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During neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP3) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo. Growth cones lacking the type 3 subunit are hypersensitive to nerve growth factor (NGF), an IP3-dependent attractive cue, and incapable of turning toward normal concentration ranges of NGF to which wild-type growth cones respond. This is due to globally, but not asymmetrically, activated Ca2+ signaling in the hypersensitive growth cones. Remarkably, lower NGF concentrations can polarize growth cones for turning if IP3 receptor type 3 is deficient. These data suggest a subtype-specific IP3 receptor function in sensitivity adjustment during axon navigation.

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During axon guidance, growth cones navigate toward attractive cues by inserting new membrane on the cue side. This process depends on Ca(2+) release from endoplasmic reticulum (ER) Ca(2+) channels, but the Ca(2+) sensor and effector governing this asymmetric vesicle export remain unknown. We identified a protein complex that controls asymmetric ER Ca(2+)-dependent membrane vesicle export. The Ca(2+)-dependent motor protein myosin Va (MyoVa) tethers membrane vesicles to the ER via a common binding site on the two major ER Ca(2+) channels, inositol 1,4,5-trisphosphate and ryanodine receptors. In response to attractive cues, micromolar Ca(2+) from ER channels triggers MyoVa-channel dissociation and the movement of freed vesicles to the cue side, enabling growth cone turning. MyoVa-Ca(2+) channel interactions are required for proper long-range axon growth in developing spinal cord in vivo. These findings reveal a peri-ER membrane export pathway for Ca(2+)-dependent attraction in axon guidance.

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Glycerophospholipids, the structural components of cell membranes, have not been considered to be spatial cues for intercellular signaling because of their ubiquitous distribution. We identified lyso-phosphatidyl-ß-D-glucoside (LysoPtdGlc), a hydrophilic glycerophospholipid, and demonstrated its role in modality-specific repulsive guidance of spinal cord sensory axons. LysoPtdGlc is locally synthesized and released by radial glia in a patterned spatial distribution to regulate the targeting of nociceptive but not proprioceptive central axon projections. Library screening identified the G protein-coupled receptor GPR55 as a high-affinity receptor for LysoPtdGlc, and GPR55 deletion or LysoPtdGlc loss of function in vivo caused the misallocation of nociceptive axons into proprioceptive zones. These findings show that LysoPtdGlc/GPR55 is a lipid-based signaling system in glia-neuron communication for neural development.

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During development, asymmetric Ca(2+) signals across the growth cone mediate bidirectional axon guidance depending on intracellular levels of cyclic AMP: Ca(2+) signals trigger attractive or repulsive turning when cyclic AMP levels are high or low, respectively. Here, we report that the cell adhesion molecule L1 elevates cyclic AMP levels in neurons via ankyrin(B), a protein that links the L1 cytoplasmic tail with the spectrin network. We also show that the loss of ankyrin(B) expression converts Ca(2+)-triggered attraction to repulsion when the growth cone migrates via an L1-dependent mechanism. These results indicate that ankyrin(B) regulates axon guidance via cyclic AMP.

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Axonal growth cones migrate along the correct paths during development, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Asymmetric Ca2+ elevations in the growth cone cytosol induce both attractive and repulsive turning in response to the guidance cues (Zheng, J.Q. 2000. Nature. 403:89-93; Henley, J.R., K.H. Huang, D. Wang, and M.M. Poo. 2004. Neuron. 44:909-916). Here, we show that CAMs regulate the activity of ryanodine receptor type 3 (RyR3) via cAMP and protein kinase A in dorsal root ganglion neurons. The activated RyR3 mediates Ca2+-induced Ca2+ release (CICR) into the cytosol, leading to attractive turning of the growth cone. In contrast, the growth cone exhibits repulsion when Ca2+ signals are not accompanied by RyR3-mediated CICR. We also propose that the source of Ca2+ influx, rather than its amplitude or the baseline Ca2+ level, is the primary determinant of the turning direction. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, which serves as a key regulator of growth cone navigation.

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The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin-actin network. This paper demonstrates that L1-CAM interactions with ankyrinB (but not with ankyrinG) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and ankyrinB continuously move toward the soma (retrograde flow). Bead-tracking experiments show that ankyrinB mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD-ankyrinB binding and the formation of stationary ankyrinB clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, ankyrinB is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that ankyrinB promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM.

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