Richard D. Fetter

Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons.

Most neurons form synapses exclusively with other neurons, but little is known about the molecular mechanisms mediating synaptogenesis in the central nervous system. Using an in vitro system, we demonstrate that neuroligin-1 and -2, postsynaptically localized proteins, can trigger the de novo formation of presynaptic structure. Nonneuronal cells engineered to express neuroligins induce morphological and functional presynaptic differentiation in contacting axons. This activity can be inhibited by addition of a soluble version of beta-neurexin, a receptor for neuroligin. Furthermore, addition of soluble beta-neurexin to a coculture of defined pre- and postsynaptic CNS neurons inhibits synaptic vesicle clustering in axons contacting target neurons. Our results suggest that neuroligins are part of the machinery employed during the formation and remodeling of CNS synapses.

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.

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

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.

wishful thinking Encodes a BMP Type II Receptor that Regulates Synaptic Growth in Drosophila

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.

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.

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.

GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems.

The identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed GRASP, a system to label membrane contacts and synapses between two cells in living animals. Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.

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.

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.