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Genetics, Vol. 172, 343-353, January 2006, Copyright © 2006
doi:10.1534/genetics.105.049650
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Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas 77843-2128
2 Corresponding author: Department of Biochemistry & Biophysics, Texas A&M University, TAMU 2128, College Station, TX 77843-2128.
E-mail: panin{at}tamu.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In mammalian organisms, one of the main targets of O-mannosylation is thought to be 
-dystroglycan (CHIBA et al. 1997), a cell adhesion glycoprotein important for the integrity of the muscle cells and neuronal migration during development (MARTIN 2003). Dystroglycan plays a central role in the dystrophin-associated glycoprotein complex (DGC) that provides a crucial link between the extracellular basal lamina and the cytoskeletal proteins (MICHELE and CAMPBELL 2003). The O-mannosyl glycans of -dystroglycan have been found to affect its binding to the extracellular matrix-associated ligand laminin, which indicates the importance of O-mannosylation for the functional integrity of DGC and its interaction with extracellular matrix (CHIBA et al. 1997; MICHELE et al. 2002).
Intriguingly, the Drosophila genome encodes homologs of all essential components necessary for the formation of DGC but with substantially less diversity (GREENER and ROBERTS 2000; DEKKERS et al. 2004), which presents an attractive possibility for using fruit flies as a model system for studying the biology of DGC and several human congenital neuromuscular diseases.
Protein O-mannosyltransferase enzymes are best characterized in yeast. A total of seven O-mannosyltransferases have been identified in Saccharomyces cerevisiae (GENTZSCH and TANNER 1996). They comprise three evolutionary conserved subfamilies of proteins, PMT1, PMT2, and PMT4 (WILLER et al. 2003). On the other hand, the family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2 that belong to PMT4 and PMT2 subfamilies, respectively (JURADO et al. 1999; WILLER et al. 2002). These enzymes are thought to modify numerous serine and threonine residues of -dystroglycan with O-linked mannose, which initiates the biosynthesis of O-mannosyl glycans of dystroglycan (WILLER et al. 2003). Recently, it was demonstrated that elevated O-mannosyltransferase activity could be detected in extracts from cultured cells only if POMT1 and POMT2 were coexpressed in these cells (MANYA et al. 2004). Although this result indicated the possibility that these enzymes function as a heterocomplex, no evidence for physical interaction between POMT1 and POMT2 proteins have been obtained so far. Thus, molecular and genetic mechanisms governing the function of these proteins remain elusive.
Similar to higher animals, Drosophila has two O-mannosyltransferases, RT (encoded by the rotated abdomen gene) and DmPOMT2, which are evolutionary related to mammalian POMT1 and POMT2, respectively (MARTIN-BLANCO and GARCIA-BELLIDO 1996; WILLER et al. 2002; ICHIMIYA et al. 2004). Several mutations in the rotated abdomen gene (rt) were previously isolated and phenotypically characterized (BRIDGES and MORGAN 1923; LINDSLEY and ZIMM 1992; MARTIN-BLANCO and GARCIA-BELLIDO 1996). Two molecularly characterized mutants, rt2 and rtP, are semiviable recessive alleles associated with disruptions of the gene coding region by P-element insertion; they possibly represent null mutations (COOLEY et al. 1988; MARTIN-BLANCO and GARCIA-BELLIDO 1996). The phenotypes of these mutations include some larval and adult muscle abnormalities, as well as a prominent, up to 90° clockwise rotation of abdominal segments in adult flies (MARTIN-BLANCO and GARCIA-BELLIDO 1996). No mutations in DmPOMT2 gene have been reported so far, although the possibility that mutations in the twisted locus might represent DmPOMT2 mutants has been suggested (J. CRUCES, cited in WILLER et al. 2002). This hypothesis was further supported by "twisted abdomen" phenotype recently obtained in RNAi-mediated DmPOMT knockdown experiments (ICHIMIYA et al. 2004). Similar to their mammalian counterparts, RT and DmPOMT2 proteins have to be coexpressed to produce O-mannosyltransferase activity (ICHIMIYA et al. 2004).
The twisted alleles represent a complementation group of viable and semiviable recessive mutations on the X chromosome that also exhibit clockwise rotated abdominal segments in the adult (LINDSLEY and ZIMM 1992). Several tw mutant alleles had been isolated (DAVIS 1980; LINDSLEY and ZIMM 1992); however, none of them was characterized in detail and no molecular data are currently available for the tw locus. Most of these tw mutants have been lost and there is only one mutant allele, tw1, available from public collections (FlyBase information, DRYSDALE et al. 2005).
In this study, we have tested the hypothesis that tw corresponds to DmPOMT2. By using an expression rescue approach, we established that the tw locus represents the DmPOMT2 gene. We also characterized the molecular nature of tw1 mutation. Our immunostaining analysis of subcellular localization of tagged RT and DmPOMT2 (TW) proteins revealed their colocalization in the endoplasmic reticulum (ER) of Drosophila cells. The pattern of embryonic tw expression was analyzed by in situ hybridization and compared to the expression of rt. Our data showed striking overlap of tw and rt expression at different stages of embryogenesis. Moreover, we found a genetic interaction between tw and rt mutant alleles. All these results are consistent with the hypothesis that RT and TW, the two Drosophila protein O-mannosyltransferases, participate in the same developmental cascade and may collaborate at the molecular level, potentially functioning as an enzymatic heterocomplex.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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AGGAT; C(472) T; T(2545) C (nucleotide numbers are relative to the beginning of coding region). The first alteration is predicted to affect the translated protein sequence [T(59) GS], while the two other mutations are silent.
DNA constructs for cell-culture expression and fly transformation
The cDNA clones were obtained from the Drosophila Genomics Resource Center at Indiana University. For cell culture expression experiments, the coding region of DmPOMT2 was PCR amplified from the DmPOMT2 cDNA (LP01681) using PCR primers containing BglII and XbaI restriction sites. Using these sites, the PCR product was inserted into the pMK33 vector (KOELLE et al. 1991). The final construct, pMK–DmPOMT2, also included a short DNA fragment encoding the HA tag (NIMAN et al. 1983); it was obtained by annealing two synthesized oligonucleotides and then introduced into DmPOMT2 cDNA using SanDI restriction site. The resulting construct encoded a DmPOMT2 (TW) protein with HA-tag inserted into nonconserved region immediately after G(650). The same construct was subcloned into the pUAST vector (BRAND et al. 1994) to obtain pUAST–DmPOMT2 for fly transformation and in vivo expression. Functionality of the construct was confirmed by Western-blot analysis (data not shown) and immunostaining of pMK–DmPOMT2-transfected S2 cells (Figure 4), as well as by an in vivo rescue assay (Figure 2). For the expression of rt in Drosophila cell culture, the coding region of rt was PCR amplified from the rt cDNA (clone RE30211) and inserted in the pRMHA3 vector (BUNCH et al. 1988). In the final expression construct, the rt coding region was modified by the addition of a short DNA fragment encoding two MYC tags (EVAN et al. 1985) immediately following the last amino acid-coding triplet of rt. As revealed by sequencing, the RE30211 clone includes a short unspliced intron preceding the last coding exon of the gene. Efficient expression of the construct and removal of the intron by in-cell splicing was confirmed using anti-MYC immunostaining of pRMHA3–rt-MYC-transfected cells (Figure 4) and Western-blot analysis of RT–MYC protein expressed in Drosophila S2 cells (data not shown). The functionality of the MYC-tagged RT protein was also confirmed in vivo by its ability to rescue the rt mutant phenotype (data not shown). The UAS–twRNAi construct was produced essentially according to the published strategy (LEE and CARTHEW 2003). Briefly, the two PCR-amplified DNA fragments of DmPOMT2 gene, both including the 6th exon (
700 bp) and one also including the following intronal sequence, were ligated together in the opposite orientation. The resulting inverted repeat was cloned into the pUAST vector using PCR-introduced EcoRI and XbaI restriction sites. The UAS–twRNAi construct is predicted to produce 700 bp hairpin dsRNA with 12 unpaired bases of loop region.
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Immunostaining and epifluorescent microscopy:
Expression of the UAST–DmPOMT2 construct was induced in salivary glands using the C155–GAL4 driver. Third instar larvae were dissected, fixed, and stained as described earlier (PANIN et al. 1997). The following primary antibodies and corresponding dilutions were used for immunostaining: rabbit anti-LVA (1:2000) (a gift from John Sisson, University of Texas, Austin, TX); mouse and rabbit anti-HA (1:1000); mouse anti-MYC (1: 1000) (BabCo, Berkeley, CA). We used the following fluorescent secondary donkey antibodies: anti-mouse-Cy3 (1:250); anti-rabbit-FITC (1:150); and anti-mouse-Cy5 (1:150) (Jackson Laboratories). Digital images were obtained using Zeiss Axioplan 2 fluorescent microscope with the ApoTome module for optical sectioning.
In situ hybridization was performed as described earlier (KOLES et al. 2004) using the tw and rt cDNA clones LP01681 and RE30211, respectively, as templates for the synthesis of DIG-labeled probes. Every in situ hybridization experiment included a negative control staining with a probe transcribed from the corresponding antiparallel cDNA sequence (data not shown).
Phenotype analysis:
All flies were grown at 25°. The rotation of abdomen was scored in CO2-anesthetized flies 1 day after eclosion (to eliminate possible influence of aging or altered morphology in very young flies). The rotation angle was measured from posterior viewpoint using Nikon SMZ microscope with a protractor reticle objective.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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GS] within the conserved PMT domain of DmPOMT2 (Figure 1), which suggests an altered functionality of the DmPOMT2 protein in the tw1 mutants. This finding further supported the hypothesis that the phenotype of tw mutants results from mutations in DmPOMT2.
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We produced UAS–DmPOMT2 transgenic flies and crossed them to flies carrying a ubiquitous Act5C–GAL4-17 driver (see MATERIALS AND METHODS). There were no visible defects in abdominal morphology of the flies from either parental stocks or UAS–DmPOMT2/+; Act5C–GAL4-17/+ progeny of the cross (data not shown). The presence of the UAS–DmPOMT2 or Act5C–GAL4 transgene alone did not modify the phenotype of tw1 mutants (Figure 3). At the same time, the complete rescue of the abdomen rotation phenotype was observed in tw1 hemizygous male and homozygous female flies carrying both UAS–DmPOMT2 and Act5C–GAL4-17 together (Figure 3). Similar results were obtained when DmPOMT2 was expressed using Act5C–GAL4-25 and tubP–GAL4 drivers (data not shown). These rescue experiments proved that tw indeed represents the DmPOMT2 gene. Thus, we suggest changing the designation of the DmPOMT2 (CG12311) gene to "twisted" (tw), the name of the originally discovered mutants that is in an agreement with the traditional nomenclature of Drosophila genetics (LINDSLEY and ZIMM 1992). Below, we use "tw" and "TW" to designate the DmPOMT2 gene and its protein product, respectively.
Subcellular localization of RT and TW proteins:
Glycosyltransferases that modify secreted glycoproteins commonly function in the Golgi apparatus. Interestingly, yeast PMT family members localize to the ER subcellular compartment (WILLER et al. 2003). The localization of O-mannosyltransferase proteins in animal cells has been reported only for human POMT2 protein expressed in human culture cells (WILLER et al. 2002). In that study, the C-terminally tagged POMT2 protein was detected in the ER by immunostaining; however, the functionality of this fusion protein remains undetermined. Thus, we decided to investigate the subcellular localization of the TW protein, the Drosophila ortholog of mammalian POMT2, using the UAS–DmPOMT2 transgenic construct that was functional in our rescue assay (Figure 2). Immunostaining for TW protein expressed in vivo in the salivary gland cells of Drosophila larvae revealed its colocalization with an ER marker, PDI-GFP (BOBINNEC et al. 2003). At the same time, the localization of TW showed minimal overlap with a Golgi marker, the LVA protein (SISSON et al. 2000), when TW was expressed in Drosophila S2 cells (Figure 4, D–F). Double immunostaining of S2 cells expressing both RT and TW proteins demonstrated their colocalization inside the cell (Figure 4, G–I). Thus, we concluded that both RT and TW proteins reside in the ER subcellular compartment.
Analysis of tw and rt expression during Drosophila embryogenesis by in situ hybridization:
Detailed analysis of the spatial and temporal patterns of gene expression by in situ hybridization can provide important information about the functioning and regulation of the gene. The pattern of embryonic rt expression was previously analyzed by in situ hybridization (MARTIN-BLANCO and GARCIA-BELLIDO 1996). The pattern of tw (DmPOMT2) expression has been reported only for embryonic stage 10, while the expression at other stages was estimated only using a real-time PCR assay (ICHIMIYA et al. 2004). Thus, we decided to perform in situ hybridization analysis of tw expression and compare it with the pattern of rt expression at different embryonic stages. In agreement with previous reports (MARTIN-BLANCO and GARCIA-BELLIDO 1996; ICHIMIYA et al. 2004), we found no significant expression of tw at early embryonic stages, while rt mRNA was detected at stages 5 and 6, which probably indicates the presence of maternally provided transcript (Figure 5, A and D). This early rt expression decays quickly and it is not readily detectable after stage 7. Although we did detect some weak staining for tw and rt expression during the stages of germband extension (stages 10 and 11), this staining was diffuse and barely detectable above the background (data not shown). Thus, we decided to concentrate our analysis on tw and rt expression at later stages, when the expression of these genes is readily detectable. The prominent expression of both genes, tw and rt, appears at early stage 14 that corresponds to the period of active muscle differentiation (Figure 5, B and E). However, we have not detected significant expression of tw or rt in the developing somatic muscle cells. Instead, the expression of these genes appeared to be pronounced in other tissues, including certain developing epidermal cells, as well as hindgut and foregut regions. At that stage, the expression of tw is also present in the developing trachea, while rt expression appears in the tracheal cells slightly later, during stage 15. Thus, remarkably overlapping patterns of rt and tw expression are established by stages 14 and 15, and this expression appears to persist through the late embryogenesis (Figure 5, C and F).
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We considered two competing explanations of these results. First, the relative excess of TW as compared to RT in the rt mutants might have a negative effect on the O-mannosylation pathway. In this case, a potential decrease of TW might explain the suppression of rt phenotype in tw1 mutant background. Alternatively, the suppression of rt phenotype by tw1 might be explained by a special feature of the tw1 allele, which would not implicate the dependence of phenotype on relative concentrations of RT and TW. To discriminate between these two possibilities, we analyzed further the genetic interaction between rt and tw.
The first possibility would predict that an increase of tw activity would increase the severity of rt mutant phenotype, while a decrease of tw would result in rt mutant phenotype suppression. To test this prediction, we varied the level of tw in rt mutants by several alternative ways: (i) by adding an extra copy of the tw locus [using duplication Dp(1;Y)y2sc], (ii) by overexpressing TW using UAS–GAL4 system, and (iii) by decreasing the tw activity via UAS–twRNAi construct expression. Neither the increase of tw expression (Figure 7A) nor the decrease of tw activity (Figure 7B) revealed the predicted sensitivity of rt mutant phenotype to varied concentrations of TW. Hence, we interpret the dominant suppression of rt phenotype by the tw1 mutation as a special feature of the tw1 allele that somehow bypasses the requirement for rt activity in the genetic pathway. This interpretation is further supported by a synergistic effect of tw RNAi and heterozygous rt mutant background (Figure 8), which revealed a positive interaction between rt and tw, indicating their close collaboration within the pathway. These data also support the hypothesis that the O-mannosylation pathway requires the simultaneous activities of both rt and tw genes (ICHIMIYA et al. 2004). This simultaneity requirement can explain the absence of any significant effect of varied TW levels on the phenotype of tested rt mutants that represent very strong hypomorphic or amorphic mutations and presumably lack the RT protein (see MARTIN-BLANCO and GARCIA-BELLIDO 1996; rt phenotypes in Figure 6A).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Interestingly, we found that tw1 could efficiently suppress the phenotype of three tested rt alleles (Figure 6). This finding was unexpected, since previously it was reported that RT and DmPOMT2 collaborate biochemically in vitro, and DmPOMT2 RNAi phenotype is enhanced in the rtP heterozygous background (ICHIMIYA et al. 2004). Yet the possibility existed that the suppression is the result of a decreased activity of tw in tw1 mutants. In this case, the suppression would suggest that an unbalanced relative increase of TW in rt mutants had a negative effect on the pathway [which, for instance, might result from a competition between nonproductive homomeric TW complexes and active RT–TW heterocomplexes (GIRRBACH and STRAHL 2003; ICHIMIYA et al. 2004)]. To test this possibility, we further analyzed the genetic interaction between tw and rt by varying the level of tw in the rt mutant background. We found that the phenotype of rt homozygous or heteroallelic mutants was neither significantly sensitive to an increase of tw expression by introducing a duplication of the tw locus or by ectopic expression of UAS–DmPOMT2 construct nor significantly sensitive to a decrease of tw activity by UAS–twRNAi expression (Figure 7 A and B). At the same time, we also confirmed the synergistic effect of the partial reduction of tw and rt activities on the mutant phenotype using UAS–twRNAi construct expression in rt heterozygotes (Figure 8). Thus, we ruled out the possibility of negative effect of tw in rt mutants and concluded that the suppression of rt phenotype is a special feature of tw1 allele.
Taken together, our results indicate that both tw and rt are involved in the same developmental pathway, where they execute nonredundant functions. Insensitivity of rt mutant phenotype to the varied level of tw expression formally characterizes rt as epistatic to tw. At the same time, the tw1 mutation could dominantly suppress the phenotype of strong (probably amorphic) rt mutations, thus revealing its epistatic position relative to rt (Figure 6). We interpret these mutually epistatic relationships between rt and tw as the evidence for possible functioning of their protein products within the same molecular complex or being involved in a regulatory interaction within the same biochemical pathway. This conclusion is in agreement with previously reported data on simultaneous requirement of RT and DmPOMT2 for their biochemical activity in vitro (ICHIMIYA et al. 2004). The conclusion is also consistent with our other observations presented here, including (i) essentially identical phenotypes of clockwise abdomen rotation in both rt and tw mutants (Figure 2), (ii) the subcellular colocalization of RT and TW proteins within the ER compartment in Drosophila cells (Figure 4), and (iii) the overlapping pattern of rt and tw expression during different stages of embryogenesis (Figure 5). Although the close relationship between RT and TW functioning is obvious from all these data, molecular events underlying this relationship remain to be elucidated. Further biochemical and cell biological experiments are necessary to discriminate between different possible molecular mechanisms, including stable physical interaction between RT and TW, their enzymatic modifications of one another, chaperone activity of these proteins, or yet other possibilities.
Our combined genetic and molecular characterization of tw1 mutant highlighted the functional importance of the conserved PMT domain of TW protein. We found that tw1 mutation should result in the expression of the TW protein with just a subtle alteration of amino acid sequence [T(59) GS] of the PMT domain (Figure 1). Despite the apparent subtleness of this mutation and the fact that T(59) is not well conserved between different species, this mutation causes decreased tw function and a pronounced rotated abdomen phenotype in tw1 homozygotes (Figures 2 and 3). In addition, the tw1 phenotype is insensitive to the decreased level of rt activity, thus indicating that TW1 mutant protein can bypass the requirement for RT activity that is obligatory for wild-type TW (Figures 6–8
). On the basis of protein sequence alignment of TW with other members of the POMT family (Figure 1 and data not shown) and the predicted topology of HsPOMT1 and ScPmt1p proteins (GIRRBACH et al. 2000; WILLER et al. 2003), the alteration in TW1 protein sequence maps to the lumenal terminus of the first transmembrane domain of TW protein. It is tempting to speculate that this protein region might be involved in TW–RT regulatory interactions; however, other possible mechanisms could also explain the properties of the tw1 mutation. Further biochemical and genetic experiments are necessary to clarify the properties of TW1 protein.
Several features of rt expression detected in our experiments are new and different from the previously reported expression of rt (MARTIN-BLANCO and GARCIA-BELLIDO 1996). In contrast to the previous report, we could not detect significant expression in the somatic muscle precursors and the midgut at stages 13 and 14. At the same time, we found the presence of the rt transcript in the developing epidermis at stages 14–16 (Figure 5), which was not reported earlier (MARTIN-BLANCO and GARCIA-BELLIDO 1996). A possible explanation for these differences arises from the difference between the rt cDNAs used in these studies. The sequence of cDNA clone used in our in situ hybridization experiments matches precisely the predicted sequence of the rt transcript reported in the FlyBase. We have also used this clone for in vivo expression experiments and found that it was able to rescue rt mutant phenotype (data not shown), which confirmed its functionality. On the other hand, the cDNA used in the previous study deviates from our sequence by 90 nucleotides (MARTIN-BLANCO and GARCIA-BELLIDO 1996), thus possibly representing a fusion with an irrelevant DNA fragment, which may explain the different in situ hybridization pattern reported earlier.
In vertebrates, O-mannosylation of -dystroglycan plays an important role in muscle and neural development (MARTIN 2003; MICHELE and CAMPBELL 2003). Drosophila Dystroglycan (Dg) is a fly homolog of the vertebrate Dystroglycan gene (GREENER and ROBERTS 2000; DENG et al. 2003). The predicted product of this gene, Drosophila DG protein, is structurally related to its vertebrate counterpart (DENG et al. 2003), thus representing a potential molecular target of RT/TW O-mannosyltransferase activity. Embryonic expression of Dg was detected in a dynamic fashion in many tissues, including visceral and somatic muscles, epidermis, nervous system, gut, and tracheal pits (DEKKERS et al. 2004). This expression has only partial overlap with the expression of rt and tw determined in our experiments, indicating that putative O-mannose modification of Drosophila DG protein may be limited to just a subset of DG-expressing cells. Interestingly, the expression of rt and tw in the developing epidermis revealed in our study corresponds to the region of epidermal segment border cells that are known to participate in the development of muscle attachment sites and to influence patterning of larval somatic muscles (VOLK and VIJAYRAGHAVAN 1994). These results indicate the intriguing possibility that these genes function in the epidermal muscle attachment cells, which would provide a novel mechanism for the involvement of O-mannosylation in muscle development. Further detailed characterization of rt and tw mutant phenotypes should shed light on this possibility and help elucidate the functions of these genes in Drosophila development.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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