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 twohybrid 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 Drosophila cortactin 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 Drosophila embryo 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 emc gene in Drosophila. The potential roles of the cortactin⅐ZO-1 complex in cell adhesion and cell signaling are discussed.Cell-cell adhesions are essential for the development of the multicellular organisms. Among the proteins composing the cell-cell adhesion complexes, members of the membrane-associated guanylate kinase homologs (MAGUKs) 1 are widely found in Hydra, Caenorhabditis elegans, Drosophila, and mammals (1-3). MAGUKs have distinctive domains including one or three copies of the PDZ domain, an SH3 domain, and a domain homologous to guanylate kinase (GUK) and implicated in both formation of cell-cell junctions and signal transduction. One of the most intensively characterized members of the MAGUKs is the mammalian ZO-1, which is known to associate with several cellular proteins including the components of cell-cell junctions (occludin, ␤-catenin, and ZO-2) and the components of cytoskeletal networks (␣-spectrin and actin filaments (F-actin)) (4 -9). While ZO-1 has been considered as a homolog of a Drosophila tumor suppresser Dlg, its biological functions in the cell-cell junction and signal transduction remain obscure (10, 11).We recently identified a new Drosophila MAGUK protein, Tamou, and reported its significant homology with ZO-1 (12). We will refer to Tamou as Drosophila ZO-1 (DZO-1) because we also found that the transgenes of mouse ZO-1 could replace the tam gene function in Drosophila.2 The DZO-1 tam1 mutant flies exhibit the supernumerary mechanosensory organs. This is a similar phenotype to that of an extramacrochaetae (emc) mutation. The emc gene encodes a helix-loop-helix type transcriptional regulator and negatively regulates specification of sensory organ precursor cells (13-16). We have previously shown that DZO-1 locates at cell-cell junctions and is involved in the signaling p...

In Drosophila sensory organ development, the balance of activities between proneural genes and repressor genes defines a proneural cluster as a population of competent cells for neural development. In this study, we report the isolation and analysis of the tamou [tain) gene that encodes a cell-cell junction-associated protein, which is homologous to mammalian ZO-1, a member of the membrane-associated guanylate kinase homolog family. The tarn mutation reduces the transcription of a repressor gene, extramacrochaetae, and causes enlargement of a proneural cluster where supernumerary precursor cells emerge, resulting in extra mechanosensory organs in the fly. These results suggest that the membrane-associated Tam protein is involved in the signaling pathway that activates emc expression.[Key Woids: Diosophila-, sensory organ formation; MAGUK protein; emc gene; ZO-1 homolog; signal transduction] Received March 15, 1996; revised version accepted May 30, 1996.The Diosophila peripheral nervous system comprises several types of sensory organs, including external mech anosensory and chemosensory organs, internal tension receptors, and touch receptors. The distribution patterns and position-dependent specifications of these sensory organs are highly reproducible (Ghysen 1980;Vandervorst and Ghysen 1980). The position of a sensory organ in the adult fly depends on the birthplace of its precursor, the sensory organ precursor (SOP) cell, which is singled out from a small group of cells called a proneural cluster in the imaginal disc. It has been shown that Diosophila sensory organ development is programmed genetically, and the framework of the genetic cascade involved has been described (Ghysen and Dambly-Chaudiere 1989;Romani et al. 1989;Cubas et al. 1991;Skeath and Carroll 1991;Campuzano and Modolell 1992;Ghysen et al. 1993). According to the scheme, cells expressing the pro neural genes acquire the potential to initiate neural de velopment, and this proneural gene activity is regulated negatively by the repressor genes. A gain-of-function mutation of the proneural gene or a loss-of-function mu-'These authois contributed equally to this work. ^Present addtess: Fujirebio, Inc., Tokyo 192, Japan. ^Corresponding author. tation of the repressor gene causes expansion of the pro neural cluster and yields extra SOP cells, resulting in supernumerary sensory organs in the fly, whereas a lossof-function mutation of the proneural gene or a gain-offunction mutation of the repressor gene has the opposite effect (Jan and Jan 1990;Ghysen et al. 1993). Thus, a balance between the activities of the proneural and re pressor genes to set up a normal proneural cluster is im portant for ensuring the formation of SOP.All known proneural genes, daughteiless [da], atonal, and achaete [ac], scute [sc], lethal of scute, and asense of the achaete-scute complex, encode basic helix-loop-he lix (bHLH) transcription factors (Villares and Cabrera 1987;Alonso and Cabrera 1988; Gaudy et al. 1988;Cronmiller et al. 1988;Gonzalez et al. 1989;Jarman et al...

From the central nervous system of Drosophila 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 in Drosophila, 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.

Northern blot and nucleotide sequence analyses of copia RNA from a transfectant made by introducing a genomic copia into copia‐free cells showed that the 2 kb RNA, one of the major transcripts of copia, is generated through splicing. Using the polymerase chain reaction (PCR), we have also found that the position of the splice sites used in Drosophila larvae and cultured cells originally containing copia is the same as that used in the transfectant. To investigate the function of the 2 kb RNA, we constructed mutant copias which harboured a single point mutation at the splice site or approximately 3 kb deletion of the internal region corresponding to the spliced out sequence. Analyses of transfectants made by introducing these mutant copias into copia‐free cells demonstrated that the spliced 2 kb RNA contains sufficient information to make copia virus‐like particles (VLPs). Furthermore, when copia RNA corresponding to the spliced RNA was translated in vitro, the major VLP protein was found to be released autocatalytically from its own precursor. A single amino acid substitution at the putative protease active site in the precursor prevented the processing, and resulted in accumulation of the mutant precursor in vitro. From these results, we conclude that copia VLPs are produced through autocatalytic processing of the precursor polyprotein encoded by the spliced copia RNA.

Background : 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.