![Figure 6. 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.](https://cdn.rupress.org/rup/content_public/journal/jcb/182/6/10.1083_jcb.200802091/6/jcb_200802091_rgb_fig6.jpeg?Expires=1771596582&Signature=jKiOQhX81DtS1fT-2YmjsX8T5Kl49V4dAgqFWV5lk~TjDCUzFgnRlxfOMM74H~WgDyVYOpOxQ9zBnQE65JNnSjyYP3uEyQF-Fb~Zk866cA2UgvdLHfqviUOJofka0k6zsn2V2HGKzNa6W0ICTsCEQGbPRpj3ZB7rA37lpoF-9o5JsHjkiHHi42dE0SD8-0BurxmKtqq91fS1QttG1Px3QgBLdRcmDQU6QR~8qsozwaMZ43YvKkonrlREkXv078V2bm9iwm6-ycE1UIbY2Faon-HeV8vpOkqViFDGKW~O-eo8YI421KF6nWF6fQner-pL-4brwGuzd8NOe4YP2W3bUQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
