Interaction of rat lin-10 with brain-enriched F-actin-binding protein, neurabin-II/spinophilin

We have recently isolated a rat homologue of the Caenorrhabditis elegans lin-10 product. Although rat lin-10 is expressed in the cytosol and membrane fractions of various tissues, it is distributed only in the membrane fraction in brain where it is enriched in the synaptic plasma membrane and postsynaptic density fractions. We have isolated here a rat lin-10-interacting protein from rat brain and identified it to be neurabin-II/spinophilin, which has recently been isolated as a protein interacting with protein phosphatase I and F-actin. Neurabin-II/spinophilin is ubiquitously expressed but enriched in brain, especially in the synaptic plasma membrane and postsynaptic density fractions. We discuss the physiological significance of the interaction of rat lin-10 with neurabin-II/spinophilin.     

The LIN-2/LIN-7/LIN-10 complex mediates basolateral membrane localization of the C. elegans EGF receptor LET-23 in vulval epithelial cells

In C. elegans, the LET-23 receptor tyrosine kinase is localized to the basolateral membranes of polarized vulval epithelial cells. lin-2, lin-7, and lin-10 are required for basolateral localization of LET-23, since LET-23 is mislocalized to the apical membrane in lin-2, lin-7, and lin-10 mutants. Yeast two-hybrid, in vitro binding, and in vivo coimmunoprecipitation experiments show that LIN-2, LIN-7, and LIN-10 form a protein complex. Furthermore, compensatory mutations in lin-7 and let-23 exhibit allele-specific suppression of apical mislocalization and signaling-defective phenotypes. These results present a mechanism for basolateral localization of LET-23 receptor tyrosine kinase by direct binding to the LIN-2/LIN-7/LIN-10 complex. Each of the binding interactions within this complex is conserved, suggesting that this complex may also mediate basolateral localization in mammals.     

Widespread expression of ecto-apyrase (CD39) in the central nervous system

We have shown that ecto-apyrase protein is expressed in primary neurons and astrocytes in cell culture (T.-F. Wang, P.A. Rosenberg, G. Guidotti, 1997. Mol. Brain Res. 1997, 47: 295-302). Here we present immunohistochemical studies showing that ecto-apyrase protein is widely distributed in rat brain, as it is present in neurons of the cerebral cortex, hippocampus and cerebellum as well as in glial cells and endothelial cells. Ecto-apyrase is enriched in brain postsynaptic density membrane fractions and is localized in proximity to synaptophysin, the marker of synaptic vesicles. These results together with the observation that P2 purinergic receptors are present throughout the brain suggest that ecto-apyrase is involved in regulating synaptic transmission mediated by extracellular ATP.     

The beta-catenin homolog BAR-1 and LET-60 Ras coordinately regulate the Hox gene lin-39 during Caenorhabditis elegans vulval development

In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Our results suggest that a Wnt signal transduced through a pathway involving the beta-catenin homolog BAR-1 controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. Our genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target LIN-39 Hox.     

Interactions of EGF, Wnt and HOM-C genes specify the P12 neuroectoblast fate in C. elegans

We investigate how temporal and spatial interactions between multiple intercellular and intracellular factors specify the fate of a single cell in Caenorhabditis elegans. P12, which is a ventral cord neuroectoblast, divides postembryonically to generate neurons and a unique epidermal cell. Three classes of proteins are involved in the specification of P12 fate: the LIN-3/LET-23 epidermal growth factor signaling pathway, a Wnt protein LIN-44 and its candidate receptor LIN-17, and a homeotic gene product EGL-5. We show that LIN-3 is an inductive signal sufficient to promote the P12 fate, and the conserved EGF signaling pathway is utilized for P12 fate specification; egl-5 is a downstream target of the lin-3/let-23 pathway in specifying P12 fate; and LIN-44 and LIN-17 act synergistically with lin-3 in the specification of the P12 fate. The Wnt pathway may function early in development to regulate the competence of the cells to respond to the LIN-3 inductive signal.     

Role of a raf proto-oncogene during Caenorhabditis elegans vulval development

During Caenorhabditis elegans vulval induction, multipotent precursors respond to an inductive signal by generating vulval cells as opposed to non-specialized epidermal cells. Both classical and 'reverse' genetic approaches have revealed that a cascade of nematode homologues of mammalian proto-oncogenes is necessary for induction of the vulva. The inductive signal is a growth factor encoded by the lin-3 gene and its candidate receptor is a tyrosine kinase encoded by the let-23 gene. let-23 acts via a Ras protein encoded by the let-60 gene. A nematode homologue of mammalian raf family protein serine/threonine kinases has been cloned and found to be encoded by the lin-45 gene. Dominant negative lin-45 raf mutants prevent vulval induction. A recessive lin-45 raf mutation prevents the excessive vulval differentiation caused by activated ras, indicating that raf might act downstream of ras during vulval induction.     

Effect of angiotensin-(1-7) on ATPase activities in several tissues

The present investigation was undertaken to determine whether Ang-(1-7) is able to modify ATPase activities in membrane fractions prepared from several tissues. In the presence of 10(-6) M Ang-(1-7), total (Na , K+, Mg2+)-ATPase activity decreased 31% in rat atrium and 13% in sheep atrium but was unmodified in sheep liver, rat ventricle or crude brain membranes. In rat brain synaptosomal membranes, Ang-(1-7) at 10(-8) and 10(-7) M concentrations activated Na+, K+-ATPase 20 and 24%, respectively. Rat kidney Na+, K+-ATPase activity decreased roughly 40-70% with 10(-10)-10(-6) M Ang-(1-7)), but increased 22% with 10(-12) M peptide concentration, thus indicating a biphasic effect. Our findings showing that ATPase from several tissues responds differently to Ang-(1-7) are attributable to enzyme tissue specificity.     

Anatomo-clinical conference: bronchiolo-alveolar cancer and confusion

Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrin-coated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cl- and could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathway - the CCV - may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.     

Sigma-1 and sigma-2 sites in rat brain: comparison of regional, ontogenetic, and subcellular patterns

Radioligand binding assay conditions were established for the selective labeling of sigma-1 and sigma-2 sites in membrane homogenates of rat brain. Selective sigma-1 assays were conducted using 5 nM(+)[3H]SKF-10,047 in the presence of 300 nM dizocilpine (MK-801). Selective sigma-2 assays were conducted using 5 nM [3H]DTG in the presence of 1 microM (+)SKF-10,047. Distributions of sigma-1 and sigma-2 binding among brain regions were found to differ. While the brain stem yields the highest level of sigma-1 binding, it yields among the lowest levels of sigma-2 binding. The reverse is true in hippocampal membranes. Different ontogenetic patterns were also observed. Sigma-2 binding decreases substantially during brain development, whereas sigma-1 binding does not vary significantly. Patterns of distribution among subcellular fractions of rat brain homogenates were found to be similar. Both sigma-1 and sigma-2 sites are most enriched in microsomal fractions, and neither is enriched in synaptosomal or mitochondrial fractions. The present results suggest that sigma-1 and sigma-2 sites are distinct entities; they do not appear to be located on a common macromolecule, and they do not represent two different affinity states of a single type of binding site. While the precise subcellular locations of sigma-1 and sigma-2 sites remain to be determined, we conclude that localization of either type of binding site to synaptic regions of plasma membrane or to mitochondria is highly unlikely.     

A novel brain-specific isoform of beta spectrin: isolation and its interaction with Munc13

Munc13 is a component of the neurotransmitter release machinery which is specifically expressed in brain. Munc13 interacts with Doc2 and syntaxin which are also implicated in the neurotransmitter release process. Here we isolated another Munc13-interacting molecule from a rat brain cDNA library by use of the yeast two-hybrid system, identified it to be a novel type of beta spectrin, and named it beta SpIII sigma 1. beta SpIII sigma 1 was specifically expressed in brain, where it was enriched in the synaptic vesicle and plasma membrane fractions. Because spectrin has been shown to interact with the actin cytoskeleton which is involved in the exocytotic process, the present results suggest that the Munc13-beta SpIII sigma 1 interactions play a role in neurotransmitter release.     

lin-14 regulates the timing of synaptic remodelling in Caenorhabditis elegans

In the nematode Caenorhabditis elegans six GABAergic motor neurons, known as DDs, remodel their patterns of synaptic connectivity during larval development. DD remodelling involves a complete reversal of the direction of information flow within nerve processes without marked changes in process morphology. We used a marker localized in vivo to DD presynaptic zones to analyse how the timing of DD remodelling is controlled. In wild-type animals, DDs remodel their synaptic outputs within a 3-5-hour period at the end of the first larval stage. We show that the heterochronic gene lin-14, which controls the timing of stage-specific cell lineages, regulates the timing of DD synaptic output remodelling. In lin-14 loss-of-function mutants, DDs remodel precociously. The degree of precocious remodelling is correlated with the level of lin-14 activity. Expression of lin-14(+) in the DDs of lin-14-null mutants rescues the precocious remodelling, indicating that lin-14 can act cell-autonomously. Consistent with this hypothesis, LIN-14 protein levels decrease in the DDs before remodelling. Our observations reveal a role of heterochronic genes in non-dividing cells, and provide an example of cell-autonomous respecification of neuronal connectivity.     

Synthesis of F pilin

Transfer of the Escherichia coli fertility plasmid, F, is dependent on expression of F pili. Synthesis of F-pilin subunits is known to involve three F plasmid transfer (tra) region products: traA encodes the 13-kDa precursor protein, TraQ permits this to be processed to the 7-kDa pilin polypeptide, and TraX catalyzes acetylation of the pilin amino terminus. Using cloned tra sequences, we performed a series of pulse-chase experiments to investigate the effect of TraQ and TraX on the fate of the traA product. In TraQ- cells, the traA gene product was found to be very unstable. While traA polypeptides of various sizes were detected early in the chase period, almost all were degraded within 5 min. Rapid traA product degradation was also observed in TraX+ cells, although an increased percentage of these products persisted during the chase. In TraQ+ cells, most of the traA product was processed to the 7-kDa pilin polypeptide within the 1-min pulse period; this product [7(Q)] was not degraded but was increasingly converted to an 8-kDa form [8(Q)] as the chase continued, suggesting that host enzymes can modify the pilin polypeptide. Similar results were observed in TraQ+ TraX+ cells, but the primary 7-kDa product appeared to be N-acetylated pilin (Ac-7). An 8-kDa product (Ac-8) was also detected, but this band did not increase in intensity during the chase. We suggest a pathway in which TraQ prevents the traA product from folding to a readily degradable conformation and assists its entry into the membrane, Leader peptidase I cleaves the traA product signal sequence, and a subset of the pilin polypeptides becomes modified by host enzymes; TraX then acetylates the N terminal of both the modified and unmodified pilin polypeptides.     

Tissue- and subcellular-distribution of the binding site of [3H]9- methyl-7-bromoeudistomin D, a potent caffeine-like Ca2+ releaser, in rabbits

Tissue and subcellular distribution of the binding site of 3H-labelled 9-methyl-7-bromoeudistomin D ([3H]MBED), a powerful caffeine-like Ca2+ releaser, were investigated in rabbits. The order of specific activities of total homogenates was liver > brain > other tissues. All binding was completely suppressed by 10 mM caffeine, indicating that all [3H]MBED binding sites are modulated by caffeine. [3H]MBED binding sites distributed mainly in membrane fractions rather than soluble fractions in most tissues. In lung and liver, [3H]MBED binding was enriched in microsomes. [3H]MBED may be useful as a probe to investigate the actions of caffeine at the molecular level not only in muscles but also in a variety of tissues including liver, kidney and lung.     

Nitric oxide signalling is required for the generation of anoxia-induced long-term potentiation in the hippocampus

The involvement of nitric oxide in anoxia-induced long-term potentiation (anoxic LTP) of synaptic transmission was investigated in CA1 neurons of rat hippocampal slices using intracellular recording techniques in vitro. In response to superfusion of an anoxic artificial cerebral spinal fluid saturated with 95% N2-5% CO2, the excitatory postsynaptic potential (EPSP) generated in hippocampal CA1 neurons by stimulation of the Schaffer collateral/commissural afferent pathway was completely abolished within 10 min of anoxia. On return to reoxygenated medium, the EPSP returned to the control value within 10 min and was subsequently and progressively potentiated to reach a plateau 15-20 min after return to oxygen. This anoxia-induced persistent increase in synaptic transmission lasted for more than 1 h. Application of the nitric oxide synthase inhibitors 7-nitroindazole (7-NI) or L-N(G)-nitroarginine (NOARG) produced no effects on the baseline EPSP amplitude, but effectively attenuated the anoxic LTP. The inhibitory effects of both 7-NI and NOARG on the anoxic LTP were blocked by L-arginine, a substrate for nitric oxide synthase. These results suggest that nitric oxide is required for the generation of anoxia-induced LTP of glutamatergic synaptic transmission in the CA1 region of the rat hippocampus.     

Identification and characterization of genes that interact with lin-12 in Caenorhabditis elegans

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.     

An ultrastructural study of the glycine transporter GLYT2 and its association with glycine in the superficial laminae of the rat spinal dorsal horn

The glycine transporter GLYT2 is present in axonal boutons throughout the spinal cord, and its laminar distribution matches that of glycine-enriched axons, which are presumed to be glycinergic. In order to determine whether boutons which possess GLYT2 are glycine-enriched, we have carried out pre-embedding immunocytochemistry with antibody raised against GLYT2, and combined this with post-embedding detection of glycine, in the rat. GLYT2 immunoreactivity was present in boutons which formed symmetrical axodendritic, axosomatic or axoaxonic synapses, and was often seen in peripheral axons of type II synaptic glomeruli. One hundred and fifty GLYT2-immunoreactive boutons were analysed quantitatively, and in 142 (94.6%) of these the density of gold particles representing glycine-like immunoreactivity exceeded the background level (over presumed glutamatergic boutons) by at least a factor of two. Within immunoreactive boutons, the GLYT2 reaction product was associated with the plasma membrane, but often appeared as discrete clumps and was generally excluded from the region of the active sites of synapses. These results confirm that GLYT2 is associated with glycine-enriched axonal boutons in the superficial dorsal horn. They also suggest that GLYT2 is unevenly distributed on the plasma membrane of these boutons, and raise the possibility that it may be excluded from synaptic clefts.     

Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors

The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions.     

Preparation and properties of mitochondria derived from synaptosomes

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.