Jonathan P. Bacon

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.

Animal escapology I: theoretical issues and emerging trends in escape trajectories

Summary Escape responses are used by many animal species as their main defence against predator attacks. Escape success is determined by a number of variables; important are the directionality (the percentage of responses directed away from the threat) and the escape trajectories (ETs) measured relative to the threat. Although logic would suggest that animals should always turn away from a predator, work on various species shows that these away responses occur only approximately 50–90% of the time. A small proportion of towards responses may introduce some unpredictability and may be an adaptive feature of the escape system. Similar issues apply to ETs. Theoretically, an optimal ET can be modelled on the geometry of predator–prey encounters. However, unpredictability (and hence high variability) in trajectories may be necessary for preventing predators from learning a simple escape pattern. This review discusses the emerging trends in escape trajectories, as well as the modulating key factors, such as the surroundings and body design. The main ET patterns identified are: (1) high ET variability within a limited angular sector (mainly 90–180 deg away from the threat; this variability is in some cases based on multiple peaks of ETs), (2) ETs that allow sensory tracking of the threat and (3) ETs towards a shelter. These characteristic features are observed across various taxa and, therefore, their expression may be mainly related to taxon-independent animal design features and to the environmental context in which prey live – for example whether the immediate surroundings of the prey provide potential refuges.

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.

The dipteran giant fibre pathway: neurons and signals

Summary1.The uniquely identifiable pair of giant descending neurons ofMusca domestica andCalliphora erythrocephala are compared and described in relation to homologues inDrosophila.2.Cobalt coupling in the giant fibre system suggests that giant descending neurons receive inputs from antennal mechanosensory afferents and small-field visual interneurons.3.InMusca andCalliphora, terminals of giant descending neurons invade several discrete areas of neuropil. This is in contrast toDrosophila where homologous neurons have a relatively simple terminal.4.Despite differences in morphology, in all three species a homologous terminal region is cobalt-coupled to the largest of three motorneurons supplying the second legs tergotrochanteral muscle.5.Sensory connections revealed anatomically were confirmed electrophysiologically by intracellular recording and dye-filling. Mechanosensory inputs from the ipsilateral antenna, and small field motion stimuli, or light ‘on’ and ‘off’ stimuli to the ipsilateral compound eyes, produce subthreshold activation in the giant descending neuron.6.As inDrosophila, light ‘on’ or ‘off’ stimuli presented to white-eyed mutants ofMusca andCalliphora elicit a spiking response in the giant descending neuron. In red-eyed flies, spikes could only be routinely induced by electrical stimulation across the head between the compound eyes or by releasing the cell from hyperpolarization.7.Double recordings from the giant (intracellular combined with lucifer or cobalt iontophoresis for cell identification) and from the tergotrochanteral motorneuron (extracellular in muscle) show that a spike in the giant neuron is usually sufficient to drive a spike in the tergotrochanteral muscle. Latencies are short, as reported inDrosophila. Releasing the giant from hyperpolarization can result in a motorneuron spike in the absence of a spike in the giant: this suggests the presence of an electrical synapse.8.Spiking in the tergotrochanteral muscle and the giant can occur independently of each other. Thus, this interneuron alone is neither necessary nor invariably sufficient for initiating ‘escape behaviour’ in the species studied. This is also corroborated by the structure of tergotrochanteral motorneurons whose extensive dendrites receive a variety of other inputs.

Animal escapology II: escape trajectory case studies

Summary Escape trajectories (ETs; measured as the angle relative to the direction of the threat) have been studied in many taxa using a variety of methodologies and definitions. Here, we provide a review of methodological issues followed by a survey of ET studies across animal taxa, including insects, crustaceans, molluscs, lizards, fish, amphibians, birds and mammals. Variability in ETs is examined in terms of ecological significance and morpho-physiological constraints. The survey shows that certain escape strategies (single ETs and highly variable ETs within a limited angular sector) are found in most taxa reviewed here, suggesting that at least some of these ET distributions are the result of convergent evolution. High variability in ETs is found to be associated with multiple preferred trajectories in species from all taxa, and is suggested to provide unpredictability in the escape response. Random ETs are relatively rare and may be related to constraints in the manoeuvrability of the prey. Similarly, reports of the effect of refuges in the immediate environment are relatively uncommon, and mainly confined to lizards and mammals. This may be related to the fact that work on ETs carried out in laboratory settings has rarely provided shelters. Although there are a relatively large number of examples in the literature that suggest trends in the distribution of ETs, our understanding of animal escape strategies would benefit from a standardization of the analytical approach in the study of ETs, using circular statistics and related tests, in addition to the generation of large data sets.

Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: A structural study

Mutations in the Drosophila shaking‐B gene perturb synaptic transmission and dye coupling in the giant fiber escape system. The GAL4 upstream activation sequence system was used to express a neuronal‐synaptobrevin‐green fluorescent protein (nsyb‐GFP) construct in the giant fibers (GFs); nsyb‐GFP was localized where the GFs contact the peripherally synapsing interneurons (PSIs) and the tergotrochanteral motorneurons (TTMns). Antibody to Shaking‐B protein stained plaquelike structures in the same regions of the GFs, although not all plaques colocalized with nsyb‐GFP. Electron microscopy showed that the GF‐TTMn and GF‐PSI contacts contained many chemical synaptic release sites. These sites were interposed with extensive regions of close membrane apposition (3.25 nm ± 0.12 separation), with faint cross striations and a single‐layered array of 41‐nm vesicles on the GF side of the apposition. These contacts appeared similar to rectifying electrical synapses in the crayfish and were eliminated in shaking‐B2 mutants. At mutant GF‐TTMn and GF‐PSI contacts, chemical synapses and small regions of close membrane apposition, more similar to vertebrate gap junctions, were not affected. Gap junctions with more vertebratelike separation of membranes (1.41 nm ± 0.08) were abundant between peripheral perineurial glial processes; these were unaffected in the mutants. J. Comp. Neurol. 404:449–458, 1999.

Cellular Colocalization of Diuretic Peptides in Locusts: A Potent Control Mechanism

Locust abdominal ganglia are shown to colocalize Locusta-diuretic peptide-, leucokinin I-, and lysine vasopressin-like immunoreactivity in posterior lateral neurosecretory cells. Extracts of abdominal ganglia were partially purified by RP-HPLC then dot immunoassay screened with the same antisera used for immunocytochemistry. Locusta-diuretic peptide-like immunoreactive material coeluted with synthetic Locusta-diuretic peptide, and leucokinin-like immunoreactive material coeluted with locustakinin. Lysine vasopressin-like material eluted in fractions that also showed Locusta-diuretic peptide and leucokinin I immunoreactivity. The diuretic activity of synthetic Locusta-diuretic peptide and locustakinin is demonstrated, and they are shown to act at least additively to promote Malpighian tubule fluid secretion. The immunoreactive neurosecretory cells are assumed to express at least these two peptides, and a model for promoting fluid secretion is proposed.

Localization of Locusta-DP in locust CNS and hemolymph satisfies initial hormonal criteria

Locusta-diuretic peptide (Locusta-DP) is a potent stimulant of fluid secretion and cyclic AMP production by locust Malpighian tubules. In this study, a polyclonal antiserum raised to the C-terminus of Locusta-DP reveals a wide distribution of immunoreactive cell bodies and processes throughout the CNS, and endings in two important neurohemal release sites: the corpora cardiaca and the perivisceral organs. HPLC fractionation of CNS, neurohemal structures, and hemolymph reveals immunoreactive material that coelutes with synthetic Locusta-DP and stimulates cyclic AMP production by locust tubules. The identity of the immunoreactive and biologically active material is confirmed as authentic Locusta-DP by mass spectrometry.