Calcium signaling in chemorepellant Slit2-dependent regulation of neuronal migration

Abstract

Migration of neuronal precursor cells in the developing brain is guided by extracellular cues, but intracellular signaling processes underlying the guidance of neuronal migration are largely unknown. By examining the migration of cerebellar granule neurons along the surface of cocultured astroglial cells, we found that an extracellular gradient of Slit2, a chemorepellant for neuronal migration in vivo, caused a reversal in the direction of migration without affecting the migration speed. A Slit2 gradient elevated the intracellular concentration of Ca 2؉ , probably due to calcium release from the internal store, led to a reversal of the preexisting asymmetric intracellular Ca 2؉ distribution in the soma of migrating neurons, and this reversal was closely related with its action of reversing the migrating direction. Asymmetric Ca 2؉ distribution in the soma was both necessary and sufficient for directing neuronal migration. These results have demonstrated an important role for Ca 2؉ in mediating neuronal responses to Slit2 and suggest a general mechanism for neuronal guidance. In the developing nervous system, a large number of neuronal precursor cells migrate from the site of birth to their destination along the surface of radial glial cells (1-3). Recent studies (4-6) have shown that several secreted or cell-surface proteins, including the netrins, Slits, and the Semaphorins can function as guidance cues for directing neuronal migration. However, most studies were performed on explants of brain tissue, and intracellular mechanisms mediating neuronal responses to these guidance cues are still poorly understood. Calcium signaling is known to be involved in regulating neuronal migration. In cultured explants of cerebellar tissue, granule cell migration correlates with intracellular Ca 2ϩ fluctuations and blockade of Ca 2ϩ influx retards migration (7-9). Voltage-gated Ca 2ϩ channels are essential for postembryonic neuronal migration in Caenorhabditis elegans (10). Slit2 is a well known secreted chemorepellant for migration of many types of neurons in the CNS (11-13). In the present study, the action of Slit2 on single-neuron migration in vitro was examined, and the role of calcium signal in mediating the repulsive action of Slit2 was explored. By using a cell culture model of migration of cerebellar granule cells on cocultured radial glial cells, we found that a gradient of Slit2 in front of the migrating neuron caused a reversal of the direction of migration without significantly affecting the subsequent migratory speed. We also found that a Slit2-induced Ca 2ϩ signaling appeared to mediate the repulsive action on neuronal migration, and that Ca 2ϩ release from intracellular store through inositol trisphosphate receptor channels played a major role in the Ca 2ϩ signaling. Materials and MethodsCoculture of Cells. The methods of coculturing granular cells and astroglia cells were similar to that described by Hatten (14). In short, astroglial cells from the cerebellum of P0-P3 SD rats isolated with a step gradient (35...

Intraislet SLIT–ROBO signaling is required for beta-cell survival and potentiates insulin secretion

“…We observed significant decreases in both ER stress-and serum starvation-induced cell death, but only under hyperglycemic conditions. Our data suggest that Ca 2+ -dependent mechanisms are important for the protective effects of Slit, which is in line with a role for Ca 2+ in Slit-Robo signaling in other cell types (24,25,27,28,(31)(32)(33). In particular, our results implicate a controlled depletion in ER Ca 2+ and an increase in the frequency of Ca 2+ oscillations.…”

“…Ca 2+ -dependent actin remodeling is induced by SLIT proteins during axon guidance (31,32). Treatment of mouse beta cells with recombinant SLIT1, SLIT2, or SLIT3 decreased cortical F-actin (Fig.…”

Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay

“…We next examined whether the distinct OEC subpopulations have different responses to the same migratory factor. Here, we developed a novel directional migration assay, which was adapted from an axon growth cone turning assay [35] and a single neuron migration assay [36]. To perform this assay, a microscopic gradient of migratory cue was produced by repetitive puffing of the solutions containing the candidate cues through a micropipette placed at a distance of 100 mm in front of the soma of migrating OECs to mimic the gradient of migratory cues in vivo.…”

“…The gradients of factors were produced as described previously [35][36][37] with some modifications. In brief, the purified OEC cells were re-plated onto square coverslips (8 mm) coated with laminin (10 µg/ml) at a low density of about 1 000 cells per coverslip.…”

Calcium Signaling in Neuronal Motility: Pharmacological Tools for Investigating Specific Pathways

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