Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Previously, we demonstrated that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Here, we identify Numb as a CRMP-2-interacting protein. Numb has been shown to interact with alpha-adaptin and to be involved in endocytosis. We found that Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb were colocalized. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibited endocytosis of L1 at axonal growth cones and suppressed axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1.

Cell-cell interactions mediated via cell adhesion molecules (CAMs) are dynamically regulated during nervous system development. One mechanism to control the amount of cell surface CAMs is to regulate their recycling from the plasma membrane. The L1 subfamily of CAMs has a highly conserved cytoplasmic domain that contains a tyrosine, followed by the alternatively spliced RSLE (Arg-Ser-Leu-Glu) sequence. The resulting sequence of YRSL conforms to a tyrosine-based sorting signal that mediates clathrin-dependent endocytosis of signal-bearing proteins. The present study shows that L1 associates in rat brain with AP-2, a clathrin adaptor that captures plasma membrane proteins with tyrosine-based signals for endocytosis by coated pits. In vitro assays demonstrate that this interaction occurs via the YRSL sequence of L1 and the mu 2 chain of AP-2. In L1-transfected 3T3 cells, L1 endocytosis is blocked by dominant-negative dynamin that specifically disrupts clathrin-mediated internalization. Furthermore, endocytosed L1 colocalizes with the transferrin receptor (TfR), a marker for clathrin-mediated internalization. Mutant forms of L1 that lack the YRSL do not colocalize with TfR, indicating that the YRSL mediates endocytosis of L1. In neurons, L1 is endocytosed preferentially at the rear of axonal growth cones, colocalizing with Eps15, another marker for the clathrin endocytic pathway. These results establish a mechanism by which L1 can be internalized from the cell surface and suggest that an active region of L1 endocytosis at the rear of growth cones is important in L1-dependent axon growth.

L1 is a neural cell adhesion molecule mainly involved in axon guidance and neuronal migration during brain development. Mutations in the human L1 gene give rise to a complex clinical picture, with mental retardation, neurologic abnormalities and a variable degree of hydrocephalus. Recently, a transgenic mouse model with a targeted null mutation in the L1 gene was generated. These knockout (KO) mice show hypoplasia of the corticospinal tract. Here we have performed further studies of these KO mice including magnetic resonance imaging of the brain, neuropathological analysis and behavioral testing. The ventricular system was shown to be abnormal with dilatation of the lateral ventricles and the 4th ventricle, and an altered shape of the Sylvius aqueduct. Additionally, the cerebellar vermis of the KO mice is hypoplastic. Their exploratory behavior is characterized by stereotype peripheral circling reminiscent of that of rodents with induced cerebellar lesions.

L1-mediated axon growth involves intracellular sig

Graded distributions of extracellular cues guide developing axons toward their targets. A network of second messengers, Ca2+ and cyclic nucleotides, shapes cue-derived information into either attractive or repulsive signals that steer growth cones bidirectionally. Emerging evidence suggests that such guidance signals create a localized imbalance between exocytosis and endocytosis, which in turn redirects membrane, adhesion and cytoskeletal components asymmetrically across the growth cone to bias the direction of axon extension. These recent advances allow us to propose a unifying model of how the growth cone translates shallow gradients of environmental information into polarized activity of the steering machinery for axon guidance.