Pauline Phelan

Innexins: a family of invertebrate gap-junction proteins.

In vertebrates, intercellular communication via gap junctions is mediated by the connexin family of molecules, which is made up of at least 13 members (reviewed in Ref. 1). These proteins, which have four transmembrane domains and intracellular C- and N-termini, oligomerize to form hemichannels. Oligomers in the adjacent membranes of two closely apposed cells ‘dock’ to form intercellular channels, through which ions and small molecules move. Intercellular communication is a fundamental function of any multicellular organism and it is odd that no obvious homologues of the connexins have been found in any invertebrate. In view of the fact that over 90% of the genomic sequence of Caenorhabditis elegans is available for analysis, it is becoming increasingly unlikely that invertebrate connexins will be found. Conventional genetic dissection of C. elegans and Drosophila, however, has identified a gene family with some role in gap-junction communication. Although they bear no sequence similarity to the connexins, these genes are predicted to encode proteins with the same topology. In C. elegans, mutations in the unc-7 gene result in an uncoordinated phenotype, and the formation of ectopic electrical junctions between some interneurons and motoneurons (J.G. White, E. Southgate and J.N. Thomson, cited in Ref. 2). A worm with an eating disorder results from mutations in the eat-5 gene; here, some pharyngeal muscles fail to establish normal electrical connections with their neighbours2. In Drosophila, one of the transcripts from the shaking-B locus [shaking-B(neural); also known as Passover ] is required for electrical synapse function between neurons of the giant-fibre escape circuit3,4 and between embryonic somatic muscles (J.P. Bacon et al., 1996, Soc. Neurosci. Abstr. 22, 38). A second transcript from this Drosophila locus, known as shaking-B(lethal), the Drosophila gene optic ganglion reduced (ogre), and several other C. elegans genes share sequence similarity with this family5 but their mutant phenotypes have not yet been fully characterized. These worm and fly data did provide some circumstantial evidence that these loci encode gap-junction proteins. They were given the name OPUS (for ogre, passover, uncoordinated, shaking-B) in a previous letter to TIG (Ref. 6). We feel that the name OPUS is confusing because we now know that Passover and shaking-B are allelic and it has recently been brought to our attention that opus is the name of a Drosophila copia-like transposable element7. In addition, the recent determination of the role of one of these genes makes it timely to rename this family in a way that reflects function. Using heterologous expression in Xenopus oocyte pairs, it has been demonstrated unequivocally that Shaking-B(lethal) protein is sufficient to form homotypic gap junctions8. Interestingly, the closely related Shaking-B(neural) protein fails to form functional junctions in this system. We suspect that either Shaking-B(neural) forms gap junctions that are closed under the particular physiological conditions of Xenopus oocytes, or that it requires a partner to form hetero-oligomeric channels; a few connexins fail to form homotypic junctions in Xenopus oocytes (reviewed in Ref. 1). Despite these remaining uncertainties about the function of Shaking-B(neural) protein, Shaking-B (lethal) is the first invertebrate gap-junction protein to be identified. It means that this family of genes, and their proteins, can be given a functional name. We propose the name innexins (invertebrate analogues of the connexins) for invertebrate gap-junction proteins. We are anxious to avoid the gratuitous proliferation of names in an already overstuffed literature but we think it better to choose a name that reflects function rather than an acronym based on an incomplete set of mutant phenotypes. It will be important to examine the function of fly and worm Shaking-B(lethal)-like proteins, using heterologous expression systems, to determine whether they truly are innexins. Judging from the vertebrate connexin data, we anticipate that the innexin family will have many members that can either work alone or in concert to build a range of gap-junction types. Another task is to look for innexins in other invertebrate phyla; so far they have been described only in insects and nematodes. Given the recent proposal that moulting animals form a new phyletic clade, the Ecdysozoa9, it remains a real possibility that innexin proteins are a molecular marker for this clade, and will not be found outside it. Interestingly, our standard BLAST searches of the protein databases at the NCBI server have revealed no vertebrate proteins with sequence similarity to Shaking-B. Whether the similar (predicted) topology, without obvious sequence similarity, of innexins and connexins is a case of convergent evolution or one of extreme sequence divergence within a protein family, remains to be determined.

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes

In most multicellular organisms direct cell–cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions,. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions,. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Gap junctions in Drosophila: developmental expression of the entire innexin gene family

Invertebrate gap junctions are composed of proteins called innexins and eight innexin encoding loci have been identified in the now complete genome sequence of Drosophila melanogaster. The intercellular channels formed by these proteins are multimeric and previous studies have shown that, in a heterologous expression system, homo- and hetero-oligomeric channels can form, each combination possessing different gating characteristics. Here we demonstrate that the innexins exhibit complex overlapping expression patterns during oogenesis, embryogenesis, imaginal wing disc development and central nervous system development and show that only certain combinations of innexin oligomerization are possible in vivo. This work forms an essential basis for future studies of innexin interactions in Drosophila and outlines the potential extent of gap-junction involvement in development.

Molecular Mechanism of Rectification at Identified Electrical Synapses in the Drosophila Giant Fiber System

Summary Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits [1–5]. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates [3–5]; innexins/pannexins encode gap-junction proteins in prechordates and chordates [6–10]. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely [11, 12]. The phenomenon of rectification was first described five decades ago [1], but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System [13] are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) [14] is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal) [15]. When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

Functional gap junction genes are encoded by insect viruses

Ichnoviruses belong to the virus family Polydnaviridae, whose members are obligately associated with certain endoparasitoid wasps. Expression of ichnovirus genes in parasitized lepidopteran hosts leads to immune suppression and is essential for successful parasitization. To date, the role of specific ichnovirus genes in alteration of host physiology has been unclear, and no cellular homologues have been described. Here, we describe the isolation of a gene family from two ichnoviruses that is homologous to the innexin gene family, which encodes gap junctions in invertebrates. Campoletis sonorensis ichnovirus (CsIV) innexins are expressed in multiple tissues in infected lepidopterans, including haemocytes, the primary immunocytes of the host. Two of the CsIV proteins have been expressed and shown to form functional gap junctions in Xenopus oocytes. To our knowledge this is the first study to describe gap junction genes in any virus. We hypothesize that the virus innexins disrupt cellular immunity in infected insects by altering normal gap junctional intercellular communication. This would represent a novel mechanism of viral alteration of host physiology, and suggests that gap junctions play a crucial role in coordinating cellular immune responses.

Innexins ogre and inx2 are required in glial cells for normal postembryonic development of the drosophila central nervous system

Summary Innexins are one of two gene families that have evolved to permit neighbouring cells in multicellular systems to communicate directly. Innexins are found in prechordates and persist in small numbers in chordates as divergent sequences termed pannexins. Connexins are functionally analogous proteins exclusive to chordates. Members of these two families of proteins form intercellular channels, assemblies of which constitute gap junctions. Each intercellular channel is a composite of two hemichannels, one from each of two apposed cells. Hemichannels dock in the extracellular space to form a complete channel with a central aqueous pore that regulates the cell–cell exchange of ions and small signalling molecules. Hemichannels can also act independently by releasing paracrine signalling molecules. optic ganglion reduced (ogre) is a member of the Drosophila innexin family, originally identified as a gene essential for postembryonic neurogenesis. Here we demonstrate, by heterologous expression in paired Xenopus oocytes, that Ogre alone does not form homotypic gap-junction channels; however, co-expression of Ogre with Innexin2 (Inx2) induces formation of functional channels with properties distinct from Inx2 homotypic channels. In the Drosophila larval central nervous system, we find that Inx2 partially colocalises with Ogre in proliferative neuroepithelia and in glial cells. Downregulation of either ogre or inx2 selectively in glia, by targeted expression of RNA interference transgenes, leads to a significant reduction in the size of the larval nervous system and behavioural defects in surviving adults. We conclude that these innexins are crucially required in glial cells for normal postembryonic development of the central nervous system.

Chapter 19: Gap Junction Communication in Invertebrates: The Innexin Gene Family

Publisher Summary Given the extent of genetic conservation through evolution, it is paradoxical that the structural components of gap junctions do not appear to be conserved throughout the animal kingdom. Electrical synapses in the escape systems of the crayfish ventral nerve cord and the goldfish spinal cord play the same basic role and, apart from subtle differences, are ultrastructurally alike; hence, they are formed from homologous proteins. Yet, despite much effort, connexins, the molecules that form gap junctions in vertebrates, have not been identified unequivocally in any invertebrate. In the wake of the sequencing of the Caenorhabditis (C.) elegans genome, this chapter discusses the evidence that intercellular channels in the nematode and the other model genetic invertebrate, Drosophila melanogaster , are formed from an apparently separate family of proteins that have been named innexins. There are still more to come in Drosophila, and there is no end of work ahead to characterize the individual proteins. Antibodies will be vital for cellular localization at LM and EM levels, and the proteins must be expressed in heterologous systems to establish their competency to form intercellular channels. As with the studies of connexins, the ultimate aim is to elucidate the functions of gap junctions in the diverse systems in which they are found.

Functional Interactions between Polydnavirus and Host Cellular Innexins

ABSTRACT Polydnaviruses are double-stranded DNA viruses associated with some subfamilies of ichneumonoid parasitoid wasps. Polydnavirus virions are delivered during wasp parasitization of a host, and virus gene expression in the host induces alterations of host physiology. Infection of susceptible host caterpillars by the polydnavirus Campoletis sonorensis ichnovirus (CsIV) leads to expression of virus genes, resulting in immune and developmental disruptions. CsIV carries four homologues of insect gap junction genes (innexins) termed vinnexins, which are expressed in multiple tissues of infected caterpillars. Previously, we demonstrated that two of these, VinnexinD and VinnexinG, form functional gap junctions in paired Xenopus oocytes. Here we show that VinnexinQ1 and VinnexinQ2, likewise, form junctions in this heterologous system. Moreover, we demonstrate that the vinnexins interact differentially with the Innexin2 orthologue of an ichnovirus host, Spodoptera frugiperda. Cell pairs coexpressing a vinnexin and Innexin2 or pairs in which one cell expresses a vinnexin and the neighboring cell Innexin2 assemble functional junctions with properties that differ from those of junctions composed of Innexin2 alone. These data suggest that altered gap junctional intercellular communication may underlie certain cellular pathologies associated with ichnovirus infection of caterpillar hosts.

Swept source optical coherence tomography Gabor fusion splicing technique for microscopy of thick samples using a deformable mirror

Abstract. We present a swept source optical coherence tomography (OCT) system at 1060 nm equipped with a wavefront sensor at 830 nm and a deformable mirror in a closed-loop adaptive optics (AO) system. Due to the AO correction, the confocal profile of the interface optics becomes narrower than the OCT axial range, restricting the part of the B-scan (cross section) with good contrast. By actuating on the deformable mirror, the depth of the focus is changed and the system is used to demonstrate Gabor filtering in order to produce B-scan OCT images with enhanced sensitivity throughout the axial range from a Drosophila larvae. The focus adjustment is achieved by manipulating the curvature of the deformable mirror between two user-defined limits. Particularities of controlling the focus for Gabor filtering using the deformable mirror are presented.