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Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier.

Gatto CL, Walker BJ, Lambert S - J. Cell Biol. (2003)

Bottom Line: In the peripheral nervous system, axo-glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier.SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes.However, a role for these contacts in node formation remains controversial.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Program in Neuroscience, University of Massachusetts Medical School, 4 Biotech, 377 Plantation St., Suite 326, Worcester, MA 01605, USA.

ABSTRACT
Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo-glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial. Using a myelinating explant culture system, we have observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth cone-like behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar component-enriched "caps" and influences the efficiency of node formation.

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Cap formation is dependent on serum and can be promoted via Rho stimulation. At d 21, cultures were supplemented with either serum or ascorbate alone, maintained for 6 d (d27/x6), and stained for (A and C) EBP50 and (B and D) neurofilament. Cap formation was found to be serum dependent. This was also shown with DRG cultures transfected with EBP50-GFP. At d27/x6/t5, various culture conditions were examined as indicated with I–L showing higher magnifications of the outlined regions shown in E–H. EBP50-GFP was found at SC cap structures (arrows) in complete myelin feed (serum + ascorbate; F and J) and cultures supplemented with serum only (G and K). Cultures were also treated with 1 μM LPA to simulate serum stimulation of Rho pathways. Quantitation revealed the presence of serum induced a five- to sixfold increase in the number of caps as compared with standard feed or ascorbate alone. LPA was able to compensate for serum in both the presence and absence of ascorbate. The Rho-kinase inhibitor Y-27632 (Y 2–20 μM, as indicated) was able to block cap formation in the presence of LPA but not complete myelin feed. Data presented as mean ± SEM. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 423 ± 41 cells per coverslip were counted. Bars, 10 μm.
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fig6: Cap formation is dependent on serum and can be promoted via Rho stimulation. At d 21, cultures were supplemented with either serum or ascorbate alone, maintained for 6 d (d27/x6), and stained for (A and C) EBP50 and (B and D) neurofilament. Cap formation was found to be serum dependent. This was also shown with DRG cultures transfected with EBP50-GFP. At d27/x6/t5, various culture conditions were examined as indicated with I–L showing higher magnifications of the outlined regions shown in E–H. EBP50-GFP was found at SC cap structures (arrows) in complete myelin feed (serum + ascorbate; F and J) and cultures supplemented with serum only (G and K). Cultures were also treated with 1 μM LPA to simulate serum stimulation of Rho pathways. Quantitation revealed the presence of serum induced a five- to sixfold increase in the number of caps as compared with standard feed or ascorbate alone. LPA was able to compensate for serum in both the presence and absence of ascorbate. The Rho-kinase inhibitor Y-27632 (Y 2–20 μM, as indicated) was able to block cap formation in the presence of LPA but not complete myelin feed. Data presented as mean ± SEM. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 423 ± 41 cells per coverslip were counted. Bars, 10 μm.

Mentions: SC caps were observed to form in response to culture conditions that promoted myelination in DRG explant cultures, namely the addition of serum and ascorbate. To determine which of these components stimulated cap formation, cultures were independently supplemented with either serum or ascorbate. It was observed that the presence of serum was required for cap formation in studies of endogenous EBP50 staining (Fig. 6, A–D). Quantitative analysis of this phenomenon using EBP50-GFP transfection experiments (Fig. 6, E–L) revealed that the presence of serum induced a five- to sixfold increase in the number of EBP50-positive caps as compared with ascorbate alone. Furthermore, ascorbate alone was as ineffective as standard feed in inducing cap formation (2.8 ± 0.8% vs. 2.3 ± 0.6%, respectively), whereas serum alone produced similar results as compared with complete myelin feed (serum + ascorbate) in promoting cap formation (11.6 ± 0.7% vs. 14.2 ± 1.1%, respectively; Fig. 6).


Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier.

Gatto CL, Walker BJ, Lambert S - J. Cell Biol. (2003)

Cap formation is dependent on serum and can be promoted via Rho stimulation. At d 21, cultures were supplemented with either serum or ascorbate alone, maintained for 6 d (d27/x6), and stained for (A and C) EBP50 and (B and D) neurofilament. Cap formation was found to be serum dependent. This was also shown with DRG cultures transfected with EBP50-GFP. At d27/x6/t5, various culture conditions were examined as indicated with I–L showing higher magnifications of the outlined regions shown in E–H. EBP50-GFP was found at SC cap structures (arrows) in complete myelin feed (serum + ascorbate; F and J) and cultures supplemented with serum only (G and K). Cultures were also treated with 1 μM LPA to simulate serum stimulation of Rho pathways. Quantitation revealed the presence of serum induced a five- to sixfold increase in the number of caps as compared with standard feed or ascorbate alone. LPA was able to compensate for serum in both the presence and absence of ascorbate. The Rho-kinase inhibitor Y-27632 (Y 2–20 μM, as indicated) was able to block cap formation in the presence of LPA but not complete myelin feed. Data presented as mean ± SEM. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 423 ± 41 cells per coverslip were counted. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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fig6: Cap formation is dependent on serum and can be promoted via Rho stimulation. At d 21, cultures were supplemented with either serum or ascorbate alone, maintained for 6 d (d27/x6), and stained for (A and C) EBP50 and (B and D) neurofilament. Cap formation was found to be serum dependent. This was also shown with DRG cultures transfected with EBP50-GFP. At d27/x6/t5, various culture conditions were examined as indicated with I–L showing higher magnifications of the outlined regions shown in E–H. EBP50-GFP was found at SC cap structures (arrows) in complete myelin feed (serum + ascorbate; F and J) and cultures supplemented with serum only (G and K). Cultures were also treated with 1 μM LPA to simulate serum stimulation of Rho pathways. Quantitation revealed the presence of serum induced a five- to sixfold increase in the number of caps as compared with standard feed or ascorbate alone. LPA was able to compensate for serum in both the presence and absence of ascorbate. The Rho-kinase inhibitor Y-27632 (Y 2–20 μM, as indicated) was able to block cap formation in the presence of LPA but not complete myelin feed. Data presented as mean ± SEM. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 423 ± 41 cells per coverslip were counted. Bars, 10 μm.
Mentions: SC caps were observed to form in response to culture conditions that promoted myelination in DRG explant cultures, namely the addition of serum and ascorbate. To determine which of these components stimulated cap formation, cultures were independently supplemented with either serum or ascorbate. It was observed that the presence of serum was required for cap formation in studies of endogenous EBP50 staining (Fig. 6, A–D). Quantitative analysis of this phenomenon using EBP50-GFP transfection experiments (Fig. 6, E–L) revealed that the presence of serum induced a five- to sixfold increase in the number of EBP50-positive caps as compared with ascorbate alone. Furthermore, ascorbate alone was as ineffective as standard feed in inducing cap formation (2.8 ± 0.8% vs. 2.3 ± 0.6%, respectively), whereas serum alone produced similar results as compared with complete myelin feed (serum + ascorbate) in promoting cap formation (11.6 ± 0.7% vs. 14.2 ± 1.1%, respectively; Fig. 6).

Bottom Line: In the peripheral nervous system, axo-glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier.SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes.However, a role for these contacts in node formation remains controversial.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Program in Neuroscience, University of Massachusetts Medical School, 4 Biotech, 377 Plantation St., Suite 326, Worcester, MA 01605, USA.

ABSTRACT
Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo-glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial. Using a myelinating explant culture system, we have observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth cone-like behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar component-enriched "caps" and influences the efficiency of node formation.

Show MeSH
Related in: MedlinePlus