Article |
Address correspondence to Nicholas H. Brown, Wellcome Trust/Cancer Research UK Institute and Dept. of Anatomy, University of Cambridge, Tennis Court Rd., Cambridge, CB2 1QR UK. Tel.: 44-1223-334128. Fax: 44-1223-334089. email: n.brown{at}welc.cam.ac.uk
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Abstract |
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Key Words: actin; cytoskeleton; cell junctions; adhesion; follicle epithelium
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Introduction |
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The NH2-terminal third of Shot contains an actin-binding domain (ABD) of the type common to both spectrin and plakin superfamily members, consisting of two calponin homology domains, but is clearly more similar to plakins than spectrin family members (see Fig. 1; Gregory and Brown, 1998). The ABD of plectin binds not only to actin but also to the unusually long cytoplasmic tail of the ß4 integrin subunit (Rezniczek et al., 1998). All plakins have a related COOH-terminal domain consisting of what are called plakin repeats or plectin repeats (Green et al., 1990; Schultz et al., 1998; Leung et al., 2001a; Bateman et al., 2002). The known function of this domain is to bind to intermediate filaments (Nikolic et al., 1996; Leung et al., 1999; Choi et al., 2002), and because intermediate filaments are not present in Drosophila it made sense that this domain was lacking in the Shot isoforms that were initially characterized. Instead, the majority of Shot was found to be composed of spectrin repeats, more related to dystrophin and spectrin (Strumpf and Volk, 1998). In addition Shot has a GAS2 domain at the COOH terminus, which has been found to bind microtubules (Lee et al., 2000; Sun et al., 2001). In embryos lacking Shot, the epidermal cells that attach to the muscles, the tendon cells, are pulled apart by muscle contractions, and the microtubules have lost their connection to the basal cell surface (Prokop et al., 1998). This appears analogous to the cell disruption in the basal layer of the epidermis when BPAG1 or plectin are missing (Guo et al., 1995; McLean et al., 1996). Thus, the region of Shot that is conserved with plectin is the portion that interacts with integrins, whereas the intermediate filament binding domain of plectin has been replaced with a microtubule binding domain. Although a role in linking integrins to the microtubules remains a likely function of Shot, several observations show that this is not the whole picture.
The identification of vertebrate orthologues of Shot rapidly demonstrated that this protein is not a specialized version of plectin unique to invertebrates (Leung et al., 2002; for review see Röper et al., 2002). Two spectraplakin genes have been found in mammals: MACF1 and BPAG1. Several of the diverse mutant phenotypes of the shot locus, or the mouse BPAG1 gene, dystonia musculorum, appear not directly related to integrin function. The discovery that prompted the work described here was the identification of a novel exon within the shot gene that encodes an extended set of plakin repeats. Integration of this domain into Shot protein isoforms could further multiply the isoform variability and potentially generate isoforms with new functions that do not involve integrins. The discovery of the plakin repeat encoding region in the shot locus is curious, as the only known function of these repeats so far is to interact with intermediate filaments. We were especially interested to see whether they had adopted a different function in the fly that could potentially shed light on additional functions of plakin repeat regions in vertebrate proteins.
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Results |
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To test whether the signals for targeting to lateral junctions are found in the plakin repeat region, we constructed fusions between segments of the plakin repeat region and GFP (Fig. 1). These were expressed in stripes in the epidermis using the GAL4 system (Brand and Perrimon, 1993). The more NH2-terminal segment was sufficient to target GFP to the lateral junctions, whereas the more COOH-terminal segment was uniform in the cytoplasm (Fig. 5, N and O). This suggests that first, the NH2-terminal segment of the plakin repeat region contained targeting information for adherens junction localization, and second, this targeting is not provided by plakin repeats per se as both fusion proteins contained plakin repeats but only one was targeted to adherens junctions.
Different alleles of shot abolish different protein isoforms
We examined alleles of shot by immunofluorescence labeling of mutant embryos to assess whether they affected the expression of different Shot protein isoforms. The allele shot3, which behaves genetically as an amorphic/null allele (Lee et al., 2000), abolished labeling with plakin repeat antibody 2 and the spectrin repeat antibody (Fig. 6 A). This confirms that these antibodies are specific for the products of the shot gene, and that shot3 is a null allele. We also examined two alleles containing an identical insertion of a P-element after transcription start sites 2 and 1, but before 3 and e, shotkakP1 and shotkakP2 (Fig. 1; Gregory and Brown, 1998). The insertion is predicted to hinder transcription from the first two start sites, blocking production of Shot forms containing the full ABD, but not affect transcription from the second two start sites. Consistent with this, these alleles eliminated staining with the anti-ABD antibody (Gregory and Brown, 1998), which also suggests that the anti-ABD antibody does not recognize the partial ABD encoded by transcripts starting at the third promoter. At stage 14, shotkakP2 mutant embryos showed reduced epidermal staining with both plakin repeat and spectrin repeat Shot antibodies, compared with a control lateral membrane marker, Fasciclin III (FasIII), but by stage 16 staining appeared close to normal (Fig. 6 B). This suggests that earlier in development most Shot protein contains the full ABD in conjunction with the plakin and spectrin repeat domains, whereas later the forms containing plakin and spectrin repeats but lacking the full ABD will be made. This temporal change in expression pattern was confirmed by Western analysis (Fig. 6 C), as was the elimination of all isoforms in shot3, and just the ABD containing isoforms, one of which being the largest isoform containing all domains, in shotkakP2 (Fig. 6 D). Previous in situ analysis (Lee et al., 2000) indicated that isoforms lacking the full ABD were only expressed in the epidermis, whereas forms containing the ABD were expressed strongly in the nervous system and the epidermis. Therefore, we predicted that nervous system expression of Shot should be eliminated in the shotkakP1 allele, and this proved to be the case (Fig. 6, E and E'). Shot alleles that lack the largest plakin repeat isoform in mid-embryogenesis (stage 14), e.g., the P-insertion alleles shotkakP1 and shotkakP2, have a weakly penetrant zygotic mutant phenotype consisting of rips in the epidermis (Gregory and Brown, 1998). This suggests that the function of the Shot isoforms containing the plakin repeats at the adherens junctions is to maintain epithelial integrity.
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The perturbation of the integrity of the epithelial layer in the absence of Shot prompted us to analyze the localization of components of the adhesion and polarity complexes that are required for epithelial integrity (for reviews see Johnson and Wodarz, 2003; Perez-Moreno et al., 2003). These include components of the adherens junction, and the apical complex, which has recently been implicated in the assembly, positioning and maintenance of the adherens junction, and consists of the transmembrane protein Crumbs and the two cytoplasmic scaffolding proteins Stardust and Discs Lost (Dlt), (Bilder et al., 2003; Tanentzapf and Tepass, 2003). The adherens junction component ß-catenin/Armadillo appeared slightly reduced in most, but not all shot3/shot3 clones (Fig. 7, H and H'). In addition, ZO-1, a PDZ-protein associated with adherens junctions in Drosophila (Takahisa et al., 1996; Takahashi et al., 1998) accumulated aberrantly, concentrating at the contacts between the double-layered mutant cells, as actin did (Fig. 7, E and E'). Apical staining for Dlt was strongly reduced (Fig. 7, F and F'), although the localization of Crumbs (Fig. 7, G and G') and Stardust (not depicted) were not altered by the absence of Shot. These results demonstrate that Shot is essential for the stable association of the proteins Armadillo to adherens junctions and Dlt to the apical complex. As these proteins are important for cell polarity, it was possible that the double layering was due to loss of epithelial polarity rather than a loss of cell adhesion. However, cell polarity appeared normal in cells lacking Shot, as judged by the normal apical distribution of ß-heavy-spectrin (Fig. 7, I and I') and lateral distribution of ß-spectrin (Fig. 7, J and J').
Previous work demonstrated that the shorter forms of Shot lacking the plakin repeats have a role in epithelial cells in linking integrin adhesive junctions to the cytoskeleton (Gregory and Brown, 1998; Prokop et al., 1998). Therefore, we tested whether the double-layering phenotype was due to the loss of a similar integrin dependent process in the follicle epithelium. Integrins are expressed on the basal surface of the follicle epithelium and are needed to align parallel actin fibers at the basal side of all follicle cells to allow oocyte elongation (Bateman et al., 2001). Loss of integrins perturbs the arrangement of the basal actin fibers, but this was not observed in the absence of Shot (Fig. 7, K and K'). Microtubule organization and levels (Fig. 7, L and L'), integrin localization and oocyte elongation (not depicted) were normal in shot3 mutant cells. In addition, Shot did not colocalize with integrins at the basal surface of follicle cells, but rather was found only at cellcell contacts. This indicates that the loss of epithelial integrity observed in the absence of Shot is due to the loss of cellcell adhesion, and not cellmatrix adhesion.
In summary, Shot is localized to adherens junctions in both the embryonic and follicular epithelia. Defects in epidermal integrity were observed as rips in the epidermis in shotkakP1 mutant embryos (Gregory and Brown, 1998), and as double layering of follicular epithelial cells mutant for shot3 or shotkakP1. The altered distribution of actin and ZO-1 and the reduction of Armadillo and apical Dlt in the absence of Shot demonstrates that Shot is essential for the organization of the apical adhesion belt protein complexes.
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Discussion |
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In the epidermis of the early embryo, reduction of the largest isoform of Shot, which is the most abundant form at this stage (with the P-insertion alleles shotkakP1 and shotkakP2) caused tears in the epidermis (Gregory and Brown, 1998), suggesting that the giant form containing all domains is required at adherens junctions to maintain cell adhesion. The low penetrance of this phenotype suggests that this function of Shot can in most cells be compensated for by other proteins of the junction. This redundancy was not found in the follicular epithelium, where loss of Shot caused the normal single layer of cells to become disorganized and double-layered in a majority of mutant clones. This double-layering phenotype is consistent with loss of lateral adhesion.
How do Shot isoforms containing the plakin repeats contribute to the integrity of cell adhesion? The loss of adhesion could arise from defects in establishing apicalbasal polarity, the initial establishment of cell adhesion, or the maintenance of cell adhesion. The absence of Shot caused a slight reduction in junctional Armadillo and a stronger reduction of the apical complex component Dlt (Fig. 7), suggesting that Shot plays a role in their recruitment or maintenance. The DltCrumbs complex is not only important for adherens junction assembly but helps to establish epithelial polarity (Medina et al., 2002; Roh et al., 2002; Bilder et al., 2003; Tanentzapf and Tepass, 2003). However, Shot does not seem to be involved in the establishment of polarity as the markers ß-spectrin and ß-heavy-spectrin were distributed normally (Fig. 7). Nor is Shot required for the initial assembly of adherens junctions because the phenotype in shot mutant clones did not appear until late during oogenesis, after adherens junctions have been established. Therefore, the loss of adhesion and the double layering observed in shot mutant clones is unlikely to be a secondary effect of a loss of apicobasal polarity or failure in the formation of adhesive junctions.
Crumbs and Dlt are linked together via Stardust and are only partially interdependent for their apical localization, so that a role for Shot in the stabilization of Dlt localization is fully consistent with previous results (Tanentzapf et al., 2000; Tepass, 2002; Bilder et al., 2003; Tanentzapf and Tepass, 2003). In some dlt mutant clones Crumbs can be retained in the apical membrane, suggesting a second mechanism to localize Crumbs, and the apical Dlt localization is only slightly reduced in crumbs clones. Reduction or absence of Armadillo does not abolish Dlt localization (Tanentzapf et al., 2000). Whether the loss of apical Dlt accumulation is cause or effect of the loss of adhesion in shot mutant clones remains to be elucidated.
Taking these findings into account, we propose that Shot aids in formation of the link between the adherens junction and the associated belt of actin filaments. Because the genetic evidence shows that the ABD is needed for function in the follicle epithelium (shot3 and shotkakP1 show the same phenotype), Shot could stabilize the adherens junction associated actin cytoskeleton by helping to link it to the membrane and/or cross-link it to microtubules that are associated with adherens junctions (Chausovsky et al., 2000; Waterman-Storer et al., 2000; Ligon et al., 2001). Our ability to visualize actin associated with adherens junctions is hindered by the high level of actin generally at the cortex and in the apical microvilli, thus, the normal appearance of actin in the absence of Shot does not rule out this proposed function. The association of part of the Shot plakin repeat domain with adherens junctions suggests that this could be the key region involved in attaching Shot to the membrane, leaving the ABD and GAS2 domain free for other interactions. Loss of Shot function may then cause a weaker link between the junction and the actin-based adhesion belt. During stage 9 of oogenesis the follicle cells undergo a rearrangement, when the cuboidal follicle cells that have surrounded the whole egg chamber up until that point start to concentrate over the oocyte and become columnar, whereas only a few anterior cells become squamous and cover the nurse cells (for review see Dobens and Raftery, 2000). The forces that occur during this follicle cell reorganization could lead to a rupture of weakened adherens junctions in the shot mutants, causing the observed double layering. The accumulation of actin could be due to the fact that the basal cell in a double layer tries to reestablish an apical surface, which is supported by the weak ß-heavy-spectrin staining in between layers. ZO-1, a component of adherens junctions, accumulates with the actin, but at higher levels than in wild-type junctions. This may be a combination of these components in both cells of the bilayer or just abnormally elevated levels at the apical surface of the basal cell. The proposed role of Shot in stabilizing adherens junctions after their initial establishment is consistent with in vitro data analyzing the vertebrate Shot orthologue MACF1/ACF7 (Karakesisoglou et al., 2000). After induction of cellcell contact in tissue culture cells, part of MACF1 localizes to sites of cellcell contact, but with slower dynamics than integral components of the adherens junctions and desmosomes, suggesting that it is associated with preformed junctions.
It is important to note that an additional function for Shot in cellcell contacts in the trachea has been described previously (Lee and Kolodziej, 2002), but that in this case Shot functions in remodelling the cytoskeleton rather than cellcell adhesion. In the absence of Shot, the tracheal cells fail to fuse and the specialized actin fibers and apical bundles of microtubules associated with cadherin contacts do not form normally. Shot isoforms containing either the ABD or the microtubule-binding GAS2 domain, but not the plakin repeats, can rescue these defects, showing that the largest isoforms containing the plakin repeats are not required. The role of Shot in the tracheal cells may, therefore, be more similar to its role in connecting microtubules to the plasma membrane in the tendon cells, rather than the function we have described in mediating the integrity of epithelial sheets.
How is the function of the largest Shot protein isoforms linked to the presence of the plakin repeats? Plakin repeatcontaining isoforms of the Drosophila spectraplakin Shot seem to behave in a peculiar way. In their proposed function, i.e., maintenance of epithelial integrity, they rather resemble members of the spectrin family of proteins that have been shown to be important in organizing cortical domains at sites of adhesion (Belkin and Burridge, 1995; Pradhan et al., 2001). In contrast, the previously described Shot isoforms that lack the plakin repeats are involved in the link between integrin receptors and the cytoskeleton, a "classical" plakin protein function. The difference in usage of these isoforms may have arisen in flies because the usual plakin repeat binding partner, cytoplasmic intermediate filaments, is missing, freeing this domain to adopt a new function. Alternatively, stabilizing adherens junctions through the plakin repeatcontaining largest isoforms may be a conserved intermediate filament-independent function of all spectraplakins.
The adherens junction recruitment signal comprises only part of the plakin repeat domain, leaving the other part of the domain available for additional functions. The plakin repeats in Drosophila appear to be always incorporated into the middle of the protein, whereas EST and cDNA data from BPAG1 in mouse show two different ways of incorporation of the repeats: in the middle in BPAG1a/b, and at the very end in BPAG1e (with no spectrin repeats or GAS2 domain being incorporated; Leung et al., 2001b). In C. elegans the orthologue of Shot, vab-10, is expressed in two distinct protein isoforms (Bosher et al., 2003 and unpublished data): one resembles the initially described dystrophin-like isoform of Shot and the other one resembles BPAG1e and ends with the plakin repeat domain. For all isoforms ending with plakin repeats, the plakin repeat region either has been shown to bind intermediate filaments, or be required for the link to intermediate filaments. We would speculate that plakin repeats have alternative binding partners if found in the middle of a protein. It will be interesting to see if the interaction of the internal plakin domains with proteins at the adherens junctions is conserved in the mammalian spectraplakins. Demonstrating that a domain makes different protein interactions depending on whether it is in the middle versus the end of a protein would provide new insight into how molecular interactions can be regulated through differential splicing of highly modular proteins.
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Materials and methods |
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In situ hybridization
In situ hybridization of whole-mount embryos was performed as described previously by Tautz and Pfeifle (1989). Images were obtained by photography on a microscope (model DMR; Leica) with a Spot digital camera (Diagnostic Instruments) and composites were assembled using Photoshop (Adobe Systems).
Production of polyclonal antibodies and GFP fusion proteins
To generate polyclonal antisera against the plakin repeat domain of Shot, antigens were prepared from DNA fragments consisting of nucleotides 9001,845 (plakin repeats 1) and 7,7558,850 (plakin repeats 2) of the large plakin repeat exon expressed in bacteria using the pGEX system. GFP fusion proteins in inducible UAS vectors were generated by subcloning nucleotides 1,4004,100 (GFPplakin repeats N) and nucleotides 6,0009,000 (GFPplakin repeats C) of the large plakin repeat exon into pUASp with GFP at the NH2 terminus.
Embryo and ovary extracts, IP, and Western blotting
Embryos were collected overnight or at the stages indicated, dechorionated in 50% bleach for 3 min and rinsed in water. Embryos were homogenized in at least five times their volume of solubilization buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) in a glass homogenizer on ice. The lysates were incubated on ice for 30 min and centrifuged for 10 min at 10,000 rpm at 4°C. The upper lipid layer formed after centrifugation was removed and the clear lysate used for immunoblotting and IPs. To obtain ovary lysates, well-fed females were dissected and the ovaries were solubilized in five volumes of solubilization buffer as described in this paragraph for embryo lysates.
Aliquots of 15 µl of embryo lysate per lane or 5 µl of ovary lysate per lane were run on a 4.2% separating gels and transferred onto PVDF membrane as described previously (Gregory and Brown, 1998). Rainbow markers (Amersham Biosciences) were used to indicate mobilities of 250 and 160 kD. Western analysis was performed as described previously in Röper et al. (2000). Anti-Shot antisera were diluted 1:200 for anti-ABD (Gregory and Brown, 1998); 1:4,000 for antiplakin repeats 1; 1:2,000 for antiplakin repeats 2; and 1:4,000 for antispectrin repeats (Strumpf and Volk, 1998). Antibodies were revealed by chemiluminescence using the Western blotting detection reagents (Amersham Biosciences).
For each IP, 400 µl of lysate diluted to 1 ml in solubilization buffer were incubated with the primary antibody (1:200 for anti-ABD; 1:200 for antiplakin repeats 1; 1:150 for antiplakin repeats 2; and 1:250 for antispectrin repeats) overnight at 4°C. Immunocomplexes were collected by incubation with protein Asepharose. Proteins were eluted from the beads by boiling in sample buffer.
Immunofluorescence and confocal analyses
Embryos were collected at the indicated stages of development, and processed for immunofluorescence using standard procedures. Ovaries were dissected from well-fed females and processed for fluorescence using standard procedures. Primary antibodies were diluted 1:200 for antiplakin repeats 2; 1:250 for antispectrin repeats (provided by T. Volk, The Weizmann Institute of Science, Israel; Strumpf and Volk, 1998); 1:500 for anti-PY (Sigma-Aldrich); 1:500 for antidisc large (provided by R. Fehon, Duke University, Durham, NC; Fehon et al., 1994); 1:150 for ZO-1 (provided by M. Takahisa, Mitsubishi Kasei Institute of Life Sciences, Machida-shi, Tokyo, Japan; Takahisa et al., 1996); 1:600 for anti-Dlt (provided by M. Bhat, Mount Sinai School of Medicine, New York, NY; Bhat et al.,1999); 1:500 for anti-Crumbs (provided by U. Tepass, University of Toronto, Toronto, Ontario, Canada; Tanentzapf et al., 2000); 1:500 for anti-Stardust (provided by E. Knust, Heinrich-Heine-Universität, Düsseldorf, Germany; Bachmann et al., 2001); 1:500 for antiß-heavy-spectrin (provided by D.P. Kiehart, Duke University, Durham, NC; Thomas and Kiehart, 1994); 1:200 for antiß-spectrin (provided by L. Goldstein, University of California San Diego, La Jolla, CA; Byers et al., 1989); and 1:6 for anti-FasIII and 1:100 for antiarmadillo (both from Developmental Studies Hybridoma Bank). Confocal images were obtained using a confocal microscope (model Radiance 2000; Bio-Rad Laboratories). Confocal laser, iris, and amplification settings in experiments comparing intensities of labeling were set to identical values. Confocal pictures were assembled in Adobe Photoshop.
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Acknowledgments |
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K. Röper has been supported by Long Term Fellowships from European Molecular Biology Organization and Human Frontiers Science Program, N.H. Brown by a Wellcome Trust Senior Fellowship.
Submitted: 14 July 2003
Accepted: 13 August 2003
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