Male Sterile Phenotype Research Articles

The role of the RING-finger protein Elfless in Drosophila spermatogenesis and apoptosis

elfless (CG15150, FBgn0032660) maps to polytene region 36DE 5' (left) of reduced ocelli/Pray for Elves (PFE) on chromosome 2L and is predicted to encode a 187 amino acid RING finger E3 ubiquitin ligase that is putatively involved in programmed cell death (PCD, eg. apoptosis). Several experimental approaches were used to characterize CG15150/elfless and test whether defects in this gene underlie the male sterile phenotype associated with overlapping chromosomal deficiencies of region 36DE. elfless expression is greatly enhanced in the testes and the expression pattern of a UAS-elfless-EGFP driven by elfless-Gal4 is restricted to the tail cyst cell nuclei of the testes. Despite this, elfless transgenes failed to rescue the male sterile phenotype in Df/Df flies. Furthermore, null alleles of elfless, generated either by imprecise excision of an upstream P-element or by FLP-FRT deletion between two flanking piggyBac elements, are fertile. In a gain-of-function setting in the eye, we found that elfless genetically interacts with key members of the apoptotic pathway including the initiator caspase Dronc and the ubiquitin conjugating enzyme UbcD1. DIAP1, but not UbcD1, protein levels are increased in heads of flies expressing Elfless-EGFP in the eye, and in testes of flies expressing elfless-Gal4 driven Elfless-EGFP. Based on these findings, we speculate that Elfless may regulate tail cyst cell degradation to provide an advantageous, though not essential, function in the testis.

Somatic ‘Soluble’ Adenylyl Cyclase Isoforms Are Unaffected in Sacytm1Lex/Sacytm1Lex ‘Knockout’ Mice

BackgroundMammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacytm1Lex/Sacytm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference.Principal FindingsWe now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which ‘escapes’ the design of the Sacytm1Lex knockout allele.Conclusions/SignificanceThese studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.

T1243, an alternative transcript of the mitochondrial T gene in Brassica juncea var. tumida, causes pollen abortion in Arabidopsis thaliana

An earlier report revealed that the mitochondrial T gene of tumorous stem mustard ( Brassica juncea var. tumida) is a cytoplasmic male sterility (CMS)-related gene. Two alternative transcripts of the T gene, T1170 and T1243, showed different expression patterns at different developmental stages. In this work, the potential roles of these two transcripts were studied in Arabidopsis thaliana through over-expression under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants over-expressing the T1243 transcript produced anthers that appeared devoid of pollen and almost fully collapsed siliques, showing a severe male sterility phenotype, while transgenic lines over-expressing T1170 were normal. To confirm the above results, we constructed T1243-RNAi lines in A. thaliana, and T1243 was over-expressed using these lines as recipients. Pollen was normal in the T1243 knocked-out lines. In the control wild type lines expressing T1243, plants showed pollen abortion. We observed a positive correlation between the sterility phenotypes and the expression levels of T1243. Test-crossing was done by pollinating T1243 transgenic lines with wild type pollen. About half of the progeny showed pollen abortion, indicating that T1243, one of the T gene transcripts, can cause a male sterility phenotype.

Mapping of the Ogura fertility restorer gene Rfo and development of Rfo allele-specific markers in canola (Brassica napus L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.

Inhibition of chalcone synthase Expression in Anthers of Raphanus sativus with Ogura Male Sterile Cytoplasm

Expression of the mitochondrial gene orf138 causes Ogura cytoplasmic male sterility (CMS) in Raphanus sativus, but little is known about the mechanism by which CMS takes place. A preliminary microarray experiment revealed that several nuclear genes concerned with flavonoid biosynthesis were inhibited in the male-sterile phenotype. In particular, a gene for one of the key enzymes for flavonoid biosynthesis, chalcone synthase (CHS), was strongly inhibited. A few reports have suggested that the inhibition of CHS causes nuclear-dependent male sterile expression; however, there do not appear to be any reports elucidating the effect of CHS on CMS expression. In this study, the expression patterns of the early genes in the flavonoid biosynthesis pathway, including CHS, were investigated in normal and male-sterile lines. In order to determine the aberrant stage for CMS expression, the characteristics of male-sterile anthers are observed using light and transmission electron microscopy for several stages of flower buds. The expression of CHS and the other flavonoid biosynthetic genes in the anthers were compared between normal and male-sterile types using real time RT-PCR. Among the flavonoid biosynthetic genes analysed, the expression of CHS was strongly inhibited in the later stages of anther development in sterility cytoplasm; accumulation of putative naringenin derivatives was also inhibited. These results show that flavonoids play an important role in the development of functional pollen, not only in nuclear-dependent male sterility, but also in CMS.

Genetic Evidence for the Role of Isopentenyl Diphosphate Isomerases in the Mevalonate Pathway and Plant Development in Arabidopsis

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C(5) units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are <50% of those in wild-type plants, and that the male-sterile phenotype is chemically complemented by squalene, a sterol precursor. In vivo isotope labeling experiments using the atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants.

HYBRID MALE STERILITY IN MIMULUS (PHRYMACEAE) IS ASSOCIATED WITH A GEOGRAPHICALLY RESTRICTED MITOCHONDRIAL REARRANGEMENT

Cytoplasmic male sterility (CMS) and nuclear fertility restoration (Rf) involves intergenomic coevolution. Although male-sterile phenotypes are rarely expressed in natural populations of angiosperms, CMS genes are thought to be common. The evolutionary dynamics of CMS/Rf systems are poorly understood, leaving gaps in our understanding of mechanisms and consequences of cytonuclear interactions. We characterized the molecular basis and geographic distribution of a CMS gene in Mimulus guttatus. We used outcrossing M. guttatus (with CMS and Rf) to self-fertilizing M. nasutus (lacking CMS and Rf) to generate hybrids segregating for CMS. Mitochondrial transcripts containing an essential gene (nad6) were perfectly associated with male sterility. The CMS mitotype was completely absent in M. nasutus, present in all genotypes collected from the original collection site, but in only two individuals from 34 other M. guttatus populations. This pattern suggests that the CMS likely originated at a single locality, spread to fixation within the population, but has not spread to other populations, indicating possible ecological or genetic constraints on dispersal of this CMS mitotype between populations. Extreme localization may be characteristic of CMS in hermaphroditic species, in contrast to geographically widespread mitotypes commonly found in gynodioecious species, and could directly contribute to hybrid incompatibilities in nature.

A male sterility‐associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST-ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.

Identification of a Third Haplotype of the Sequence Linked to the Restorer-of-fertility (Rf) Gene and Its Implications for Male-Sterility Phenotypes in Peppers (Capsicum annuum L.)

Identification of a Third Haplotype of the Sequence Linked to the Restorer-of-fertility (Rf) Gene and Its Implications for Male-Sterility Phenotypes in Peppers (Capsicum annuum L.)

Genetic-phytohormonal interactions in male fertility and male sterility phenotype expression in sunflower (Helianthus annuus L.)

SUMMARY Quantitative ratio of IAA/GA 3 has been studied during the growth and development of different sunflower genotypes that represent a cms -R f system. It was shown that the IAA/GA 3 ratio was variable and it depended on ontogenetic stage, plant organ and genotype. Thus, the IAA/GA 3 ratio the maximal value in the male sterile line while the minimal ratio was found in the restorer line RW 637 R f . The highest IAA/GA 3 ratio was registered in cotyledon leaves, which decreased subsequently in the course of ontogenesis. During reproductive stages, the ratio was higher in inflorescences than in leaves. The exogenously applied gibberellic acid (GA3) increased the ratio in the fertile line. Specificity of the IAA/GA 3 balance in the male sterility-fertility phenotype expression and in GA-induced pollen sterility is discussed. RESUMEN Se estudió la relación cuantitativa IAA/GA 3 durante el crecimiento y desarrollo de diferentes genotipos de girasol que representan un sistema cms - R f. Se ha demostrado que la relación IAA/GA 3 es variable y depende de los estados ontogénicos, órganos y genotipo. Así la relación IAA/GA 3 presentó valores máximos para la línea androestéril y mínimos para la línea restauradora RW637R f . La más alta relación IAA/GA 3 se registró en hojas cotiledonares que decrecieron subsecuentemente durante la ontogénesis. La relación hormonal en estadíos reproductivos fue mayor en inflorescencias que en hojas. El ácido giberélico (GA 3 ) aplicado en forma exógena incrementó la relación hormonal en la línea fértil. Se discute la especificidad del balance IAA/GA 3 en la expresión de los fenotipos androfértiles y androestériles y en la esterilidad de polen inducida por GA. RÉSUMÉ On a réalisé l’étude du rapport entre la quantité des auxines et gibbérelline dans le processus du croissance et développement des différents génotypes de tournesol qui constituent le système de la stérilité cytoplasmique masculine. Les résultats des recherches ont montré que le rapport entre les auxines et les gibbérellines est dépendant de la phase ontogénétique, les organes étudiés et le génotype de tournesol. La ligne androstérile est caractérisée par un rapport maximal entre les auxines et gibbérelline pendant que la ligne RW 637 R f est caractérisée par un rapport minimal. L’analyse de ce paramètre a montré que le rapport entre les auxines et gibbérellines est plus élevé dans les inflorescences que dans les feuilles. La valeur plus grande de rapport auxines/ gibbérellines a été enregistré dans les feuilles de cotylédon. On atteste une diminution du rapport dans l’ontogenèse. Chez les lignes fertiles le traitement avec les gibbérellines cause une croissance du rapport auxines/ gibbérellines. On dispute sur la problème vis-à-vis du rapport spécifique auxines/ gibbérellines dans la manifestation phénotypique de l’androstérilité- androfertilité, ainsi que dans l’induction phénotypique de la stérilité de du grain de pollen par le traitement par gibbérellines.

Genetic-phytohormonal interactions in male fertility and male sterility phenotype expression in sunflower (Helianthus annuus L.)

Amounts of indole-3-acetic acid (IAA) and gibberellic acid (GA3) in vegetative and reproductive tissues of diverse sunflower genotypes have been investigated using gas-liquid chromatography. Quantification of endogenous GA3 content in the homozygous line MB 514, characterized by cytoplasmic male sterility (cms), revealed a lower level of the hormone in contrast to the fertility restorer line RW 637 Rf. The highest amount of IAA was found in the heterozygous F1 hybrid obtained by crossing these lines, regardless of tissues and ontogenetic phases analyzed. Similar features were found in leaves, apices, inflorescences and disk flowers in most of the investigated variants.

Cloning and Characterization of a PI-like MADS-Box Gene in Phalaenopsis Orchid

The highly evolved flowers of orchids have colorful sepals and fused columns that offer an opportunity to discover new genes involved in floral development in monocotyledon species. In this investigation, we cloned and characterized the homologous PISTALLATA-like (PI-like) gene PhPI15 (Phalaenopsis PI STILLATA # 15), from the Phalaenopsis hybrid cultivar. The protein sequence encoded by PhPI15 contains a typical PI-motif. Its sequence also formed a subclade with other monocot PI-type genes in phylogenetic analysis. Southern analysis showed that PhPI15 was present in the Phalaenopsis orchid genome as a single copy. Furthermore, it was expressed in all the whorls of the Phalaenopsis flower, while no expression was detected in vegetative organs. The flowers of transgenic tobacco plants ectopically expressing PhPI15 showed male-sterile phenotypes. Thus, as a Class-B MADS-box gene, PhPI15 specifies floral organ identity in orchids.

Global analysis of gene expression in flower buds of Ms-cd1 Brassica oleracea conferring male sterility by using an Arabidopsis microarray

The dominant male sterility gene Ms-cd1 is identified in Brassica oleracea. Electron microscopical observations revealed that abortion of pollen development starts after tetrad formation. This important male sterility phenotype is characterized by lack of degradation of the primary pollen mother cell (PMC) wall and delayed degradation of callose surrounding the tetrads and thus arrest of microspore release. Gene expression of the male sterile and fertile buds was analyzed by heterologous hybridization of Brassica oleracea cRNA onto an Arabidopsis whole genome oligonucleotide microarray. A total of 277 suppressed genes including 40 kinase-, 32 cell wall modification and 29 transport related genes were found to be significantly down regulated >3-fold in the male sterile mutant. The vast majority of the differentially expressed transcripts are found to present late pollen stage specific genes. Kinase genes, cell wall modification genes and ion transport genes were greatly over-represented when compared to their percentage of all flower bud expressed genes and represent 36.5% of the genes suppressed by Ms-cd1. Our results also suggest that Ms-cd1 may blocks an anther developmental pathway with a small number of genes suppressed in tapetum cells which prevent the degradation of callose and PMC wall, which further leads to the suppression of a large number of genes involved in signaling pathways, cell wall modification and ion transport in pollen grains.

Mitochondrial regulation of flower development

Flower development in plants depends not only on a set of nuclear genes but also on the coordinate action of the mitochondrion. Certain mitochondrial genomes in combination with certain nuclear genomes lead to the expression of cytoplasmic male-sterility (CMS). Both mitochondrial genes that determine male-sterility and nuclear Restorer-of-fertility genes that suppress the male-sterile phenotype have been cloned. Lately, the interactions between mitochondrial and nuclear genes through retrograde signalling in CMS-systems have been dissected. Of special interest are the altered expression patterns of floral homeotic genes in certain CMS-systems. Here, we review the mitochondrial influence on flower development and give examples from CMS-systems developed in Brassica, Daucus carota, Nicotiana tabacum and Triticum aestivum.

Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis

Downregulation of the transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration during anther development in Arabidopsis thaliana. This paper describes the functional analysis of the AtMYB103 gene in three knock-out mutants. Two male sterile mutants, ms188-1 and ms188-2, were generated by ethyl-methane sulfonate (EMS) mutagenesis. A map-based cloning approach was used, and ms188 was mapped to a 95.8-kb region on chromosome 5 containing an AtMYB103 transcription factor. Sequence analysis revealed that ms188-1 had a pre-mature stop codon in the AtMYB103 coding region, whereas ms188-2 had a CCT-->CTT base-pair change in the first exon of AtMYB103, which resulted in the replacement of a proline by a leucine residue in the R2R3 domain. The third mutant, an AtMYB103 transposon-tagging line, also showed a male sterile phenotype. Allelism tests indicated that MS188 and AtMYB103 belong to the same locus. Cytological observation revealed defective tapetum development and altered callose dissolution in ms188 plants. Additionally, most of the microspores in mature anthers were degraded and surviving microspores lacked exine. AtMYB103 encoded an R2R3 MYB protein that is predominantly located in the nucleus. Real-time RT-PCR analysis indicated that the callase-related gene A6 was regulated by AtMYB103. Expression of the exine formation gene MS2 was not detected in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.

CYP703 Is an Ancient Cytochrome P450 in Land Plants Catalyzing in-Chain Hydroxylation of Lauric Acid to Provide Building Blocks for Sporopollenin Synthesis in Pollen

CYP703 is a cytochrome P450 family specific to land plants. Typically, each plant species contains a single CYP703. Arabidopsis thaliana CYP703A2 is expressed in the anthers of developing flowers. Expression is initiated at the tetrad stage and restricted to microspores and to the tapetum cell layer. Arabidopsis CYP703A2 knockout lines showed impaired pollen development and a partial male-sterile phenotype. Scanning electron and transmission electron microscopy of pollen from the knockout plants showed impaired pollen wall development with absence of exine. The fluorescent layer around the pollen grains ascribed to the presence of phenylpropanoid units in sporopollenin was absent in the CYP703A2 knockout lines. Heterologous expression of CYP703A2 in yeast cells demonstrated that CYP703 catalyzes the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids, with a preferential hydroxylation of lauric acid at the C-7 position. Incubation of recombinant CYP703 with methanol extracts from developing flowers confirmed that lauric acid and in-chain hydroxy lauric acids are the in planta substrate and product, respectively. These data demonstrate that in-chain hydroxy lauric acids are essential building blocks in sporopollenin synthesis and enable the formation of ester and ether linkages with phenylpropanoid units. This study identifies CYP703 as a P450 family specifically involved in pollen development.

Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants

Stability of the mitochondrial genome is controlled by nuclear loci. In plants, nuclear genes suppress mitochondrial DNA rearrangements during development. One nuclear gene involved in this process is Msh1. Msh1 appears to be involved in the suppression of illegitimate recombination in plant mitochondria. To test the hypothesis that Msh1 disruption leads to the type of mitochondrial DNA rearrangements associated with naturally occurring cytoplasmic male sterility in plants, a transgenic approach for RNAi was used to modulate expression of Msh1 in tobacco and tomato. In both species, these experiments resulted in reproducible mitochondrial DNA rearrangements and a condition of male (pollen) sterility. The male sterility was, in each case, heritable, associated with normal female fertility, and apparently maternal in its inheritance. Segregation of the transgene did not reverse the male sterile phenotype, producing stable, nontransgenic male sterility. The reproducible transgenic induction of mitochondrial rearrangements in plants is unprecedented, providing a means to develop novel cytoplasmic male sterile lines for release as non-GMO or transgenic materials.

Isolation and characterization of the cytoplasmic male sterility-associated orf456 gene of chili pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3'-end of the coxll gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45 % of the T1 transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T1 plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene--from among the many CMS-associated mitochondrial genes--for determining the male-sterile phenotype of CMS in chili pepper.

Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers

To gain new insights into the mechanism underlying cytoplasmic male sterility (CMS), we compared the nuclear gene expression profiles of flowers of a Brassica napus CMS line with that of the fertile B. napus maintainer line using Arabidopsis thaliana flower-specific cDNA microarrays. The CMS line used has a B. napus nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of both B. napus and A. thaliana DNA. Gene expression profiling revealed that a large number of genes differed in expression between the two lines. For example, nuclear genes coding for proteins that are involved in protein import into organelles, genes expressed in stamens and pollen, as well as genes implicated in either cell-wall remodeling or architecture, were repressed in the CMS line compared with B. napus. These results show that the mt genome of the CMS line strongly influences nuclear gene expression, and thus reveal the importance of retrograde signalling between the mitochondria and the nucleus. Furthermore, flowers of the CMS line are characterized by a replacement of stamens with carpelloid organs, and thus partially resemble the APETALA3 (AP3) and PISTILLATA (PI) mutants. In accordance with this phenotype, AP3 expression was downregulated in the stamens, shortly before these organs developed carpelloid characteristics, even though it was initiated correctly. Repression of PI succeeded that of AP3 and might be a consequence of a loss of AP3 activity. These results suggest that AP3 expression in stamens depends on proper mt function and a correct nuclear-mt interaction, and that mt alterations cause the male sterility phenotype of the CMS line.

The expression pattern and cellular localisation of Myosin VI during the Drosophila melanogaster life cycle

Myosin VI is a motor protein which is necessary for the morphogenesis of epithelial tissues during Drosophila development. The spatial and temporal expression of Myosin VI was examined by expressing a GFP (Green Fluorescent Protein) tagged Myosin VI molecule (PGM), under the control of a Myosin VI-Gal4 line. PGM was present in tissues that were shown previously to express Myosin VI, such as the ovarian follicle epithelium, and the individualization complex; and in other tissues, including the trachea, the midgut, the salivary glands and the imaginal discs. The GFP-tagged Myosin V1 rescued the male sterile phenotype of Jaguar showing it is functional in vivo. Within individual cells, the role of the head and neck domain and the tail domain in targeting of the Myosin V1 molecule was examined by investigating the localisation of the separate domains tagged to GFP. In salivary glands and follicle cells the head and neck domains were concentrated in the cell nucleus, where the minus end of each actin filament is located. We found that the tail domain anchors the whole molecule outside of the nucleus. Similarly, in the individualization complex in the testes, the tail anchors the whole molecule to the base of the complex while the separated head with neck domain becomes scattered along the entire actin molecule suggesting the cellular location may be determined by cargo proteins that bind to the tail domain rather than by the movement of Myosin VI along the actin filaments.