The formation of complicated but precise neuronal circuits depends crucially on the spatial accuracy of axon growth and navigation. Each extending axon in the developing nervous system is tipped by a growth cone, a specialized structure that is able to interpret extracellular guidance cues and migrate along the correct path toward the appropriate post-synaptic target. Extracellular cues attract or repel a growth cone via localized elevations in cytosolic Ca2+ concentration on the side of the growth cone facing higher cue concentrations. My laboratory has found that the source of Ca2+ is a primary determinant of the growth cone turning direction: Ca2+ microdomains on the endoplasmic reticulum (ER) are sufficient to initiate attractive turning whereas Ca2+ influx from the extracellular space causes repulsive turning. We also have accumulated evidence that Ca2+-regulated membrane trafficking is a decisive effector process: centrifugal transport of membrane vesicles and their subsequent exocytosis drive attraction whereas membrane retrieval via endocytosis causes repulsion. Based on these results, we have proposed a model of axon guidance in which Ca2+-regulated membrane trafficking organizes the polarized subcellular distribution of driving machinery components that generate asymmetric traction and protrusive forces for growth cone turning (Nat Rev Neurosci 2011).
In this single-cell project, we have demonstrated how a growth cone can interpret Ca2+ microdomains on the ER and asymmetrically activate the membrane transport machinery for axon guidance (Cell Rep 2016). To tackle this problem, we combined two complementary approaches: single-cell imaging technology and proteomic analysis. We applied photo-activation/inhibition strategies to control molecular functions within a small subcellular region and visualize growth cone responses including molecular/organellar dynamics (Fig. 1). This study led to the identification of a protein complex that specifically detects ER-derived Ca2+ and controls asymmetric membrane vesicle export for growth cone attractive steering. The Ca2+-dependent motor protein myosin Va tethered VAMP2-positive vesicles to the ER via a common binding site on the two major ER Ca2+ channels, inositol 1,4,5-trisphosphate and ryanodine receptors. In response to attractive cues, Ca2+ release through these channels triggered myosin Va dissociation from the channels and the movement of freed vesicles to the cue side enabling growth cone attraction (Fig. 2). These findings revealed a peri-ER membrane export pathway for precise Ca2+-dependent turning in axon guidance.
We are further investigating subcellular dynamics of signaling systems including lipid mediators that polarize the growth cone for its navigational response during circuit formation. These revelations will help elucidate the fundamental principles of axon tract development and establish the molecular and cellular biological basis for axon tract repair after brain damage.