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Myosin II has distinct functions in PNS and CNS myelin sheath formation.

Wang H, Tewari A, Einheber S, Salzer JL, Melendez-Vasquez CV - J. Cell Biol. (2008)

Bottom Line: We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL).In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II.Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065, USA.

ABSTRACT
The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

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Related in: MedlinePlus

Blebbistatin promotes OPC differentiation in vitro and increases the complexity of OL branching in cultures. (A) Purified OPC kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. Staining for MBP revealed a slight but significant increase in the percentage of MBP+ cells in myosin II–inhibited cultures. Examples of OL exhibiting extensive membrane expansion are indicated (arrowhead). Data in graph represent the mean ± SEM of four independent experiments (two cultures per condition per experiment). Bar, 20 μm. (B) Fractal analysis of OPC morphological complexity was performed in cultures kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. The mean fractal dimension (D) in blebbistatin-treated cultures was significantly higher than control (1.37 ± 0.12 [control] vs. 1.46 ± 0.08 [treated]; mean ± SD; P < 0.0001; t test), which indicates more complex cytoskeleton branching in the absence of myosin II activity. Representative examples of low- and high-complexity OPC stained with O4 are shown on the right. Bars, 10 μm. (C) Western blot of OPC cultures kept in proliferating media (PDGF) for 3 d or differentiating media (T3) for 2–7 d. A significant reduction in the levels of myosin II (isoforms A and B) was found between cultures kept in proliferating conditions compared with T3. As OPC differentiate, the levels of myosin II are further down-regulated. (D) Developmental expression profile of myosin II, actin-binding proteins, and MBP in rat brain lysates. The levels of myosin IIB and the phosphorylated regulatory chain (pMLC2) are down-regulated as myelination progresses in rat CNS, whereas total MLC2 remain unchanged. Total actin and Arp2 also remain constant, but the levels of WAVE1 and N-WASP, proteins that are involved in actin polymerization, are up-regulated at the onset of myelination (P15). In addition, the levels of p-cofilin are down-regulated at this stage, which is consistent with the role of the nonphosphorylated form in promoting actin polymerization.
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fig6: Blebbistatin promotes OPC differentiation in vitro and increases the complexity of OL branching in cultures. (A) Purified OPC kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. Staining for MBP revealed a slight but significant increase in the percentage of MBP+ cells in myosin II–inhibited cultures. Examples of OL exhibiting extensive membrane expansion are indicated (arrowhead). Data in graph represent the mean ± SEM of four independent experiments (two cultures per condition per experiment). Bar, 20 μm. (B) Fractal analysis of OPC morphological complexity was performed in cultures kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. The mean fractal dimension (D) in blebbistatin-treated cultures was significantly higher than control (1.37 ± 0.12 [control] vs. 1.46 ± 0.08 [treated]; mean ± SD; P < 0.0001; t test), which indicates more complex cytoskeleton branching in the absence of myosin II activity. Representative examples of low- and high-complexity OPC stained with O4 are shown on the right. Bars, 10 μm. (C) Western blot of OPC cultures kept in proliferating media (PDGF) for 3 d or differentiating media (T3) for 2–7 d. A significant reduction in the levels of myosin II (isoforms A and B) was found between cultures kept in proliferating conditions compared with T3. As OPC differentiate, the levels of myosin II are further down-regulated. (D) Developmental expression profile of myosin II, actin-binding proteins, and MBP in rat brain lysates. The levels of myosin IIB and the phosphorylated regulatory chain (pMLC2) are down-regulated as myelination progresses in rat CNS, whereas total MLC2 remain unchanged. Total actin and Arp2 also remain constant, but the levels of WAVE1 and N-WASP, proteins that are involved in actin polymerization, are up-regulated at the onset of myelination (P15). In addition, the levels of p-cofilin are down-regulated at this stage, which is consistent with the role of the nonphosphorylated form in promoting actin polymerization.

Mentions: To evaluate differentiation, OPC cultures were kept for 3 d in media supplemented with T3 with or without blebbistatin and then stained for MBP expression. Unlike OPC in coculture with neurons, we found a modest but significant increase in the percentage of cells expressing MBP in blebbistatin-treated cultures compared with control (40 ± 1.7% [control] vs. 49 ± 2% [treated]; mean ± SEM; P < 0.005; Fig. 6 A). OL in the blebbistatin-treated group also exhibit extensive membrane expansion (Fig. 6 A), a change that has been correlated with myelin sheath formation (Kachar et al., 1986; Kim et al., 2006; Sloane and Vartanian, 2007). We next examined the effects of blebbistatin treatment in OL morphological complexity and branching. We measured the fractal dimension of individual OL in control and blebbistatin-treated cultures. An increase in fractal dimension has been correlated with OL maturation in culture (Behar, 2001). We found that treatment with blebbistatin significantly increased the mean fractal dimension of OL in cultures (1.37 ± 0.12 [control] vs. 1.46 ± 0.08 [treated] mean ± SEM; P < 0.001; Fig. 6 B), which indicates more complex cytoskeleton branching in the absence of myosin II activity. Real-time studies also revealed a dramatic increase in protrusive activity along the OPC cell body and branches, with extensive formation of filopodia and ruffling lamellipodia. We also observed the development of new branches within 15–30 min of treatment with blebbistatin via the coalescence of some of these processes (Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200802091/DC1). These results suggest that myosin II inhibition influences OL morphogenesis by promoting complex branching and lamella formation, changes that have been correlated with myelin formation in vitro (Kachar et al., 1986) and in vivo (Kim et al., 2006; Sloane and Vartanian, 2007).


Myosin II has distinct functions in PNS and CNS myelin sheath formation.

Wang H, Tewari A, Einheber S, Salzer JL, Melendez-Vasquez CV - J. Cell Biol. (2008)

Blebbistatin promotes OPC differentiation in vitro and increases the complexity of OL branching in cultures. (A) Purified OPC kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. Staining for MBP revealed a slight but significant increase in the percentage of MBP+ cells in myosin II–inhibited cultures. Examples of OL exhibiting extensive membrane expansion are indicated (arrowhead). Data in graph represent the mean ± SEM of four independent experiments (two cultures per condition per experiment). Bar, 20 μm. (B) Fractal analysis of OPC morphological complexity was performed in cultures kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. The mean fractal dimension (D) in blebbistatin-treated cultures was significantly higher than control (1.37 ± 0.12 [control] vs. 1.46 ± 0.08 [treated]; mean ± SD; P < 0.0001; t test), which indicates more complex cytoskeleton branching in the absence of myosin II activity. Representative examples of low- and high-complexity OPC stained with O4 are shown on the right. Bars, 10 μm. (C) Western blot of OPC cultures kept in proliferating media (PDGF) for 3 d or differentiating media (T3) for 2–7 d. A significant reduction in the levels of myosin II (isoforms A and B) was found between cultures kept in proliferating conditions compared with T3. As OPC differentiate, the levels of myosin II are further down-regulated. (D) Developmental expression profile of myosin II, actin-binding proteins, and MBP in rat brain lysates. The levels of myosin IIB and the phosphorylated regulatory chain (pMLC2) are down-regulated as myelination progresses in rat CNS, whereas total MLC2 remain unchanged. Total actin and Arp2 also remain constant, but the levels of WAVE1 and N-WASP, proteins that are involved in actin polymerization, are up-regulated at the onset of myelination (P15). In addition, the levels of p-cofilin are down-regulated at this stage, which is consistent with the role of the nonphosphorylated form in promoting actin polymerization.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2542477&req=5

fig6: Blebbistatin promotes OPC differentiation in vitro and increases the complexity of OL branching in cultures. (A) Purified OPC kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. Staining for MBP revealed a slight but significant increase in the percentage of MBP+ cells in myosin II–inhibited cultures. Examples of OL exhibiting extensive membrane expansion are indicated (arrowhead). Data in graph represent the mean ± SEM of four independent experiments (two cultures per condition per experiment). Bar, 20 μm. (B) Fractal analysis of OPC morphological complexity was performed in cultures kept for 3 d in differentiation-promoting media in the absence or presence of 10 μM blebbistatin. The mean fractal dimension (D) in blebbistatin-treated cultures was significantly higher than control (1.37 ± 0.12 [control] vs. 1.46 ± 0.08 [treated]; mean ± SD; P < 0.0001; t test), which indicates more complex cytoskeleton branching in the absence of myosin II activity. Representative examples of low- and high-complexity OPC stained with O4 are shown on the right. Bars, 10 μm. (C) Western blot of OPC cultures kept in proliferating media (PDGF) for 3 d or differentiating media (T3) for 2–7 d. A significant reduction in the levels of myosin II (isoforms A and B) was found between cultures kept in proliferating conditions compared with T3. As OPC differentiate, the levels of myosin II are further down-regulated. (D) Developmental expression profile of myosin II, actin-binding proteins, and MBP in rat brain lysates. The levels of myosin IIB and the phosphorylated regulatory chain (pMLC2) are down-regulated as myelination progresses in rat CNS, whereas total MLC2 remain unchanged. Total actin and Arp2 also remain constant, but the levels of WAVE1 and N-WASP, proteins that are involved in actin polymerization, are up-regulated at the onset of myelination (P15). In addition, the levels of p-cofilin are down-regulated at this stage, which is consistent with the role of the nonphosphorylated form in promoting actin polymerization.
Mentions: To evaluate differentiation, OPC cultures were kept for 3 d in media supplemented with T3 with or without blebbistatin and then stained for MBP expression. Unlike OPC in coculture with neurons, we found a modest but significant increase in the percentage of cells expressing MBP in blebbistatin-treated cultures compared with control (40 ± 1.7% [control] vs. 49 ± 2% [treated]; mean ± SEM; P < 0.005; Fig. 6 A). OL in the blebbistatin-treated group also exhibit extensive membrane expansion (Fig. 6 A), a change that has been correlated with myelin sheath formation (Kachar et al., 1986; Kim et al., 2006; Sloane and Vartanian, 2007). We next examined the effects of blebbistatin treatment in OL morphological complexity and branching. We measured the fractal dimension of individual OL in control and blebbistatin-treated cultures. An increase in fractal dimension has been correlated with OL maturation in culture (Behar, 2001). We found that treatment with blebbistatin significantly increased the mean fractal dimension of OL in cultures (1.37 ± 0.12 [control] vs. 1.46 ± 0.08 [treated] mean ± SEM; P < 0.001; Fig. 6 B), which indicates more complex cytoskeleton branching in the absence of myosin II activity. Real-time studies also revealed a dramatic increase in protrusive activity along the OPC cell body and branches, with extensive formation of filopodia and ruffling lamellipodia. We also observed the development of new branches within 15–30 min of treatment with blebbistatin via the coalescence of some of these processes (Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200802091/DC1). These results suggest that myosin II inhibition influences OL morphogenesis by promoting complex branching and lamella formation, changes that have been correlated with myelin formation in vitro (Kachar et al., 1986) and in vivo (Kim et al., 2006; Sloane and Vartanian, 2007).

Bottom Line: We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL).In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II.Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065, USA.

ABSTRACT
The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

Show MeSH
Related in: MedlinePlus