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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Time-course of process extension in the presence or absence of mSlit2.(A, B) Differentiated CAD cells were plated at low density and cultured for the indicated period of time in the absence or presence of recombinant mSlit2 (25 nM). Cells were fixed and stained for tubulin at three different time points (24 hr, 48 hr, 72 hr). Representative images are shown in (A), and quantification of the lengths of neurite-like processes is shown in (B). Bar, 200 µm.
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pone-0051895-g005: Time-course of process extension in the presence or absence of mSlit2.(A, B) Differentiated CAD cells were plated at low density and cultured for the indicated period of time in the absence or presence of recombinant mSlit2 (25 nM). Cells were fixed and stained for tubulin at three different time points (24 hr, 48 hr, 72 hr). Representative images are shown in (A), and quantification of the lengths of neurite-like processes is shown in (B). Bar, 200 µm.

Mentions: Further investigation revealed that Slit2 inhibited process extension in a dose-dependent manner (Figures 4A, 4B). When the lengths of processes in control and Slit2-treated cells were compared at 24, 48, and 72 hr after serum deprivation, the processes of Slit2-treated cells were shorter than those of the control cells at all time points (Figures 5A, 5B). Moreover, we observed that Slit2-treated cells continued to grow neurite-like processes throughout the time of serum deprivation, albeit at a slower rate compared to control cells (Figures 5A, 5B).


Slit2 inactivates GSK3β to signal neurite outgrowth inhibition.

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

Time-course of process extension in the presence or absence of mSlit2.(A, B) Differentiated CAD cells were plated at low density and cultured for the indicated period of time in the absence or presence of recombinant mSlit2 (25 nM). Cells were fixed and stained for tubulin at three different time points (24 hr, 48 hr, 72 hr). Representative images are shown in (A), and quantification of the lengths of neurite-like processes is shown in (B). Bar, 200 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526488&req=5

pone-0051895-g005: Time-course of process extension in the presence or absence of mSlit2.(A, B) Differentiated CAD cells were plated at low density and cultured for the indicated period of time in the absence or presence of recombinant mSlit2 (25 nM). Cells were fixed and stained for tubulin at three different time points (24 hr, 48 hr, 72 hr). Representative images are shown in (A), and quantification of the lengths of neurite-like processes is shown in (B). Bar, 200 µm.
Mentions: Further investigation revealed that Slit2 inhibited process extension in a dose-dependent manner (Figures 4A, 4B). When the lengths of processes in control and Slit2-treated cells were compared at 24, 48, and 72 hr after serum deprivation, the processes of Slit2-treated cells were shorter than those of the control cells at all time points (Figures 5A, 5B). Moreover, we observed that Slit2-treated cells continued to grow neurite-like processes throughout the time of serum deprivation, albeit at a slower rate compared to control cells (Figures 5A, 5B).

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