A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila.

Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. Here, we uncover a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism. This process involves the formation of membrane complexes of phosphorylated, activated rhodopsin and its inhibitory protein arrestin, and subsequent clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment. Together, these data define the proapoptotic molecules in Drosophila photoreceptors and indicate a novel signaling pathway for light-activated rhodopsin molecules in control of photoreceptor viability.

Evidence that Argos is an antagonistic ligand of the EGF receptor.

Argos, the inhibitor of the Drosophila epidermal growth factor (EGF) receptor, remains the only known extracellular inhibitor of this family of receptors in any organism. The functional domain of Argos includes an atypical EGF domain and it is not clear whether it binds to the EGF receptor or if it acts via a distinct receptor to reduce Egfr activity indirectly. Here we present two lines of evidence that strongly suggest that Argos directly interacts with the EGF receptor. First, Argos is unable to inhibit a chimeric receptor that contains an extracellular domain from an unrelated RTK, indicating the need for the EGF receptor extracellular domain. Second, Argos can inhibit the Drosophila EGF receptor even when expressed in human cells, implying that no other Drosophila protein is necessary for inhibition. We also report that Argos and the Drosophila activating ligand, Spitz, can influence mammalian RTK activation, albeit in a cell-type specific manner. This includes the first evidence that Argos can inhibit signalling in mammalian cells, raising the possibility of engineering an effective human EGF receptor/ErbB antagonist. Oncogene (2000) 19, 3560 - 3562

Sprouty, an intracellular inhibitor of Ras signaling.

Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty- eyes have excess photoreceptors, cone cells, and pigment cells. Sprouty's function is, however, more widespread. We show that it also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, we show by biochemical analysis that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. Our results indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction.

Troponin-T is a calcium-binding protein in insect muscle: in vivo phosphorylation, muscle-specific isoforms and developmental profile in Drosophila melanogaster.

Two sets of muscle polypeptides showing calcium-binding capacity and intense labelling in vivo with 32P were purified and characterized from Drosophila melanogaster adult extracts. The polypeptides exhibit crossed immunoreactivity and share similar biochemical properties such as those involved in purification. They have been identified as isoforms of troponin-T (TnT) by sequence analysis of a cDNA clone isolated from an embryonic library. The two sets of TnT polypeptides correspond to the fibrillar and non-fibrillar muscle isoforms, respectively. The non-fibrillar muscle isoforms separate into two bands which are differentially expressed during development. Analysis of TnT isoforms in bee thoraces indicates that the expression of the fibrillar muscle isoform correlates with the acquisition of functional flight capability. In vivo labelling experiments reveal that the two TnT sets are readily phosphorylated. The Drosophila TnTs show calcium-binding properties by three different types of assays. Our results suggest that this property could be specific to insect TnTs and may be related to the long, extremely acidic polyglutamic carboxy-terminus present in these polypeptides, which does not occur in non-arthropod TnTs.

A G protein-coupled receptor phosphatase required for rhodopsin function.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors are phosphorylated by kinases that mediate agonist-dependent receptor deactivation. Although many receptor kinases have been isolated, the corresponding phosphatases, necessary for restoring the ground state of the receptor, have not been identified. Drosophila RDGC (retinal degeneration C) is a phosphatase required for rhodopsin dephosphorylation in vivo. Loss of RDGC caused severe defects in the termination of the light response as well as extensive light-dependent retinal degeneration. These phenotypes resulted from the hyperphosphorylation of rhodopsin because expression of a truncated rhodopsin lacking the phosphorylation sites restored normal photoreceptor function. These results suggest the existence of a family of receptor phosphatases involved in the regulation of G protein-coupled signaling cascades.

Autophosphorylating protein kinase activity in titin-like arthropod projectin.

The function of the high molecular weight structural proteins from muscle, namely vertebrate titin, arthropod projectin and nematode twitchin, remains to be established. Using a simple method for the purification of projectin from crayfish and Drosophila melanogaster, a polyclonal antibody has been raised against crayfish projectin, and shown to immunocrossreact with Drosophila projectin but not with rat titin. In this study, evidence is presented that projectin and twitchin may share functional protein kinase domains, indicating a possible relationship between them. Projectin has a serine/threonine protein kinase activity. This supports the relationship with twitchin since, in sequence analysis of the latter, a protein-kinase-like domain has been found. Moreover, projectin is capable of autophosphorylation in vitro. These kinase activities imply regulatory functions for this group of proteins, extending its previously assumed structural role in the sarcomere. We also show here that projectin is phosphorylated in vivo at serine residues, as described for titin.

Drosophila melanogaster paramyosin: developmental pattern, mapping and properties deduced from its complete coding sequence.

Several cDNA clones encoding the complete Drosophila paramyosin sequence, including two potential polyadenylation sites, have been obtained. Southern analysis and in situ hybridization to polytene chromosomes indicate that in Drosophila the paramyosin gene is single copy, located on the left arm of the third chromosome at region 66D14. Northern analyses show predominantly two different RNAs which are the products of the choice between the two alternative polyadenylation sites. The two species begin to be synthesized around 10 h of development when embryonic muscles are formed, expression peaking at the end of embryogenesis. The protein is first expressed at germ band shortening in association with muscle precursor cells. A second maximum of paramyosin RNA expression occurs at late pupal stages when the higher molecular weight form becomes more abundant. In young adults this species becomes the main transcript detected. The 102 kDa polypeptide sequence is highly similar to that of Caenorhabditis elegans paramyosin. The protein has a central alpha-helical coiled-coil rod, organized in 29 groups of four typical seven-residue repeats and flanked by two short non-alpha-helical regions. Several leucine zippers are located on the hydrophobic face of the alpha-helix in paramyosin which, together with disulfide bonds between cysteines, are probably involved in the stabilization of the dimer. The structural and functional properties of Drosophila paramyosin deduced from the sequence are compared with those of known invertebrate myosins and paramyosins.

Identification and characterization of Drosophila melanogaster paramyosin.

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, alpha-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a lambda gt11 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.

Insects as test systems for assessing the potential role of microgravity in biological development and evolution.

Gravity and radiation are undoubtedly the two major environmental factors altered in space. Gravity is a weak force, which creates a permanent potential field acting on the mass of biological systems and their cellular components, strongly reduced in space flights. Developmental systems, particularly at very early stages, provide the larger cellular compartments known, where the effects of alterations in the size of the gravity vector on living organisms can be more effectively tested. The insects, one of the more highly evolved classes of animals in which early development occurs in a syncytial embryo, are systems particularly well suited to test these effects and the specific developmental mechanisms affected. Furthermore, they share some basic features such as small size, short life cycles, relatively high radio-resistance, etc. and show a diversity of developmental strategies and tempos advantageous in experiments of this type in space. Drosophila melanogaster, the current biological paradigm to study development, with so much genetic and evolutionary background available, is clearly the reference organism for these studies. The current evidence on the effects of the physical parameters altered in space flights on insect development indicate a surprising correlation between effects seen on the fast developing and relatively small Drosophila embryo and the more slowly developing and large Carausius morosus system. In relation to the issue of the importance of developmental and environmental constraints in biological evolution, still the missing link in current evolutionary thinking, insects and space facilities for long-term experiments could provide useful experimental settings where to critically assess how development and evolution may be interconnected. Finally, it has to be pointed out that since there are experimental data indicating a possible synergism between microgravity and space radiation, possible effects of space radiation should be taken into account in the planning and evaluation of experiments designed to test the potential role of microgravity on biological developmental and evolution.

Drosophila melanogaster contains a set of polypeptides capable of polymerizing into intermediate-like filaments.

A partial purification scheme applied to the Triton X-100 insoluble pellet of adult flies homogenates yielded a fraction which upon polymerization reproducibly produces filaments which in the electron microscope have all the typical properties of intermediate filaments. This is the first report of the presence of protein components with such properties in Drosophila. Thus, it is highly possible that insects and arthropodes, like other lower invertebrates, may contain the third extremely insoluble element of the cellular cytoskeleton.

Signals in the phi 29 DNA-terminal protein template for the initiation of phage phi 29 DNA replication.

The protein-free terminal fragments HindIII B and L, from the left and right ends of phi 29 DNA, respectively, but not internal fragments of similar size, were active as templates in the formation of the p3-dAMP initiation complex in an in vitro system containing purified phi 29 terminal protein p3 and DNA polymerase p2, although the activity was lower than that obtained with the phi 29 DNA-p3 complex. These results indicate the existence of specific sequences at the ends of phi 29 DNA that allow the initiation of phi 29 DNA replication. The template activity of the protein-free terminal fragments was size dependent. The protein-free single strands of the HindIII L fragment were much less active than the corresponding double-stranded fragment. Terminal protein-DNA complexes of phages PZA and phi 15, with a terminal protein closely related to the phi 29 protein p3, were more active as templates in the initiation reaction with the purified phi 29 proteins than the corresponding protein-free DNAs, as it happens in the case of phi 29. However, the terminal protein-DNA complexes of phages Nf, B103, and GA-1, with a terminal protein less related or unrelated to the phi 29 protein p3, were essentially inactive and became active after removal of the parental terminal protein. These results strongly suggest that the parental terminal protein is the major signal in the template for the initiation of phi 29 DNA replication.