Jonathan A. Raper

Collapsin: A protein in brain that induces the collapse and paralysis of neuronal growth cones

Repulsive guidance cues can steer neuronal growth cones during development and prevent mature axons from regenerating. We have identified a 100 kd glycoprotein in the chick brain that is a good candidate for a repulsive cue. Since it induces the collapse and paralysis of neuronal growth cones in vitro, we have named it collapsin. It is effective at concentrations of approximately 10 pM. The C-terminal half of collapsin contains a single immunoglobulin-like domain and an additional highly basic region. The N-terminal half of collapsin shares significant homology with fasciclin IV, a growth cone guidance protein in grasshopper. Recombinant collapsin causes sensory ganglion growth cones to collapse but not retinal ganglion cell growth cones. We propose that collapsin could serve as a ligand that guides specific growth cones by a motility-inhibiting mechanism.

Semaphorins and their receptors in vertebrates and invertebrates

The semaphorins are a family of intercellular signaling proteins that has grown to include 19 identified members in higher vertebrates. Several of its members act as axonal guidance molecules. One participates in signaling in the immune system. The majority, however, do not yet have known biological functions. Recent studies have shown that neuropilins and plexins act as receptors for semaphorins. The most important challenge for the future is to define the biological roles of semaphorins in vivo.

The enrichment of a neuronal growth cone collapsing activity from embryonic chick brain

We have devised a simple bioassay for the identification of molecules that inhibit growth cone motility. Chick dorsal root ganglion (DRG) growth cones extending on laminin collapse when exposed to a suspension of embryonic brain membranes. Detergent-solubilized membranes from which the detergent has been removed collapse DRG growth cones extending on either laminin or chick L1. Collapse occurs over a time course of minutes and is fully reversible. Solubilized liver, primary fibroblast, or RN22 schwannoma cell membranes do not collapse DRG or retinal growth cones. Solubilized PC12 membranes cause retinal but not DRG growth cones to collapse. The collapsing activity from embryonic brain is heat-labile, is trypsin-sensitive, and behaves as a macromolecule on a sizing column. It can be enriched 100-fold by chromatography on heparin and hydroxylapatite. These results are consistent with the idea that growth cone motility is inhibited by specific membrane-associated proteins in the developing nervous system.

Localized collapsing cues can steer growth cones without inducing their full collapse

Collapsing factors are proteins that induce growth cone collapse and paralysis when added in a soluble form to cultured embryonic neurons. Here we examine the responses of growth cones to localized collapsing signals. Temporal retinal ganglion cell growth cones exposed to a localized collapsing stimulus from nasal retinal ganglion cell axons frequently turn smoothly away from the axons without collapsing. Turning is rare on contact with retinal axons that are unable to induce collapse. In a separate series of experiments, dorsal root ganglion growth cones tend to turn away from beads coated with a brain extract enriched for the motility-inhibiting protein collapsin. Many turns are accomplished with filopodial contact alone. Growth cones do not turn away from control beads coated with heat-inactivated collapsin. These results suggest that inhibitory guidance cues can steer growth cones through a localized inhibition of lamellipodial protrusion.

A FAMILY OF MOLECULES RELATED TO COLLAPSIN IN THE EMBRYONIC CHICK NERVOUS SYSTEM

Signaling molecules with either attractive or repulsive effects on specific growth cones are likely to play a role in guiding axons to their appropriate targets. A chick brain glycoprotein, collapsin, has been shown to be a good candidate for a repulsive guidance cue. We report here the discovery of four new molecules related to collapsin in chick brains. All contain a semaphorin domain. One is structurally very similar to collapsin but is only 50% identical in its amino acid sequence. We have named it collapsin-2. The collapsin-related genes exhibit distinct but overlapping patterns of mRNA expression in the developing spinal cord and the developing visual system. This family of collapsin-related molecules could potentially act as repulsive cues toward specific neuronal populations.

DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension

We have identified a 95 kd cell surface protein, DM-GRASP, that is expressed on a restricted population of axons. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons. We have cloned and sequenced the cDNA corresponding to this protein and find that it is a new member of the immunoglobulin superfamily of adhesion molecules. Consequently we have named this protein DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein that is expressed in the dorsal funiculus and ventral midline of the chick spinal cord.

Secreted Chick Semaphorins Bind Recombinant Neuropilin with Similar Affinities but Bind Different Subsets of Neurons In Situ

Collapsin-1, a member of the semaphorin family, activates receptors on specific growth cones, thereby inhibiting their motility. Neuropilin, a previously cloned transmembrane protein, has recently been identified as a candidate receptor for collapsin-1. We have completed the cloning of chick collapsin-3 and -5 and show that collapsin-1, -2, -3, and -5 bind to overlapping but distinct axon tracts. We infer that in situ, there are distinct receptors with different affinities for collapsin-1, -2, -3, and -5. In contrast, these four collapsins all bind recombinant neuropilin with similar affinities. Strong binding to neuropilin is mediated by the carboxy third of the collapsins, while the semaphorin domain confers their unique binding patterns in situ. We propose that neuropilin is a common component of a semaphorin receptor complex, and that additional differentially expressed receptor components interact with the semaphorin domains to confer binding specificity.

Slit2 Is a Repellent for Retinal Ganglion Cell Axons

We set out to isolate inhibitory guidance cues that affect retinal ganglion cell (RGC) axons in vitro and that could potentially be involved in RGC pathfinding decisions. Here we describe the biochemical purification of an RGC growth cone collapsing factor from bovine brain membranes and its identification as Slit2. Recombinant human Slit2 collapses and repels RGC growth cones from all quadrants of the chick retina. In the developing mouse visual system, slit2 is expressed in the eye, in the optic stalk, and in the ventral diencephalon. Slit2 expression is strong in anterior ventral diencephalic structures but is absent from the ventral midline where the optic chiasm forms. The putative receptors for Slits, robo1 and robo2, are expressed in the inner retinal layer in which RGCs are located. A comparison of the expression patterns of Slit2 and retinal axon trajectories suggests that slit2 acts as a short range repellent for retinal ganglion cell axons.

A 70 Amino Acid Region within the Semaphorin Domain Activates Specific Cellular Response of Semaphorin Family Members

The semaphorin family contains secreted and transmembrane signaling proteins that function in the nervous, immune, and cardiovascular systems. Chick collapsin-1 is a repellent for specific growth cones. Two other secreted members of the semaphorin family, collapsin-2 and -3, are structurally similar to collapsin-1 but have different biological activities. Semaphorins contain a 500 amino acid family signature semaphorin domain. We show in this study that (1) the semaphorin domain of collapsin-1 is both necessary and sufficient for biological activity, (2) the semaphorin domain contains a 70 amino acid region that specifies the biological activity of the three family members, and (3) the positively charged carboxy terminus potentiates activity without affecting specificity. We propose that semaphorins interact with their receptors through two independent binding sites: one that mediates the biological response and one that potentiates it.

Neurofascin: A novel chick cell-surface glycoprotein involved in neurite-neurite interactions

We have identified neurofascin, a novel chick cell-surface glycoprotein involved in neurite-neurite interactions. Neurofascin is defined by its reactivity with monoclonal antibody (MAb) F6, which detects two polypeptides (160 and 185 kd) in immunotransfers of brain plasma membrane proteins. Immunoaffinity chromatography using immobilized MAb F6 yields major molecular mass bands at 185, 160, 135-110, and 92 kd. Fingerprint analyses show that these polypeptides are related. Neurofascin is expressed primarily in fiber-rich areas of embryonic cerebellum, spinal cord, and retina. Fab fragments of polyclonal antibodies to neurofascin interfere with the outgrowth of retinal and sympathetic axons in two different in vitro bioassays. Neurofascin is immunologically distinct from other known neurite-associated surface glycoproteins.