1 Department of Cell and Developmental Biology, Center for Molecular
Neuroscience, Program in Developmental Biology, Vanderbilt University Medical
Center, Nashville TN 37232-2175, USA
2 School of Biological Sciences, College of Natural Sciences, Seoul National
University, Seoul 151-742, Republic of Korea
3 College of Dentistry, Seoul National University, Seoul 110-740, Republic of
Korea
Author for correspondence (e-mail:
kolodzp{at}ctrvax.vanderbilt.edu)
Accepted 20 August 2003
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SUMMARY |
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Key words: E-cadherin, Drosophila, Cytoskeleton
![]() |
Introduction |
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These remodeling processes require the adherens junction to organize the
cytoskeleton. E-cadherin interacts with the F-actin cytoskeleton via the
ß-catenin/-catenin complex
(Rimm et al., 1995
), and may
also regulate F-actin assembly via Rho family GTPases
(Kuroda et al., 1998
;
Anastasiadis and Reynolds,
2001
; Kovacs et al.,
2002a
; Lampugnani et al.,
2002
) and the ARP2/3 complex
(Kovacs et al., 2002b
).
E-cadherin can also organize and stabilize microtubules in cultured cells via
an unknown mechanism (Chausovsky et al.,
2000
; Waterman-Storer et al.,
2000
). However, cytoskeletal structures associated with adherens
junctions during development are not well characterized, nor are their
functions understood.
Tracheal tube fusion in the Drosophila embryo provides a powerful
system for investigating how cells remodel their E-cadherin contacts
(Hogan and Kolodziej, 2002).
During the fusion process, specialized cells at tracheal branch tips, fusion
cells, meet their partners in the adjacent hemisegment at precise locations
along segment boundaries (Samakovlis et
al., 1996b
) (Fig.
1A). A new E-cadherin contact forms between these fusion cells
(Tanaka-Matakatsu et al.,
1996
; Uemura et al.,
1996
), and is associated with a track of F-actin and the plakin
Short Stop (Shot) (Lee and Kolodziej,
2002
). The evolutionarily conserved Shot proteins bind F-actin and
microtubules to promote fusion (Lee and
Kolodziej, 2002
). Presumably, microtubules are also in the track,
although they have only been detected indirectly
(Lee and Kolodziej, 2002
).
This track then grows to span the two cells. These cells become doughnut
shaped as lumenal connections between the branches form along the track
(Lee and Kolodziej, 2002
).
Tracheal tube fusion may reveal mechanisms underlying tube formation in other
systems. Similar doughnut-shaped cells exist in the mammalian vasculature
(Wolff and Bar, 1972
), and
VE-cadherin is required for blood vessel formation
(Lampugnani et al., 2002
).
|
We have further investigated how E-cadherin and Shot regulate track
development during tracheal tube fusion. The formation of a Shot-containing
track and its subsequent maturation are obligate intermediate steps in fusion.
Surprisingly, E-cadherin controls both of these steps using distinct sites in
its cytoplasmic domain. E-cadherin binding of ß-catenin is required for
fusion track formation. The juxtamembrane site, previously thought to be
dispensable in Drosophila, controls track maturation. When expressed
in wild-type tracheal cells, an E-cadherin bearing a mutation in this site
causes fusion tracks to form that contain F-actin and Shot, but lack
detectable microtubules. Fusion involves direct interactions between Shot and
microtubules (Lee and Kolodziej,
2002). We propose that distinct sites in the E-cadherin
cytoplasmic domain mediate the initial assembly of F-actin and recruitment of
Shot to the fusion track, and subsequent microtubule-dependent track
maturation.
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Materials and methods |
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Molecular biology
Using the Quikchange Multi kit (Stratagene), mutations were introduced into
a full-length E-cadherin cDNA (Accession Number D28749) from T. Uemura (Kyoto
University), that changed residues 1607-1609 to alanines (AAA-JXT) or
deleted residues 1672-1696 (-arm). Wild-type and mutant cDNAs
were cloned into the EcoRI/NotI sites of pUASt, a P-element
containing GAL4 expression vector (Brand
and Perrimon, 1993
).
Monoclonal production
A DNA fragment encoding residues 1454-1909 amplified from the Shot L(A)
cDNA (Accession Number CAA09869), subcloned into pGEX6P1 (Amersham) was used
to produce GST fusion protein for immunization. Mouse hybridomas (Vanderbilt
Hybridoma Facility) were screened in pools of 10 for immunoreactivity with fly
embryos and mAb Rod1 isolated by limiting dilution of a reactive pool.
Immunohistochemistry
Fusion tracks were scored in fillets using confocal microscopy. Fillets
were staged using gut morphology prior to dissection and neuronal morphology
after dissection. Scored embryos had well formed commissural and some
longitudinal CNS axon tracts, and fusion cells had made contact by
membrane-GFP staining. To visualize microtubules, wild-type btl-GAL4
UAS-GAP43-GFP or mutant btl-GAL4 UAS-GAP43-GFP/+;
UAS-E-cad-AAA-JXT embryos were filleted in a silicone gel well on a glass
slide and quickly fixed by exposure to 90% methanol, 5 mM sodium bicarbonate
pH 9, 3% formaldehyde chilled to 70°C for 10 minutes
(Rogers et al., 2002). The
fillets were carefully rehydrated in PBS/0.1% Triton containing 0.2% BSA
(bovine serum albumin). Otherwise, fillets were fixed in 4% formaldehyde in
Ringer's solution at room temperature for 20 minutes and processed as
described (Lee and Kolodziej,
2002
). Schneider S2 cells were transfected with CellFECTIN
(Invitrogen). pRmHA3-p120 was used to express p120
(Pacquelet et al., 2003
), and
pActin-GAL4 and pUASt-AAA-JXT-E-cadherin were used to express AAA-JXT
E-cadherin.
Primary antibodies were: rabbit anti-EB1
(Rogers et al., 2002) (1:500),
rabbit anti-CLIP190 (Lantz and Miller,
1998
) (1:1000), rabbit anti-ß-galactosidase (Sigma), rat
anti-tubulin (Serotec), rat mAb anti-cadherin (1:5)
(Oda et al., 1994
), mouse mAb
anti-Shot mAbRod1 (1:10), mouse mAb 2A12 (1:10), and mouse mAb 12CA5
(anti-hemagglutinen (HA) epitope tag, 1:200). Images were acquired on a Zeiss
LSM 510 confocal microscope using filter sets and excitation frequencies
specific for FITC, Cy3 or Cy5. Live images were acquired from dechorionated,
late stage 12 embryos expressing Shot L(A)-GFP (UAS-Shot-L(A)-GFP/+;
btl-GAL4/+) (Lee and Kolodziej,
2002
) mounted laterally on a Matek coverslip/culture dish and
covered with modified insect saline solution
(Kim et al., 2002
) containing
2% methylcellulose (Sigma). Argon laser power was set at an average of 10%
(adjusted progressively downward to avoid saturating signal); a pinhole
yielding 0.5 µm slices was used with a GFP filter set. Fluorescence signals
were acquired in the linear range, and optimized for signal to noise by
manually adjusting computer generated levels. To compensate for embryo
movement or curvature, images were composited manually from serial sections
using Adobe Photoshop 6.0 (Adobe Systems), and compared to 3D reconstructions
made with Metamorph (Universal Imaging). A median filter was applied to remove
noise.
E-cadherin was extracted for western blotting from embryos quick frozen on
dry ice, and ground with a pestle in 1% NP40 extraction buffer
(McNeill et al., 1993).
Samples adjusted for equal amounts of protein were transferred to Immobilon-P
(Millipore), western blotted and probed with rat anti-E-cadherin
(Oda et al., 1994
) at 1:1000.
The membrane was further probed with HRP conjugated-anti-rat (Jackson
Immunochemicals) at 1:5000 and developed with Supersignal (Pierce).
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Results |
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To address these issues, we imaged Shot-GFP during tracheal tube fusion in
the dorsal trunk. Movies of several fusion events share similar kinetics and
reveal details of the fusion process. In the first phase, contact, tracheal
cells at branch tips touch, but there is no sign of Shot-GFP at the interface
between the tips (Fig. 1B). The
second phase, track initiation, begins 10 minutes after initial contact.
A faint track of Shot-GFP is detected in the anterior fusion cell, consistent
with observations in fixed tissue that detect new E-cadherin
(Tanaka-Matakatsu et al.,
1996
) and associated cytoskeletal assembly first in the anterior
cell (Lee and Kolodziej, 2002
)
(Fig. 1C). Forty minutes later,
this track is centered on the fusion cell contact
(Fig. 1D). This cytoskeletal
intermediate persists for
1 hour, and accumulates progressively more Shot
(Fig. 1D-G). Regions of the
existing apical surface, defined by Shot-GFP accumulation, appear to change in
intensity during this second phase (insets of fusion site in
Fig. 1F-G).
In the third phase, track maturation and surface invagination, the existing apical surfaces invaginate and the track shrinks, eventually forming a bottleneck at the narrowest point (Fig. 1H). The track to bottleneck transition occurs within five minutes. In cross-section, the apical surfaces appear connected on one side and open on the other (Fig. 1H). In a final phase, fusion and expansion, the bottleneck expands to form a tube (Fig. 1I). In cross-section, two tracks of Shot-GFP are now clearly visible (Fig. 1I). Fusion and expansion is also very fast, occurring in five minutes or less, although expansion to the same diameter as the rest of the trachea takes longer (Fig. 1K). During this period, Shot becomes concentrated into a ring at the adherens junction between the fusion cells (Fig. 1J).
The new lumenal connection between the branches appears to develop as the existing apical surfaces approach closer along the track. We do not observe the development of a cavity internal to the fusion cells that subsequently fuses with its neighbors, an alternative possibility.
Shot colocalizes with dynamic microtubules in the fusion track
Our previous study suggested that the interactions of Shot with
microtubules promote adherens junction and track assembly in fusion cells
(Lee and Kolodziej, 2002).
However, microtubules were not detected as fusion track components under the
fixation conditions used in this earlier study. In order to determine whether
microtubules are present, we dissected live embryos at early stage 13, a stage
where fusion is ongoing in the dorsal trunk, and fixed them rapidly in
methanol/formaldehyde at 70°C, a treatment that preserves more
dynamic microtubules. Under these conditions, microtubules are clearly
detected apically in all tracheal cells even at these early stages (late stage
12 to late 13), and colocalize with the Shot/F-actin containing fusion track
(Fig. 2A-F). Microtubules in
tracheal cells at this stage of development appear especially sensitive to
fixation conditions, as they were not observed using formaldehyde fixation at
room temperature (Lee and Kolodziej,
2002
).
|
Short Stop is associated with adherens junctions
Short Stop (Shot) is required for fusion cells to form the new E-cadherin
contacts that drive fusion, and a Shot-GFP fusion localizes to the
cytoskeletal track in fusion cells that is associated with the new E-cadherin
contact (Lee and Kolodziej,
2002) (Fig. 1).
These data suggest that Shot may be associated with adherens junctions, at
least in tracheal cells. To visualize the endogenous protein in tracheal
cells, we raised a monoclonal antibody against a region of the rod domain,
mAbRod1. mAbRod1 recognizes Shot protein in wild-type
(Fig. 3A-H), but not in
shot3 null mutant embryos
(Fig. 3J-L). Co-staining of
tracheal cells with a rat monoclonal antibody directed against E-cadherin
(Oda et al., 1994
) reveals
that Shot colocalizes with adherens junctions
(Fig. 3A-C). In shot
mutant embryos, adherens junctions between tracheal branches frequently fail
to form, and the branches therefore fail to fuse
(Fig. 3J).
|
ß-Catenin, but not p120, is required for track formation
As E-cadherin is required for fusion
(Uemura et al., 1996), it is
likely to recruit Shot to the fusion track. To investigate this possibility,
we examined track formation in wild-type
(Fig. 4A-C) and
E-cadherin/shotgun (shg)
(Fig. 4D-F) mutant embryos
using phalloidin to label F-actin, and Shot-GFP. In these mutant embryos, some
residual maternal E-cadherin function is present, and they are able to form
trachea and in some cases, to complete fusion
(Uemura et al., 1996
).
However, at segment boundaries where fusion did not occur, F-actin and
Shot-GFP containing fusion tracks were absent
(Fig. 4D-F). In these cases,
fusion cells appeared to be in contact
(Fig. 4D-F). These data
therefore suggest that the failure to form adherens junction associated
cytoskeletal structures in shg mutant embryos reflect a requirement
for E-cadherin signaling in track formation, and not possible earlier roles
for E-cadherin in enabling fusion cells to contact each other.
|
Recently, a null mutation in the single Drosophila p120 related
gene has been described (Myster et al.,
2003). Though homozygous null mutant p120 flies are
viable and fertile, we nonetheless examined tracheal fusion in these mutant
embryos, as it is not known whether tracheal fusion is an essential process.
Tracheal development appeared normal in all homozygous null mutant
p120 embryos examined (n=20)
(Fig. 4M). Thus, p120
is not essential for fusion or other aspects of tracheal development.
Mutations in the E-cadherin juxtamembrane domain dominantly interfere
with track maturation and microtubule track assembly
We further investigated the signaling pathway required to localize Short
Stop to the fusion track by structure/function analysis of E-cadherin. Two
domains in E-cadherin affect cell adhesion in tissue culture models: the
juxtamembrane domain and the C-terminal ß-catenin-binding site
(Ozawa and Kemler, 1998;
Yap et al., 1998
). We
therefore constructed flies expressing wild-type E-cadherin (UAS-WT)
(Uemura et al., 1996
), or
E-cadherins mutant in the ß-catenin binding sites
(UAS-
arm) or in the juxtamembrane domain
(UAS-AAA-JXT). These transgenes are under the control of a
GAL4-dependent promoter, allowing expression to be targeted to particular
tissues during development (Brand and
Perrimon, 1993
). The juxtamembrane domain mutant mutates the
evolutionarily conserved residues 1607-1609 (ERD in fly; EED in mouse) to
alanine. In mammalian E-cadherin, this mutation disrupts binding to p120 in
tissue culture (Thoreson et al.,
2000
) and also affects activation of Rac by E-cadherin
(Goodwin et al., 2003
). These
residues immediately follow the conserved glycine triplet required for p120
binding and recruitment to E-cadherin contacts in cultured Drosophila
S2 cells, but not for E-cadherin function in Drosophila
(Pacquelet et al., 2003
).
Expression of wild-type (Fig.
5A) or arm E-cadherin
(Fig. 5B) in wild-type tracheal
cells did not detectably affect tracheal development. By contrast, expression
of AAA-JXT E-cadherin from either of two independently derived
transgenic lines dominantly disrupted fusion, without detectable effects on
other aspects of tracheal development such as branch migration
(Fig. 5C). Expression of the
E-cadherin altered in the adjacent juxtamembrane glycine residues
(Pacquelet et al., 2003
) had
no effect on fusion (Fig. 5D).
Surprisingly, the AAA-JXT mutant E-cadherin concentrated at cell contacts when
introduced into Drosophila S2 cells and colocalized with
epitope-tagged p120 (Fig. 5E).
As S2 cells do not normally express E-cadherin, these data suggest that the
AAA-JXT mutant E-cadherin functions as a homophilic adhesion molecule and
recruits p120 to cell contacts. E-cadherin mutant in the adjacent glycine
triplet does not bind to p120 or recruit it to cell contacts
(Pacquelet et al., 2003
).
|
To investigate further the fusion defect in the embryos expressing AAA-JXT E-cadherin in tracheal cells, we examined track formation using phalloidin and anti-Shot. Confocal microscopy revealed that fusion cells in these AAA-JXT mutant embryos generally assemble F-actin and Shot at the site of cell contact (79%, n=41) (Fig. 5K,L,N). However, in these cells, F-actin appears abnormally organized (Fig. 5K; 5%) or forms weaker tracks (Fig. 5L,N; 74%) than in wild type, suggesting a defect in track maturation. Visualization of the plasma membrane (Fig. 5M) indicates that fusion cells still contact each other in AAA-JXT mutant embryos. Thus, these defects do not appear to reflect failure of fusion cells to contact each other.
We therefore used confocal microscopy to examine track development in live
embryos expressing Shot-GFP and AAA-JXT in tracheal cells. The fusion track
persisted longer in mutant fusion cells, and the Shot-GFP accumulations at
existing apical surfaces failed to exhibit the remodeling
(Fig. 5P-T) observed in
wild-type (Fig. 1). In confocal
sections along the plane of the track, persistent gaps are observed in the
distribution of Shot-GFP along the existing apical surfaces
(Fig. 5P-S). In other cases,
the track resolved and the existing apical surfaces approached closer
together, but remained blind-ended (Fig.
5T). In AAA-JXT mutant embryos, successful fusion events
took 2-3 hours to complete after track initiation (data not shown), instead of
1 hour in wild-type embryos (Fig.
1).
To address the basis of the maturation defect, we used confocal fluorescent microscopy to examine microtubules in fusion cell pairs in wild-type and mutant embryos. In stage 13 wild-type embryos, 97% (n=59) of fusion cell pairs had microtubules associated with the point of contact, often forming a track (Fig. 5U). By contrast, in 73% of fusion cell pairs examined in stage 13 embryos expressing AAA-JXT E-cadherin in tracheal cells (n=72), microtubule tracks are absent (Fig. 5V). In the remaining fraction, microtubules are often abnormally assembled (Fig. 5V). These data suggest that embryos expressing AAA-JXT E-cadherin can initiate F-actin, but not microtubule track assembly. Alternatively, they may be defective in capturing or stabilizing microtubules. We propose that microtubules are important for later steps in tube fusion, including track maturation, and possibly remodeling of the existing apical surfaces.
The cytoskeletal defects observed in AAA-JXT embryos may reflect
defects in the localization of AAA-JXT E-cadherin. Delocalization of the
receptor may delocalize signals that control F-actin and microtubule assembly.
To test this possibility, we also examined tracheal fusion in embryos that
express AAA-JXT E-cadherin under the control of hairy-GAL4
(Fig. 5W-Y).
Hairy-GAL4 drives AAA-JXT E-cadherin expression in all cells
in odd-numbered segments (T2, A1, A3, A5, A7) and at a lower level
(Brand and Perrimon, 1993).
Cells in tracheal branches expressing AAA-JXT E-cadherin appeared to localize
most of their E-cadherin to adherens junctions, but still frequently failed to
form fusion tracks (Fig. 5W-Y)
or to complete fusion (data not shown). However, dorsal closure, another
E-cadherin dependent process involving epithelial migration, appeared normal
in affected segments (data not shown). These results suggest that fusion
defects do not arise solely from defects in E-cadherin localization or
expression levels of the transgene, and that fusion cell development may be
particularly sensitive to pathways disrupted by the juxtamembrane mutation or
to reductions in E-cadherin function. Fusion tracks also frequently failed to
form on both sides of the affected segment, suggesting that E-cadherin
signaling must occur in both fusion cells for fusion to proceed.
Distinct sites in the E-cadherin cytoplasmic domain control track
formation and maturation
Tracheal expression of wild-type and mutant E-cadherins in wild-type
embryos suggested distinct functions for the juxtamembrane and
ß-catenin-binding domains. To determine the activity of these domains
with respect to track formation and maturation, we examined tracheal fusion,
track formation and maturation in shotgun (shg) mutant
embryos expressing wild-type or mutant E-cadherin transgenes in tracheal
cells. The allelic combination (shg2/shgIH)
selected for these experiments represents a partial loss of cadherin function
(Uemura et al., 1996),
allowing both enhancement and suppression to be scored
(Fig. 6A-E; Table 1). Tracheal expression
of wild-type E-cadherin largely rescued fusion defects in shg mutant
embryo (Fig. 6C;
Table 1). Expression of either
arm or AAA-JXT mutant E-cadherin enhanced the frequency and severity of
fusion defects (Fig. 6D,E;
Table 1). Thus, both the
ß-catenin binding site and the juxtamembrane site are required for
fusion.
|
|
We then investigated the ability of these mutant transgenes to localize
Shot to the fusion track. In shg mutant embryos expressing arm
mutant E-cadherins in tracheal cells, fusion cells fail to form
Shot-containing tracks (Fig.
6G). Thus, the ß-catenin-binding domain is required for
cytoskeletal track assembly in the fusion cells. However, other tracheal cells
localize Shot apically and continue to maintain this organization at later
developmental stages (data not shown).
shg2/shgIH mutant embryos express little
detectable E-cadherin, so the E-cadherin observed in tracheal cells in these
embryos is almost exclusively due to the transgene. In
shg2/shgIH embryos, -arm E-cadherin was
delocalized (Fig. 6H),
suggesting that the ß-catenin binding domain is required for the
localization of E-cadherin to adherens junctions in tracheal cells.
In shg mutant embryos expressing AAA-JXT mutant E-cadherin in tracheal cells, Shot and F-actin fusion tracks are weak or abnormally oriented (Fig. 6J) or absent (Fig. 6P,R) from fusion cells. The fragility of these mutant embryos prevented us from examining microtubule track formation. At all stages (Fig. 6K,N), E-cadherin in these embryos appears delocalized. These data suggest that the juxtamembrane domain is required for E-cadherin localization to adherens junctions and fusion track maturation, which we define as a step occurring after initial track assembly. In these embryos, Shot initially localized apically in other tracheal cells (Fig. 6J,P). However, as development progressed, Shot became delocalized (Fig. 6M). Continued expression of the juxtamembrane site E-cadherin mutant disrupts the apical polarization of the cytoskeleton, suggesting that this site normally actively maintains this evolutionarily conserved feature of tubular epithelia.
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Discussion |
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We show here that in selected cell types, Shot localizes with proteins of
the adherens junction and may play a role in adherens junction-mediated
organization of the cytoskeleton. We propose that Shot and E-cadherin form a
feedback loop in which E-cadherin, via ß-catenin, recruits Shot to new
contacts between the fusion cells and Shot stabilizes the contacts. The
cytoskeleton organizes around these contacts because adherens junction
associated Shot promotes the assembly of an F-actin/microtubule-rich track
(Lee and Kolodziej, 2002).
This track grows to span the fusion cells, extending the reach of the
junctions through the cells. The recruitment mechanism may be indirect in that
new adherens junctions in fusion cells are centers for cytoskeletal assembly,
and Short Stop binds F-actin and microtubules. Alternatively, Shot may
associate directly with E-cadherin or associated proteins. The assembly of
Shot with F-actin and microtubules may stabilize E-cadherin contacts simply by
bringing in cytoskeletal proteins that bind E-cadherin or associated proteins
(Karakesisoglou et al., 2000
).
For example, EB1, which is present in the fusion track, co-immunoprecipitates
with a C-terminal fragment of Shot in cultured cells
(Subramanian et al., 2003
) and
associates with APC (Su et al.,
1995
). APC interacts with ß-catenin to control tubulogenesis
in vitro (Pollack et al.,
1997
).
Distinct sites in the E-cadherin cytoplasmic domain control F-actin
and microtubule assembly
We propose that the assembly and maturation of a cytoskeletal intermediate
are two E-cadherin-dependent steps in tracheal cell fusion
(Fig. 7). Imaging of fixed and
live embryos suggests that fusion proceeds through the assembly and maturation
of a cytoskeletal track associated with adherens junctions. The track forms
after contact between the fusion cells, and persists for 1 hour before
fusion occurs.
|
Both the ß-catenin and juxtamembrane binding sites are required for
E-cadherin localization to adherens junctions, although only the juxtamembrane
mutation seems to interfere with endogeneous E-cadherin localization. Our
results suggest that like mammalian E-cadherin, an evolutionarily conserved
juxtamembrane site is required for some E-cadherin functions. Similar effects
of mutations in the juxtamembrane site were observed in mammalian tissue
culture cells (Yap et al.,
1998). However, juxtamembrane site function in Drosophila
E-cadherin probably does not require p120.
Dominant effects on localization appear sensitive to expression levels,
whereas effects on fusion are less so, suggesting that defects in localization
are not enough to explain the defects in track maturation. Possibly, effects
on localization also reflect defects in organizing the cytoskeleton, as has
been observed in studies in which dominant alleles of Rho family GTPases
affect cadherin localization in culture
(Braga et al., 1997;
Jou and Nelson, 1998
) and
during tracheal development (Chihara et
al., 2003
).
We propose that the ß-catenin-binding site and ß-catenin are required for track assembly, and that the juxtamembrane site regulates other proteins involved in a later maturation step (Fig. 7). This later step likely requires microtubules. The microtubules or associated proteins may reinforce the initial F-actin assembly in the track, as F-actin in fusion tracks appears to be abnormally or poorly assembled in embryos expressing AAA-JXT mutant E-cadherin in tracheal cells. The microtubules appear also required for remodeling the fusion cell apical surfaces and also for bringing them together to fuse. In embryos expressing AAA-JXT mutant E-cadherin in tracheal cells, fusion cell apical surfaces do not develop or seal gaps at appropriate times, and fusion tracks persist substantially longer, if they resolve at all.
The microtubule regulated steps during fusion therefore likely involve
effects on F-actin dynamics. Microtubule-associated factors that may regulate
the F-actin cytoskeleton include Rac GTPase
(Waterman-Storer et al., 1999)
and exchange factors for Rho GTPase (Ren
et al., 1998
; van Horck et
al., 2000
). Rac1 affects E-cadherin dependent adhesion in tracheal
cells (Chihara et al., 2003
)
and a mutation in the juxtamembrane site in mammalian E-cadherin analogous to
the one described here affects Rac activation
(Goodwin et al., 2003
). RhoA
activation inhibits fusion track assembly
(Lee and Kolodziej, 2002
).
Downstream interactions between F-actin and microtubules, such as those
mediated by Shot, may vary with cell type to produce distinct morphogenetic
outcomes. Further studies of tracheal tube fusion, a genetic system in which
adherens junction associated structures can be visualized in living embryos,
promises to identify the regulatory molecules that allow E-cadherin to direct
F-actin and microtubule assembly from the ß-catenin binding and
juxtamembrane domains.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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