Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development.

VAMP is a synaptic vesicle membrane protein required for fusion. Synaptic vesicle targeting was measured for mutants of an epitope-tagged form of VAMP in transfected PC12 cells. A signal within a predicted amphipathic alpha helix is essential for targeting to synaptic vesicles. Cellubrevin, a nonneural VAMP homolog, contains this signal and is also targeted to synaptic vesicles. Amino acid substitutions within the synaptic vesicle targeting signal either enhance or inhibit sorting of VAMP to synaptic vesicles, but do not affect the ability of VAMP to form complexes with syntaxin and SNAP-25.

The C. elegans SAX-3/Robo receptor acts in anterior-posterior, dorsal-ventral and midline guidance decisions. Here we show that SAX-3 signaling involves the C. elegans Enabled protein UNC-34 and an unexpected Netrin-independent function of the Netrin receptor UNC-40/DCC. Genetic interactions with gain- and loss-of-function mutations suggest that unc-34 and unc-40 act together with sax-3 in several guidance decisions, but the C. elegans Netrin gene unc-6 does not act in the same genetic pathways. Within the migrating axon, sax-3, unc-34 and unc-40 all act cell-autonomously. Our results support a role for UNC-34/Enabled proteins in SAX-3-mediated repulsion, and show that UNC-40/DCC can potentiate SAX-3/Robo signaling via a mechanism that may involve direct binding of the two guidance receptors. A combinatorial logic dictates alternative functions for UNC-40/DCC, which can act in attraction to UNC-6/Netrin, repulsion from Netrin (with UNC-5), or repulsion from Slit (with SAX-3).

SUMMARY Neurons innervate multiple targets by sprouting axon branches from a primary axon shaft. We show here that the ventral guidance factor unc-6 (Netrin), its receptor unc-40 (DCC), and the gene madd-2 stimulate ventral axon branching in C. elegans chemosensory and mechanosensory neurons. madd-2 also promotes attractive axon guidance to UNC-6 and assists unc-6- and unc-40-dependent ventral recruitment of the actin regulator MIG-10 in nascent axons. MADD-2 is a tripartite motif protein related to MID-1, the causative gene for the human developmental disorder Opitz syndrome. MADD-2 and UNC-40 proteins preferentially localize to a ventral axon branch that requires their function; genetic results indicate that MADD-2 potentiates UNC-40 activity. Our results identify MADD-2 as an UNC-40 cofactor in axon attraction and branching, paralleling the role of UNC-5 in repulsion, and provide evidence that targeting of a guidance factor to specific axonal branches can confer differential responsiveness to guidance cues.

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A⅐SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and aminoand carboxyl-terminal regions of SNAP-25. Furthermore, separate amino-and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino-and carboxyl-terminal regions of SNAP-25 may function as independent domains.Neurotransmitter release represents a specialized form of regulated secretion wherein neurotransmitter-containing synaptic vesicles selectively dock and fuse with the plasma membrane. This process constitutes an essential step in chemical synaptic transmission and is a likely target for regulatory reactions that modulate the strength of synaptic signaling. The identification and biochemical characterization of numerous synaptic vesicle and presynaptic plasma membrane proteins have provided fundamental insight into the molecular mechanisms underlying neurotransmitter release (for reviews see Refs 1 and 2).Among the important findings that have emerged from molecular studies of neurotransmitter release is the central role of a protein complex composed of two presynaptic plasma membrane proteins, syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), 1 along with one synaptic vesicle protein, vesicle-associated membrane protein 2 (VAMP2; also known as synaptobrevin). Several lines of evidence suggest a primary role for syntaxin 1, SNAP-25, and VAMP2 in synaptic vesicle exocytosis. First, each represents a substrate for cleavage by distinct clostridial neurotoxins, toxins that irreversibly inhibit neurotransmitter release in vivo (3, 4). Furthermore, genetic studies in Drosophila have shown that loss of syntaxin 1 or VAMP2 prevents Ca 2ϩ -dependent exocytosis (5). Finally, the ternary complex formed by all three proteins acts as a receptor for soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins (SNAPs), cytosolic proteins required in multiple membrane trafficking events (6). Consequently, the syntaxin 1⅐SNAP-25⅐VAMP2 ternary complex is commonly referred to as the synaptic SNA...

The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosomeassociated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

Motor neuron identity and target selection are primarily genetically controlled. Significance and context Different subtypes of motor neurons not only display characteristic properties of cell migration and axon guidance, but also express unique combinations of LIM homeodomain transcription factors that may act as intrinsic regulators of these processes. For example, the MMCm (medial half of the median motor column) motor neurons, which innervate axial muscles and express the LIM genes Isl1, Lhx3 and others, occupy a ventral position in the spinal cord and project axons to axial muscles. However, the PGC (preganglionic motor column) motor neurons, which do not express Lhx3, migrate to settle along the intermediolateral edge of the spinal cord and project axons onto sympathetic neurons. Although studies have shown that displaced motor neurons project to the appropriate targets, the axons of neurons in a completely novel environment often passively follow the axonal pathways in that region. It is therefore apparent that both intrinsic and extrinsic mechanisms contribute to proper motor neuron connectivity, but little is known about their relative contributions in controlling target selection. Sharma et al. show that forcing all motor neurons to express the MMCm-specific LIM gene Lhx3 is sufficient to convert their cell migration, gene-expression profile, and axonal projections to that of the MMCm subtype of motor neurons. Despite the strong correlation between LIM gene expression and motor neuron identity, elevated occupancy of the MMCm axonal pathway can override the genetic program, causing some Lhx3-expressing axons to project to other targets. This suggests that environmental factors may also regulate target selection. Key results The authors converted all motor neurons to an axial-muscle-innervating MMCm identity by ectopically expressing Lhx3 under the control of a pan-motor-neuronal promoter in a transgenic mouse line. In these ectopic Lhx3-expressing embryos, Lhx4, another LIM gene normally found only in MMCm motor neurons, was expressed by all motor neurons. Likewise, markers for other motor neuron subtypes were downregulated. These results provide strong evidence that Lhx3 expression is sufficient to drive all motor neurons to display the molecular attributes of MMCm cells.

The chemotropic factor semaphorin 3A has been shown to act in both the axonal and dendritic guidance of cortical pyramidal neurons. Significance and context The axons and dendrites of a developing neuron must grow in the proper orientation in order to make the right connections with the appropriate targets. The mechanisms by which axons and dendrites achieve proper orientation and guidance during development are not fully known, especially in the case of dendrites. Diffusible extracellular signals affect axon guidance, as well as dendritic growth and branching, suggesting that such signals may also guide dendrites. Here, Polleux et al. provide the first evidence that different compartments of the same neuron can respond in opposite ways to an identical secreted signal. Axons of pyramidal neurons are repelled by a source of semaphorin 3A (Sema3A), whereas their dendrites are attracted. This suggests a new role for Sema3A, a member of a large family of secreted and transmembrane proteins once thought to be involved only in axonal chemorepulsion. This study is also one of the first to demonstrate, in a physiological setting, the role of cyclic nucleotides in neurite guidance. The results strongly suggest that the patterning of dendrites can be regulated by extracellular chemotropic cues, and that the different response of axons and dendrites to such cues can determine neuronal morphology. Key results Cortical pyramidal neurons extend apical dendrites towards the marginal zone of the developing cortex, while their axons project in the opposite direction, towards the white matter. Polleux et al. show that the majority of apical dendrites from pyramidal neurons plated on a cortical slice are preferentially oriented towards the marginal zone of the slice, and that this effect is dependent on the distance between the neuron and the marginal zone. These results support the role of a signal emanating from or near the marginal zone that acts to orient and attract dendrites. Evidence that this signal is Sema3A comes from SEMA3A null mice, in which axonal processes, normally repelled from a Sema3A source, project randomly. The authors show that SEMA3A null mice also have random orientation of dendritic processes. These results suggest that in normal circumstances a dendrite and an axon from a given cortical neuron behave in an opposite manner to the same cue secreted near the marginal zone: dendrites