Article |
Address correspondence to André Le Bivic, Laboratoire de Neurogenèse et Morphogenèse dans le Développement et l'Adulte, Institut de Biologie du Développement de Marseille, Faculté des Sciences de Luminy, case 907, Université de la Méditerranée, 13288 Marseille, cedex 09, France. Tel.: 33-4 91-26-97-41. Fax: 33-4-91-26-97-48. E-mail: lebivic{at}ibdm.univ-mrs.fr
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Abstract |
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Key Words: epithelial polarity; zonula adherens; Drosophila; spectrin; DMoesin
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Introduction |
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Surprisingly, most of the polarity functions in crumbs mutants are rescued by expression of its transmembrane and short cytoplasmic domains, suggesting that the major interactions regulating cell polarity and shape in the embryo are mediated by the 37 intracellular amino acids of this large (2,139 amino acids) protein (Wodarz et al., 1995). This hypothesis is reinforced by the observation that a nonsense mutation in the crumbs8F105 allele, preventing the translation of the last 23 amino acids of the cytoplasmic tail, produces a severe loss of function phenotype (Wodarz et al., 1993). Furthermore, it has been shown that the MAGUK family member Stardust (Sdt) binds to the last four amino acid residues (ERLI) of the cytoplasmic tail of Crumbs, along with the PSD-95/DLG/ZO-1 (PDZ) domain protein Discs-lost (Dlt) (Bhat et al., 1999; Klebes and Knust, 2000; Bachmann et al., 2001; Hong et al., 2001). These two proteins are both required for epithelial polarity, and thus Crumbs, together with Dlt and Sdt, defines a membraneassociated complex in the apical cytocortex of epithelial cells that is necessary for the proper generation of the polarized phenotype. Additional contributions from lateral proteins below the ZA, such as Scribble, are also involved in maintaining polarity and the integrity of the ZA. Because loss of scribble function results in a phenotype reminiscent of Crumbs overexpression, it has been suggested that the position and integrity of the ZA arises from a balance between the Crumbs-Dlt/Sdt complex at the apical border and the Scribble network on its basal side (Bilder and Perrimon, 2000).
Spectrins are long, tetrameric, F-actincrosslinking proteins comprised of two and two ß subunits (for review see Bennett and Baines, 2001). The spectrin-based membrane skeleton (SBMS) is a branching cytoskeletal network of spectrin-crosslinked F-actin associated with the various membrane compartments in the cell. Each SBMS is bound to the membrane via interaction with integral membrane proteins and phospholipids (De Matteis and Morrow, 2000). At the plasma membrane, spectrin, in conjunction with cortical F-actin, provides a structural basis for modulating cell shape and membrane stability in both epithelial and nonepithelial cells. In Drosophila, a single
-spectrin isoform combines with either of two, structurally distinct ß-isoforms (ß-spectrin and ßHeavy-spectrin [ßH]) to produce (
ß)2 and (
ßH)2 tetramers, respectively. In epithelial cells of Drosophila, (
ß)2 tetramers are restricted to the basolateral membrane, while the (
ßH)2 tetramers localize to the apical membrane and the ZA (Dubreuil et al., 1997; Lee et al., 1997; Thomas et al., 1998; Thomas and Williams, 1999).
All three spectrin subunits are essential for normal development. ßH, encoded by the karst locus, is an essential protein that is required for epithelial morphogenesis (Thomas et al., 1998). karst mutant cells exhibit altered shapes and disruption of the ZA indicating that (ßH)2 contributes to the integrity of the latter, but is not necessary for apicobasal polarity per se (Zarnescu and Thomas, 1999). Similarly, complex phenotypes are caused by mutations in the fly
- and ß-spectrin genes as well as in the orthologous genes in Caenorhabditis elegans (Lee et al., 1993; de Cuevas et al., 1996; Dubreuil et al., 1998; McKeown et al., 1998; Dubreuil et al., 2000; Moorthy et al., 2000). Together, these studies indicate that the SBMS has an essential role in cell structure and morphogenesis (for review see Thomas, 2001), making the identification of proteins that recruit and/or organize this structure of considerable interest.
Spectrins are generally recruited to the membrane via adapter proteins that link the SBMS to integral membrane proteins (Bennett and Baines, 2001). Two families of such adapter proteins have been well characterized: ankyrins and protein 4.1 family members. The former binds to the midregion of the ß-spectrin spectrin repeat array (Lombardo et al., 1994), whereas the latter forms a ternary complex between the actin-binding domain of ß-spectrin and F-actin itself (Marfatia et al., 1997). Protein 4.1 is part of a larger superfamily of proteins containing protein 4.1/ezrin/radixin/moesin (FERM) domains (Chishti et al., 1998) that function to attach cortical F-actin to a variety of integral membrane proteins (Tsukita and Yonemura, 1999). The existence of multiple adapter protein genes, as well as alternatively spliced isoforms, generates great diversity in the number of proteins to which an SBMS can be attached (see De Matteis and Morrow, 2000 for a list of almost 50 spectrin associated proteins). The recruitment of conventional ß-spectrins by adapter proteins is well characterized (e.g., Jenkins and Bennett, 2001); however, the cues recruiting spectrin to the apical domain are currently uncharacterized, as are the adapter proteins that associate with the ßH isoform.
Overexpression of Crumbs in the embryonic ectoderm causes an enlargement of the apical membrane and a concomitant expansion in the distribution of ßH staining (Wodarz et al., 1995). This result suggested that this apical polarity cue might also be responsible for recruiting and/or organizing the apical SBMS. To investigate this possibility, we looked for genetic and physical interactions between ßH and Crumbs. In this paper, we report that at least one allele of crumbs is a dominant enhancer of the karst phenotype, and that whereas the Crumbs distribution is unaffected in karst mutants, ßH is mislocalized in the epithelial cells of crumbs8F105 mutants. Furthermore, overexpression of Crumbs led to redistribution of ßH, DMoesin, and actin, indicating that Crumbs acts upstream of ßH in organizing the apical SBMS. We also demonstrate that clustering of a chimeric-tagged form of Crumbs in Schneider 2 (S2) cells induces cocapping of ßH and DMoesin. This provides evidence for a relationship between Crumbs and these two proteins under physiological conditions. This interaction is dependent on a consensus motif for the binding of proteins of the FERM family in the cytoplasmic tail of Crumbs. Finally, we show that Dlt, Crumbs, ßH, and DMoesin coimmunoprecipitate, indicating that a multiprotein complex is recruited by Crumbs. These results indicate that Crumbs mediates a novel coordination between cell polarity, junctional stabilization, and morphogenesis.
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Results |
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crumbs is a dominant enhancer of karst
Given the close functional and spatial relationship between ßH and the ZA (Thomas and Williams, 1999; Zarnescu and Thomas, 1999), we looked for a genetic interaction between karst and crumbs. Such an interaction is likely to be modest due to the existence of multiple pathways for recruiting ßH (see Discussion). Furthermore, all karst alleles isolated to date exhibit variable expressivity necessitating a statistical approach. The interaction test was thus limited to the most readily quantified feature of the pleiotropic karst phenotype, the degree of lethality. Comparison of viability rates between karst crumbs/karst + and karst/karst genotypes reveals a statistically significant enhancement of lethality in the presence of one mutant crumbs allele (Fig. 2). Thus, halving the level of Crumbs further reduces the remaining functionality of the mutant ßH protein. This defines crumbs as a dominant enhancer of karst and is the expected result if Crumbs lies upstream in the organization of the apical SBMS.
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A Crumbs construct in which most of the extracellular domain was replaced by an epitope of vesicular stomatitis virusprotein G (VSV-G) (Fig. 6 A; recognized by the monoclonal antibody [mAb] P5D4), was stably expressed in S2 cells. The encoded crumbs (CRB)VSV-G WT protein is transported to the cell surface where it is recognized by both the P5D4 mAb and a polyclonal antibody raised against the cytoplasmic domain of Crumbs (Fig. 6 B; see Materials and methods). This protein is equivalent to the Crumbsmyc-intra protein that we used in the overexpression experiments in embryos (Fig. 5). ßH accumulates at the plasma membrane in adherent S2 cells (Dubreuil et al., 1997), but not when these cells are grown in suspension (Fig. 7, untransfected cells), whereas DMoesin is always associated with the plasma membrane. CRBVSV-G WT expression caused no conspicuous change in the distribution of DMoesin or ßH in adherent cells with both proteins colocalizing at the plasma membrane (unpublished data).
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To investigate the role of the cytoplasmic domain of Crumbs for the interaction with DMoesin or ßH, we expressed the truncated construct CRBVSV-G S6 in which only the first five amino acids of the cytoplasmic domain remained (Fig. 6 A). This mutation prevented binding to Dlt in a GST pulldown assay (unpublished data) and essentially eliminated the ability to cluster Dlt as expected (Fig. 7 A, bottom). CRBVSV-G S6 was also unable to efficiently recruit DMoesin and ßH, indicating that the cytoplasmic domain of Crumbs was also necessary for the interaction with ßH and DMoesin (Fig. 7 B and C, bottom).
The Crumbs FERM domain binding site is required to efficiently recruit both DMoesin and ßH
The capping technique provides a readily quantifiable assay for interactions between the Crumbs cytoplasmic domain and other proteins. Thus, we next used it to determine which part of the cytoplasmic domain of Crumbs is necessary for the interaction with DMoesin and ßH. A second truncation mutant, CRBVSV-G 8F105 (Fig. 6 A), that mimics the crumbs8F105 allele with a stop codon at position 15 of the cytoplasmic domain (Wodarz et al., 1993), was able to recruit ßH and DMoesin just as efficiently as CRBVSV-G WT, but could no longer bind to Dlt as predicted (Fig. 8). These results indicate that the distal part of the Crumbs cytoplasmic domain is not crucial for the DMoesin/ßHCrumbs interaction, in contrast to the interaction between Crumbs and Dlt (Bhat et al., 1999; Klebes and Knust, 2000) and Crumbs and Sdt (Bachmann et al., 2001; Hong et al., 2001).
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Discussion |
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crumbs lies upstream of karst, stabilizing the apical spectrin membrane skeleton
Several lines of evidence indicate that Crumbs can recruit ßH into its complex: (a) ßH is mislocalized in embryos mutant for the truncation allele crumbs 8F105, in which the mutant Crumbs protein itself is mislocalized; (b) ßH mislocalization can be induced by overexpression of the Crumbs transmembrane and cytoplasmic domains in vivo; (c) ßH is recruited to Crumbs protein clusters in an S2 cell cocapping assay; (d) we can coimmunoprecipitate Crumbs with ßH; and (e) the protein-null allele crumbs11A22 acts as a dominant enhancer of hypomorphic karst alleles, strongly indicating that a reduction in the normal amount of Crumbs reduces the level of partially functional ßH at the membrane. Moreover, because the karst mutant alleles all produce COOH-terminally truncated proteins (see Materials and methods), these results further suggest that the Crumbs-ßH interaction site lies in the NH2-terminal portion of the latter. Finally, in a paper that came out while this manuscript was under review, it was shown that loss of Crumbs eliminates ßH from the stalk membrane of photoreceptors in the adult eye (Pellikka et al., 2002).
Current evidence indicates that ßH can be recruited to the membrane in several additional ways. First, it can associate with the specialized basal adherens junctions during cellularization in a Crumbs-independent manner (Thomas and Williams, 1999). Second, it is found in the terminal web subtending brush borders in the midgut epithelium (Thomas et al., 1998) that does not express Crumbs (Tepass, 1997). Finally, it has also been shown that ßH is only partially reduced in crumbs11A22 mutant follicle cell clones (Tanentzapf et al., 2000), indicating that in this Crumbs-expressing epithelium there are multiple mechanisms to recruit ßH. These data provide a compelling explanation for the modest nature of the karst-crumbs genetic interaction. By reducing Crumbs, we are discretely affecting only one of these pathways. The observation that the karst1 allele produces readily detectable quantities of truncated product (Thomas et al., 1998), most of which is not recruited to the membrane in any of these epithelia, suggests that there is a general and essential role of the COOH-terminal half of ßH in its stable membrane localization (Zarnescu and Thomas, 1999). Together, the above data are consistent with the multifunctional nature of spectrin membrane skeletons and with the idea that specific pathways recruit the SBMS to establish spatially distinct polarized membrane domains, whereas general COOH-terminal membrane association domains permit tight membrane association and network integration (Lombardo et al., 1994; Bennett and Baines, 2001).
The cytoplasmic domain of Crumbs recruits DMoesin and ßH
The previously reported partial rescue of crumbs mutants by the crumbsmyc-intra construct (Wodarz et al., 1995) suggested that the transmembrane and cytoplasmic domains of Crumbs might be sufficient to concentrate ßH to some areas of the apical membrane. We have confirmed and extended this result, showing that the critical region for recruiting ßH is just 9 amino acids from position 6 through 14 of the cytoplasmic domain in the putative FERM domain binding site (Klebes and Knust, 2000). Within this region, a conserved tyrosine residue at cytoplasmic domain position 10 (crucial for Crumbs function in vivo; Klebes and Knust, 2000) and an arginine at position 7 are both required for this activity. It is worth noting that all Crumbs genes cloned so far contain a charged amino acid residue at position 7 in the cytoplasmic domain (see Klebes and Knust, 2000), suggesting that this is an evolutionarily conserved interaction site.
FERM domains are found in the protein 4.1 family of proteins which link the SBMS to cell-surface receptors (Hoover and Bryant, 2000) as well as several other proteins which organize the cortical actin (ezrin/radixin/moesin; Bretscher, 1999; Tsukita and Yonemura, 1999). The founding member of this group, protein 4.1, was originally identified as a major component of the erythrocyte SBMS where it facilitates the interaction of spectrin with actin and the transmembrane protein Glycophorin C (Marfatia et al., 1997). Therefore, the presence of a conserved FERM binding domain in the Crumbs cytoplasmic domain suggests that Crumbs may bind to ßH via a FERM domain protein.
In Drosophila, the FERM domain family includes the proteins Coracle, DMerlin, DMoesin, and Expanded (McCartney and Fehon, 1996). Of these four proteins, Coracle is an unlikely candidate to bind to the Crumbs juxtamembrane domain since it is localized to the septate junctions basal to the ZA (Fehon et al., 1994). However, the DMerlin, DMoesin, and expanded proteins are localized in part or in whole at the ZA region in epithelia (McCartney and Fehon, 1996; Boedigheimer et al., 1997), and could thus be involved in the interaction between Crumbs and ßH. The fact that none of protein 4.1 family members known in Drosophila contains a spectrin-binding domain as defined by the archetypal protein 4.1 does not necessarily abrogate this hypothesis. ßH-spectrin is clearly recruited to the membrane by different mechanisms than its basolateral counterpart (Dubreuil and Grushko, 1999), and this specificity would likely be reflected in divergent interaction domains. In this work, we have found that ßH and DMoesin can both coimmunoprecipitate Crumbs. Furthermore, our capping assay and embryo expression evidence provide in vivo support for this result. Not only will DMoesin cocap with the Crumbs cytoplasmic domain, it is dependent on exactly the same sequences that recruit ßH. These results, together with the existence of the consensus binding site for a FERM domain protein in Crumbs, strongly support the hypothesis that DMoesin forms a bridge between Crumbs and the SBMS (see model in Fig. 9). A functional test of this relationship must wait until mutations in the DMoesin locus become available. Thus, the current data, although highly suggestive, do not formally distinguish between the possibility of a DMoesin bridge between Crumbs and the SBMS, and the existence of two separate complexes with direct interaction between Crumbs and ßH or Dmoesin in each. Significantly, actin did not cap consistently with Crumbs in S2 cells and was not present in our immunoprecipitates (unpublished data). This suggests that other components present in epithelial cells are necessary for stabilization of the actin skeleton around the Crumbs complex. It also indicates that ßH is specifically recruited to the proposed complex and is not merely a passive arrival along with bulk actin.
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A model for Crumbs action in apical network formation
Because both the crumbs and karst phenotypes disrupt the ZA (Grawe et al., 1996; Zarnescu and Thomas, 1999), we hypothesize that Crumbs promotes the accumulation of ßH to the apicolateral region during gastrulation to orchestrate the fusion of spot adherens junctions and/or to stabilize the ZA. Moreover, the observation that karst mutants exhibit morphogenetic defects without any loss of epithelial polarity (Zarnescu and Thomas, 1999), whereas dlt mutants exhibit a strong polarity phenotype (Bhat et al., 1999), suggests that the polarization and junction building functions of Crumbs are separate and parallel pathways. In support of this hypothesis, a paper appeared while this manuscript was under review indicating that the FERM domain binding region of Crumbs is indeed required for correct organization of the ZA (Izaddoost et al., 2002).
The loss of ßH function causes defects in cell shape change that are associated with apical contraction driven by an apically located actomyosin contractile ring (McKeown et al., 1998; Zarnescu and Thomas, 1999; for review see Thomas, 2001). In this context the discovery that this spectrin isoform is complexed with DMoesin is particularly provocative, as the activity of the latter is strongly correlated with modulation of cell shape and the actin cytoskeleton (Edwards et al., 1997; Tsukita and Yonemura, 1999). Furthermore, the activity of moesin is modulated by phosphorylation in response to activation of Rho-associated kinase (ROK) in parallel with myosin II. Both Moesin and myosin light chain are activated by ROK phosphorylation and by ROK mediated inhibition of the myosin/moesin phosphatase (e.g., Fukata et al., 1998; Eto et al., 2000). Therefore, we speculate that ßH is part of the cytoskeletal network that facilitates such cell shape changes, and that in organizing spectrin at the membrane, Crumbs would appear to be acting as a molecular coordinator of polarity and morphogenesis. Furthermore, the finding that in human, mutations in CRB1 lead to pathologies such as retinitis pigmentosa (RP12) (den Hollander et al., 1999) emphasizes the importance of deciphering the molecular networks associated with Crumbs in Drosophila. The human orthologue of ßH, ßV-spectrin, is strongly expressed in photoreceptor cells (Stabach and Morrow, 2000). This raises the exciting possibility that a similar interaction between CRB1 and ßV-spectrin exists in these cells. This will be examined in future work.
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Materials and methods |
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Statistical analysis
The karst phenotype exhibits variable expressivity (Thomas et al., 1998; Zarnescu and Thomas, 1999), and thus enhancer/suppressor interactions must be characterized in replicate experiments with appropriate statistical comparisons. In this paper, viability to adulthood is expressed as a lethal fraction of the Mendelian expectation estimated using a maximum likelihood model to determine the cost of each allelic combination of karst. Because karst cannot be maintained for many generations over the TM3 chromosome (because the 63CD region is not effectively balanced and karst is rapidly lost through recombination), the more effective TM6 balancer is routinely used. However, TM6 itself exhibits a low level of dominant lethality. Thus, to accurately estimate karst viabilities in our crosses, we first estimated the cost of the TM6 chromosome (0.321 ± 0.138 [95% confidence interval]; 17 crosses; 6, 153 flies scored) and this figure was used in our estimates of karst viabilities. Testing for any increased lethality of karst crumbs/karst + versus karst alone utilized Microsoft Excel 98 (Microsoft Corporation) to perform a one tailed t-test on the mean lethality appropriate for equal or unequal variances (assessed using an F-test).
Antibodies
A serum raised against the cytoplasmic domain of Crumbs was affinity purified and used at a dilution of 1:50 for immunofluorescence. A mouse monoclonal anti-Crumbs antibody MabCq4 (provided by Dr. E. Knust) was used at a dilution of 1:2 for immunostaining and immunoblotting. A rabbit polyclonal anti Dlt antibody provided by Dr. M. Bhat (Mount Sinai School of Medicine, New York, NY) (Bhat et al., 1999) was used at a dilution of 1:3,000 and 1:300 for immunoblotting and immunofluorescence, respectively. A mouse monoclonal anti-VSV-G antibody P5D4 (Sigma-Aldrich) was used at a dilution of 1:500 and 1:300 for immunoprecipitation and immunofluorescence, respectively, and at 1:400 for capping experiments. Affinity-purified anti ßH serum (#243) was prepared as previously described (Thomas and Kiehart, 1994) and used at 1:1,000 or 1:500 for immunoblots and immunofluorescence, respectively. Antibodies against DMoesin were provided by D. Kiehart (Duke University, Durham, NC), prepared as described (Edwards et al., 1997), and used at 1:500 for immunoprecipitations, 1:2,000 for immunofluorescence, and 1:20,000 for immunoblotting. The anti-myc mouse monoclonal antibody 9E10 (Santa Cruz Biotechnology, Inc.) was used at a dilution of 1:50 and TRITC-phalloidin (Sigma-Aldrich) was used at a dilution of 1:100.
Immunofluorescent staining of embryos and cells
Immunostaining of embryos (from 2 to 14 h) was performed as described (Muller and Wieschaus, 1996) using fluorescein isothiocyanate-conjugated goat antimouse IgG or rhodamine-conjugated goat antirabbit IgG as appropriate (Jackson ImmunoResearch Laboratories, Inc.) at a dilution of 1:100. Procedures for indirect immunofluorescence of S2 cells were as described for mammalian cells (Le Bivic et al., 1989). For intracellular staining, fixed cells were permeabilized with 0.05% saponin. Fluorescent secondary antibodies were used at a dilution of 1:200. For phalloidin staining, embryos were devitellinized in 80% ethanol.
Immunoblots and immunoprecipitations
For immunoprecipitations, 2 to 14 h wild-type Drosophila embryos (1 g) were homogenized in 6 ml of purification buffer (10 mM Tris, pH 7.5, 0.32 M sucrose, 3 mM MgCl2) supplemented with anti-proteases (1/1,000) and orthovanadate (0.2 mM) and centrifuged for 10 min at 1,500 g. Supernatant was collected and the pellet was resuspended in 4 ml of purification buffer, centrifuged and the supernatants were pooled. Supernatant was ultracentrifuged for 1 h at 40,000 rpm (Ti 70 rotor; Beckman Coulter) and pellet was resuspended in 3 ml of lysis buffer (1% Igepal, 50 mM Tris, pH 7.5, 10 mM EDTA, 3 mM MgCl2) supplemented with anti-proteases and orthovanadate as described above. After incubation for 30 min at 4°C, the lysate was centrifuged for 10 min at 14,500 g, incubated for 1 h with Pansorbin, and centrifuged at 14,500 g for 15 min. Lysates were immunoprecipitated for 2 h at 4°C using the anti-Moesin or anti-ßH or rabbit antimouse antibodies (1:500; Compiègne) preabsorbed on protein ASepharose beads (Amersham Biosciences). Precipitates were fractionated by SDS-PAGE, electrophoretically transferred to nitrocellulose (Schleicher and Schuell GmbH), and incubated with appropriate primary and peroxidase-conjugated secondary antibodies (1:10,000; Immunotech SA). ßH was analyzed as described previously (Thomas and Kiehart, 1994).
DNA constructs, transfections and cell culture
The chimeric construct CRBVSV-G WT was obtained by amplifying a COOH-terminal Crumbs fragment containing the stalk region, transmembrane domain and cytoplasmic domain of Crumbs (amino acid 20742146) using the full-length crumbs cDNA as template, a gift of Dr. E. Knust, and cloning it into the pUC19 vector containing the VSV-G tag, a gift of Dr. P. Boquet (University of Nice, Nice, France). This fusion construct was subsequently subcloned into the EcoRV-BamHI sites of the pMK33/pMtHy plasmid with a metallothionein promoter, a gift of Dr. M. Koelle (Yale University, New Haven, CT). Mutant CRBVSV-G constructs (Fig. 5) were derived by PCR and subcloned in the same vector. All constructs were verified by sequencing (Genome Express SA).
Drosophila S2 cells were transiently or stably transfected with constructs in pMK33/pMtHy plasmid using FuGENE 6 Transfection Reagent according to the manufacturer instructions (Roche Diagnostics GmbH). Stably transfected cells were selected and maintained with Hygromycin B (Roche Diagnostics GmbH) used at a concentration of 250 and 100 µg/ml, respectively. Expression of CRBVSV-G constructs was induced by the addition of 1 mM CuCl2 to the growth medium for 1724 h.
Capping experiments
Stably transfected S2 cells were processed as described (Jefford and Dubreuil, 2000), except that fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:200 in Drosophila Ringer's) was added for 5 min before being transferred to polylysine-coated slides. Once on slides, the cells were fixed and stained as above for Dlt, DMoesin, or ßH and cells were examined with a Zeiss LSM 410 confocal microscope. Capped S2 cells expressing the CRBVSV-G constructs were scored for the presence of fluorescent antibody-stain caps using the fluorescein channel and for Dlt, actin, DMoesin, or ßH colocalization at caps using the rhodamine channel. About 50 VSV-Gpositive cells were scored in each experiment, and results are expressed as a percentage of the cocapped cells found for each protein with the CRBVSV-G S6 construct normalized at 0% (actual capping percentage, 25% for CRBVSV-G S6 and 75% for CRBVSV-G WT).
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Footnotes |
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Acknowledgments |
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This work was supported by Centre National de la Recherche Scientifique CNRS 6156, Université de la Méditerranée, Institut de Biologie du Développement de Marseille, Fondation de France and Association pour la Recherche sur le Cancer 9297, and an EC grant (Crumbs therapeutics) to A. Le Bivic, and by National Institutes of Health grant #GM52506 to G. Thomas and a National Institutes of Health predoctoral fellowship GM20906 to J. Williams. This paper is dedicated to C. Goridis.
Submitted: 19 March 2002
Revised: 25 July 2002
Accepted: 25 July 2002
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