Guiding neuronal growth cones using Ca2+ signals

Abstract

Pathfinding by growing axons in the developing or regenerating nervous system is guided by gradients of molecular guidance cues. The neuronal growth cone, located at the ends of axons, uses surface receptors to sense these cues and to transduce guidance information to cellular machinery that mediates growth and turning responses. Cytoplasmic Ca2+ signals have key roles in regulating this motility. Global growth cone Ca2+ signals can regulate cytoskeletal elements and membrane dynamics to control elongation, whereas Ca2+ signals localized to one side of the growth cone can cause asymmetric activation of effector enzymes to steer the growth cone. Modulating Ca2+ levels in the growth cone might overcome inhibitory signals that normally prevent regeneration in the central nervous system.

Figure 1

Growth cone turning induced by diffusible factors. Phase-contrast images show Xenopus spinal neuron growth cones actively extending in culture during a 1-hr exposure to an external gradient (arrow) of netrin-1 (left) or MAG (middle and right). Assays were done in normal saline (left and middle) or low Ca²⁺ saline (with EGTA) after preloading the neuron with BAPTA-AM (right) to suppress Ca²⁺ signals. Cross hairs indicate the position of the growth cone central domain before the onset of the gradient. Assays were performed as previously reported [44]. Scale bar, 25 μm.

Figure 2

Localized Ca²⁺ signals in the growth cone induced by an extracellular gradient of the guidance cue netrin-1. Confocal images depict [Ca²⁺]_i in the growth cone of a cultured Xenopus spinal neuron after injection with the Ca²⁺-sensitive fluorescence indicator Oregon Green BAPTA-dextran. In this pseudocolor scheme, blue and white represent the lowest and highest [Ca²⁺]_i, respectively, and time (in min) before and after the onset of the netrin-1 gradient (arrows) is indicated. Imaging was done as previously published [43]. Scale bar, 10 μm. The full-length movie can be accessed by the internet (http://www.sciencedirect.com/science/journal/09628924).

Figure 3

The growth cone actin and microtubule cytoskeleton. Fluorescence image shows the distribution of F-actin (red) and microtubules (green) in the growth cone of a Xenopus spinal neuron that was extending in culture before being fixed and processed for immunofluorescence microscopy. Actin filaments (stained by rhodamine-phalloidin) are predominant in the peripheral domain, whereas bundles of microtubules (labeled with a tubulin antibody) localize to the central domain. Free microtubules may also penetrate into filopodia and lamellipodia (arrowheads). Scale bar, 10 μm. (Courtesy of Xiaobin Yuan and Ming Jin.)

Figure 4

Model for growth cone steering mediated by localized Ca²⁺ signals. An extracellular gradient of guidance cue (green) can induce localized Ca²⁺ signals on one side of the growth cone. The [Ca²⁺]_i is highest near open Ca²⁺ channels (red) in the plasma membrane and ER (pink), establishing a Ca²⁺ gradient across the growth cone. These localized Ca²⁺ signals activate Ca²⁺-sensitive kinases (e.g. CaMKII and PKC), phosphatases (e.g. calcineurin) and other effectors (e.g. Rho-family GTPases, see text and Tables 2 and 3), which in turn regulate the dynamics of actin- and microtubule-elements and membrane-bound vesicles (purple) to steer the growth cone. Higher amplitude Ca²⁺ signals trigger a local increase in cytoskeletal dynamics and filopodial protrusions to mediate attractive steering, whereas lower amplitude Ca²⁺ signals reduce filopodial activity, leading to repulsive steering.