Yoshihiro H. Inoue

A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila

D-raf, a Drosophila homolog of Raf-1, plays key roles in multiple signal transduction pathways. Dsor1, a putative factor downstream of D-raf, was genetically identified by screening of dominant suppressors of D-raf. Dsor1Su1 mapped on X chromosome significantly suppressed the D-raf mutant phenotypes, and the loss-of-function mutations of Dsor1 showed phenotypes similar to those of the D-raf null mutations. Dsor1Su1 also significantly suppressed the mutations of other terminal class genes acting further upstream of D-raf. Molecular cloning of Dsor1 revealed its product with striking similarity to the microtubule-associated protein (MAP) kinase activator and yeast PBS2, STE7, and byr1. Our genetic results demonstrate the connection between raf and the highly conserved protein kinase cascade involving MAP kinase in vivo.

Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression

We address the relative roles of astral and central spindle microtubules (MTs) in cytokinesis of Drosophila melanogaster primary spermatocytes. Time-lapse imaging studies reveal that the central spindle is comprised of two MT populations, “interior” central spindle MTs found within the spindle envelope and “peripheral” astral MTs that probe the cytoplasm and initiate cleavage furrows where they contact the cortex and form overlapping bundles. The MT-associated protein Orbit/Mast/CLASP concentrates on interior rather than peripheral central spindle MTs. Interior MTs are preferentially affected in hypomorphic orbit mutants, and consequently the interior central spindle fails to form or is unstable. In contrast, peripheral MTs still probe the cortex and form regions of overlap that recruit the Pav-KLP motor and Aurora B kinase. orbit mutants have disorganized or incomplete anillin and actin rings, and although cleavage furrows initiate, they ultimately regress. Our work identifies a new function for Orbit/Mast/CLASP and identifies a novel MT population involved in cleavage furrow initiation.

Identification of ter94 , Drosophila VCP , as a modulator of polyglutamine-induced neurodegeneration

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.

Ectopic Expression of DREF Induces DNA Synthesis, Apoptosis, and Unusual Morphogenesis in the Drosophila Eye Imaginal Disc: Possible Interaction with Polycomb and trithorax Group Proteins

ABSTRACT The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5′-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs′ chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-lessenhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection ofDrosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

Drosophila Larp associates with poly(A)-binding protein and is required for male fertility and syncytial embryo development

As the influence of mRNA translation upon cell cycle regulation becomes clearer, we searched for genes that might specify such control in Drosophila. A maternal-effect lethal screen identified mutants in the Drosophila gene for Larp (La-related protein) which displayed maternal-effect lethality and male sterility. A role for La protein has already been implicated in mRNA translation whereas Larp has been proposed to regulate mRNA stability. Here we demonstrate that Larp exists in a physical complex with, and also interacts genetically with, the translation regulator poly(A)-binding protein (PABP). Most mutant alleles of pAbp are embryonic lethal. However hypomorphic pAbp alleles show similar meiotic defects to larp mutants. We find that larp mutant-derived syncytial embryos show a range of mitotic phenotypes, including failure of centrosomes to migrate around the nuclear envelope, detachment of centrosomes from spindle poles, the formation of multipolar spindle arrays and cytokinetic defects. We discuss why the syncytial mitotic cycles and male meiosis should have a particularly sensitive requirement for Larp proteins in regulating not only transcript stability but also potentially the translation of mRNAs.

Ectopic expression of human p53 inhibits entry into S phase and induces apoptosis in the Drosophila eye imaginal disc.

Transgenic flies in which ectopic expression of human p53 was targeted to the Drosophila eye imaginal disc were established. On sectioning of adult fly eyes which displayed a severe rough eye phenotype, most ommatidia were found to be fused and irregular shapes of rabdomeres were observed. In addition, many pigment cells were lost. In the developing eye imaginal disc, photoreceptor cell differentiation was initiated normally despite the ectopic expression of p53. However, expression of p53 inhibited cell cycle progression in eye imaginal disc cells and the S phase zone (the second mitotic wave) behind the morphogenetic furrow was almost completely abolished. Furthermore, expression of p53 induced extensive apoptosis of eye imaginal disc cells, and co-expression of baculovirus P35 in the eye imaginal disc suppressed the p53-induced rough eye phenotype. These results are consistent with the known functions of human p53 and indicate the existence of signaling systems with elements corresponding to human p53 in Drosophila eye imaginal disc cells. Genetic crosses of transgenic flies expressing p53 to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the p53-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify novel targets of p53 and its positive or negative regulators in Drosophila.

Orbit/Mast, the CLASP orthologue of Drosophila, is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis

Drosophila oocyte differentiation is preceded by the formation of a polarised 16-cell cyst from a single progenitor stem cell as a result of four rounds of asymmetric mitosis followed by incomplete cytokinesis. We show that the Orbit/Mast microtubule-associated protein is required at several stages in the formation of such polarised 16-cell cysts. In wild-type cysts, the Orbit/Mast protein not only associates with the mitotic spindle and its poles, but also with the central spindle (spindle remnant), ring canal and fusome, suggesting it participates in interactions between these structures. In orbit mutants, the stem cells and their associated fusomes are eventually lost as Orbit/Mast protein is depleted. The mitotic spindles of those cystocytes that do divide are either diminutive or monopolar, and do not make contact with the fusome. Moreover, the spindle remnants and ring canals fail to differentiate correctly in such cells and the structure of fusome is compromised. The Orbit/Mast protein thus appears to facilitate multiple interactions of the fusome with mitotic spindles and ring canals. This ensures correct growth of the fusome into a branched asymmetrically distributed organelle that is pre-determinative of 16-cell cyst formation and oocyte fate specification. Finally the Orbit/Mast protein is required during mid-oogenesis for the organisation of the polarised microtubule network inside the 16-cell cyst that ensures oocyte differentiation. The localisation of CLIP-190 to such microtubules and to the fusome is dependent upon Orbit/Mast to which it is complexed.

Over-expression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype.

Background DNA replication‐related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication‐ and proliferation‐related genes in Drosophila. While the effects on the mutation in the DNA replication‐related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild‐type DREF activity in developing tissues have hitherto remained unclear.

Transcriptional regulation of the Drosophila rfc1 gene by the DRE–DREF pathway

The DNA replication‐related element (DRE) is a common 8‐bp sequence (5′‐TATCGATA) found in the promoters of many DNA replication‐related genes, to which DRE‐binding factor (DREF) specifically binds to activate transcription. Replication factor C (RFC) is an essential five‐subunit complex in DNA replication, the largest subunit being RFC140. We first identified the gene (rfc1) encoding the Drosophila RFC140 (dRFC140) protein and then isolated a mutant. The phenotypes suggested that the gene is essential for cell‐cycle progression, and immunocytochemical studies also indicated a relation between its expression and the cell cycle. The rfc1 gene contains three DRE‐like sequences in its 5′‐flanking region, one of them perfectly matching DRE and the other two demonstrating a match in seven of eight nucleotides. These sequences were named DRE1 (−63 to −69), DRE2 (−378 to −385), and DRE3 (−1127 to −1134), respectively. Immunostaining of polytene chromosomes in third‐instar larvae using anti‐DREF sera detected a specific band in 82E2 of 3R chromosome, containing the rfc1 gene region. Band‐mobility shift assays using Drosophila Kc cell nuclear extracts revealed that DREF binds to DRE1, ‐2, and ‐3 in vitro, and chromatin immunoprecipitation using anti‐DREF IgG confirmed that this occurs in vivo. Luciferase transient expression assays in S2 cells further suggested that DREs in the rfc1 promoter are involved in transcriptional regulation of the gene. Moreover, rfc1 promoter activity was reduced by 38% in DREF double‐stranded RNA‐treated S2 cells. These results indicate that DREF positively regulates the rfc1 promoter.

Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF).

The transcription factor DREF regulates proliferation-related genes in Drosophila. With two-hybrid screening using DREF as a bait, we have obtained a clone encoding a protein homologous to human myelodysplasia/myeloid leukemia factor 1 (hMLF1). We termed the protein Drosophila MLF (dMLF); it consists of a polypeptide of 309 amino acid residues, whose sequence shares 23.1% identity with hMLF1. High conservation of 54.2% identity over 107 amino acids was found in the central region. The dMLF gene was mapped to 52D on the second chromosome by in situ hybridization. Interaction between dMLF and DREF in vitro could be confirmed by glutathione S-transferase pull-down assay, with the conserved central region appearing to play an important role in this. Northern blot hybridization analysis revealed dMLF mRNA levels to be high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae. This fluctuation of mRNA during Drosophila development is similar to that observed for DREF mRNA, except in the pupa and adult male. Using a specific antibody against the dMLF, we performed immunofluorescent staining of Drosophila Kc cells and showed a primarily cytoplasmic staining, whereas DREF localizes in the nucleus. However, dMLF protein contains a putative 14-3-3 binding motif involved in the subcellular localization of various regulatory molecules, and interaction with DREF could be regulated through this motif. The transgenic fly data suggesting the genetic interaction between DREF and dMLF support this possibility. Characterization of dMLF in the present study provides the molecular basis for analysis of its significance in Drosophila.