Shin Togashi

Cortactin associates with the cell-cell junction protein ZO-1 in both Drosophila and mouse

Cortactin is an actin filament-binding protein localizing at cortical regions of cells and a prominent substrate for Src family protein-tyrosine kinases in response to multiple extracellular stimuli. Human cortactin has been identified as a protein product of a putative oncogene, EMS1. In this report, we describe the identification of a Drosophila homolog of cortactin as a molecule that interacts with Drosophila ZO-1 using yeast two-hybrid screening. Drosophila cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. Deletion and substitution mutant analyses revealed that the SH3 domain of Drosophilacortactin binds to a PXXP motif in the proline-rich domain of Drosophila ZO-1. Colocalization of these proteins at cell-cell junction sites was evident under a confocal laser-scanning microscope. In vivo association was confirmed by coimmunoprecipitation of cortactin and ZO-1 from Drosophilaembryo lysates. We also demonstrate an association for each of the murine homologs by immunoprecipitation analyses of mouse tissue lysates. Our previous work has demonstrated the involvement of ZO-1 in a signaling pathway that regulates expression of the emcgene in Drosophila. The potential roles of the cortactin·ZO-1 complex in cell adhesion and cell signaling are discussed.

Newly established cell lines from Drosophila larval CNS express neural specific characteristics.

SummaryFrom the central nervous system ofDrosophila melanogaster 3rd instar larvae, eight continuous cell lines have been established (named ML-DmBG1 to 8). Using ML-DmBG2, single colony isolation was carried out and six colonial clones were obtained. All reacted to the antibody to horseradish peroxidase, which is a neuronal marker in insects. Acetylcholine, a known neurotransmitter inDrosophila, was detected in three of the colonial clones by high performance liquid chromatography. Therefore, it is concluded that the established colonial clones are neural cells originating in the larval central nervous system. Among them, some variation was observed with respect to morphology, acetylcholine content, and reactivity to anti-HRP. The variation may reflect the heterogeneity of cells composing the central nervous system.

DCRY is a Drosophila photoreceptor protein implicated in light entrainment of circadian rhythm

: Light is the major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, the period(per) and timeless (tim) genes are required for circadian behavioural rhythms and their expression levels undergo circadian fluctuations. Light signals can entrain these rhythms by shifting their phases. However, little is known about the molecular mechanism for the perception and transduction of the light signal. The members of the photolyase/cryptochrome family contain flavin adenine dinucleotide (FAD) as chromophore and are involved in two diverse functions, DNA repair and photoreception of environmental light signals.

Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.

Identification of Xenopus Cortactin: Two Isoforms of the Transcript and Multiple Forms of the Protein

Abstract Cortactin was initially identified as a substrate for Src tyrosine kinase. It interacts with the filamentous actin in the cell cortex through the tandem repeats of 37-amino acid. In this report, we describe the identification of a Xenopus homolog of cortactin. The deduced amino acid sequence shares over 70% identity with human, mouse, and chicken cortactin. Northern and Western blot analyses revealed that Xenopus cortactin is widely expressed in Xenopus tissues. Analysis of the transcripts using polymerase chain reaction revealed two isoforms being different in the number of the tandem repeats. The major isoform has 6.5 tandem repeats but the minor one has 5.5 tandem repeats. As the sixth repeat, which is missed in the minor isoform, is encoded by a single exon flanked by introns on both sides, these two isoforms are likely to be generated by alternative splicing. We propose that cortactin regulates the construction of actin cytoskeleton by altering the number of its tandem repeats.

Developmental profile of P element transposition in Drosophila somatic cells

Transposition of the P element duringDrosophila ontogenesis was monitored. A modified P element was transposed by the PΔ2-3 transposase source. P elements inserted into the genome were cloned by the plasmid rescue at various developmental stages of the G1 hybrid to trace events in somatic cells. The transposed elements were directly counted by analyzing RFLP of genomic DNA fragments flanking the P elements. Transposition began from the late embryonic stage, but occurred rarely. Frequent transposition was observed from the late third instar to early pupal stage. From these results, transposition of the P element would appear to be affected by the developmental state of somatic host cells.