* Department of Anatomy, and Department of Biochemistry and Cardiovascular Research Institute, University of California,
San Francisco, California 94143-0452; and Department of Molecular and Cellular Physiology, Stanford University School of
Medicine, Stanford, California 94305-5426
Epithelial tubulogenesis involves complex
cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms
regulating these processes during tubule development
are not well understood. Interactions of the cytoplasmic protein, -catenin, with several molecular partners
have been shown to be important for cell signaling and
cell-cell adhesion. To examine if
-catenin has a role in
tubulogenesis, we tested the effect of expressing
NH2-terminal deleted
-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/
SF)-stimulated MDCK cysts initiated tubulogenesis by
forming many long cell extensions. Expression of
NH2-terminal deleted
-catenins inhibited formation of
these cell extensions. Both
N90
-catenin, which
binds to
-catenin, and
N131
-catenin, which does
not bind to
-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of
-catenin distinct from its role in cadherin-mediated cell-cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous
polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions.
Inhibition of cell extension formation correlated with
the colocalization and accumulation of NH2-terminal
deleted
-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/
SF treatment of parental cell MDCK cysts resulted in
cell proliferation and reorganization of cell extensions
into multicellular tubules. Similar HGF/SF treatment of
cysts derived from cells expressing NH2-terminal deleted
-catenins resulted in cells that proliferated but
formed cell aggregates (polyps) within the cyst rather
than tubules. Our results demonstrate an unexpected
role for
-catenin in cell migration and indicate that dynamic
-catenin-APC protein interactions are critical
for regulating cell migration during epithelial tubulogenesis.
Morphogenetic movements of cells are critical to
normal development of organs and tissues
(Trinkaus, 1984 Binding of To examine the function(s) of Cell Culture
The clones of MDCK cells used in this study are described in detail in
Barth et al. (1997) Production of MRC5 Fibroblast-Conditioned Medium
Containing Hepatocyte Growth Factor/Scatter Factor
MRC5 human lung fibroblasts (CCL171; American Type Culture Collection, Rockville, MD) were grown to confluence in DME containing 10%
FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, in tissue culture flasks
(T75; Corning, Acton, MA). The medium was replaced with 30 ml fresh
medium, and cells were cultured for 3 d. Conditioned medium was collected, centrifuged to remove cell debris, and stored frozen at Stimulation of Tubulogenesis
in MDCK Cysts with Hepatocyte Growth Factor/
Scatter Factor-Conditioned Medium
Medium was removed from three-dimensional collagen gels containing
MDCK cysts and replaced with MRC5-conditioned medium diluted 1:1
with MEM-EBSS, 5% FBS. Treatment was continued for different periods (see Fig. 1), during which time cultures were refed daily with freshly
diluted, conditioned medium. (Virtually identical results were obtained
using purified recombinant hepatocyte growth factor/scatter factor [HGF/
SF], a kind gift from R. Schwall, Genentech, So. San Fransisco, CA).
Nomarski interference contrast images of cysts and developing tubules
were obtained using a microscope (BH2-RFCA; Olympus Corp., Park
Success, NY) fitted with a 20× objective (LWD CD Plan; Olympus) and
connected to an MTI-CCD video camera. Images were captured with
Scion 1.59 onto a Power MacIntosh (7500; Apple Computers, Cupertino,
CA) and processed using NIH Image 1.6 and Adobe Photoshop 3.0.5 (Adobe Systems Inc., Mountain View, CA). For quantifying tubule formation, phase contrast images of 24-h cultures ±HGF/SF and ±Dox were
photographed at 20 to 35 random sites throughout each culture dish using
a microscope (Axiovert 35; Zeiss, Inc., Thornwood, NY) with a 20× LP
Acrostigmat objective; focus was adjusted to the midline of cysts in the
field so that the monolayer of cells in the cyst wall was in focus. Three different cultures were analyzed from each of two independent experiments for each clone ±HGF/SF and ±Dox. Images were printed, and the number of cell extensions from each cyst in the field was counted manually,
binned, and tabulated. Pooled counts from three cultures were used to determine the percentage of cysts with a certain number of extensions within
each bin or the average number of extensions per cyst; statistical significance was calculated by Student's t test.
Immunofluorescence Confocal Microscopy
MDCK cysts were rinsed with PBS, pH 7.4, containing 1 mM CaCl2, 0.5 mM MgCl2 (PBS+), fixed for 20 min with 4% paraformaldehyde in PBS+
and 10 min with 4% paraformaldehyde in PBS+/0.025% saponin, permeabilized for 30 min with 0.025% saponin in PBS+, rinsed with PBS+, and
quenched for 10 min with 75 mM NH4Cl, 20 mM glycine in PBS+, pH 8.0. Nonspecific binding sites for antibodies were blocked by incubating collagen gels for 30 min in blocking buffer (BB) containing PBS+, 0.025% saponin, 0.7% fish skin gelatin, followed by 10 min in BB containing 0.1 mg/
ml boiled RNase A. Samples were then rocked for 1-3 d at 4°C in primary
antibody diluted in BB containing 0.02% Sodium-azide. Primary antibodies included: rr1 mAb supernatant (anti-E-cadherin; Gumbiner and Simons, 1986 MDCK cells seeded in a collagen gel matrix grow clonally
to form cysts that comprise a monolayer of polarized cells
that surround a fluid-filled lumen (Wang et al., 1990 To examine functions of We first examined whether addition of Dox affected tubulogenesis. MDCK parental cell line (T23) cysts were
grown and stimulated with MRC5 cell-conditioned medium in the presence or absence of Dox. Nomarski interference contrast images show that formation of initial cell
extensions (day 1), complex reorganization of cells into
cords (day 4), and development of tubules (data not shown) is similar for parental (T23) cells in the presence or absence of Dox (Fig. 1). Thus, Dox does not affect the timing or extent of morphological changes in cell organization
during tubulogenesis.
Expression of The following clones of MDCK cells were seeded in collagen gel matrices in the presence or absence of Dox: parental cell line (T23), luciferase-D (Lu-D),
To determine if expression of an exogenous neutral protein affected tubulogenesis, we examined cysts formed
from cells expressing Lu. Lu-D cysts cultured in either the
presence or absence of Dox formed multiple cell extensions and cell chains when stimulated for 1 d with HGF/SF
(Fig. 2, Lu, compare +Dox with
To quantify numbers of cell extensions in cysts derived
from different MDCK clones, randomly selected cysts
throughout each culture were photographed with the midline plane of cells in the cyst wall in focus, and the number
of cell extensions in this focal plane was counted. Cysts
were examined from parental (T23) cells, clone The mean number of extensions for each condition
(± Dox) and clone was calculated (Fig. 3 B). Results show
that control cysts from parental cells, Inhibition of Tubulogenic Activity Is Affected by the
Level of Mutant Mutant
The heterogeneity in exogenous gene expression proved
useful in assessing the effects of mutant To determine if the inverse correlation between protein
expression levels and inhibition of tubulogenesis was specific for mutant We have shown previously that in MDCK cell monolayers
Cysts from parental,
APC protein staining in cysts derived from cells expressing Formation of Cell Aggregates (Polyps) in the
Absence of Formation of Long Cell Extensions in Cysts
Stimulated with HGF/SF for Four Days
4 d after addition of HGF/SF to parental cells, developing
tubules consist of multilayered cords of cells that have begun to rearrange into lumen-containing tubules (Fig. 6,
left; Pollack, A.L., and K.E. Mostov, manuscript submitted
for publication); the bottom left panels of Fig. 6 show that
In contrast to parental cells, cysts derived from cells expressing Tubulogenesis involves complex cellular rearrangements
that are important for the formation of many organs and
tissues during embryogenesis. For example, after mesenchymal to epithelial conversion in kidney development,
cell aggregates reorganize into long tubules. These tubules
are comprised of a closed epithelial monolayer surrounding a fluid-filled lumen and develop into differentiated
nephrons (Saxen and Sariola, 1987 To analyze the role(s) of Cysts formed from cells expressing Although neither How does expression of The localization of APC protein in clusters at the tip of
actively protruding membranes has led to the suggestion
that APC protein may have a function in cell migration
(Näthke et al., 1996 What function of APC protein is regulated by After prolonged treatment with HGF/SF (4 d), cysts expressing mutant ). During embryogenesis, folding and rearrangement of sheets of cells are required for gastrulation and neurulation (Keller and Winklbauer,
1992
; Keller et al., 1992a
,b
). During organogenesis, cell rearrangements have a central role in branching morphogenesis and tubulogenesis, processes that are essential for
formation of many organs, including lung, kidney, pancreas, and mammary gland (Spooner and Wessells, 1970
; Wessells, 1970
; Wessells, 1977
; Hogg et al., 1983
; Coleman
et al., 1988
; Saxen, 1987
; Hisaoka et al., 1992
, 1993
). Cell
rearrangements require coordinate regulation of cell-cell
adhesion and cell migration through extracellular matrix
(Gumbiner, 1992
). To understand the molecular mechanisms involved in cell rearrangements during development, it is important to define molecules that regulate and
coordinate cell adhesion and migration during morphogenesis.
-catenin/armadillo plays a critical role in morphogenesis during embryonic development (McCrea et al., 1993
;
Peifer et al., 1993b
; Funayama et al., 1995
; Haegel et al.,
1995
; Cox et al., 1996
). Originally,
-catenin was identified
in a complex with cadherins, a family of Ca2+-dependent
cell adhesion proteins (Nagafuchi and Takeichi, 1989
; Ozawa et al., 1989
; McCrea and Gumbiner, 1991
; McCrea
et al., 1991
).
-catenin binds to the cytoplasmic domain of
cadherins and to another cytoplasmic protein,
-catenin,
which in turn links the cadherin-catenin complex to the
actin cytoskeleton (Aberle et al., 1994
; Hulsken et al.,
1994
; Jou et al., 1995
; Rimm et al., 1995
). Disruption of
-catenin binding to cadherin (Ozawa et al., 1990
; Oyama
et al., 1994
), or deletion of the
-catenin gene (Peifer et al.,
1993b
; Haegel et al., 1995
; Cox et al., 1996
) results in loss
of cell-cell adhesion and disorganization of cells and tissues.
-catenin is also part of a signal transduction pathway initiated by the extracellular ligand, wingless/Wnt
(McCrea et al., 1993
; Peifer et al., 1993a
; Noordermeer et
al., 1994
; Siegfried and Perrimon, 1994
; Siegfried et al.,
1994
). Activation of this pathway results in localization of
-catenin to the nucleus, indicating a role for
-catenin in
regulating gene expression (Funayama et al., 1995
; Gumbiner, 1995
). Recently,
-catenin has been shown to bind
to LEF-1, a DNA-binding transcription factor, and affects LEF-1-induced DNA bending (Behrens et al., 1996
; Huber et al., 1996
; Molenaar et al., 1996
).
-catenin also binds
to the product of the adenomatous polyposis coli (APC)1
tumor suppressor gene in a complex that is independent of
-catenin binding to E-cadherin (Hulsken et al., 1994
; Rubinfeld et al., 1995
). Mutations in the APC gene constitute
the genetic defect in the inherited colon cancer syndrome,
familial adenomatous polyposis, and represent an early
event in a high percentage of sporadic colon cancers (Polakis, 1995
). Mutations in APC protein result in accumulation of enterocytes near the crypt-villus boundary in the
colon (Polakis, 1995
). Näthke et al. (1996)
showed that APC protein is localized in punctate clusters near the ends
of microtubules that protrude into actively migrating
membrane structures, suggesting a role for APC protein in
cell migration.
-catenin to cadherin, APC protein, and
LEF-1 indicates divergent functions of
-catenin. However, these interactions are interrelated. Participation of
-catenin in signal transduction is dependent on a high cytoplasmic level of
-catenin that may be regulated, in part,
by cadherin and APC protein (Hulsken et al., 1994
). Binding of
-catenin to cadherin antagonizes
-catenin signaling function by sequestering
-catenin to the cadherin
complex (Heasman et al., 1994
; Fagotto et al., 1996
). Binding of
-catenin to APC protein has been suggested to increase
-catenin turnover, thereby decreasing
-catenin
levels (Munemitsu et al., 1995
). That
-catenin forms complexes with both cadherin and APC protein also raises the
possibility that
-catenin is involved in regulating functions of both proteins during epithelial morphogenesis.
-catenin in epithelial
morphogenesis, we expressed mutant
-catenins containing NH2-terminal deletions of 90 (
N90) or 131 (
N131)
amino acids in MDCK epithelial cells. We have determined recently that in MDCK cells grown as monolayers,
N90 and
N131
-catenin are significantly more stable and strongly colocalize in complexes with APC protein at
the tips of membrane protrusions. Colocalization of mutant
-catenin and APC protein correlated with a more fibroblastic cell morphology and a delay in colony formation (Barth et al., 1997
). In the present study, we examined
the effects of mutant
-catenin on tubulogenesis in an in
vitro assay for epithelial tubule development. This assay provides an experimentally tractable system with which to
analyze the molecular mechanisms underlying the role of
-catenin in cell adhesion and migration during morphogenetic cell rearrangements. Our results reveal that stable
interaction of mutant
-catenin with APC protein inhibits
cell migration during tubulogenesis. We provide evidence
that dynamic interaction of
-catenin with APC protein is
modulated by the NH2-terminal domain of
-catenin and
may regulate directed cell movements that are critical for tubulogenesis.
Materials and Methods
. The parental (T23) MDCK cell clone was derived from
a polymeric immunoglobulin receptor-expressing cell line (Mostov et al.,
1987
) that was cotransfected with plasmid pUHD15-1 coding for expression of the tetracycline-repressible transactivator, tTA (Gossen and Bujard, 1992
), and a plasmid pSV2-puro, containing a gene for resistance to
puromycin. The parental T23 cell line was cotransfected with an expression vector under the control of a tetracycline-repressible promoter for
full length
-catenin (
-cat*), luciferase (Lu), or
-catenin mutant proteins containing NH2-terminal deletions of either 90 (
N90) or 131 (
N131) amino acids (Barth et al., 1997
) and plasmid pHMR272 carrying a gene for resistance to hygromycin (Gossen and Bujard, 1992
). The following clones of MDCK cells were used in this study: parental (T23);
-cat*-10; luciferase-C and -D;
N90-2 and -A;
N131-5, -7, and -D. Multiple clones were used to ensure that our results are not due to clonal variation. All cells were passaged at subconfluent density on plastic petri
dishes in minimal essential medium (MEM) containing Earle's balanced
salt solution (MEM-EBSS; Cellgro; Mediatech, Inc., Hendron, VA) supplemented with 5% FCS (Hyclone, Logan, UT), 100 U/ml penicillin, and
100 mg/ml streptomycin in 5% CO2/95% air. Cells were grown in the presence or absence of Doxycyclin (Dox; 20 ng/ml; Sigma Chemical Co., St.
Louis, MO). For growth in three-dimensional collagen gels, cells were isolated with trypsin and triturated into a single-cell suspension. Cells were
diluted to ~2 × 104 cells/ml in a type I collagen solution containing 66%
Vitrogen 100 (3 mg/ml; Celtrix, Palo Alto, CA), 1 × MEM, 2.35 mg/ml
NaHCO3, 0.02 M Hepes, pH 7.6, and 12% dH2O. The cell suspension was
pipetted onto NUNC filters (10 mm, 0.02-0.2 µm pore size; 162243; Applied Scientific, So. San Francisco, CA). The culture was incubated at
37°C, 5% CO2/95% air to allow the type I collagen solution to gel and
then overlayered with medium. The medium was changed every 2-3 d.
Cultures were grown for 10-12 d, at which time cysts comprising a closed
monolayer of cells surrounding a fluid-filled lumen had formed.
20°C.
Fig. 1.
Tubulogenesis is
not affected by Dox. Cysts
derived from the parental
(T23) clone of MDCK cells
were grown and treated with HGF/SF in the presence
(+ Dox) or absence ( Dox)
of doxycycline. Cultures grown
(0 d) and treated for either 1 or 4 d with HGF/SF demonstrate that Dox had no effect
on cyst formation or tubule development. Bar, 50 µm.
[View Larger Version of this Image (86K GIF file)]
); affinity purified KT3 mAb supernatant (KT3 epitope tag on
mutant
-catenins; Barth et al., 1997
); and a mouse mAb to
-catenin
(Transduction Labs, Lexington, KY). Rabbit polyclonal antibodies to
APC protein were kindly provided by P. Polakis (ONYX Pharmaceuticals, Richmond, CA) and I. Näthke (Stanford University, Stanford, CA;
Näthke et al., 1996
). Collagen gels were washed extensively in PBS+/saponin and BB and incubated for 18 h at 4°C in BB containing FITC- or Texas
red-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:100 dilution) and/or propidium iodide (ppI; Sigma
Chemical Co.; 1:1,000 dilution from a 3-4 mg/ml stock). After extensive
washing with PBS+/saponin and BB, collagen gels were postfixed with 4%
paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, and mounted in
Vectashield (Vector Labs, Burlingame, CA). Confocal images were collected using a krypton-argon laser with K1 and K2 filter sets coupled to a
confocal head (MRC600; BioRad, Richmond, CA) and a Nikon microscope (Optiphot II) with a Plan Apo 60× 1.4 NA objective. Individual sections were selected from sets of serial sections collected in the x-y plane
that completely spanned through cysts. Data were analyzed using Cosmos
software and NIH Image 1.6. Images were converted to TIFF format, contrast levels of the images adjusted, and composites of contrast-corrected
images were prepared using Adobe Photoshop (Adobe Co.) on a Power
Macintosh (7200/75; Apple Computers).
Results
; Montesano et al., 1991b
; Santos et al., 1993
; Fig. 1, 0 d). In the
presence of conditioned medium from MRC5 cells that
contains HGF/SF, MDCK cysts are stimulated to form
branching tubules (Montesano et al., 1991a
,b; Fig. 1, 1 and
4 d). Tubulogenesis of MDCK cells in vitro can be subdivided into 4 major stages: (1) extension of cell processes
from the wall of the cysts; (2) formation of cell chains that
remain in contact with the cyst; (3) lengthening and conversion of chains of cells to thickened cords; and (4) formation of long, blind-ended tubules that connect with the
lumen of the cyst (Pollack, A.L., K.E. Mostov, manuscript
submitted for publication). These events require localized
changes in cell adhesion and migration through which cells
emigrate and rearrange into new structures without loss of
cell-cell contact (Pollack, A.L., and K.E. Mostov, manuscript submitted for publication). To investigate the role of
-catenin in tubule development we analyzed two time periods after addition of HGF/SF (Fig. 1): day 1 (stages 1 and
2), when cell extensions form and chains of cells are initiated; and day 4 (stages 3 and 4), when cords of cells have
been established and some tubule lumens have formed.
-catenin in MDCK cell tubulogenesis, we expressed mutant
-catenins with NH2-terminal deletions of either 90 (
N90) or 131 (
N131) amino
acids.
N90
-catenin retains binding sites for
-catenin,
E-cadherin, and APC protein, whereas
N131
-catenin
binds E-cadherin and APC protein but not
-catenin (Barth et al., 1997
). For comparison, we also examined tubulogenesis of MDCK cysts derived from parental (T23)
cells, cells expressing
-cat*, and cells expressing a control
protein, luciferase. Expression of exogenous genes was under the control of the Dox repressible transactivator; in
the presence of Dox, gene expression was repressed, and
in the absence of Dox
-cat*, mutant
-catenins or luciferase was expressed (for details of
-catenin expression
see Barth et al., 1997
).
-catenin Mutants (
N90,
N131)
Decreases HGF/SF-stimulated Cell Extension and Cell
Chain Formation (Day 1)
N90-A
-catenin,
and
N131-D
-catenin. MDCK cysts formed from cells
expressing mutant
-catenin or luciferase appear morphologically identical to cysts derived from parental cells;
phase contrast microscopy shows that all cysts comprise a
closed monolayer of cells surrounding a central lumen
(data not shown). To stimulate tubulogenesis, cyst medium
was exchanged with conditioned medium from MRC5
cells containing HGF/SF, and cysts were examined after 1 d
by Nomarski interference contrast microscopy (Fig. 2).
Fig. 2.
Expression of NH2-terminal deleted -catenin
inhibits early stages of tubulogenesis. Cysts were grown
in the presence or absence of
Dox from T23 (Parental)
MDCK cells or from T23
cells transfected with Dox-
repressible expression vectors for Lu or mutant
-catenins that contain NH2-terminal
deletions of 90 (
N90) or 131 (
N131) amino acids. Nomarski interference contrast microscopy images of cysts
treated 1 d with HGF/SF are
shown. Top images (+ Dox) show typical examples of
cysts in which expression of
transfected proteins was inhibited with Dox. Bottom
images (
Dox) show cysts in which Lu,
N90, or
N131
are expressed. Note that cysts
expressing
N90 or
N131
-catenins have fewer extensions. Representative images
are shown. Experiments were
repeated two to four times for
each clone. Bar, 50 µm.
[View Larger Version of this Image (173K GIF file)]
Dox), similar to the parental (T23) cyst response (Fig. 2, Parental, + and
Dox). Cysts derived from cells overexpressing exogenous
-cat*
also formed multiple cell extensions that were indistinguishable from those formed by parental (T23) cells (data
not shown; Fig. 3); note, however, that cells accumulate
higher amounts of mutant
-catenins than full length
-catenin, because deletion of the NH2-terminus of
-catenin
results in increased protein stability (Barth et al., 1997
).
Cysts derived from
N90-A or
N131-D cells, in which
mutant
-catenin expression was repressed by Dox, also formed numerous cell extensions similar to those of parental (T23) and Lu-D cysts (Fig. 2, +Dox, compare Lu with
N90 and
N131). In contrast, cysts derived from cells expressing
N90 or
N131
-catenin in the absence of Dox
had significantly fewer and shorter cell extensions and
chains compared to the same clones grown in the presence
of Dox (Fig. 2,
N90 and
N131, compare +Dox with
Dox), suggesting that extension formation was inhibited
in the presence of mutant
-catenin. Note that expression
of either
N90 or
N131
-catenin inhibited tubulogenesis,
demonstrating that the inhibitory effect was independent
of the ability of mutant
-catenins to bind
-catenin.
Fig. 3.
Quantification of tubulogenetic response after 1 d of treatment with HGF/SF. Cysts derived from T23 (Parental) MDCK cells, clone -cat*-10 expressing full length
-catenin, two different luciferase clones (Lu-C and Lu-D), two different
N90 clones (
N90-A and -2), and three different clones of
N131 (
N131-D, -5, -7) were grown and treated for 1 d in the presence or absence of Dox. The
number of extensions per cyst for each clone, ±Dox were counted in the midline focal plane of each cyst. Histograms in A show binned
numbers of extensions per cyst. A representative experiment for each clone is shown. n, total number of cysts counted for each clone
and experimental condition. The graph in B shows the average number of extensions per cyst (mean ± SEM). Quantification was obtained from two experiments for each clone.
, + Dox;
,
Dox.
[View Larger Versions of these Images (42 + 48K GIF file)]
-cat*-10
expressing full length
-catenin, Lu-C and -D clones, two
independent clones of cells expressing
N90
-catenin
(
N90-2 and -A), and three independent clones of cells expressing
N131
-catenin (
N131-D, -5, and -7). For each
clone, data were compiled from the analysis of ~25-30
cysts/culture in three separate cultures for each of two independent experiments; the data were binned and plotted
as histograms (Fig. 3 A). Detailed analysis of numbers of
cell extensions in cysts derived from parental (± Dox),
-cat*-10 (± Dox), Lu-C and -D (± Dox), and
N90
(+ Dox) and
N131 (+ Dox) clones reveals that the majority of cysts contained >20 cell extensions, and very few
had <9 cell extensions (Fig. 3 A). In contrast, detailed
analysis of the numbers of cell extensions in cysts derived
from cells expressing
N90 and
N131
-catenin (i.e., in
the absence of Dox) reveals that the majority of cysts had
<9 cell extensions, and very few had >20 cell extensions
(Fig. 3 A).
-cat*-10, and Lu
clones that were formed and stimulated in the presence of
Dox had pooled averages of 26 ± 1, 23 ± 1, and 24 ± 2 cell
extensions, respectively. In the absence of Dox, parental
cells,
-cat*-10, and Lu clones had pooled averages of 25 ± 1, 25 ± 1, and 24 ± 1 cell extensions, respectively. This
confirms quantitatively that neither Dox, full length
-catenin expression, nor Lu expression affected the formation of
cell extensions during initial stages of HGF/SF-induced tubulogenesis. Similarly, cysts derived from
N90 (
N90
plus Dox) and
N131 (
N131 plus Dox) cells in which mutant
-catenin expression was repressed with Dox had
pooled averages of 27 ± 2 and 27 ± 1 extensions, respectively. However, cysts derived from cells expressing
N90
-catenin (
N90 minus Dox) had a pooled average of 8 ± 3 extensions, and cysts derived from cells expressing
N131
-catenin (
N131 minus Dox) had a pooled average of 14 ± 2 extensions. The difference in number of cell extensions from cysts expressing mutant
-catenin compared to
controls is significant (P < 0.01; Student's t test). These results demonstrate that expression of mutant
-catenins containing NH2-terminal deletions inhibits cell extension
and chain formation.
-Catenin Expression
-catenin expression in cysts stimulated for 1 d
with HGF/SF was analyzed by confocal immunofluorescence microscopy. Mutant
-catenin was distinguishable
from endogenous
-catenin, because both
N90 and
N131
mutants were tagged with an epitope recognized specifically by the KT3 monoclonal antibody. In the presence of
Dox, expression of
N90 and
N131
-catenin was completely repressed, and we observed diffuse background
staining with KT3 antibody (KT3; Fig. 4, A and F); as expected, these cysts formed many cell extensions, as detected by staining with propidium iodide (ppI; Fig. 4, A
and F
). In the absence of Dox, cells within cysts had heterogeneous levels of mutant
-catenin expression; in some
cells KT3 staining was strong (KT3; Fig. 4, B, C, G, and H,
closed arrowheads) and in other cells, levels of KT3 staining were low (KT3; Fig. 4, B, C, G and H, open arrowheads).
The reasons for heterogeneity in expression of mutant
-catenin are unknown. We note, however, that heterogeneity of expression was observed only with polarized cells
in monolayer cultures (data not shown) or cysts (Fig. 4)
but not in low density cultures of the same clones (Barth et
al., 1997
). Thus, heterogeneity in exogenous gene expression appears to be related to the differentiation status of
the cells.
Fig. 4.
Expression of N90
or
N131
-catenin inhibits
formation of cell extensions.
Cysts derived from
N90,
N131, and luciferase clones
were grown and treated for 1 d with HGF/SF in the presence (A, F, and K) or absence (B-E, G-J, and L-O)
of Dox and processed for
double immunofluorescence
labeling of KT3 and ppI (A-
C
and F-H
) or anti-luciferase (anti-Lu) and ppI
(K-M
), KT3 alone (D, E, I,
and J), or anti-Lu alone (N
and O). In A, F, and K, the weak KT3 and anti-Lu staining shows that Dox inhibits
expression of mutant
-catenins and luciferase. ppI
staining in A
, F
, and K
shows that cysts from all
clones treated with HGF/SF
plus Dox formed multiple extensions. Cultures in B-J and
L-O were treated with HGF/
SF minus Dox. Staining with
KT3 or anti-Lu detects cells with high (closed arrowheads) and low (open arrowheads)
N90,
N131, and luciferase expression. (B-J)
Cells expressing high amounts
of mutant
-catenins do not
form extensions. Extensions formed from cells with low
mutant
-catenin expression
are detected with ppI. Subcellular clusters of mutant
-catenins are shown at tips of cells adjacent to extending cells (D and G; closed arrows) and at tips of cell extensions (D and I; open arrows). (L-O) Cells with both high and low luciferase expression form extensions.
Bars: (A, E, F, J-O) 20 µm; (B-D and G-I) 10 µm.
[View Larger Version of this Image (95K GIF file)]
-catenin expression on formation of cell extensions. All cells expressing
high levels of
N90 or
N131
-catenin did not form cell
extensions. However, cells that expressed much lower levels of
N90 or
N131
-catenin formed cell extensions
(compare KT3 and ppI; Fig. 4, B, B
, C, C
, G, G
, H, and
H
). An even stronger correlation between expression of
mutant
-catenins and lack of formation of cell extensions is shown in slightly lower magnification images (Fig. 4, E
and J). At multiple sites along each cyst wall, extensions
formed from cells that had very low
N90 or
N131
-catenin expression (Fig. 4, E and J, open arrowheads) but did
not form from any of the adjacent cells expressing high
levels of
N90 or
N131
-catenin.
-catenins, we examined expression levels
of a control protein, Lu, versus formation of HGF/SF-
induced cell extensions. Fig. 4 shows representative examples of cysts derived from a Lu-expressing clone that had
been stimulated with HGF/SF for 1 d in the presence (Fig.
4, K and K
) or absence (Fig. 4, L-O) of Dox. Cysts
formed multiple cell extensions when grown in either the
presence or absence of Dox (Figs. 2 and 3). Immunofluorescence staining revealed that Lu expression levels in individual cells from cysts cultured in the absence of Dox are
quite heterogeneous; some cells exhibit high levels of
staining (Fig. 4, L-O, closed arrowheads), while others exhibit low, background levels of staining (Fig. 4, L-O, open
arrowheads). Note that there is no correlation between the level of Lu expression and formation of cell extensions;
cell extensions formed from cells that express high, medium, or low levels of Lu. These results establish that: (a)
the factor(s) causing heterogeneous gene expression in these
cysts do not affect formation of cell extensions; and (b)
tubulogenic activity is specifically affected by the level of
expression of NH2-terminal-deleted
-catenins. Thus, some
function of the NH2-terminal region of endogenous
-catenin
is essential to support early stages of tubulogenesis.
N90 and
N131
-Catenins Colocalize with APC
Protein at the Tips of Cell Extensions
N-
-catenin accumulates in APC protein clusters at the
tips of extending cell membranes (Barth et al., 1997
). In
addition, results from Näthke et al. (1996)
suggest that
these APC protein clusters have a function in the formation of stable, microtubule-containing membrane extensions. Close inspection of the subcellular distribution of
N90 and
N131
-catenin during early stages of tubulogenesis revealed that mutant
-catenins are localized in
punctate clusters at the tips of the short cell extensions
formed from cells expressing low levels of mutant
-catenin (Fig. 4, D and I; open arrows). Therefore, we sought to
determine if mutant
-catenin and APC protein colocalize
in these punctate structures and if their codistribution correlated with observed defects in the development of cell
extensions during HGF/SF-induced tubulogenesis.
N90- and
N131-expressing clones
were grown in the absence of Dox and treated for 1 d with
HGF/SF. In parental cells, immunofluorescence staining of
APC protein revealed diffuse, finely granular staining in
cells of the cyst wall (Fig. 5 A
), intense linear staining
within cell extensions (Fig. 5 A
, open arrows), and localized accumulation in puncta at the tip of cell extensions
(Fig. 5, B and C; closed arrowheads). Double immunofluorescence revealed little or no overlap in the staining patterns of endogenous
-catenin and APC protein at either
cell-cell contacts or along the shaft of cell extensions (Fig.
5 A
). Colocalization of APC protein and endogenous
-catenin was observed occasionally in puncta at the tip of
cell extensions (Fig. 5 A
, open arrowhead), although the
majority of APC protein clusters was free of detectable endogenous
-catenin (Fig. 5, B and C).
Fig. 5.
N90 and
N131
-catenins accumulate in clusters with APC protein. Parental cell cysts (A, A
, A
, B, and C) and cysts expressing either
N90 (D, D
, D
) or
N131 (E, E
, E
)
-catenin were treated for 1 d with HGF/SF. Cultures were double labeled for APC protein (A
, D
, and E
) and either endogenous
-catenin (A) or KT3 (D and E). Merged images are shown in A
, B, C, D
, and E
. Extensions from parental cysts contained bright, linear staining of APC (A
, open arrows) that did not colocalize with endogenous
-catenin (A
, B, C). Punctate APC protein staining is sometimes detected at tips of parental cell extensions (B and C, closed arrowheads) and occasionally colocalizes with endogenous
-catenin (A, open arrowheads). Punctate clusters of KT3 staining (D and E,
closed arrows) are strikingly detected at tips of extensions of cells expressing
N90 and
N131
-catenin. Punctate clusters of APC protein detected in D
and E
(closed arrowheads) colocalize with clusters of mutant
-catenin staining (D
and E
, open arrowheads). Bar,
10 µm.
[View Larger Version of this Image (37K GIF file)]
N90 and
N131
-catenin was diffuse in the cytoplasm of cells within the cyst wall and was localized in
punctate clusters at the tips of short cell extensions (Fig. 5,
D
and E
, closed arrowheads), similar to APC protein
staining in parental cells. Double immunofluorescence revealed that
N90 and
N131
-catenin (Fig. 5, D and E;
closed arrows) colocalized with APC protein in intensely
stained punctate clusters at the tips of cell extensions (Fig.
5, D
and E
, open arrowheads). In contrast to APC protein staining in parental cell extensions, staining of linear
arrays of APC protein was absent in short extensions
formed from cells expressing mutant
-catenins (Fig. 5, D
and E
). Linear staining in control extensions might be due
to the association of APC with a cellular structure (e.g., cytoskeleton) or may result from confinement of diffuse APC protein within long, narrow extensions. We note,
however, that in control cell extensions, linear APC protein staining can be distinguished from diffuse staining
near the cell membrane (Fig. 5 A
, and data not shown),
suggesting that APC protein is associated with a distinct
cellular structure. The lack of linear arrays of APC protein
in short extensions formed from mutant
-catenin-expressing cells likely reflects perturbation of these arrays. In addition, these results show that accumulation of
N90 and
N131
-catenin in the APC protein clusters correlated
with the inhibition of formation of long membrane extensions.
-catenin and ppI staining are not continuous in the center
of the extension, indicating that a small lumen has formed
between the layers of cells. Endogenous
-catenin staining
is strongly localized to cell-cell contacts within the cyst
wall and between cells of the cords.
Fig. 6.
Stimulation of tubulogenesis from cysts, in which
cell extension is inhibited, results in the formation of cell
aggregates (polyps). Cysts
derived from Parental (left
column) and N131
-catenin clones (right column)
were stimulated for 4 d with
HGF/SF in the absence of
Dox. Double immunofluorescence images of endogenous
-catenin and ppI or
KT3 and ppI show that cells
accumulate in aggregates at
the base of short cell extensions formed from cells expressing low levels of
N131
-catenin (arrowheads) but
not at the base of tubules developing from parental cysts.
Bars, 10 µm.
[View Larger Version of this Image (68K GIF file)]
N131 (Fig. 6, right),
N90 (data not shown), and
-catenin formed short cell extensions that had not developed further into multilayered cords of cells after 4 d in
the presence of HGF/SF. These extensions were formed
from cells that expressed low levels of mutant
-catenin,
whereas cells expressing relatively high levels of mutant
-catenin did not form extensions (Fig. 4). In cysts formed
from cells expressing
N131
-catenin, we noticed the occasional accumulation of cells that were not integrated into the wall of the cyst but instead appeared to form
grape-like aggregates (polyps) within the wall of the cyst
(Fig. 6, right, arrowheads). These cell clusters were invariably found at the base of cells that were forming short
plasma membrane extensions into the surrounding collagen gel matrix. In some cases, these cell aggregates protruded into the lumenal space of the cyst. Occasionally,
cells in mitosis were detected in these cell aggregates (Fig.
6, ppI, bottom right). These cell aggregates were never found in control cysts (Fig. 6, Parental, left), nor in cysts derived from cells expressing
N90
-catenin (data not
shown).
Discussion
). Cellular rearrangements during tubulogenesis likely involve the coordinate
regulation of cell-cell interactions and cell migration
(Trinkaus, 1984
; Gumbiner, 1992
). In this study, we demonstrate for the first time that
-catenin, a cytoplasmic protein involved in both cell-cell adhesion and cell signaling, has a role in regulating tubulogenesis. Although
-catenin binds to functionally divergent proteins, including E-cadherin,
-catenin, APC protein, fascin, and LEF-1
(Nagafuchi and Takeichi, 1989
; Ozawa et al., 1989
; McCrea and Gumbiner, 1991
; McCrea et al., 1991
; Rubinfeld
et al., 1993
; Behrens et al., 1996
; Tao et al., 1996
), we have
shown that expression of NH2-terminal deleted
-catenin inhibits tubulogenesis coincident with the colocalization
and accumulation of mutant
-catenin with APC protein.
We suggest that dynamic interaction of
-catenin with
APC protein is regulated by the NH2-terminal domain of
-catenin and is essential for normal tubule development.
-catenin in epithelial tubulogenesis we expressed mutant
-catenins that contain a deletion of the NH2 terminus either before (
N90
-catenin)
or including (
N131
-catenin) the binding site for
-catenin. Binding sites for E-cadherin and APC protein were
retained in these mutants (Barth et al., 1997
). Mutant
-catenin expression was controlled by the Dox-repressible transactivator (Gossen and Bujard, 1992
), which allowed
us to examine the same clone of cells with (
Dox) or without (+ Dox) mutant
-catenin expression. To examine
stages of tubule development we exploited an in vitro tubulogenesis assay in which three-dimensional cysts formed
from kidney epithelial MDCK cells are induced with HGF/SF. This assay has been shown previously to mimic
cellular rearrangements that accompany tubulogenesis in
vivo (Montesano et al., 1991b
; Gumbiner, 1992
).
N90 and
N131
-catenin were similar in size and structure to cysts derived from parental (T23) cells and "control" cysts derived
from Lu or full length
-catenin-expressing cells and
N90-
or
N131-
-catenin cells grown under Dox repression.
Control cysts and cysts derived from cells expressing
N90
or
N131
-catenin had similar growth rates, dimensions,
and comprised a closed monolayer of cells surrounding a
fluid-filled lumen. Thus, expression of neither
N90 nor
N131
-catenin affected formation of a polarized cell
monolayer during cyst development.
N90
-catenin binds
to both E-cadherin and
-catenin and, therefore, may not
interfere with E-cadherin-based adhesion.
N131
-catenin, on the other hand, binds E-cadherin and not
-catenin
and might be expected to disrupt binding of the cadherin
complex to the actin cytoskeleton. However, levels of
N131
-catenin expression may not be sufficient to compete with and disrupt the function of endogenous
-catenin in E-cadherin-based adhesion.
N90 nor
N131
-catenin had detectable effects on cell-cell adhesion in polarized cysts,
both mutant
-catenins had strong inhibitory effects on tubulogenesis. Tubulogenesis begins with directed, protrusive plasma membrane activity of individual cells of the
cyst wall, such that long membrane extensions formed as
cell processes migrate in the direction of subsequent tubule development (Montesano et al., 1991b
; Pollack, A.L.,
and K.E. Mostov, manuscript submitted for publication).
Cysts derived from cells expressing
N90 and
N131
-catenin had significantly fewer cell extensions than control cysts after 1 d, and many cysts had little or no evidence
of migration of cell processes from the cyst wall. The few
cell extensions that formed from cysts expressing
N90
and
N131
-catenin were initiated exclusively from cells that expressed low levels of mutant
-catenin. Note that
inhibition of cell extensions by these mutant
-catenins is
independent of their ability to bind
-catenin. Therefore,
NH2-terminal deletions of
-catenin disrupt a function of
-catenin in tubulogenesis that is distinct from its role as a
functional link between E-cadherin,
-catenin, and the actin cytoskeleton.
N90 and
N131
-catenin inhibit directed cell extension and migration from MDCK
cysts? Recently,
-catenin has been shown to bind to a
transcription factor (Behrens et al., 1996
; Huber et al.,
1996
; Molenaar et al., 1996
). It is possible that mutant
-catenins interfere with the cellular response to HGF/SF
by altering the expression of genes that are important for
signal transduction. However, our results indicate that mutant
-catenin inhibits the motogenic but not mitogenic response to HGF/SF. Taken together with the distinctive
subcellular localization of mutant
-catenin (see below),
we suggest that these mutant
-catenins affect a structural
component of cell migration that is required for tubulogenesis.
). Accordingly, during tubulogenesis
we observed that APC protein was prominently localized
in clusters at the tips of membrane extensions, in cells that
were actively protruding from the cyst wall and their nearest neighbors but not in cells of the cyst wall that were not
forming extensions. In our previous study (Barth et al.,
1997
) we showed that both
N90 and
N131
-catenin were significantly more stable in APC protein complexes than
full length
-catenin. This increased stability resulted in
the accumulation of mutant
-catenin within APC protein
clusters at the tips of membrane processes. Endogenous
-catenin and overexpressed full length
-catenin were
rarely found in these APC protein clusters (Näthke et al.,
1996
; Barth et al., 1997
). During tubulogenesis, full length
-catenin was only occasionally found colocalized in distinct clusters with APC protein in cell extensions from control cysts. Inhibition of cell extension formation during
tubulogenesis, however, correlated with the accumulation
of mutant
-catenin in APC protein clusters and the absence of linear cytoplasmic arrays of APC protein in the
few short extensions that did form from cells expressing
mutant
-catenin. Therefore, we suggest that
-catenin
binding to APC protein regulates a function of APC protein that is important in the development of cell extensions during tubulogenesis.
-catenin? It is noteworthy that in actively migrating epithelial
cells, bundles of microtubules invade cell extensions and
coalesce at clusters of APC protein that are localized at
the leading edge of cell protrusions (Näthke et al., 1996
).
In vitro, APC protein binds to and bundles microtubules
(Munemitsu et al., 1994
), and in transfected cells, exogenous APC protein codistributes along the length of microtubules (Smith et al., 1994
). Significantly, addition of nocodazole to cells results in disruption of both microtubules and APC protein localization to the tips of membrane extensions and inhibition of directed cell migration (Näthke
et al., 1996
). An interesting corollary to these observations
is that during the formation of stable extensions in growth
cone outgrowth, individual microtubules actively invade
cell protrusions and are subsequently organized into bundles that stabilize the direction of migration (Sabry et al.,
1991
; Tanaka and Kirschner, 1991
; Tanaka and Sabry, 1995
). Formation of cell extensions during epithelial tubulogenesis may involve similar processes in which establishing and stabilizing the direction of migration involves the
reorganization and stabilization of microtubules. As evidence of this, disruption of microtubule organization or
dynamics, respectively, with colcemid or taxol, inhibits
elongation and directed cell migration during HGF/SF- induced tubulogenesis (Dugina et al., 1995
). We suggest
that
-catenin regulates a function of APC protein in organizing microtubules that is required for the formation and/
or stabilization of cell extensions during tubulogenesis. We
propose that
-catenin-APC protein interactions are normally transient, thereby allowing APC protein to function.
Stabilization of the
-catenin-APC complex perturbs APC
protein function, resulting in inhibition of cell migration.
-catenin formed aggregates (polyps) that
accumulated within the cyst wall subjacent to cell extensions that were inhibited in migration. A few mitotic cells
were observed in these aggregates as well as in normally
developing tubules of control cysts in response to mitogenic signaling by HGF/SF. We suggest that a normal proliferative response of mutant
-catenin-expressing cells to HGF/SF, in the absence of cell migration, results in the
formation of cell aggregates rather than tubules. This phenotype has an intriguing parallel to intestinal epithelium
organization after mutations in APC protein. During differentiation of mammalian enterocytes, cells migrate from
the crypt to the villus as a sheet of cells and are then
sloughed from the villus tip (Gordon and Hermiston,
1994
). However, mutations in APC protein that delete the
microtubule binding site result in the accumulation of cells in polyps at the crypt/villus boundary (Polakis et al., 1995). Our results indicate that altering the function of APC protein in cell migration, through stabilization of dynamic interactions of APC protein with
-catenin, inhibits cell rearrangements during tubulogenesis. Furthermore, in the
presence of continued proliferation, inhibition of cell migration may lead to the piling up of cells and the formation
of cell aggregates (polyps). Taken together with the results
of other studies, we suggest that in vivo disruption of the
function of APC protein, either through abnormalities in binding to microtubules or alterations in the dynamics of
-catenin-APC protein interactions, may modify the timing and progression of cell rearrangements that affect the
final outcome of epithelial morphogenesis.
Received for publication 20 December 1996 and in revised form 17 March 1997.
A.L. Pollack and A.I.M. Barth contributed equally to this paper.We thank Dr. Ray Runyan (University of Arizona, Tucson, AZ) for help
with digital capture of Nomarski images. We are also thankful to Drs.
Inke Näthke (Stanford University, Stanford, CA) and Paul Polakis,
(ONYX Pharmaceuticals, Richmond, CA) for providing APC protein antisera and for helpful discussions, to Dr. Rolf Kemler (Max Planck Institute for Immunobiology, Frieburg, Germany) for providing the -catenin
cDNA, to Dr. Hermann Bujard, (ZMDH, Heidelberg, Germany) for providing the plasmids for the tetracycline repressible expression system, and
to Dr. Gernot Walter (University of California, San Diego, CA) for providing the monoclonal antibody KT3.
This work was supported by grants to W.J. Nelson from the National Institutes of Health (DK45573) and The Mathers Charitable Foundation, and by grants to K.E. Mostov from the National Institutes of Health (AI25144, AI36953, HL55980). A. Barth was supported by a North Atlantic Treaty Organization (NATO) fellowship from the Deutscher Akademischer Austauschdienst and an American Heart Association fellowship.
APC, adenomatous polyposis coli;
-cat*, full length
-catenin;
Dox, doxycycline;
HGF/SF, hepatocyte growth
factor/scatter factor;
Lu, luciferase;
ppI, propidium iodide.
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