any redistribution from basolat-
formation (Fig. 6B). eral to apical plasma membranes would be due to apical cell-
Previous results indicated that in polarized MDCK cells b 1 collagen interactions. The cultures were processed up to the 12
integrins localize only to basolateral and not to apical cell hour timepoint when layering and lumen formation are sparse
surfaces (Schoenenberger et al., 1994) and that they are (see Fig. 2B).
targeted directly to basolateral plasma membranes (Boll et al., We found that in the absence of collagen overlay, polyclonal
1991; G. M. Zinkl and K. S. Matlin, unpublished observation). anti-b 1 integrin antibody (Giancotti and Ruoslahti, 1990)
Thus, the observation that an anti-b 1 integrin antibody added immunoprecipitates a 2b 1 and a 3b 1 integrins from basolateral
to collagen at the time of overlay inhibits tubulocyst formation plasma membranes at the 0, 6 and 12 hour timepoints under
raised the question of how b 1 integrins that are normally baso- nondenaturing conditions (lanes 2, 6, 10, Fig. 7). Surprisingly,
lateral can exert an effect at apical plasma membranes. We the same matrix receptors are also detected apically (lanes 1,
hypothesized that apical application of type I collagen gel 5, 9, Fig. 7), although in amounts much less than those seen
mobilizes b 1integrins to apical plasma membranes to mediate basolaterally. Levels of the b 1 family at all times are signifi-
the formation of tubulocysts. In response to collagen overlay, cantly greater on basolateral cell surfaces (lanes 2, 6, 10, Fig.
b 1 integrins would be redistributed from basolateral to apical 7) than on apical cell surfaces (lanes 1, 5, 9, Fig. 7). Six and
cell surfaces and/or newly synthesized integrins would be 12 hours after overlay, apical collagen does not increase the
delivered to apical plasma membranes. Even though we did not amount of b 1 integrins on apical plasma membranes (lanes 7,
detect the b 1 family on apical surfaces of polarized MDCK
cells (Schoenenberger et al., 1994), we also considered the pos-
sibility that low levels are endogenously expressed on apical
cell surfaces that were previously undetected.
Fig. 7. Autoradiograms of SDS-PAGE (6%, reduced) gels analyzing
surface expression of b 1 integrins in HD MDCK cells labeled to
steady-state with [35S]methionine [35S]cysteine and overlaid with
collagen gel. Samples were processed at 0, 6 and 12 hours after
overlay. Surface biotinylated integrins were immunoprecipiated with
polyclonal anti-b 1 integrin antibody under non-denaturing
conditions; immunoprecipitates were resolubilized and reprecipitated
with streptavidin-agarose to capture surface biotinylated proteins.
Fig. 6.Light micrographs of transverse sections (0.5 m m) of HD HD MDCK cells express the b 1 integrin family, including a 2b 1 and
MDCK cells cultured on Transwell filters and overlaid from the a 3b 1, on basolateral (b) cell surfaces (lanes 2, 4, 6, 8, 10, 12) in the
apical surface with type I collagen gel. Cells were incubated in the absence (lanes 2, 6, 10) or presence (lanes 4, 8, 12) of collagen
presence of antibody for 48 hours. Sections were stained with 1% overlay. Interestingly, HD MDCK cells express a small pool of a 2b 1
toluidine blue. The rat monoclonal anti-human b 1 integrin and a 3b 1 on apical (a) plasma membranes with (lanes 3, 7, 11) or
antibody, AIIB2, which is function-blocking, inhibits layering and without (lanes 1, 5, 9) collagen overlay. There is no increase in levels
lumen formation of HD MDCK in response to apical collagen of apical b 1 integrins with collagen overlay (compare lanes 1 vs 3;
overlay (A) whereas the control antibody AD7 (B) does not. In the lanes 5 vs 7; lanes 9 vs 11). Identification of integrin a subunits was
presence of anti-b 1 antibody (A), the collagen gel does not attach based on mobility differences observed by SDS-PAGE after
to apical cell surfaces; the polarized epithelial phenotype is immunoprecipitation with subunit specific antibodies
maintained. Hybridoma culture supernatants were used at a l:5 (Schoenenberger et al., 1994). The band indicated by the arrowhead
dilution. Bar, 10 m m. tentatively identifies the a 1 subunit.
Apical b 1 integrin in polarized MDCK cells 1883
11, Fig. 7) when levels are compared to non-overlaid apical
controls (lanes 5, 9, Fig. 7). Levels of b 1 integrins on basolat-
eral cell surfaces are the same with (lanes 4, 8, 12, Fig. 7) or
without (lanes 2, 6, 10, Fig. 7) collagen overlay. Thus, it
appears that a small pool of the b 1 integrin family is endoge-
nously expressed on apical plasma membranes of HD MDCK
cells and that the steady-state levels of b 1 integrins on apical
surfaces do not increase within 12 hours of collagen overlay.
Because no major differences in levels of b 1 integrins are
detected on apical and basolateral plasma membranes in
response to collagen, the results further indicate that b 1
integrins do not redistribute from basolateral to apical cell
surfaces after collagen overlay.
These observations did not rule out the possibility that
apical application of collagen would induce increased
targeting of newly synthesized integrins to apical cell
surfaces. To test this, we pulse labeled HD MDCK cells 12
hours after collagen overlay and differentially surface biotiny-
lated and immunoprecipitated b 1 integrins under non-
denaturing conditions after chase incubations. At 0 minutes
chase, integrin is not detected on HD MDCK cell surfaces
regardless of collagen overlay (lanes 2, 3, Fig. 8A). Only the
precursor to b 1 integrin is synthesized at this time (lanes 1¢-
3¢, Fig. 8B). After 90 minutes chase, all members of the b 1
family, including a 2b 1, a 3b 1 and the b 1 precursor are made
(lanes 4¢-9¢, Fig. 8B). As previously reported (Schoenenberger
et al., 1994), it was difficult to resolve the a xb 1. The a 2b 1
and a 3b 1 are detected on basal plasma membranes in the
absence or presence of collagen (lanes 7, 9, respectively; Fig. Fig. 8. Autoradiograms of SDS-PAGE (6%, reduced) gels analyzing
8A) after 90 minutes chase. In agreement with our conclusion pulse-chase labeling of b 1 integrins in HD MDCK cells twelve hours
after collagen overlay. Cells were pulsed for 10 minutes and chased
from steady-state labeling experiments, we found that in the
absence of collagen overlay (lane 6, Fig. 8A), the b 1 family, for 90 minutes. HD MDCK cells were then differentially surface
biotinylated from either the apical (a), basolateral (b) or both (ab)
primarily a 2b 1, is detected on apical cell surfaces in amounts cell surfaces and immunoprecipitated with polyclonal anti-b 1
equivalent to that seen after steady-state labeling. Moreover,
antibody under non-denaturing conditions. (A) Surface expression of
collagen overlay does not stimulate delivery of additional b 1 integrins. At 0 minutes chase, integrins are not detected on
newly synthesized integrin to apical plasma membranes (lane MDCK cell surfaces with (lane 3) or without (lane 2) collagen
8, Fig. 8A). overlay. After 90 minutes chase, the b 1 integrin family, including the
The results indicate that confluent HD MDCK cells grown a 2b 1 and a 3b 1, are detected on basal cell surfaces in the absence or
in transfilter culture and polarized with respect to apical and presence of collagen (lanes 7, 9, respectively). In the absence of
basolateral membrane markers endogenously express low collagen (lane 6), b 1 integrins, primarily the a 2b 1, are detected on
levels of b 1 integrins on apical plasma membranes and that apical cell surfaces. Collagen overlay does not mobilize additional
newly synthesized integrins to apical plasma membranes (lane 8).
these mediate tubulocyst formation in response to collagen
Sorting of the a 2b 1 appears to be less stringent than the a 3b 1
overlay. b 1 integrins are not mobilized to apical plasma
explaining its predominance on apical plasma membranes.
membranes either by redistribution from basolateral to apical
(B)Immunoprecipitable antigen of the respective samples shown in
cell surfaces or by new synthesis and delivery to apical plasma (A). Aliquots (5 m l) were taken after immunoprecipitation with b 1
membranes. integrin and Protein A Trisacryl but prior to precipitation of
biotinylated integrins with streptavidin agarose. The band indicated
The C6 MDCK cell line does not form tubulocysts by the arrowhead has been tentatively identified as the a 1 integrin
We previously reported that b 1 integrins are not detected on subunit. The a 3integrin subunit is difficult to resolve from the
apical cell surfaces in another clonal MDCK cell line known precursor to b 1 (pb 1).
as C6 (Schoenenberger et al., 1994). C6 MDCK cells contain
the empty retroviral vector pMV7 and are identical to HD
MDCK cells with respect to polarization of apical and baso- membranes of HD MDCK (lane 3, Fig. 9). The profile of b 1
lateral membrane markers, transepithelial electrical resistance integrins expressed on basolateral plasma membranes are
and ultrastructure (Schoenenberger et al., 1991). They differ, identical for both cell lines (lanes 2 and 4, Fig. 9).
however, in the polarization of b 1 integrins. Steady-state The b 1 integrins found on apical plasma membranes of HD
labeling of both cell lines followed by differential cell surface MDCK cells are sufficient to mediate tubulocyst formation
biotinylation and immunoprecipitation of b 1 integrins under without additional receptor mobilization to the apical domain.
non-denaturing conditions confirms that b 1 integrins are not To complement these observations, we used the C6 MDCK
expressed on apical plasma membranes of C6 cells (lane 1, Fig. cell line which does not express apical b 1 to determine whether
9), whereas the a 2b 1 and a 3b 1 are detected on apical plasma in the absence of this receptor family, cell-collagen interactions
1884 A. Zuk and K. S. Matlin
Fig. 9.Autoradiograms of SDS-PAGE (6%, reduced) gels analyzing
surface expression of b 1 integrins in C6 and HD MDCK cells
labeled to steady-state with [35S]methionine [35S]cysteine in the
absence of collagen overlay. Surface biotinylated proteins were
immunoprecipitated with rabbit polyclonal anti-b 1 integrin antibody
under non-denaturing conditions. C6 cells do not express b 1
integrins on apical plasma membranes (lane 1), in contrast to HD
MDCK cells that express the a 2b 1 and a 3b 1 on apical cell surfaces
(lane 3). b 1 integrins localize to basolateral plasma membranes of Fig. 10. Autoradiograms of SDS-PAGE (6%, reduced) gels
analyzing pulse-chase labeling of b 1 integrins in C6 MDCK cells
both C6 and HD MDCK cells (lanes 2 and 4, respectively). The
profile of b 1 expression on basolateral surfaces are identical between twelve hours after collagen overlay. Cells were pulsed for 10 minutes
and chased for 90 minutes. C6 cells were then differentially surface
the two cell types. The band indicated by the arrowhead has been
tentatively identified as the a 1 subunit. biotinylated from either the apical (a), basolateral (b) or both (ab)
cell surfaces and immunoprecipitated with polyclonal anti-b 1
integrin antibody under non-denaturing conditions. (A) Surface
expression of b 1 integrins. At 0 minutes chase, integrins are not
depolarize b 1 integrins to apical plasma membranes. Twelve
detected on cell surfaces with (lane 4) or without (lane 3) collagen
hours after overlay, C6 cells were pulse-chased, differentially overlay. After 90 minutes chase, the a 2b 1 and a 3b 1 are detected on
surface biotinylated and immunoprecipitated with polyclonal basal (b) cell surfaces in the absence or presence of collagen (lanes 8,
anti-b 1 integrin antibody. Confirming our steady-state labeling 10, respectively). b 1 integrins are not detected on apical (a) surfaces
data (Fig. 9, lanes 1, 2), we found that the b 1 integrin family of C6 cells (lane 7) and collagen overlay does not mobilize newly
is expressed only on basolateral cell surfaces after a 90 minute synthesized integrins to apical domains (lane 9). Faint expression of
chase (lane 8, Fig. 10A) and is not detected at appreciable the b 1 subunit after 90 minutes chase is due to inefficient labeling
during the pulse owing to the pool in the ER.
levels on apical plasma membranes (lane 7, Fig. 10A). Low
(B)Immunoprecipitable antigen of the respective samples shown in
levels are detected with prolonged exposure of autoradi-
(A). Aliquots (5 m l) were taken after immunoprecipitation with anti-
ograms; however, the signal is significantly reduced when b 1 integrin antibody and Protein A Trisacryl but prior to
compared to the apical signal on HD MDCK cells (Figs 7, 8).
precipitation of biotinylated proteins with streptavidin agarose. The
Apical application of collagen on C6 cells does not depolarize band indicated by the arrowhead has been tentatively identified as
b 1 integrins to apical plasma membranes (lane 9, Fig. 10A). the a 1 integrin subunit.
Ninety minutes after pulse-labeling, newly synthesized b 1
integrins are not detected on apical cell surfaces in response to
collagen overlay. cell line. Complementing the data which establishes a role for
Light microscopy of C6 cells overlaid with type I collagen apical b 1 integrins in tubulocyst formation, experiments with
gel further indicates that in the absence of apical b 1integrins, C6 cells suggest that in the absence of apical b 1 tubulocysts
this cell line does not form tubulocysts. Forty-eight hours after do not develop in response to collagen.
overlay (Fig. 11B), tubulocysts do not develop across the entire
extent of the filter as they do in overlaid HD cells (compare to
Fig. 2D and E); cell layering is negligible and lumena are DISCUSSION
absent. During processing for light microscopy, we observed
that collagen gels were not attached to apical plasma We demonstrate that overlay of HD MDCK cells with type I
membranes of C6 cells. collagen gel initiates changes in organization and cell polarity
These data indicate that collagen overlay does not stimulate leading to the formation of tubulocysts. In particular, we show
new synthesis or delivery of b 1 integrins to apical plasma that b 1 integrins pre-existing on apical plasma membranes are
membranes, confirming our observation using the HD MDCK necessary for tubulocyst formation. In the polarized HD