Article |
Address correspondence to Michel Labouesse, IGBMC, CNRS/INSERM/ULP, BP10142, CU de Strasbourg, Illkirch Cedex F-67404, France. Tel.: (33) 3-88-65-33-93. Fax: (33) 3-88-65-32-01. E-mail: lmichel{at}igbmc.u-strasbg.fr
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Abstract |
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Key Words: plakin; cytoskeleton; hemidesmosome; cell adhesion; morphogenesis
The online version of this article includes supplemental material.
B.-S. Hahn's present address is National Institute of Agricultural Biotechnology, Metabolic Engineering, Division 225, Seodun-Dong, Suwon, 441-707, South Korea.
R. Legouis' present address is CNRS-CGM, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
* Abbreviations used in this paper: ABD, actin-binding domain; BPAG, bullous pemphigoid antigen; FO, fibrous organelle; GAR, growth-arrest protein 2related homology; IF, intermediate filament; MCAF, microtubule actin cross-linking factor; MF, microfilament; MT, microtubule; RNAi, RNA interference.
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Introduction |
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Motors molding tissues generate forces that can have shearing effects. Thus, it is likely that they are coupled to molecules acting to preserve cell and tissue integrity. The mechanisms that protect cells against mechanical stresses have seldom been investigated in the context of morphogenesis. Their dissection has instead relied primarily on the analysis of human autoimmune or genetic diseases affecting tissue integrity, and on mouse knockout models. Biochemical and genetic studies have outlined the key function played by integrin- and dystrophin-associated adhesion complexes, plakins, and intermediate filaments (IFs;* Fuchs and Cleveland, 1998; De Arcangelis and Georges-Labouesse, 2000; Spence et al., 2002). In the skin, for instance, the plectin and bullous pemphigoid antigen (BPAG)-e plakins were shown to link the keratin filaments to 6ß4-integrin located in hemidesmosomes (Nievers et al., 1999). Mutations in any of these proteins cause skin blistering disorders on tension or outer abrasion (Pulkkinen and Uitto, 1999; McGrath and Eady, 2001).
We are analyzing the molecular basis and the mechanical aspects of morphogenesis using a genetic strategy in Caenorhabditis elegans. C. elegans embryos elongate more than fourfold along their anteriorposterior axis in the absence of cell division. This process mainly depends on the epidermis surrounding the embryo and the underlying body wall musculature (Chin-Sang and Chisholm, 2000). The initial phase of elongation, which corresponds to a twofold increase in embryonic length, is driven by the contraction of circumferentially oriented actin microfilaments (MFs) causing changes of epidermal cell shapes (Priess and Hirsh, 1986). Mutations affecting proteins that anchor MFs (-catenin, ß-catenin, E-cadherin) or that organize the MF bundles and regulate their contractions (rho kinase, myosin light chain, spectrins) disrupt embryonic elongation (for review see Chin-Sang and Chisholm, 2000). The subsequent phase of elongation requires the activity of muscle cells, which assemble sarcomeres at the plasma membrane facing epidermal cells during the initial phase (Hresko et al., 1994). Genetic analysis has identified most components involved in building a functional sarcomere (Williams and Waterston, 1994; Mackinnon et al., 2002). Mutations in the corresponding genes generally prevent embryonic elongation beyond the twofold stage, resulting in a terminal phenotype termed Pat, for "paralyzed at twofold" (Williams and Waterston, 1994).
The molecular mechanism underlying the mechanical coupling between muscle and epidermal cells is poorly understood. In C. elegans, the external cuticle acts as an exoskeleton onto which muscles attach in order to transform their contractions into body movements. These attachments comprise two distinct entities. Within muscle cells, dense bodies at the level of thin filaments and the M-line at the level of thick filaments anchor sarcomeres to the muscle plasma membrane. They are functionally and molecularly related to vertebrate adhesion plaques, including, for instance, an integrin dimer called PAT-2/PAT-3 (Williams and Waterston, 1994; Gettner et al., 1995; Mackinnon et al., 2002). Within epidermal cells, a series of small electron-dense plaques are found at the basal and apical membranes facing muscles and the cuticle, respectively. These plaques are reminiscent of vertebrate hemidesmosomes because they are connected to IFs (Francis and Waterston, 1985). The entire unit (plaques and IFs) is known as a fibrous organelle (FO). Besides IFs, another potential FO component, based on its distribution, corresponds to the uncharacterized protein recognized by the mAb MH5 that was generated by immunizing mice against muscle- and cuticle-associated extracts (Francis and Waterston, 1985, 1991). Molecules that might connect FOs to the ECM and the cuticle include myotactin at the epidermal basal surface, and MUP-4 and MUA-3 at the epidermal apical surface (Hresko et al., 1999; Bercher et al., 2001; Hong et al., 2001). Inactivation of known FO components causes a detachment of muscle cells from the epidermis and/or a detachment of the epidermis from the cuticle, suggesting that FOs are essential to maintain the musclecuticle attachment (Hresko et al., 1999; Bercher et al., 2001; Hong et al., 2001; Karabinos et al., 2001).
A screen for mutants with elongation defects led us to characterize a gene known as vab-10. We report that vab-10 corresponds to the C. elegans spectraplakin locus, a recently recognized plakin subfamily defined by vertebrate BPAG1 and MACF1, and by Drosophila shot loci (Roper et al., 2002). The roles of BPAG1 and microtubule actin cross-linking factor (MACF) 1 during morphogenesis, if any, have not yet been described (Fuchs and Karakesisoglou, 2001; Leung et al., 2002). Shot is known to form complexes with integrins and play a function very similar to that of plectin/BPAG1-e in vertebrate epidermal cells (Gregory and Brown, 1998; Prokop et al., 1998; Strumpf and Volk, 1998). However, besides its role in controlling actin remodeling in tracheal cells, its function during morphogenesis has not been fully investigated (Lee and Kolodziej, 2002a). We show that in C. elegans, vab-10 encodes several protein isoforms related either to plectin and BPAG1-e, or to MACF and BPAG1-a, with distinct functions in the epidermis. Our work shows that molecules initially described for their role in protecting cells against mechanical stress are essential for epithelial and embryonic morphogenesis. We suggest that spectraplakins protect epidermal cells against external forces exerted by muscles, and against internal forces resulting from cell shape changes occurring in the epidermis.
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Results |
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Genetic data validate the molecular model described Fig. 2 A, which predicts that vab-10A and vab-10B are two distinct transcription units sharing a common 5' region. The h1356 mutation affects a splice donor site in the common region and should truncate all isoforms; e698 and ju281 are missense mutations affecting vab-10A isoforms, and mc44 is a 1-kb deletion in exons 2425 that should eliminate the last 1,400 amino acids of VAB-10B isoforms (Fig. 2 A). Consistent with their positions, h1356 failed to complement the other vab-10 alleles, whereas ju281 and e698 did complement mc44 (Fig. 2 B). Hereafter, we refer to these alleles as vab-10(h1356), vab-10A(ju281), vab-10A(e698) and vab-10B(mc44). We suggest that vab-10(h1356) and vab-10B(mc44) are very strong or null vab-10 and vab-10B alleles, respectively, as their phenotypes did not become more severe in trans to the deficiency hDf17. In contrast, we suggest that vab-10A(ju281) and vab-10A(e698) are not vab-10A null alleles, as their phenotypes became more severe in trans to vab-10(h1356) (Fig. 2 B).
VAB-10A and VAB-10B have nonoverlapping distributions in the epidermis
The preceding molecular and genetic data predict that VAB-10A and VAB-10B isoforms should fulfil distinct functions. To address this hypothesis, we set out to examine whether their cellular and subcellular distributions are similar or different. We raised pAbs against one VAB-10Aspecific and two VAB-10Bspecific domains (Fig. 3). In addition, we used the mAb MH5, known to recognize an FO component (Francis and Waterston, 1991). While characterizing vab-10, we noticed that MH5 failed to stain vab-10(h1356) embryos (Fig. 4 D), suggesting that it might recognize a VAB-10 isoform. Several lines of evidence establish the specificity of these antibodies and demonstrate that MH5 specifically recognizes VAB-10A. First, rabbit polyclonal VAB-10A antibodies detected a band on Western blots that co-migrated with the band detected by MH5 in the 300400 kD range, in agreement with previous estimates (Francis and Waterston, 1991). Second, VAB-10B antibodies detected a band that migrated above VAB-10A (Fig. 3), consistent with the predicted sizes of both plakins. Third, addition of the polypeptide used to raise the antibodies eliminated the signal recognized by VAB-10A and VAB-10B pAbs (Fig. 3), attesting to their specificity. Last, we could further map the epitope recognized by MH5 within the VAB-10Aspecific alternative exon 16 (Fig. S3).
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VAB-10A is necessary for epidermis attachment to the ECM and VAB-10B to link the apical and basal epidermal plasma membranes
The distinct distributions of VAB-10A and VAB-10B suggest that these isoforms carry out different functions. Consistent with this notion, vab-10A and vab-10B mutants exhibit different terminal phenotypes. Specifically, embryos with no (or strongly reduced) VAB-10A activity arrested before the two-fold stage of elongation showed no or very little muscle contractions, but had an apparently normal intestine and pharynx (Fig. 1, C and D). vab-10B(mc44) mutants also failed to elongate beyond the 2.5-fold stage; however, most could hatch (Fig. 1 E) to generate larvae that generally died during the L1 stage with a very irregular and lumpy body morphology. The phenotype of vab-10B(RNAi) embryos was more variable, but not more severe (Fig. 1 F).
The paralysis of vab-10A mutants indicates that muscles are not functional, and the lumpiness of vab-10B mutants primarily unveils epidermal defects. Because VAB-10A is associated with FOs, which are envisioned as key structures for muscleepidermiscuticle attachment, loss of VAB-10A function would be expected to cause the epidermis to detach from the ECM, whereas loss of VAB-10B function should have different consequences. To address this issue, we analyzed vab-10A and vab-10B mutants by light and electron microscopy.
To examine the morphology of muscles and the epidermis, we visualized these tissues with specific antibodies. We used an mAb against a muscle protein with the same distribution as paramyosin (Schnabel, 1995), and VAB-10A antibodies (in vab-10Bdeficient embryos) or VAB-10B antibodies (in vab-10Adeficient embryos); we also used VAB-10A antibodies to stain vab-10A(ju281) embryos, as ju281 does not affect VAB-10A synthesis. In wild-type animals, muscles are closely apposed to the epidermis at early stages of elongation (Fig. 6, AC). Similarly, we found that in VAB-10Adeficient embryos, the positions and shapes of muscle and epidermal cells were normal until the 1.5-fold stage of elongation (Fig. 6, GJ). However, in vab-10B(RNAi) (Fig. 6, DF) and vab-10B(mc44) (unpublished data), at a stage when muscles are not yet functional, muscle cells were located at an unusually large distance from the edge of the embryo on its dorsal side; in these embryos, VAB-10A was detected at two positions, along muscles and along the edge of the embryo. Because VAB-10A is basal and apical in younger wild-type embryos (Fig. 4 A), we interpret this image to mean that muscles still adhere to the epidermis, but that the thickness of epidermal cells dramatically increased.
As muscles become functional (normally at the 1.7/1.8-fold stages), they remain closely apposed to the epidermis in wild-type embryos (Fig. 6 M). However, in vab-10(h1356), vab-10A(RNAi), and the most severely affected vab-10A(ju281) embryos, they gradually collapsed to a central position (Fig. 6 K) while remaining attached to each other and organized into four enlarged quadrants. We believe that muscle activity is directly responsible for this phenotype because muscles were still found attached in the head and tail regions when muscle activity was blocked by simultaneously inducing RNA interference (RNAi) against vab-10A and against myo-3 (Fig. 6 L), the main myosin heavy chain gene (Williams and Waterston, 1994). In addition, VAB-10A and VAB-10B became mutually dependent for their maintenance in the epidermis. VAB-10A was absent in the bodies of most vab-10A(ju281) (Fig. 6 N) and vab-10B(mc44) (Fig. 6 O) embryos where muscles had detached. Similarly, VAB-10B was locally undetectable in some vab-10A(ju281) embryos mainly in areas where muscles had detached (Fig. 6, PR, arrows), and was essentially absent at the end of embryogenesis (Fig. 6 K).
The results described (along with Fig. 6) suggest that VAB-10A is essential to anchor muscle cells to the epidermis, and that VAB-10B is required to maintain the distance between the apical and basal plasma membranes of the epidermis. EM analysis allowed us to refine these conclusions. In wild-type embryos, sarcomeres are small relative to larvae and adults, and the dense bodies resemble electron-dense plaques at the plasma membrane instead of the elongated plaques spanning the sarcomeres of larvae and adults (Fig. 7 A). However, in the overlying epidermis, FOs had the same appearance as in adults, looking like regularly spaced electron-dense dots found between the developing cuticle or the basal lamina and the epidermis (Fig. 7 A). In vab-10(h1356) (Fig. 7 E) and vab-10A(RNAi) (Fig. 7 F), sarcomeres appeared severely disorganized and were not closely apposed to the epidermis. Some greyish material, which probably corresponds to ECM material, accumulated between muscles and the epidermis. Within epidermal cells, we observed fewer FOs than normal in vab-10A(ju281), vab-10A(RNAi), and vab-10(h1356) embryos with gaps between the cuticle and the epidermis (Fig. 7, B, C, and E). One reason why the cuticleepidermis gap was not wider, except occasionally (Fig. 1 C, arrow), could be that once muscles have pulled away, there is no more tension exerted to widen it. Because we failed to observe similar defects in control embryos, we conclude that VAB-10A is essential to assemble or maintain FOs. In contrast, muscles remained closely apposed to epidermal cells in vab-10B(RNAi) embryos, but the thickness of epidermal cells was increased even in areas where the number of FOs was normal (Fig. 7 D).
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Discussion |
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FOs are molecularly and functionally homologous to hemidesmosomes
Genetic analysis of vab-10 mutants strongly suggests that VAB-10A is an essential component of FOs, which have been proposed to mediate musclecuticle attachment (Francis and Waterston, 1985). Our data provide the first unambiguous demonstration that FOs are indeed essential to anchor muscles to the cuticle. First, we show by immunoelectron microscopy that VAB-10A is concentrated in FOs. Next, we demonstrate that in the absence of VAB-10A, the number of FOs is reduced, and the epidermis detaches from the cuticle apically and from muscle cells basally. Finally, we illustrate that a loss of VAB-10A strongly affects the stability of IFs and myotactin, two FO components. Because converse mutations inactivating myotactin (MUA-3 and MUP-4) do not drastically affect IFs or VAB-10A distribution (Hresko et al., 1999; Bercher et al., 2001; Hong et al., 2001), VAB-10A likely acts upstream of these proteins in FO assembly.
FOs have been compared with vertebrate type I hemidesmosomes due to their subcellular position and linkage to IFs. Our analysis establishes that FOs and hemidesmosomes are not only structurally related, but are also functionally and molecularly related. Mutations affecting vertebrate hemidesmosomal components are characterized by a detachment of the epidermis from the ECM (Nievers et al., 1999). Among them, mouse plectin and BPAG1 knockouts lead to the loss of keratin filaments, although they leave some hemidesmosomes intact (Guo et al., 1995; Andra et al., 1997). Likewise, in VAB-10Adeficient embryos, the epidermis is not attached to the ECM, and the stability of IFs is strongly compromised, although some FOs remain visible. The loss-of-function phenotypes of nematode VAB-10A and these mouse plakins are thus similar in these detailed aspects.
Given the considerable evolutionary distance between nematodes and vertebrates, we suggest that a hemidesmosomal-like structure was present in their common ancestor where it probably also mediated resistance to mechanical stress at sites of connection with the ECM. Among vertebrate type I hemidesmosome components (Nievers et al., 1999), plectin appears to be the sole constituent conserved in C. elegans (Hutter et al., 2000) because C. elegans integrins have not so far been found to be expressed in the epidermis during embryonic elongation (Gettner et al., 1995; Baum and Garriga, 1997). We suggest that a plectin-like protein forms the evolutionarily conserved core component of hemidesmosomal-like structures acting at an early step in the assembly of this ancestral structure.
VAB-10B maintains epidermal integrity
Our results demonstrate that VAB-10B and VAB-10A roles differ. Loss of VAB-10B function leads to increased epidermal thickness at a time when muscle cells are not yet functional, a phenotype that is not observed in VAB-10Adeficient embryos of the same age. This change was visible predominantly on the dorsal side of the embryo, where the epidermis is strongly bent and formed by several cells that fuse at approximately the same time to form a large syncytium called hyp7. Thus, one appealing possibility could be that VAB-10B acts to protect cells against mechanical forces generated within cells that change their shapes. VAB-10B could do so in several ways. For example, Drosophila Shot is concentrated at the end of MT bundles in neurons and tendon cells, and is required to anchor these bundles (Gregory and Brown, 1998; Prokop et al., 1998; Lee and Kolodziej, 2002b). By analogy, VAB-10B, which has well conserved actin- and MT-binding domains, could attach to cortical actin and anchor MTs, which are generally oriented along the apicobasal axis in epithelial cells (Fig. 9). An alternative possibility, which takes into account the large size of VAB-10B (4950 aa), the thinness of the epidermis in muscle contact areas (200 nm in early embryos and 50 nm in later embryos), and plakin potential to form dimers through their rod domains (Leung et al., 2002), is that VAB-10B molecules located on the basal and apical membranes dimerize to maintain epidermal thickness (Fig. 9).
Localized change of epidermal thickness might explain why muscles also detach from the epidermis in VAB-10Bdeficient embryos. IFs might be unable to maintain a connection between FO plaques when the epidermal thickness increases. In addition, as muscle cells are known to help maintain FOs in the adjacent epidermis (Hresko et al., 1999), perhaps localized weakening of FO integrity triggers a chain reaction, whereby muscle partially detaches, causing further FO instability and increased muscle detachment.
Organization of the cytoskeleton
The mechanism that controls the formation of alternating VAB-10A and VAB-10B circumferential bands is intriguing. The fact that both VAB-10B and actin filament bundles are localized at furrows separating annuli, and the observation that the actin cytoskeleton is perturbed in VAB-10Bdeficient embryos, suggest that VAB-10B could play a key role in the initial events that organize VAB-10 and actin distribution. Because VAB-10A and VAB-10B are mutually dependent on each other for their stabilization, the establishment of the alternating pattern of VAB-10B with VAB-10A could be governed by rules of self-organization as often observed for the cytoskeleton (for a discussion on self-organization, see Misteli, 2001).
Elongation of the C. elegans embryo depends on the epidermal cytoskeleton and on a communication between muscles and epidermal cells. We have shown that VAB-10 isoforms are essential for embryonic elongation. Future analysis of vab-10 function should be invaluable to understand how spectraplakins can modulate cytoskeletal dynamics and signaling between tissues during morphogenesis.
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Materials and methods |
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vab-10 mutations and cloning
The ethyl methane sulfonateinduced lethal mutation h1356 belongs to a collection of eight lethal mutations isolated in the Rose laboratory (University of British Columbia, Vancouver, Canada). The mutation ju281 was isolated after ethyl methane sulfonate mutagenesis in screens for larval lethal mutations performed in the Chisholm laboratory (University of California, Santa Cruz, Santa Cruz, CA). We recovered the allele vab-10(mc44) by PCR screening after trimethylpsoralen/UV mutagenesis using two pairs of nested primers located 2.3 kb apart in vab-10 exons 2324 (VAB-10Bspecific). One pool carrying a 1-kb deletion (mc44), among 96 pools of 25 F1 mutagenized animals, was sib-selected; heterozygous animals segregating mc44 were outcrossed six times to wild-type animals and balanced. The molecular lesions affecting vab-10 alleles were identified by sequencing each predicted vab-10 exon after PCR amplification from h1356- and ju281-arrested embryos or e698 adults. Potential mutations were confirmed on an independent embryo/animal. The sequence of mc44 was determined from mc44-arrested larvae. Complementation tests between vab-10 alleles were performed by crossing vab-10(x)/+ males with vab-10(y)/+ hermaphrodites. Mating partners were transferred to fresh plates twice daily, and the number of unhatched eggs, arrested larvae, normal males, and hermaphrodites was counted; retarded larvae (putative trans-heterozygotes) were placed on separate plates and were examined separately.
To molecularly identify vab-10, we used RNAi (Fire et al., 1998) against predicted genes mapping under hDf17 (www.wormbase.org). RNAi against ZK1151.1, ZK1151.2, and ZK1151.3, the three genes predicted within cosmid ZK1151 (GenBank/EMBL/DDBJ accession no. Z93398), resulted in phenotypes similar or almost identical to those of strong vab-10 alleles, suggesting that they define a single locus. RT-PCR experiments were performed using the one-step RT-PCR kit (QIAGEN) starting from total RNA (see Fig. S1). The GenBank/EMBL/DDBJ accession nos. for vab-10 sequences are AJ505815 and AJ505816.
RNAi
Double-stranded RNA (dsRNA) used for RNAi was prepared and injected as described previously (McMahon et al., 2001). RNAi against vab-10 common exons (positions 2563526464 of ZK1151) generated a phenotype indistinguishable from that of h1356 mutant embryos. RNAi against vab-10Aspecific exons was obtained with either of two regions from the large vab-10Aspecific exon (positions 14630 to 15756, or 19490 and 20417 of ZK1151); both dsRNAs produced the phenotypes shown in Fig. 1 E. RNAi against vab-10Bspecific exons was obtained by co-injecting two dsRNAs from different areas of vab-10B (positions 5807 of Y47H9B and 518 of ZK1151, plus 2018 and 3136 of ZK1151). This resulted in mc44-like phenotypes within 2430 h after injection, then phenotypes became weaker; injection of each dsRNA separately resulted in weaker and less penetrant phenotypes.
Generation of VAB-10 antibodies
To produce antibodies, GST fusions with RT-PCR fragments encoding VAB-10A residues G2837Q3436, VAB-10B residues D2697R3862, or VAB-10B residues E3863K4955 were purified using DEAE-Sepharose followed by glutathione-Sepharose 4B columns, and were used to immunize two male New Zealand rabbits. Sera were purified by incubation for 3 h with fusion proteins that had been transferred onto nitrocellulose filters, followed by washing with 0.15 M NaCl for 30 min and PBS for 5 min. Specific antibodies were eluted by 0.1 M glycine-HCl (pH 2.5) for 10 min, and 1 M Tris-HCl (pH 8.0) was added to neutralize the solution. These antibodies were used to detect VAB-10 isoforms (1/1,000 dilution) on Western blots of total worm extracts separated by electrophoresis on 5% acrylamide gels.
Fluorescence microscopy
Embryos were fixed and stained by indirect immunofluorescence or by rhodamine-conjugated phalloidin as described elsewhere (Costa et al., 1997). Primary antibodies were diluted 1:1,000 for VAB-10A and VAB-10Bimmunopurified pAbs, or 1:50 for MH5, MH4, MH46, MH3, MH24, NE8/4C6, and 5.6.1.1 mAbs (Miller et al., 1983; Francis and Waterston, 1991; Schnabel, 1995). MH-series antibodies were a gift from Chelly Hresko and Bob Waterston (Washinton University, St. Louis, MO), NE8/4C6 from the Medical Research Council, and 5.6.1.1 from David Miller (Vanderbilt University, Nashville, TN). Primary antibodies were detected using Cy3- or FITC-conjugated secondary antibodies. Stacks of images every 0.5 µm (0.2 µm for images in Fig. 5 and Fig. 8, EH) were captured using a confocal microscope (model Sp1; Leica); generally, 1215 confocal sections were projected using the Tcstk software (McMahon et al., 2001) and then processed using Adobe Photoshop®.
EM
Mutant embryos laid between 6 and 9 h before processing were prepared for EM as described elsewhere (McMahon et al., 2001). Only nonruptured embryos that had reached the 1.51.7-fold stage were fixed. Thin sections were examined with a microscope (model EM208; Philips). We examined wild-type or heterozygous mutant/+ background, three embryos and five larvae; h1356, four embryos; ju281, four embryos; vab-10A(RNAi) three embryos; and vab-10B(RNAi), three embryos and six larvae. In each case, at least 1015 sections at different levels were examined using a 10,00015,000 magnification.
Immunoelectron microscopy
Wild-type adults were prepared for post-embedded immunolabeling as previously described for studies in other invertebrates (McDonald, 1999). In brief, young adults were immobilized by high pressure freeze, fixed in anhydrous methanol with 4% PFA and 1.5% uranyl acetate at -90°C, and infiltrated at -45°C with lowicryl HM20 (Polyscience, Inc.) that was then polymerized by UV exposure. Thin sections (80 nm) were incubated with either VAB-10A (1:2,000), VAB-10B K22 (1:500), or no primary antibody, and were labeled with gold colloidal suspensions of a 15-nm diameter coupled to GAR-IgG (BBInternational) according to manufacturer's recommendations (Aurion). Specificity of staining was judged based on comparing the number of gold beads localized in muscle, epidermis, cuticle, and resin in primary antibodystained and control grids.
Online supplemental material
Fig. S1 presents some of the RT-PCR experiments that lead us to propose the structure of vab-10 transcripts shown in Fig. 2. Fig. S2 presents, in further details, alignments between the cytoskeleton-binding domains of VAB-10 and other plakins. Fig. S3 describes how we mapped the epitope recognized by the mAb MH5 to vab-10A exon 16. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200302151/DC1.
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Acknowledgments |
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J.M. Bosher was supported by fellowships from the Fondation por la Recherche Médicale and the AFM, B.-S. Hahn by the Ministère de la Recherche, and R.M. Weimer by the INSERM "postes verts" program. This work was supported by institutional funds from CNRS, INSERM, Hôpitaux Universitaires de Strasbourg, and grants to M. Labouesse from the Association pour le Recherche sur la Cancer and the EEC-TMR program. Light camera equipment was purchased with a grant from the Ligue Nationale Centre le Cancer. Work in the laboratory of A.D. Chisholm is supported by a grant from the National Institutes of Health (GM54657)
Submitted: 24 February 2003
Revised: 7 April 2003
Accepted: 10 April 2003
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