Limits...
Slit2 inactivates GSK3β to signal neurite outgrowth inhibition.

Byun J, Kim BT, Kim YT, Jiao Z, Hur EM, Zhou FQ - PLoS ONE (2012)

Bottom Line: Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth.Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons.Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

ABSTRACT
Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

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Expression of Robo receptors in CAD cells.(A) Representative images of a differentiated CAD cell and a dorsal root gangion (DRG) neuron immunostained for tubulin and actin cytoskeleton. Note that in general, the cytoskeletal organization of a neurite-like process in a differentiated CAD cell is similar to that of a primary neuron. In both cells, microtubules labeled the neurite or the neurite-like process along the length, whereas the actin cytoskeleton was enriched at the distal end. Bar, 25 µm. (B) Undifferentiated and differentiated CAD cells were tested for expression of the robo gene using RT-PCR. The PCR products of robo1 and robo2 were sequenced to confirm their identity. (C) CAD cells were transiently transfected with an HA-tagged Robo1 construct. Immunostaining with anti-HA antibodies revealed that ectopically expressed Robo1 was enriched at the growth cone (arrows). The cells were co-transfected with green fluorescent protein (GFP) to illustrate cell morphology. Bar, 25 µm.
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pone-0051895-g001: Expression of Robo receptors in CAD cells.(A) Representative images of a differentiated CAD cell and a dorsal root gangion (DRG) neuron immunostained for tubulin and actin cytoskeleton. Note that in general, the cytoskeletal organization of a neurite-like process in a differentiated CAD cell is similar to that of a primary neuron. In both cells, microtubules labeled the neurite or the neurite-like process along the length, whereas the actin cytoskeleton was enriched at the distal end. Bar, 25 µm. (B) Undifferentiated and differentiated CAD cells were tested for expression of the robo gene using RT-PCR. The PCR products of robo1 and robo2 were sequenced to confirm their identity. (C) CAD cells were transiently transfected with an HA-tagged Robo1 construct. Immunostaining with anti-HA antibodies revealed that ectopically expressed Robo1 was enriched at the growth cone (arrows). The cells were co-transfected with green fluorescent protein (GFP) to illustrate cell morphology. Bar, 25 µm.

Mentions: CAD cell line is a variant of Cath.a, a CNS catecholaminergic cell line derived from a brain tumor that arose in a transgenic mouse [20]. In response to serum deprivation, CAD cells undergo neuronal differentiation by expressing neuron-specific biochemical markers, such as class III β-tubulin, GAP-43, and synaptotagmin [19]. CAD cells also undergo morphological differentiation upon serum-withdrawal by sending out long neurite-like processes that are tipped with growth cones (Figure 1A). We observed that in differentiated CAD cells, microtubules labeled neurite-like processes along their lengths, whereas the actin cytoskeleton was located primarily at the periphery, similar to the cytoskeletal organization of neurites from primary neurons (Figure 1A).


Slit2 inactivates GSK3β to signal neurite outgrowth inhibition.

Byun J, Kim BT, Kim YT, Jiao Z, Hur EM, Zhou FQ - PLoS ONE (2012)

Expression of Robo receptors in CAD cells.(A) Representative images of a differentiated CAD cell and a dorsal root gangion (DRG) neuron immunostained for tubulin and actin cytoskeleton. Note that in general, the cytoskeletal organization of a neurite-like process in a differentiated CAD cell is similar to that of a primary neuron. In both cells, microtubules labeled the neurite or the neurite-like process along the length, whereas the actin cytoskeleton was enriched at the distal end. Bar, 25 µm. (B) Undifferentiated and differentiated CAD cells were tested for expression of the robo gene using RT-PCR. The PCR products of robo1 and robo2 were sequenced to confirm their identity. (C) CAD cells were transiently transfected with an HA-tagged Robo1 construct. Immunostaining with anti-HA antibodies revealed that ectopically expressed Robo1 was enriched at the growth cone (arrows). The cells were co-transfected with green fluorescent protein (GFP) to illustrate cell morphology. Bar, 25 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526488&req=5

pone-0051895-g001: Expression of Robo receptors in CAD cells.(A) Representative images of a differentiated CAD cell and a dorsal root gangion (DRG) neuron immunostained for tubulin and actin cytoskeleton. Note that in general, the cytoskeletal organization of a neurite-like process in a differentiated CAD cell is similar to that of a primary neuron. In both cells, microtubules labeled the neurite or the neurite-like process along the length, whereas the actin cytoskeleton was enriched at the distal end. Bar, 25 µm. (B) Undifferentiated and differentiated CAD cells were tested for expression of the robo gene using RT-PCR. The PCR products of robo1 and robo2 were sequenced to confirm their identity. (C) CAD cells were transiently transfected with an HA-tagged Robo1 construct. Immunostaining with anti-HA antibodies revealed that ectopically expressed Robo1 was enriched at the growth cone (arrows). The cells were co-transfected with green fluorescent protein (GFP) to illustrate cell morphology. Bar, 25 µm.
Mentions: CAD cell line is a variant of Cath.a, a CNS catecholaminergic cell line derived from a brain tumor that arose in a transgenic mouse [20]. In response to serum deprivation, CAD cells undergo neuronal differentiation by expressing neuron-specific biochemical markers, such as class III β-tubulin, GAP-43, and synaptotagmin [19]. CAD cells also undergo morphological differentiation upon serum-withdrawal by sending out long neurite-like processes that are tipped with growth cones (Figure 1A). We observed that in differentiated CAD cells, microtubules labeled neurite-like processes along their lengths, whereas the actin cytoskeleton was located primarily at the periphery, similar to the cytoskeletal organization of neurites from primary neurons (Figure 1A).

Bottom Line: Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth.Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons.Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

ABSTRACT
Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

Show MeSH
Related in: MedlinePlus