A voltage-dependent gap junction in Drosophila melanogaster.

Steady-state and kinetic analyses of gap junctional conductance, gi, in salivary glands of Drosophila melanogaster third instar larvae reveal a strong and complex voltage dependence that can be elicited by two types of voltages. Voltages applied between the cells, i.e., transjunctional voltages, Vj, and those applied between the cytoplasm and the extracellular space, inside-outside voltages, Vi,o, markedly alter gj. Alteration of Vi-o while holding Vj = O,i.e., by equal displacement of the voltages in the cells, causes gj to increase to a maximum on hyperpolarization and to decrease to near zero on depolarization. These conductance changes associated with Vi-o are fit by a model in which there are two independent gates in series, one in each series, one in each membrane, where each gate is equally sensitive to Vi-o and exhibits first order kinetics. Vj's generated by applying voltage steps of either polarity to either cell, substantially reduce gj. These conductance changes exhibit complex kinetics that depend on Vi-o as well as Vj. At more positive Vi-o's, the changes in gj have two phases, an early phase consisting of of a decrease in gj for either polarity of Vj and a later phase consisting of an increase in gj on hyperpolarizing either cell and a decrease on depolarizing either cell. At negative Vi-o's in the plateau region of the gj-Vi-o relation, the later slow increase in gj is absent on hyperpolarizing either cell. Also, the early decrease in gj for either polarity of Vj is faster the more positive the Vi-o. The complex time course elicited by applying voltage steps to one cell can be explained as combined actions of Vi-o and Vj, with the early phase ascribable to Vj, but influenced by Vi-o, and the later phase to the changes in Vi-o associated with the generation of Vj. The substantially different kinetics and sensitivity of changes in gj by Vi-o and Vj suggests that the mechanisms of gating by these two voltages are different. Evidently, these gap-junction channels are capable of two distinct, but interactive forms of voltage dependence.     

Heteromerization of innexin gap junction proteins regulates epithelial tissue organization in Drosophila

Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.     

Innexin genes and gap junction proteins in the locust frontal ganglion

Gap junctions (GJs) belong to one of the most conserved cellular structures in multicellular organisms. They probably serve similar functions in all Metazoa, providing one of the most common forms of intercellular communication. GJs are widely distributed in embryonic cells and tissues and have been attributed an important role in development, modulating cell growth and differentiation. These channels have been also implicated in mediating electrical synaptic signaling; Coupling through GJs is now accepted as a major pathway that supports network behavior and contributes to physiological rhythms. Here we focus on the physiology and molecular biology of GJs in a recently established model for the study of rhythm-generating networks and their role in behavior: the frontal ganglion (FG) of the desert locust, Schistocerca gregaria. Four novel genes of the invertebrate GJs (innexin) gene family were found to be expressed in the FG: Sg-inx1, Sg-inx2, Sg-inx3 and Sg-inx4. Immunohistochemistry revealed that some of the neurons in the FG express at least one innexin protein, INX1. We also established the presence of functional gap junction proteins in the FG and demonstrated functional electrical coupling between the neurons in the FG. This study forms the basis for further investigation of the role of GJs in network development and behavior.     

Expression of gap junction genes during postnatal neural development.

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.     

Gastrointestinal development in the Drosophila embryo requires the activity of innexin gap junction channel proteins

Cell to cell communication plays an essential role during pattern formation and morphogenesis of the diverse tissues and organs of the body. In invertebrates, such as the fruitfly Drosophila, the direct communication of closely apposed cells is mediated by gap junctions which are composed of oligomers of the innexin family of transmembrane channel proteins. Few data exist about the developmental role of the eight innexin genes which have been found in the Drosophila genome. We have investigated the role of the innexin 2 and ogre genes during gastrointestinal development of the fly embryo. Our findings suggest that innexins are involved in the formation of the proventriculus, an organ that develops at the foregut/midgut boundary by migration of primordial cells and subsequent infolding of epithelial tissue layers.     

Expression of gap junction genes during postnatal neural development

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.     

Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins.     

Functional reconstitution of lens gap junction proteins into proteoliposomees

Summary Membranes rich in junction complexes were prepared from bovine lens, and the fragments of the membranes were reconstituted into proteoliposomes with a large excess of phosphatidylcholine and dicetylphosphate. The osmotic swelling behavior of these liposomes showed that the lens junction membranes contributed protein components that produced channels with a nominal diameter of 1.4 nm. Most preparations of lens junctions produced rates of osmotic swelling much slower than those found in proteoliposomes containing equivalent amounts ofEscherichia coli porin, and we discuss several possible explanations for this observation.     

Calcium-dependent binding of calmodulin to neuronal gap junction proteins

We examined the interactions of calmodulin with neuronal gap junction proteins connexin35 (Cx35) from perch, its mouse homologue Cx36, and the related perch Cx34.7 using surface plasmon resonance. Calmodulin bound to the C-terminal domains of all three connexins with rapid kinetics in a concentration- and Ca2+-dependent manner. Dissociation was also very rapid. K(d)'s for calmodulin binding at a high-affinity site ranged from 11 to 72 nM, and K(1/2)'s for Ca2+ were between 3 and 5 microM. No binding to the intracellular loops was observed. Binding competition experiments with synthetic peptides mapped the calmodulin binding site to a 10-30 amino acid segment at the beginning of the C-terminal domain of Cx36. The micromolar K(1/2)'s and rapid on and off rates suggest that this interaction may change dynamically in neurons, and may occur transiently when Ca2+ is elevated to a level that would occur in the near vicinity of an activated synapse.     

The diversity of connexin genes encoding gap junctional proteins.

The multigene family of connexins is larger than previously anticipated. Ten different connexin homologous sequences have been characterized in the mouse genome, five of which are probably the mouse analogues of the known rat connexins26, -31, -32, -43, and -46. Since the additional 5 sequences have been isolated as cDNAs or hybridize specifically to distinct mRNA species, they most likely represent functional connexin genes. Since seven of the genomic connexin sequences have been shown to contain no intron in the coding sequence, this may apply to all mammalian connexin genes. Some of the structural features based on amino acid sequences deduced from cDNA or genomic sequences and the RNA expression pattern of the new connexins are compared with previously described connexins. The structural diversity of the connexin genes suggests that they fulfill different functions coordinated with, and perhaps required for, different programs of cellular differentiation.     

Spontaneous fragmentation of several proteins in Drosophila pupae

Autoproteolysis is an essential activity in the expression of the entire genomes of a number of viruses. That is, new viruses can be produced only after large polyprotein products translated from the genome or from subgenomic mRNA degrade themselves to the polypeptides necessary for RNA replication or for the construction of new virus particles. We have recently shown that the major heat shock protein of Drosophila and a mouse cell line (70 kDa) also undergoes autoproteolysis with the production of specific patterns of smaller polypeptides. We show now that many other proteins in eucaryotic tissues also have a potential for self-degradation. We suggest that special coding regions in many genes may have important roles in both protein turnover and in the production of regulatory peptides.     

Gating of gap junction channels.

Gap junctional conductance ( gj ) in various species is gated by voltage and intracellular pH (pHi). In amphibian embryos, gj is reduced to half by a 14 mV transjunctional voltage ( Vj ), a change that in fish embryo requires approximately 28 mV. Crayfish septate axon and pairs of dissociated rat myocytes show no voltage dependence of gj over a range of Vj greater than +/- 50 mV. In fish and amphibian blastomeres , gj is steeply decreased by decrease in pHi (n, Hill coefficient: 4.5) and the apparent pKH (7.3) is in the physiological range. In crayfish septate axon the pKH is lower (6.7) and the curve is less steep (n = 2.7). Rises in cytoplasmic Ca can also decrease gj but much higher concentrations are required (greater than 0.1 mM in fish blastomeres). Voltage and pH gates on gap junctions in amphibian embryos appear independent. In squid blastomeres pH gates exhibit some sensitivity to potential, both transjunctional and between inside and outside. A pharmacology of gap junctions is being developed: certain agents block gj directly (aldehydes, alcohols, NEM in crayfish); others block by decreasing pHi (esters that are hydrolyzed by intrinsic esterases, NEM in vertebrates, and, as in the experiments demonstrating the effect of pHi, weak acids). Certain agents block pH sensitivity without affecting voltage dependence (retinoic acid, glutaraldehyde, EEDQ), further indicating separateness of pH and voltage gates. These studies demonstrate a dynamics of gap junctional conductance and variability in gating in a series of possibly homologous membrane channels.     

The tight junction protein ZO-2 has several functional nuclear export signals

The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.     

Evidence that nuclear genes code for several chloroplast ribosomal proteins

Electrophoresis of the proteins of the 50S chloroplast ribosome subunit reveals that there are at least two detectable differencesbetween Nicotiana tabacum and N. glauca. Nuclear genes contain the information for these two polypeptides since they are transmitted to F₁ interspecific hybrids independently of the maternal parent.