Department of Cell Biology and Center for Molecular Neuroscience, C-2210 Medical Center North,Vanderbilt University Medical Center, Nashville, TN 37232-0295, USA
*Author for correspondence (e-mail: kolodzp{at}ctrvax.vanderbilt.edu)
Accepted 17 December 2001
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SUMMARY |
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Key words: Anastomosis, Discs lost, Cadherin, F-actin, Microtubules, Drosophila
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INTRODUCTION |
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The tracheal lumen is initially closed at branch tips. Concurrent with branching morphogenesis, specialized cells at branch tips, known as fusion cells, join branches into a continuous tubular network. This process of anastomosis requires each fusion cell to recognize its partner in the adjacent hemisegment and to form a lumen that connects the two branches (Samakovlis et al., 1996b; Tanaka-Matakatsu et al., 1996
). Shotgun, the Drosophila homolog of the cell adhesion molecule E-cadherin is integral to the initial fusion cell contact (Uemura et al., 1996
). Mutations in shotgun affect tracheal branch extension and lumen formation at anastomosis sites, as do mutations in armadillo, the Drosophila homolog of its effector ß-catenin (Uemura et al., 1996
; Beitel and Krasnow, 2000
). E-cadherin and ß-catenin control cell polarity (McNeill et al., 1990
) and tube extension in culture (Yap et al., 1995
; Matsumura et al., 1997
; Pollack et al., 1997
), suggesting an evolutionarily conserved role for cadherin-mediated cell adhesion in apical surface regulation.
Tracheal cells face the lumen with their apical surfaces, suggesting that apically localized molecules play a role in lumen formation. Mutations in discs lost (dlt), which encodes an apically localized PDZ domain family protein (Bhat et al., 1999; Tanentzapf et al., 2000
), disrupt epithelial cell polarity, as do mutations in crumbs, which encodes a Dlt-associated (Bhat et al., 1999
), EGF repeat family transmembrane protein (Tepass et al., 1990
). crumbs mutant embryos are defective in forming mature zonula adherens (ZAs) (Tepass, 1996
), structures at apical/lateral contacts between cells that contain E-cadherin.
Several studies suggest that these apical surface determinants and E-cadherin regulate the cytoskeleton and therefore control lumen formation and morphology. For example, Crumbs may attach ßH-spectrin, an F-actin cross-linker, to the apical membrane in Drosophila (Wodarz et al., 1995) and mutations in a C. elegans ßH-spectrin moderately enlarge the lumen of the excretory canal (Buechner et al., 1999
). E-cadherin interacts with F-actin via multiple mechanisms (Gumbiner, 2000
). These include binding to p120 catenin (p120ctn), a negative regulator of the RhoA GTPase (Anastasiadis et al., 2000
; Noren et al., 2000
). RhoA controls the formation of F-actin-containing focal adhesions and stress fibers in cultured cells (Ridley and Hall, 1992
; Jou and Nelson, 1998
). These interactions with RhoA also potentially regulate apical membrane protein targeting (Jou and Nelson, 1998
), a process important for lumen development in culture (Lipschutz et al., 2000
).
Little is known about the cytoskeletal structures required for lumen formation and how apical surface determinants are localized. We identify here an F-actin-rich track that is associated with E-cadherin-dependent contacts between fusion cells that appears to guide deposition of apical surface determinants and lumen formation. During anastomosis, Short Stop (Shot), an evolutionarily conserved plakin (Gregory and Brown, 1998; Strumpf and Volk, 1998
; Leung et al., 1999
; Lee et al., 2000a
), accumulates at these contacts and transiently along the track. Mutations in shot and constitutively active alleles of the Drosophila RhoA (RhoA) GTPase specifically disrupt this contact and the associated track. Remarkably, the interactions of Shot with F-actin and its binding to microtubules are functionally redundant in organizing the track, suggesting that Shot acts with other pathways to organize F-actin and microtubules, rather than as an F-actin/microtubule cross-linker. We propose that in fusion cells, RhoA antagonizes Shot to regulate E-cadherin-associated cytoskeletal structures required for apical surface determinant localization and lumen formation.
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MATERIALS AND METHODS |
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Molecular biology and genetics
pUAST-C-Shot L-GFP contains a DNA fragment that encodes a GFP fusion with the Shot long isoforms C-terminal microtubule binding domain (C-Shot L) (Lee and Kolodziej, 2002) in the GAL4 expression pUAST vector (Brand and Perrimon, 1993
). pUAST-Shot L(C)-GFP expresses a GFP fusion with a full-length type C Shot long isoform (Lee and Kolodziej, 2002
). Type C isoforms do not bind F-actin. pUAST-Shot L(C)-
GAS2-GFP also lacks DNA sequences encoding most of the GAS2 motif (amino acids 4859 to 4905; Accession Number, AAF24343).
Flies bearing these transgenes were obtained by standard methods (Ashburner, 1989); other transgenic flies have been previously described (Lee and Kolodziej, 2002
). The btl-GAL4 and esg-GAL4 enhancer trap lines (chromosome II) obtained from Mark Krasnow (Stanford University) were used to drive pUAST transgenes in tracheal and fusion cells, respectively. In most cases, pUAST transgenes were expressed in shot3 mutant embryos by crossing shot3 pUAST recombinants to btl-GAL4 shot3 or esg-GAL4 shot3 stocks. To visualize tracheal microtubule organization in wild-type and shot3 mutant embryos, shot3/+; pUAST-C-Shot L-GFP/+ flies were crossed to btl-GAL4 shot3/CyO276 flies. The shot3 mutant embryos lack epidermal and CNS Shot protein (Lee et al., 2000a
). Second chromosome pUAST-actin-GFP and pUAST-GAP43-GFP lines were obtained from Akira Chiba (University of Illinois, Urbana-Champaign); esg-lacZ lines were obtained from Shigeo Hayashi (National Institute of Genetics, Mishima, Japan). Homozygous third chromosome pUAST-RhoAV14, pUAST-Rac1N17 and pUAST-Cdc42N17 lines were obtained from Liqun Luo (Stanford University) and pUAST-RhoAN19 was obtained from Jeffrey Settleman (Massachusetts General Hospital). These flies were crossed to btl-GAL4 pUAST-actin-GFP or btl-GAL4/+; pUAST-C-Shot L-GFP/+ flies to visualize F-actin and microtubules in tracheal cells expressing dominant negative or constitutively active Rho family GTPases.
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RESULTS |
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Mutations in shot disrupt apical cytoskeletal structures in tracheal cells
shot is allelic to kakapo (Gregory and Brown, 1998; Strumpf and Volk, 1998
) and encodes a family of plakin-related proteins (Lee et al., 2000a
). Long isoforms of Shot provide essential cross-links between F-actin and microtubules during axon extension (Lee and Kolodziej, 2002
) and stabilize microtubule arrays in neuronal support and muscle attachment cells (Prokop et al., 1998
). Our observation of lumen formation defects in shot mutant embryos led us to investigate the organization of the tracheal cell cytoskeleton after anastomosis in wild-type and shot mutant embryos. We focused on cells in the dorsal trunk, the largest tracheal branch and therefore the easiest in which to visualize cytoskeletal structures.
We visualized F-actin both by labeling dissected embryos with fluorescent phalloidin and by detecting actin-GFP (Verkhusha et al., 1999) specifically expressed in tracheal cells (Fig. 2). In wild-type tracheal cells at anastomosis sites, F-actin accumulates apically so that it appears as a continuous tube (Fig. 2A,G). In shot mutant tracheal cells at abnormal anastomosis sites, F-actin accumulates apically, but surrounds two blind-ended lumens (Fig. 2D,I).
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We investigated the identity of the cells that fail to form a lumen at anastomosis sites by labeling fusion cells with the esg-lacZ enhancer trap (Tanaka-Matakatsu et al., 1996) and labeling all tracheal cells with GAP43-GFP, a membrane-associated GFP derivative (Ritzenthaler et al., 2000
). After anastomosis is complete in wild-type embryos (stage 14), the fusion cells form a compact doughnut around the lumen (Fig. 2K) (Samakovlis et al., 1996b
) and flanking tracheal cells are drawn close to each other at the anastomosis site (Fig. 2K). The apically concentrated actin surrounds the tube and appears continuous through the fusion cells (Fig. 2K). In stage 14 shot mutant embryos, the fusion cells remain extended (Fig. 2L), creating a wide gap between flanking tracheal cells. The fusion cells are the only tracheal cells in the gap. The posterior cell often appears intercalated into the anterior branch, so that it stretches between the two branches (Fig. 2L). All tracheal cells accumulate F-actin apically, including the fusion cells, but the apical surface in each fusion cell does not develop into a bridging tube. Their apical surfaces remain facing the blind-ended lumens in their respective branches (Fig. 2L). Fusion cells therefore fail to remodel their F-actin cytoskeletons in shot mutant embryos.
Shot accumulates at E-cadherin containing contacts between fusion cells as it associates with an F-actin-rich track that marks the future axis of lumen formation
Antibodies against the long Shot isoforms (Strumpf and Volk, 1998) reveal that these proteins concentrate cortically in all tracheal cells, and also assemble in fusion cells into a transient track (Fig. 3A) that marks the future axis of the lumen bridging the two branches (Fig. 3D). Shot is not detected cortically in tracheal cells or in a track in fusion cells in shot3 null mutant embryos (Fig. 3E,H). However, anti-Shot also non-specifically labels the tracheal lumen (Fig. 3E), precluding visualization of apically localized endogenous Shot with this reagent.
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We investigated whether Shot L(A) can associate with E-cadherin contacts via its C-terminal domain (C-Shot L) or whether these associations primarily occur via its F-actin binding domain. C-Shot L-GFP expressed in tracheal cells accumulates at sites of E-cadherin localization (Fig. 4U-X), suggesting that it also mediates interactions with E-cadherin-associated cytoskeletal structures. As C-Shot L-GFP colocalizes with microtubules in cultured cells (Lee and Kolodziej, 2002), microtubules may also be concentrated near these sites. However, we were unable to visualize microtubules consistently during anastomosis using anti-tubulin staining and a variety of fixation methods, though we were able to detect microtubules in other tissues at this developmental stage (data not shown).
The Shot- and F-actin-containing track in fusion cells (Fig. 5L,Q) appears before apical surface determinants Dlt are detectable along the track (Fig. 5G). As the lumen forms within the fusion cells, Dlt accumulates along the track and then the new apical surface (Fig. 5H-J). Dlt levels are initially lower in the fusion cells relative to the flanking tracheal cells (Fig. 5I), suggesting that new apical surface is made in the fusion cells and existing apical surfaces do not simply stretch to connect with each other. Thus, a E-cadherin-associated track of F-actin and Shot appears to mark the site of new apical surface formation in fusion cells.
The interactions of Shot with the cytoskeleton are required for E-cadherin contacts that are essential for lumen formation in fusion cells
In shot mutant embryos, fusion cells frequently fail to form new E-cadherin contacts (Fig. 7E) and an associated F-actin track (Fig. 7G), but sometimes form inappropriate or misoriented E-cadherin contacts with tracheal cells other than their fusion partner (Fig. 7I,M). These aberrant contacts are not detectably associated with F-actin (Fig. 7K,N). These findings suggest that Shot is required to form or maintain the F-actin-containing structures associated with E-cadherin contacts, and to direct the formation of these contacts to the appropriate part of the fusion cells.
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Shot L(A)-GFP accumulates apically in tracheal cells (Fig. 8E). Mutant derivatives that lacked either the F-actin (Fig. 8F) or the microtubule binding site (Fig. 8G) are largely apically concentrated, but are partially delocalized. However, Shot molecules that lack both the F-actin and microtubule-binding domains no longer accumulate apically (Fig. 8H). Thus, Shot concentrates apically via interactions with the cytoskeleton.
To investigate further the role of Shot in organizing the cytoskeleton, we examined F-actin and microtubule distributions in tracheal cells in rescued shot mutant embryos. Tracheal expression of Shot proteins that lack either the F-actin or the microtubule binding sites restores both normal F-actin distribution in fusion cells (Fig. 9A,B) and the apical accumulation of C-Shot L-GFP, a microtubule binding protein, in all tracheal cells (Fig. 9C,D). Normal Dlt localization in fusion cells was also restored in rescued embryos (data not shown). F-actin remodeling in fusion cells and apical accumulation of microtubules in tracheal cells can therefore occur provided that Shot can directly interact with either F-actin or microtubules. A Shot L(A) derivative that lacks both the F-actin and the microtubule binding domains does not rescue F-actin remodeling in fusion cells (Fig. 9E), or apical accumulation of microtubules in all tracheal cells (Fig. 9F). Thus, the F-actin- and microtubule-binding domains of Shot are required for these processes, but appear functionally redundant. The interactions of Shot with the cytoskeleton may therefore facilitate additional organizing interactions between F-actin and microtubules.
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We therefore examined the effect of expressing constitutively active RhoAV14 in tracheal cells. Tracheal defects in these mutant embryos strongly resemble those in shot mutant embryos. Lumen formation is blocked at all anastomosis sites, but occurs normally in cells interior to branches (Fig. 10A). The effects of RhoAV14 on new apical cytoskeletal structures in fusion cells are also similar to those observed in shot mutant embryos, but more penetrant. In embryos expressing RhoAV14 in tracheal cells, F-actin (Fig. 10D) and Dlt (Fig. 10G) accumulate apically in tracheal cells, but surround blind-ended tubes terminating at anastomosis sites, rather than continuous lumenal connections. E-cadherin does not form a three ringed structure at anastomosis sites (Fig. 10J), and Shot L(A)-GFP is distributed apically around blind-ended tubes (Fig. 10L). Tracheal expression of RhoAV14 also disrupts apical accumulation of C-Shot L-GFP throughout the trachea (Fig. 10N), another similarity with shot mutant embryos (Fig. 2J). Fusion cells in embryos that express RhoAV14 in tracheal cells fail to form initial E-cadherin-containing contacts (Fig. 10P) and associated F-actin tracks (Fig. 10Q). RhoAV14 expression also modestly attenuates branch migration, but does not detectably affect the pattern of primary and secondary branching (Fig. 10A).
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RhoA, Rac and Cdc42 make up a family of related GTPases with specific roles in diverse F-actin-based morphogenetic processes (Hall, 1998). To determine whether the role of RhoA is specific, we also expressed wild-type RhoA, dominant negative Drosophila Rac1 (Luo et al., 1994
) or Cdc42 (Luo et al., 1994
) in tracheal cells and examined tracheal development. None of these alleles detectably affects lumen formation or apical structures (Fig. 10C,F,I and data not shown), though Cdc42N17 and Rac1N17 embryos exhibit modest defects in branch migration (data not shown). Thus, the role of RhoA role in controlling the formation of apical cytoskeletal structures is likely to be specific among these Rho family GTPases.
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DISCUSSION |
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Loss-of-function shot and gain-of-function RhoA alleles have similar phenotypes in fusion cells, and RhoA disrupts Shot localization. We therefore propose that RhoA negatively regulates track assembly and E-cadherin contact remodeling by Shot. Apically organized F-actin and adherens junctions in other tracheal cells appear to develop normally in shot mutant and RhoAV14 embryos, suggesting specific requirements for shot and RhoA during new apical surface formation in fusion cells. We propose that Shot and RhoA regulate E-cadherin-dependent cell adhesion in selected developmental contexts.
Redundant interactions between Shot and cytoskeletal elements organize the fusion cell cytoskeleton
We and others have shown that shot is required in neurons for growth cone motility (Van Vactor et al., 1993; Kolodziej et al., 1995
; Prokop et al., 1998
; Gao et al., 1999
; Lee et al., 2000a
). We show here that shot is required to remodel E-cadherin-containing contacts between tracheal fusion cells. Surprisingly, Shot proteins perform these distinct morphogenetic roles using different combinations of the same cytoskeletal interaction domains. In fusion cells, the binding sites for F-actin and microtubules appear functionally redundant. The F-actin binding domain is essential when the GAS2 microtubule binding site is absent, and the GAS2 microtubule binding site is essential when the F-actin binding site is absent. By contrast, during axon extension, the Shot behaves as an F-actin/microtubule cross-linker because the cytoskeletal interaction domains are both individually essential and required in the same molecule (Lee and Kolodziej, 2002
).
These observations suggest that direct interactions between Shot and cytoskeletal proteins organize the cytoskeleton in fusion cells. The F-actin and microtubule domains may directly enable the accumulation of their cytoskeletal partners at the E-cadherin contact. In support of this hypothesis, the structurally similar F-actin binding domain of plectin alters F-actin organization (Andra et al., 1998) and the GAS2 motif stabilizes associated microtubules against depolymerization (Sun et al., 2001
) in cultured cells. Since Shots interactions either with F-actin or with microtubules suffice to organize both cytoskeletal elements, binding to either F-actin or microtubules may then enhance other organizing interactions between F-actin and microtubules.
These other interactions may involve molecules required for E-cadherin signaling. E-cadherins are physically linked to F-actin via the ß-catenin/-catenin complex (Gumbiner, 2000
) and to dynein, a microtubule-based motor, via ß-catenin (Ligon et al., 2001
). They can further regulate actin dynamics via association with p120, a RhoA antagonist (Anastasiadis et al., 2000
; Noren et al., 2000
); E-cadherins also stabilize microtubule minus ends in cultured cells (Chausovsky et al., 2000
). E-cadherin signaling may therefore affect other proteins mediating interactions between F-actin and microtubules. Candidates include other F-actin/microtubule cross-linkers (Fuchs and Yang, 1999
; Goode et al., 2000
), regulators of Rho family GTPases that bind to microtubules (Ren et al., 1998
; Glaven et al., 1999
; Waterman-Storer et al., 1999
) and F-actin-based motors that form complexes with microtubule-based motors (Huang et al., 1999
). Further analysis will be necessary to identify these other molecules in fusion cells; these other cytoskeletal regulators may permit residual anastomoses in shot mutant embryos.
RhoA controls the formation of apical cytoskeletal structures in tracheal cells
Our analysis also indicates that RhoA is required for lumen formation, most probably by regulating the apical cytoskeleton or by affecting the transport of lumenal antigens. Similarities between RhoAV14 and shot mutant phenotypes suggest that RhoA could work either to antagonize Shot activity, or through parallel pathways acting on F-actin and microtubules. RhoA has many effectors that control F-actin distribution (Hall, 1998). RhoAV14 has been reported to stabilize subsets of microtubules in fibroblasts in culture via an F-actin-independent pathway (Cook et al., 1998
). Shot localizes apically via its interactions with the cytoskeleton, and either these interactions or the cytoskeletal structures themselves may be RhoA-regulated.
In cells throughout the trachea, reduced RhoA activity disrupts lumen formation and partially disrupts Dlt localization. Tracheal expression of RhoAN19 does not appreciably affect E-cadherin localization. In cultured epithelial cells, E-cadherin localization is also resistant to RhoAN19 (Jou and Nelson, 1998). These findings are consistent with RhoA functioning downstream of or parallel to E-cadherin. E-cadherin-associated p120ctn negatively regulates RhoA (Anastasiadis et al., 2000
; Noren et al., 2000
), but whether a similar pathway operates in Drosophila is unknown.
In fusion cells, RhoA can also function upstream of E-cadherin, as constitutively active RhoAV14 affects E-cadherin localization selectively in these cells. E-cadherin distribution is more dynamic in fusion cells than in other tracheal cells (Tanaka-Matakatsu et al., 1996; Uemura et al., 1996
), and may therefore be more sensitive to RhoAV14. RhoAV14 also affects new E-cadherin contacts in culture (Jou and Nelson, 1998
). Further experiments will reveal whether Shot, RhoA and E-cadherin function in a common, evolutionarily conserved pathway to regulate apical surface remodeling in fusion cells.
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ACKNOWLEDGMENTS |
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