Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix.

Mutations in dpix were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dpix encodes dPix, a Rho-type guanine nucleotide exchange factor (RtGEF) homologous to mammalian Pix. Here we show that dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations lead to decreased synaptic levels of the PDZ protein Dlg, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Thus, a Rho-type GEF and Rho-type effector kinase regulate postsynaptic structure.

Presynaptic target of Ca2+ action on neuropeptide and acetylcholine release in Aplysia californica.

1. When buccal neuron B2 of Aplysia californica is co-cultured with sensory neurons (SNs), slow peptidergic synapses are formed. When B2 is co-cultured with neurons B3 or B6, fast cholinergic synapses are formed. 2. Patch pipettes were used to voltage clamp pre- and postsynaptic neurons and to load the caged Ca2+ chelator o-nitrophenyl EGTA (NPE) and the Ca2+ indicator BTC into presynaptic neurons. The relationships between presynaptic [Ca2+]i and postsynaptic responses were compared between peptidergic and cholinergic synapses formed by cell B2. 3. Using variable intensity flashes, Ca2+ stoichiometries of peptide and acetylcholine (ACh) release were approximately 2 and 3, respectively. The difference did not reach statistical significance. 4. ACh quanta summate linearly postsynaptically. We also found a linear dose-response curve for peptide action, indicating a linear relationship between submaximal peptide concentration and response of the SN. 5. The minimum intracellular calcium concentrations ([Ca2+]i) for triggering peptidergic and cholinergic transmission were estimated to be about 5 and 10 microM, respectively. 6. By comparing normal postsynaptic responses to those evoked by photolysis of NPE, we estimate [Ca2+]i at the release trigger site elicited by a single action potential (AP) to be at least 10 microM for peptidergic synapses and probably higher for cholinergic synapses. 7. Cholinergic release is brief (half-width approximately 200 ms), even in response to a prolonged rise in [Ca2+]i, while some peptidergic release appears to persist for as long as [Ca2+]i remains elevated (for up to 10 s). This may reflect differences in sizes of reserve pools, or in replenishment rates of immediately releasable pools of vesicles. 8. Electron microscopy revealed that most synaptic contacts had at least one morphologically docked dense core vesicle that presumably contained peptide; these were often located within conventional active zones. 9. Both cholinergic and peptidergic vesicles are docked within active zones, but cholinergic vesicles may be located closer to Ca2+ channels than are peptidergic vesicles.

Highwire regulates synaptic growth in Drosophila.

The formation, stabilization, and growth of synaptic connections are dynamic and highly regulated processes. The glutamatergic neuromuscular junction (NMJ) in Drosophila grows new boutons and branches throughout larval development. A primary walking behavior screen followed by a secondary anatomical screen led to the identification of the highwire (hiw) gene. In hiw mutants, the specificity of motor axon pathfinding and synapse formation appears normal. However, NMJ synapses grow exuberantly and are greatly expanded in both the number of boutons and the extent and length of branches. These synapses appear normal ultrastructurally but have reduced quantal content physiologically. hiw encodes a large protein found at presynaptic terminals. Within presynaptic terminals, HIW is localized to the periactive zone surrounding active zones; Fasciclin II (Fas II), which also controls synaptic growth, is found at the same location.

Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position.

Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. Here we show that a combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region).

Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.

The glutamatergic neuromuscular junction (NMJ) in Drosophila adds new boutons and branches during larval development. We generated transgenic fruit flies that express a novel green fluorescent membrane protein at the postsynaptic specialization, allowing for repeated noninvasive confocal imaging of synapses in live, developing larvae. As synapses grow, existing synaptic boutons stretch apart and new boutons insert between them; in addition, new boutons are added at the ends of existing strings of boutons. Some boutons are added de novo, while others bud from existing boutons. New branches form as multiple boutons bud from existing boutons. Nascent boutons contain active zones, T bars, and synaptic vesicles; we observe no specialized growth structures. Some new boutons exhibit a lower level of Fasciclin II, suggesting that the levels of this synaptic cell adhesion molecule vary locally during synaptic growth.

Wrapper, a novel member of the Ig superfamily, is expressed by midline glia and is required for them to ensheath commissural axons in Drosophila.

The midline glia are specialized, nonneuronal cells at the midline of the Drosophila central nervous system (CNS). During development, the midline glia provide guidance cues for extending axons. At the same time, they migrate and help separate the two axon commissures. They then wrap around and ensheath the commissural axons. In many segments, a few of the glia do not enwrap the axons, and these cells die. The wrapper gene encodes a novel member of the immunoglobulin (Ig) superfamily. Wrapper protein is expressed specifically on the surface of midline glia. In wrapper mutant embryos, the midline glia express their normal guidance cues and migrate normally. However, they do not ensheath the commissural axons, and as a result, the glia die. In the absence of Wrapper, the two axon commissures are not properly separated.

Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors.

The robo gene in Drosophila was identified in a large-scale mutant screen for genes that control the decision by axons to cross the CNS midline. In robo mutants, too many axons cross and recross the midline. Here we show that robo encodes an axon guidance receptor that defines a novel subfamily of immunoglobulin superfamily proteins that is highly conserved from fruit flies to mammals. For those axons that never cross the midline, Robo is expressed on their growth cones from the outset; for the majority of axons that do cross the midline, Robo is expressed at high levels on their growth cones only after they cross the midline. Transgenic rescue experiments reveal that Robo can function in a cell-autonomous fashion. Robo appears to function as the gatekeeper controlling midline crossing.

Synaptic clustering of Fascilin II and Shaker: essential targeting sequences and role of Dlg.

Previous studies have shown that both the Fasciclin II (Fas II) cell adhesion molecule and the Shaker potassium channel are localized at the Drosophila neuromuscular junction, where they function in the growth and plasticity of the synapse. Here, we use the GAL4-UAS system to drive expression of the chimeric proteins CD8-Fas II and CD8-Shaker and show that the C-terminal sequences of both Fas II and Shaker are necessary and sufficient to drive the synaptic localization of a heterologous protein. Moreover, we show that the PDZ-containing protein Discs-Large (Dlg) controls the localization of these proteins, most likely through a direct interaction with their C-terminal amino acids. Finally, transient expression studies show that the pathway these proteins take to the synapse involves either an active clustering or a selective stabilization in the synaptic membrane.

Genetic analysis of myoblast fusion: blown fuse is required for progression beyond the prefusion complex.

The events of myoblast fusion in Drosophila are dissected here by combining genetic analysis with light and electron microscopy. We describe a new and essential intermediate step in the process, the formation of a prefusion complex consisting of "paired vesicles." These pairs of vesicles from different cells align with each other across apposed plasma membranes. This prefusion complex resolves into dense membrane plaques between apposed cells; these cells then establish cytoplasmic continuity by fusion of small areas of plasma membrane followed by vesiculation of apposed membranes. Different steps in this process are specifically blocked by mutations in four genes required for myoblast fusion. One of these genes, blown fuse, encodes a novel cytoplasmic protein expressed in unfused myoblasts that is essential for progression beyond the prefusion complex stage.

Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release.

Postsynaptic sensitivity to glutamate was genetically manipulated at the Drosophila neuromuscular junction (NMJ) to test whether postsynaptic activity can regulate presynaptic function during development. We cloned the gene encoding a second muscle-specific glutamate receptor, DGluRIIB, which is closely related to the previously identified DGluRIIA and located adjacent to it in the genome. Mutations that eliminate DGluRIIA (but not DGluRIIB) or transgenic constructs that increase DGluRIIA expression were generated. When DGluRIIA is missing, the response of the muscle to a single vesicle of transmitter is substantially decreased. However, the response of the muscle to nerve stimulation is normal because quantal content is significantly increased. Thus, a decrease in postsynaptic receptors leads to an increase in presynaptic transmitter release, indicating that postsynaptic activity controls a retrograde signal that regulates presynaptic function.

Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth.

The glutamatergic neuromuscular synapse in Drosophila forms and differentiates into distinct boutons in the embryo and grows by sprouting new boutons throughout larval life. We demonstrate that two axons form approximately 18 boutons on muscles 7 and 6 by hatching and grow to approximately 180 boutons by third instar. We further show that, after synapse formation, the homophilic cell adhesion molecule Fasciclin II (Fas II) is localized both pre- and postsynaptically where it controls synapse stabilization. In FasII null mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and resulting lethality are rescued by transgenes that drive Fas II expression both pre- and postsynaptically; driving Fas II expression on either side alone is insufficient. Fas II can also control synaptic growth; various FasII alleles lead to either an increase or decrease in sprouting, depending upon the level of Fas II.

Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity.

Increased neuronal activity (eag Shaker mutants) and cAMP concentration (dunce mutants) lead to increased synaptic structure and function at the Drosophila neuromuscular junction. Here, we show that the increase in synaptic growth is accompanied by an approximately 50% decrease in synaptic levels of the cell adhesion molecule Fasciclin II (Fas II). This decrease in Fas II is both necessary and sufficient for presynaptic sprouting; FasII mutants that decrease Fas II levels by approximately 50% lead to sprouting similar to eag Shaker and dunce, while transgenes that maintain synaptic Fas II levels suppress sprouting in eag Shaker and dunce. However, FasII mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons.

Genetic analysis of Laminin A in Drosophila: extracellular matrix containing laminin A is required for ocellar axon pathfinding.

Genetic analysis of the Laminin A (LamA) gene in Drosophila reveals that distinct classes of sensory axons have different requirements for extracellular matrix (ECM) containing laminin A versus epithelial cell surfaces. In the eye-antenna imaginal disc, the nerve from the three simple eyes (ocelli) to the brain is pioneered by a population of transient ocellar neurons whose axons extend on an ECM that covers and connects the disc epithelium and brain. Axons from neighboring mechanosensory (bristle) neurons extend under the ECM in direct contact with the surface of the disc cells, and pioneer a different axon pathway that enters the brain in a different location. In LamA mutants, the ocellar pioneer axons display striking pathfinding defects, while neighboring bristle axons appear normal; the ocellar pioneers usually extend in the proper direction, adhering to the epithelium and sometimes fasciculating with mechanosensory axons, but they invariably fail to reach the brain.

glial cells missing: a genetic switch that controls glial versus neuronal fate.

The glial cells missing (gcm) gene in Drosophila encodes a novel nuclear protein that is transiently expressed early in the development of nearly all glia. In loss-of-function gcm mutant alleles, nearly all glia fail to differentiate, and, where we can follow them in the PNS, are transformed into neurons. In gain-if-function gcm conditions using transgenic constructs that drive ectopic gcm expression, many presumptive neurons are transformed into glia. Thus, gcm appears to function as a binary genetic switch for glia versus neurons. In the presence of gcm protein, presumptive neurons become glia, while in its absence, presumptive glia become neurons.

Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila.

Peripheral glia help ensure that motor and sensory axons are bathed in the appropriate ionic and biochemical environment. In Drosophila, peripheral glia help shield these axons against the high K+ concentration of the hemolymph, which would largely abolish their excitability. Here, we describe the molecular genetic analysis of gliotactin, a novel transmembrane protein that is transiently expressed on peripheral glia and that is required for the formation of the peripheral blood-nerve barrier. In gliotactin mutant embryos, the peripheral glia develop normally in many respects, except that ultrastructurally and physiologically they do not form a complete blood-nerve barrier. As a result, peripheral motor axons are exposed to the high K+ hemolymph, action potentials fail to propagate, and the embryos are nearly paralyzed.

Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation.

The Drosophila neural cell adhesion molecule Fasciclin II (Fas II) is expressed dynamically on a subset of embryonic CNS axons, many of which selectively fasciculate in the vMP2, MP1, and FN3 pathways. Here we show complementary fasII loss-of-function and gain-of-function phenotypes. Loss-of-function fasII mutations lead to the complete or partial defasciculation of all three pathways. Gain-of-function conditions, using a specific control element to direct increased levels of Fas II on the axons in these three pathways, rescue the loss-of-function phenotype. Moreover, the gain-of-function can alter fasciculation by abnormally fusing pathways together, in one case apparently by preventing normal defasciculation. These results define an in vivo function for Fas II as a neuronal recognition molecule that controls one mechanism of growth cone guidance-selective axon fasciculation--and genetically separates this function from other aspects of outgrowth and directional guidance.

Three-dimensional membrane crystals in amphibian cone outer segments. 1. Light-dependent crystal formation in frog retinas.

When frog retinas are exposed to light, a series of three-dimensional crystals develop within the outer segment disk system of cones but not rods. The crystals involve components that span cytoplasmic, disk membrane, and intradisk domains of the outer segment. The crystalline membrane domains are directly continuous with adjacent, noncrystalline lamellar regions. In axial extent, the crystals may involve as few as 1 or 2 disks or as many as 30 disks. However, within each disk, only one crystalline domain typically is observed. Within a crystal, the membranes are more planar in shape and more uniform in axial spacing then adjacent, noncrystalline lamellar regions. Furthermore, as crystalline domains expand laterally, one observes increased axial spacing disorder in noncrystalline lamellar regions, along with an increase in the width of the intradisk compartment. Thus, crystals appear to grow laterally by depleting adjacent lamellar regions of components that influence the normal membrane pair separation and axial spacing of cone outer segment disks. In isolated retinas, the crystalline domains appear to be randomly distributed along the length of the outer segment and show no preference for association with either the closed or open margins of the disk. After 45 min in the light, the crystals occupy approximately 10% of the cone outer segment volume. On the basis of comparative structural, biochemical, and physiological data, cone outer segment crystals may represent a cocrystal between bleached, phosphorylated opsin (providing transmembrane and intradisk elements) and the cytoplasmic protein, arrestin (providing trans-cytoplasmic elements). Thus, crystal formation may provide one mechanism of light adaptation within the cone outer segment. The spontaneous, bleaching-induced formation of these crystals in situ offers the possibility that cocrystals of cone outer segment components can be prepared in vitro for higher resolution crystallographic analyses.

Modulation of disk margin structure during renewal of cone outer segments in the vertebrate retina.

In the process of disk renewal in retinal cone outer segments (COSs), apical displacement of disks must be coupled to systematic reductions in disk area and perimeter in order to retain overall conical geometry. We have quantified these changes in disk area and perimeter segment lengths by morphometric analyses of cross sections of fully formed disks taken from basal to apical ends of COSs. Specifically excluded from these analyses are data arising from partial or incomplete disks within the COS, which do not conform to the conical geometry and which constitute a minor fraction of the COS disk population. Thus, our results address the long-range pattern of structural changes affecting the major population of disks along the length of the COS. Our data indicate that decreases in total disk margin length associated with apical displacement of fully formed disks are due to decreases in the length of the margin opposite the cilium, i.e., the open margin segment. In contrast, the average length of the closed margin segment remains constant or increases slightly in the apical direction. The open margins of frog COS disks have recently been shown to possess a distinctive lattice of membrane-associated components (Fetter and Corless: Invest. Ophthalmol. Vis. Sci. 28:646-657, '87). We have also examined COSs by the freeze-fracture, deep-etch technique for evidence of a mechanism whereby measured changes in open margin length may be accommodated while maintaining the overall organization of the open margin segments. In regions of membrane continuity between open margins and the COS plasma membrane, we have observed elevated ridges on the plasma membrane that 1) tend to lie parallel to the open margin segments, 2) have a similar axial spacing, 3) occasionally demonstrate interconnecting filaments similar to those of the open margin lattice, and 4) appear to have a particulate substructure. The mechanism proposed for reducing open margin length involves tangential displacement of the lateral edges of the open margin lattice to the adjacent plasma membrane. These shifted lattice domains initially give rise to the plasmalemmal ridges, which subsequently disassemble, and whose components become redistributed in the COS plasma membrane. These structural features of COS open margins suggest several revisions of our earlier model of disk morphogenesis (Corless and Fetter: J. Comp. Neurol. 257:24-38, '87), which was based on the margin structure of ROS disks alone. Eckmiller (J. Cell Biol. 105:2267-2277, '87) has recently proposed that partial-disks observed within the COS represent sites of new disk formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase.

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.

Morphological components associated with frog cone outer segment disc margins.

The two margin morphologies of frog retinal cone outer segment (COS) discs were examined by thin sectioning, freeze-fracture, and deep-etch, rotary shadowing techniques. The disc margin adjacent to the connecting cilium, which morphologically resembles the terminal loop of rod outer segment (ROS) discs, exhibited a distinctive staining density tightly apposed to the membrane surface facing the lumen. This density was crescent-shaped in longitudinal sections, and a continuous band in cross-sections. Associated with the disc margin opposite the cilium, two additional extracellular structures were observed: a globular staining density located at the outer edge of the membrane loop forming the margin, and filaments axially interconnecting adjacent margins. The globular densities and filaments were spaced at regular intervals along the margin. Where these margins were adjacent to calycal processes, the globular densities appeared to span the extracellular gap and interconnect the membranes of the COS discs and the calycal process. Distinctive intramembrane particles were observed along both margins by freeze-fracture. The distribution of the globular and filamentous elements suggests that they may have a role in maintaining the radial dimensions and axial spacing of the associated disc margin by forming an extracellular framework.