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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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Efficient node formation is linked to cap formation. Cultures maintained with either ascorbate alone or complete myelin feed were stained for MBP, AnkG, and phospho-ERM. Myelin segments were identified and each nodal region associated was examined (A and D; closed arrowhead, left-most tip of segment; open arrowhead, right-most tip of segment). Each segment was classified based on the (E and F) presence or (B and C) absence of associated nodes. It was observed that serum stimulation in complete myelin feed resulted in a mean of 1.4 times more segments having two nodes, whereas ascorbate alone produced a fourfold increase in the mean number of segments formed in the absence of nodes. In addition, cultures were treated with LPA + ascorbate. Although LPA was able to moderately compensate for serum, there was a twofold increase in the mean number of segments without nodes and a 16% decrease in segments with two nodes. Finally, cultures were treated with complete myelin feed + Y-27632. Here, a threefold increase in the mean number of segments without nodes and a 30% decrease in segments with two nodes were observed. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 297 ± 41 (mean ± SEM) segments per coverslip were counted. Bars, 10 μm.
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fig7: Efficient node formation is linked to cap formation. Cultures maintained with either ascorbate alone or complete myelin feed were stained for MBP, AnkG, and phospho-ERM. Myelin segments were identified and each nodal region associated was examined (A and D; closed arrowhead, left-most tip of segment; open arrowhead, right-most tip of segment). Each segment was classified based on the (E and F) presence or (B and C) absence of associated nodes. It was observed that serum stimulation in complete myelin feed resulted in a mean of 1.4 times more segments having two nodes, whereas ascorbate alone produced a fourfold increase in the mean number of segments formed in the absence of nodes. In addition, cultures were treated with LPA + ascorbate. Although LPA was able to moderately compensate for serum, there was a twofold increase in the mean number of segments without nodes and a 16% decrease in segments with two nodes. Finally, cultures were treated with complete myelin feed + Y-27632. Here, a threefold increase in the mean number of segments without nodes and a 30% decrease in segments with two nodes were observed. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 297 ± 41 (mean ± SEM) segments per coverslip were counted. Bars, 10 μm.

Mentions: Polarization of microvillar components appears to be an early event in myelination. As microvilli are observed to form contacts with developing nodes of Ranvier, we examined whether SC cap formation was linked to the clustering of proteins at the nodal membrane. Cultures were induced to myelinate under various conditions and myelin segments with associated nodal clusters of ankyrinG detected by triple staining with antibodies to MBP, ankyrinG, and phospho-ERM (Fig. 7, A–F). 65.6 ± 3.6% of myelin segments exhibited associated ankyrinG staining at both ends of the segment in cultures treated with both serum and ascorbate. Cultures treated with ascorbate only exhibited 47.2 ± 3.6% of myelin segments with associated ankyrinG staining and a fourfold increase in the number of myelin segments that showed no associated ankyrinG staining (17.4 ± 2.8% of myelin segments vs. 4.1 ± 0.8% of segments in cultures treated with serum and ascorbate). In contrast with a previous paper (Eldridge et al., 1987), the average number of myelin segments formed in each culture appeared independent of serum. However, the addition of serum did result in segments of greater average length than those observed in ascorbate alone (Fig. 7, A and D). No clusters of ankyrinG staining were observed without accompanying phospho-ERM staining (Fig. 7, D–F).


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)

Efficient node formation is linked to cap formation. Cultures maintained with either ascorbate alone or complete myelin feed were stained for MBP, AnkG, and phospho-ERM. Myelin segments were identified and each nodal region associated was examined (A and D; closed arrowhead, left-most tip of segment; open arrowhead, right-most tip of segment). Each segment was classified based on the (E and F) presence or (B and C) absence of associated nodes. It was observed that serum stimulation in complete myelin feed resulted in a mean of 1.4 times more segments having two nodes, whereas ascorbate alone produced a fourfold increase in the mean number of segments formed in the absence of nodes. In addition, cultures were treated with LPA + ascorbate. Although LPA was able to moderately compensate for serum, there was a twofold increase in the mean number of segments without nodes and a 16% decrease in segments with two nodes. Finally, cultures were treated with complete myelin feed + Y-27632. Here, a threefold increase in the mean number of segments without nodes and a 30% decrease in segments with two nodes were observed. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 297 ± 41 (mean ± SEM) segments per coverslip were counted. Bars, 10 μm.
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Related In: Results  -  Collection

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fig7: Efficient node formation is linked to cap formation. Cultures maintained with either ascorbate alone or complete myelin feed were stained for MBP, AnkG, and phospho-ERM. Myelin segments were identified and each nodal region associated was examined (A and D; closed arrowhead, left-most tip of segment; open arrowhead, right-most tip of segment). Each segment was classified based on the (E and F) presence or (B and C) absence of associated nodes. It was observed that serum stimulation in complete myelin feed resulted in a mean of 1.4 times more segments having two nodes, whereas ascorbate alone produced a fourfold increase in the mean number of segments formed in the absence of nodes. In addition, cultures were treated with LPA + ascorbate. Although LPA was able to moderately compensate for serum, there was a twofold increase in the mean number of segments without nodes and a 16% decrease in segments with two nodes. Finally, cultures were treated with complete myelin feed + Y-27632. Here, a threefold increase in the mean number of segments without nodes and a 30% decrease in segments with two nodes were observed. n equals the number of coverslips analyzed from at least two independent cultures each having two DRGs per coverslip. An average of 297 ± 41 (mean ± SEM) segments per coverslip were counted. Bars, 10 μm.
Mentions: Polarization of microvillar components appears to be an early event in myelination. As microvilli are observed to form contacts with developing nodes of Ranvier, we examined whether SC cap formation was linked to the clustering of proteins at the nodal membrane. Cultures were induced to myelinate under various conditions and myelin segments with associated nodal clusters of ankyrinG detected by triple staining with antibodies to MBP, ankyrinG, and phospho-ERM (Fig. 7, A–F). 65.6 ± 3.6% of myelin segments exhibited associated ankyrinG staining at both ends of the segment in cultures treated with both serum and ascorbate. Cultures treated with ascorbate only exhibited 47.2 ± 3.6% of myelin segments with associated ankyrinG staining and a fourfold increase in the number of myelin segments that showed no associated ankyrinG staining (17.4 ± 2.8% of myelin segments vs. 4.1 ± 0.8% of segments in cultures treated with serum and ascorbate). In contrast with a previous paper (Eldridge et al., 1987), the average number of myelin segments formed in each culture appeared independent of serum. However, the addition of serum did result in segments of greater average length than those observed in ascorbate alone (Fig. 7, A and D). No clusters of ankyrinG staining were observed without accompanying phospho-ERM staining (Fig. 7, D–F).

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