A novel synaptic vesicle fusion path in the rat cerebral cortex: the "saddle" point hypothesis.

We improved freeze-fracture electron microscopy to study synapses in the neuropil of the rat cerebral cortex at ∼2 nm resolution and in three-dimensions. In the pre-synaptic axon, we found that "rods" assembled from short filaments protruding from the vesicle and the plasma membrane connects synaptic vesicles to the membrane of the active zone. We equated these "connector rods" to protein complexes involved in "docking" and "priming" vesicles to the active zone. Depending on their orientation, the "rods" define two synaptic vesicle-fusion paths: When parallel to the plasma membrane, the vesicles hemi-fuse anywhere ("randomly") in the active zone following the conventional path anticipated by the SNARE hypothesis. When perpendicular to the plasma membrane, the vesicles hemi-fuse at the base of sharp crooks, called "indentations," that are spaced 75-85 nm center-to-center, arranged in files and contained within gutters. They result from primary and secondary membrane curvatures that intersect at stationary inflection ("saddle") points. Computer simulations indicate that this novel vesicle-fusion path evokes neurotransmitter concentration domains on the post-synaptic spine that are wider, shallower, and that reach higher average concentrations than the more conventional vesicle fusion path. In the post-synaptic spine, large (∼9× âˆ¼15 nm) rectangular particles at densities of 72±10/ µm2 (170-240/spine) match the envelopes of the homotetrameric GluR2 AMPA-sensitive receptor. While these putative receptors join clusters, called the "post-synaptic domains," the overwhelming majority of the rectangular particles formed bands in the "non-synaptic" plasma membrane of the spine. In conclusion, in the neuropil of the rat cerebral cortex, curvatures of the plasma membrane define a novel vesicle-fusion path that preconditions specific regions of the active zone for neurotransmitter release. We hypothesize that a change in the hybridization of the R-SNARE synaptobrevin from parallel to antiparallel swings the synapse into this novel vesicle-fusion path.

The three-dimensional distribution of αA-crystalline in rat lenses and its possible relation to transparency.

Lens transparency depends on the accumulation of massive quantities (600-800 mg/ml) of twelve primary crystallines and two truncated crystallines in highly elongated "fiber" cells. Despite numerous studies, major unanswered questions are how this heterogeneous group of proteins becomes organized to bestow the lens with its unique optical properties and how it changes during cataract formation. Using novel methods based on conical tomography and labeling with antibody/gold conjugates, we have profiled the 3D-distribution of the αA-crystalline in rat lenses at ∼2 nm resolutions and three-dimensions. Analysis of tomograms calculated from lenses labeled with anti-αA-crystalline and gold particles (∼3 nm and ∼7 nm diameter) revealed geometric patterns shaped as lines, isosceles triangles and polyhedrons. A Gaussian distribution centered at ∼7.5 nm fitted the distances between the ∼3 nm diameter gold conjugates. A Gaussian distribution centered at ∼14 nm fitted the Euclidian distances between the smaller and the larger gold particles and another Gaussian at 21-24 nm the distances between the larger particles. Independent of their diameters, tethers of 14-17 nm in length connected files of gold particles to thin filaments or clusters to ∼15 nm diameter "beads." We used the information gathered from tomograms of labeled lenses to determine the distribution of the αA-crystalline in unlabeled lenses. We found that αA-crystalline monomers spaced ∼7 nm or αA-crystalline dimers spaced ∼15 nm center-to-center apart decorated thin filaments of the lens cytoskeleton. It thus seems likely that lost or gain of long-range order determines the 3D-structure of the fiber cell and possible also cataract formation.

Conical tomography of a ribbon synapse: structural evidence for vesicle fusion.

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ∼470 vesicles that were hemi-fused and ∼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.

Conical electron tomography of a chemical synapse: polyhedral cages dock vesicles to the active zone.

In this study, we tested the hypothesis that the structure of the active zone of chemical synapses has remained uncertain because of limitations of conventional electron microscopy. To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical "average" synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of approximately 3 nm and eliminates the need to impose symmetry or use averaging methods to increase signal-to-noise ratios. Analysis of 17 reconstructions by semiautomatic density segmentation indicated that the active zone was constructed of a variable number of distinct "synaptic units" comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured approximately 60 nm in diameter, with a density of approximately 44/microm2 and were associated with vesicles at the active zone ("first tier"). Vesicles in this first-tier position represented approximately 7.5% of the total number of vesicles in the terminal and were contiguous, hemifused (approximately 4% of total), or fully fused (approximately 0.5% of total) to the plasma membrane. Our study supports the hypothesis that rat neocortical synapses are constructed of variable numbers of distinct synaptic units that facilitate the docking of vesicles to the active zone and determine the number of vesicles available for immediate release.

Protein kinase C-gamma activation in the early streptozotocin diabetic rat lens.

PURPOSE: The purpose of this study is to demonstrate the early activation of the protein kinase C-gamma (PKC-gamma) pathway in the streptozotocin (STZ)-induced diabetic rat lens. METHODS: Twelve-week-old male and female Sprague-Dawley rats were injected with 80 mg/kg (body weight) of STZ (N-[methylnitrosocarbamoyl]-D-glucosamine) intraperitoneally. Very high glucose (VHG) diabetes was defined as a nonfasting blood glucose level of at least 450 mg/dl, confirmed by daily monitoring with Accu-Check Advantage test strips, and occurred about 2 weeks after STZ administration. All assayed lenses were from VHG or age-matched control rats, harvested within 24 hr of VHG detection. PKC-gamma activation was measured by enzyme activity assay and by Western blotting to show autophosphorylation on Thr514. Cellular insulin-like growth factor-1 (IGF-1), PKC-gamma phosphorylation of Cx43 on Ser368, and activation of phospholipase C-gamma 1 (PLC-gamma 1), extracellular signal-regulated kinase (ERK1/2), and caspase-3 were determined by Western blotting. Endogenous diacylglycerol (DAG) levels were measured with a DAG assay kit. Lens gap junction activity was determined by the microinjection/Lucifer yellow dye transfer assay. Electron microscopy was applied to affirm fiber cell damage in the VHG diabetic lenses. RESULTS: In the lenses of VHG diabetic rats, PKC-gamma enzyme was activated. PKC-gamma could be further activated by 400 nM phorbol-12-myristate-13-acetate (PMA), but the PKC-gamma protein levels remained constant. No elevation of IGF-1 level was observed. Western blots showed that activation of PKC-gamma may be due to activation of PLC-gamma 1, which synthesized endogenous DAG, a native PKC activator. The level of PKC-gamma -catalyzed phosphorylation of Cx43 on Ser368 and resulting inhibition of lens gap junction dye transfer activity was increased in the VHG diabetic lenses. At this early time period, the diabetic lens showed no activation of either caspase-3 or ERK1/2. Only a single fiber cell layer deep within the cortex (approximately 90 cell layers from capsule surface) showed vacuoles and damaged cell connections. CONCLUSIONS: Early activation of PLC-gamma 1 and elevated DAG were observed within VHG diabetic lenses. These were correlated with activation of PKC-gamma, phosphorylation of Cx43 on Ser368, and inhibition of dye transfer. Abnormal signaling from PKC-gamma to Cx43 in the epithelial cells/early fiber cells, observed within VHG diabetic lenses, may be responsible for fiber cell damage deeper in the lens cortex.

PKCgamma knockout mouse lenses are more susceptible to oxidative stress damage.

Cataracts, or lens opacities, are the leading cause of blindness worldwide. Cataracts increase with age and environmental insults, e.g. oxidative stress. Lens homeostasis depends on functional gap junctions. Knockout or missense mutations of lens gap junction proteins, Cx46 or Cx50, result in cataractogenesis in mice. We have previously demonstrated that protein kinase Cgamma (PKCgamma) regulates gap junctions in the lens epithelium and cortex. In the current study, we further determined whether PKCgamma control of gap junctions protects the lens from cataractogenesis induced by oxidative stress in vitro, using PKCgamma knockout and control mice as our models. The results demonstrate that PKCgamma knockout lenses are normal at 2 days post-natal when compared to control. However, cell damage, but not obvious cataract, was observed in the lenses of 6-week-old PKCgamma knockout mice, suggesting that the deletion of PKCgamma causes lenses to be more susceptible to damage. Furthermore, in vitro incubation or lens oxidative stress treatment by H(2)O(2) significantly induced lens opacification (cataract) in the PKCgamma knockout mice when compared to controls. Biochemical and structural results also demonstrated that H(2)O(2) activation of endogenous PKCgamma resulted in phosphorylation of Cx50 and subsequent inhibition of gap junctions in the lenses of control mice, but not in the knockout. Deletion of PKCgamma altered the arrangement of gap junctions on the cortical fiber cell surface, and completely abolished the inhibitory effect of H(2)O(2) on lens gap junctions. Data suggest that activation of PKCgamma is an important mechanism regulating the closure of the communicating pathway mediated by gap junction channels in lens fiber cells. The absence of this regulatory mechanism in the PKCgamma knockout mice may cause those lenses to have increased susceptibility to oxidative damage.

Regulation of lens cell-to-cell communication by activation of PKCgamma and disassembly of Cx50 channels.

PURPOSE: Lens fiber gap junctions comprise approximately equal molar amounts of connexin46 (Cx46) and connexin50 (Cx50), both of which contribute significantly to coupling in the lens cortex and nucleus. The current study was conducted to test the hypothesis that regulation of lens coupling by activation of protein kinase Cgamma (PKCgamma) affects the number of channels composed of Cx46, Cx50, or both connexins. METHODS: Whole rat lenses were treated with phorbol-12-myristate-13-acetate (TPA) to activate PKCgamma or the inactive analogue 4alpha-phorbol,12,13-didecaneote (PDD) as a control. The superficial cortical fibers were studied morphologically by quantitative freeze-fracture immunolabeling (FRIL); functionally by Lucifer yellow dye transfer assay; and chemically by measuring PKCgamma activity, connexin phosphorylation and coimmunoprecipitation. RESULTS: Treatment with TPA activated PKCgamma and uncoupled the lens cortex by approximately 60%. PDD had no effect. Activation of PKCgamma decreased the density of Cx50 channels assembled in gap junctions, increased the density of Cx50 hemichannels in the plasma membrane and induced circular voids measuring 22 to 300 nm in diameter within the remaining plaques. Coimmunoprecipitation studies indicated that the soluble PKCgamma was translocated into membrane fractions that contained Cx46, Cx50, and the lipid raft marker caveolin (Cav)-1. In the membrane environment, PKCgamma phosphorylated Cx50 at serines and threonines and Cx46 only at threonines. CONCLUSIONS: The studies provide experimental support for the hypothesis that gap junctions comprising mixtures of Cx46 and Cx50 channels provide malleable communicating pathways between the lens nucleus and the metabolically active fibers in the surface. The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains.

Coupled sodium/glucose cotransport by SGLT1 requires a negative charge at position 454.

Na(+)/glucose cotransport by SGLT1 is a tightly coupled process that is driven by the Na(+) electrochemical gradient across the plasma membrane. We have previously proposed that SGLT1 contains separate Na(+)- and glucose-binding domains, that A166 (in the Na(+) domain) is close to D454 (in the sugar domain), and that interactions between these residues influence sugar specificity and transport. We have now expressed the mutant D454C in Xenopus laevis oocytes and examined the role of charge on residue 454 by replacing the Asp with Cys or His, and by chemical mutation of D454C with alkylating reagents of different charge (MTSES(-), MTSET(+), MMTS(0), MTSHE(0), and iodoacetate(-)). Functional properties were examined by measuring sugar transport and cotransporter currents. In addition, D454C was labeled with fluorescent dyes and the fluorescence of the labeled transporter was recorded as a function of voltage and ligand concentration. The data shows that (1) aspartate 454 is critically important for the normal trafficking of the protein to the plasma membrane; (2) there were marked changes in the functional properties of D454C, i.e., a reduction in turnover number and a loss of voltage sensitivity, although there were no alterations in sugar selectivity or sugar and Na(+) affinity; (3) a negative charge on residue 454 increased Na(+) and sugar transport with a normal stoichiometry of 2 Na(+):1 sugar. A positive charge on residue 454, in contrast, uncoupled Na(+) and sugar transport, indicating the importance of the negative charge in the coordination of the cotransport mechanism.

Distribution of connexin50 channels and hemichannels in lens fibers: a structural approach.

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.

Structure of functional single AQP0 channels in phospholipid membranes.

Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Two-dimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (approximately 73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.

Micro-domains of AQP0 in lens equatorial fibers.

To understand why the water channel aquaporin-0 (AQP0) replaces aquaporin-1(AQP1) during lens development, we studied its spatial arrangement and interactions with proteins in the plasma membrane of equatorial fibers. We used freeze-fracture-labelling; a method that can identify the individual intramembrane particle representing the AQP0 channel. We found that AQP0 was arranged in micro-domains that extended along the long axis of the equatorial fiber cell. One micro-domain consisted of AQP0 channels intermingled with the normal complement of integral proteins of the fiber plasma membrane. We found that the density of AQP0 channels varied along the long axis of the fiber. At the apical end of the fiber, the density was barely above background noise (approximately 50 microm(-2)). It increased first to 345=109 microm(-2) and then to 719+/-35 microm(-2) in the region of the plasma membrane facing adjacent fibers (the lateral surface). Another micro-domain, located at the apical end of the fiber, was composed of AQP0 channels within gap junctions ('mixed' junctions). This micro-domain contained approximately 1.5 x 10(5) cell-to-cell channels and approximately 3500 AQP0 channels. A third micro-domain, located exclusively in the lateral surface of the fiber, was composed of clusters of channels abutted against an opposing, particle-free plasma membrane (AQP0 junction). In equatorial fibers, the intramembrane particles in the AQP0 junctions were densely packed (6747+/-1007 microm(-2)), but were not arranged in orthogonal arrays that are characteristic of equaporins. This micro-domain occupied 20-25% of the lateral surface of equatorial fibers and, more importantly, it was arranged in 'ribbons' that extended for long stretches (30-40 microm) along the apical-basal axis. We concluded that the ability of AQP0 to arrange itself in micro-domains conferred functional properties that might contribute to the maintenance of lens transparency and homeostasis.

Nerve growth factor signals via preexisting TrkA receptor oligomers.

Nerve growth factor (NGF) promotes neuronal survival and differentiation by activating TrkA receptors. Similar to other receptor tyrosine kinases, ligand-induced dimerization is thought to be required for TrkA receptor activation. To study this process, we expressed TrkA receptors in Xenopus laevis oocytes and analyzed their response to NGF by using a combination of functional, biochemical, and structural approaches. TrkA receptor protein was detected in the membrane fraction of oocytes injected with TrkA receptor cRNA, but not in uninjected or mock-injected oocytes. Application of NGF to TrkA receptor-expressing oocytes promoted tyrosine phosphorylation and activated an oscillating transmembrane inward current, indicating that the TrkA receptors were functional. Freeze-fracture electron microscopic analysis demonstrated novel transmembrane particles in the P-face (protoplasmic face) of oocytes injected with TrkA cRNA, but not in uninjected or mock injected oocytes. Incubating TrkA cRNA-injected oocytes with the transcriptional inhibitor actinomycin D did not prevent the appearance of these P-face particles or electrophysiological responses to NGF, demonstrating that they did not arise from de novo transcription of an endogenous Xenopus oocyte gene. The appearance of these particles in the plasma membrane correlated with responsiveness to NGF as detected by electrophysiological analysis and receptor phosphorylation, indicating that these novel P-face particles were TrkA receptors. The dimensions of these particles (8.6 x 10 nm) were too large to be accounted for by TrkA monomers, suggesting the formation of TrkA receptor oligomers. Application of NGF did not lead to a discernible change in the size or shape of these TrkA receptor particles during an active response. These results indicate that in Xenopus oocytes, NGF activates signaling via pre-formed TrkA receptor oligomers.