Asymmetric Clathrin-Mediated Endocytosis Drives Repulsive Growth Cone Guidance

Abstract

Asymmetric Ca(2+) elevations across the axonal growth cone mediate its turning responses to attractive and repulsive guidance cues. Here we show that clathrin-mediated endocytosis acts downstream of Ca(2+) signals as driving machinery for growth cone turning. In dorsal root ganglion neurons, the formation of clathrin-coated pits is facilitated asymmetrically across the growth cone by a directionally applied chemorepellent, semaphorin 3A, or by Ca(2+) signals that mediate repulsive guidance. In contrast, coated pit formation remains symmetric in the presence of attractive Ca(2+) signals. Inhibition of clathrin-mediated endocytosis abolishes growth cone repulsion, but not attraction, induced by Ca(2+) or extracellular physiological cues. Furthermore, asymmetric perturbation of the balance of endocytosis and exocytosis in the growth cone is sufficient to initiate its turning toward the side with less endocytosis or more exocytosis. With our previous finding that growth cone attraction involves asymmetric exocytosis, we propose that the balance between membrane addition and removal dictates bidirectional axon guidance.

Calcium signaling in axon guidance

“…Tojima et al [17] found that calcium entry involving CICR and IICR produced an attractive response, whereas Ca 2+ signals without store entry invoked a repulsive response [17]. Furthermore, the method of calcium entry also affects axon outgrowth and growth cone motility [40,41].…”

“…A calmodulin-dependent protein kinase II (CaMKII)-calcineurin (CaN) switch often initiates either an attractive (turning towards the highest guidance cue concentration) or repulsive (turning away from the highest guidance cue concentration) response to these intracellular calcium elevations [8]. These responses are effected via the regulation of cytoskeletal components, such as microtubules and actin filaments [11][12][13][14][15], and by membrane dynamics, including vesicle trafficking [16][17][18]. In the past few years many new insights have emerged regarding the central and multifaceted roles of calcium in mediating axon growth and guidance.…”

Regulation of dendritic development by semaphorin 3A through novel intracellular remote signaling

“…60,62 Sema3A-induced growth cone collapse or repulsive turning was shown to be mediated through Ca 2C -triggered clathrin-mediated endocytosis. 60 Sema3A-induced growth cone collapse is suppressed by introduction of GSK3b mutants, GSK3b-R96A, L128A, or K85M, into DRG neurons, indicating that unprimed and primed substrates are involved in stimulating endocytosis of the growth cone plasma membrane. 63 Either Axin-1 or b-catenin RNAi knockdown suppresses the internalization of venus-tagged Sema3A.…”

“…55,56 Sema3A-induced growth cone collapse was inhibited by hanatoxin (a voltage-gated K C or Ca 2C channel blocker), 57 voltage-dependent L-type Ca 2C channel blockers such as nimodipine, and a reduction in extracellular Ca 2C . 54,56,[58][59][60] Sema3A enhances intracellular Ca 2C in the growth cones, 54,56,60,61 through which Sema3A can enhance both endocytosis and exocytosis. 60,62 Sema3A-induced growth cone collapse or repulsive turning was shown to be mediated through Ca 2C -triggered clathrin-mediated endocytosis.…”

“…54,56,[58][59][60] Sema3A enhances intracellular Ca 2C in the growth cones, 54,56,60,61 through which Sema3A can enhance both endocytosis and exocytosis. 60,62 Sema3A-induced growth cone collapse or repulsive turning was shown to be mediated through Ca 2C -triggered clathrin-mediated endocytosis. 60 Sema3A-induced growth cone collapse is suppressed by introduction of GSK3b mutants, GSK3b-R96A, L128A, or K85M, into DRG neurons, indicating that unprimed and primed substrates are involved in stimulating endocytosis of the growth cone plasma membrane.…”