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First published online 8 May 2007
doi: 10.1242/jcs.03448

Journal of Cell Science 120, 1877-1887 (2007)
Published by The Company of Biologists 2007
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Nogo enhances the adhesion of olfactory ensheathing cells and inhibits their migration

Zhida Su*, Li Cao*, Yanling Zhu, Xiujie Liu, Zhihui Huang, Aijun Huang and Cheng He{ddagger}

Department of Neurobiology, Second Military Medical University, Shanghai 200433, China


Figure 1
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  		Fig. 1. Nogo-66 inhibits the migration of OECs. (a-e) Photomicrograph of cultured OECs that have transmigrated through transwell membranes coated either with laminin (e, control) or with various proteins (100 µg/ml each, diluted in laminin): GST (a), GST–Nogo-66 (b), His (c) and His–Nogo-66 (d). (f) Quantitative assessment of cells transmigrated through the transwell membranes. Data are presented as mean ± s.d./visual field. **P<0.01 versus laminin.

 

Figure 2
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  		Fig. 2. Nogo-66 enhances the adhesion of OECs. (A) Photomicrographs of OECs plated for 3 hours, 6 hours and 12 hours on spots of 100 µg/ml GST, GST–Nogo-66, His or His–Nogo-66, respectively. (B) Quantitative assessment of the number of cells attaching to the protein spots at various time points. Values are reported as mean ± s.d./visual field. *P<0.05, **P<0.01 versus vehicle. (C) Enhancement of adhesion of OECs by Nogo-66 was dose-dependent. After 6 hours, OECs attached to GST, GST–Nogo-66, His or His–Nogo-66 spotted for a series of concentrations (µg/ml) were quantitatively assessed.

 

Figure 3
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  		Fig. 3. NgR is expressed on plasma membrane of cultured OECs. (A) OECs were double-stained with antibodies against S-100 and NgR after treatment with Triton X-100 (a-c). OECs (d) and Schwann cells (e) were surface stained (without treatment with Triton X-100) with anti-NgR antibody. (B) Cell lysates from SCs (negative control), OECs and PC12 (positive control) cells were subjected to immunoblotting with anti-NgR (upper panel) and anti-GAPDH (lower panel). The arrowhead points to NgR (80 kDa). (C) Immunostaining for NgR on plasma membrane of OECs before (–) and after (+) treatment with PI-PLC (0.1 U/ml) at 37°C for 2 hours. (D) The supernatant (S) and membrane fractions (P) of OECs treated (+) or untreated (–) with PI-PLC (0.1 U/ml) at 37°C for 40 minutes were subjected to immunoblotting with anti-NgR (upper panel) and anti-GAPDH (lower panel). Bars, 50 µm.

 

Figure 4
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  		Fig. 4. The effects of Nogo-66 on the migration and adhesion of OECs are mediated by NgR. (A) OECs were pre-treated with or without PI-PLC (0.1 U/ml), NgR antibody (1:100) or goat IgG (1:100) for 2 hours and applied to Boyden chamber assays. The trans-well membranes were coated with laminin, GST, GST–Nogo-66, His or His–Nogo-66 (100 µg/ml each). SCs were served as a negative control. (B) After pre-treatment with or without either PI-PLC, NgR antibody or goat IgG, OECs or SCs were plated for 6 hours on spots of GST, GST–Nogo-66, His or His–Nogo-66. Quantitative assessment was obtained by determining the percentage of the cell numbers within the spots compared with outside the spots (vehicle, 100%). (C) Dose-dependency of the effect of PI-PLC. After being exposed to PI-PLC at the indicated concentration for 2 hours, cells were harvested and used in an adhesion assay. Values were reported as mean ± s.d./visual field. **P<0.01 versus laminin or vehicle.

 

Figure 5
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  		Fig. 5. RhoA activation is critical for mediating the effect of Nogo-66 on OECs. (A,B) Y-27632 treatment significantly attenuates the enhancement of Nogo-66 on adhesion of OECs. OECs (a,c,e,g,i) and Y-27632-treated OECs (b,d,f,h,j) were plated on dishes coated with laminin (a,b), GST (c,d), GST–Nogo-66 (e,f), His (g,h) or His–Nogo-66 (i,j). After incubation for 30 minutes with gentle shaking, adherent cells were photographed and counted. The values were normalized with respect to the values obtained for laminin. (C) Y-27632 treatment significantly attenuates the inhibition of Nogo-66 on migration of OECs. OECs were pre-treated with or without Y-27632 and applied to Boyden chamber assays. The trans-well membranes were coated with laminin, GST, GST–Nogo-66, His or His–Nogo-66 (100 µg/ml each). Values were reported as mean ± s.d./visual field. *P<0.05, **P<0.01 versus laminin or vehicle. (D) RhoA activation in OECs was assessed with pull-down assays with the Rho-binding domain of rhotekin. The amount of GTP-bound RhoA was normalized to the amount of total RhoA. Results are means ± s.d. from three independent experiments. *P<0.05 versus GST or His. (E) Nogo-66 promotes the formation of focal adhesions and inhibits membrane protrusion of OECs through RhoA activation. OECs and Y-27632-treated OECs were plated for 60 minutes on coverslips coated with GST (a-d), GST–Nogo-66 (e-l), His (m-p) or His–Nogo-66 (q-x). Cultures were then fixed, permeabilized, and stained with anti-paxillin to visualize focal adhesions (green) and rhodamine-conjugated phalloidin to visualize filamentous actin (red). Arrows (in c,k,o,w) indicate the thick bundles of actin in the membrane protrusions of OECs. Arrowheads (in h,t) indicate the stress fibers terminating at the adhesion plaques. Bar, 10 µm. (F) Quantification of the paxillin punctae. The number of punctae/cell were counted for 40 cells selected randomly in each group. The number of punctae was normalized by comparing with GST or His, and the percentage of punctae in control and treated cells was compared. **P<0.01.

 

Figure 6
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  		Fig. 6. Effects of OLs on the adhesion and migration of OECs. (A) OLs were identified by staining with anti-O1 and MBP(a-d). MBP-negative cell as the arrowhead points to (c,d) may be oligodendrocyte precursor cell. Nogo-A expression on OECs is revealed by immunostaining without permeabilization (e-f). (B) OECs adhere well to oligodendrocyte monolayers (OLs-MON). DiI-labelled OECs were placed over laminin or OLs-MON. The values are normalized against the laminin group. (C) Photomicrographs of OEC migration from the edge of the coverslip fragment. Coverslip fragments covered with Di-I-labbled OECs were inverted with cells facing downward onto laminin, GST, GST–Nogo-66, His, His–Nogo-66 or OLs-MON. (D) Average maximum distance traveled from the edge of the inverted fragment by OECs upon each substrate compared with laminin (100%). Migration index is the ratio of maximum migration distance traveled by OECs on a particular substrate to the maximum migration distance traveled by theses cells on laminin. (E) Distribution of the number of migrated cells occupying successive 50 µm x 1 mm areas from the edge of an inverted fragment for each substrate. **P<0.01 versus laminin. Bar, 50 µm.

 

Figure 7
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  		Fig. 7. The effect of OLs on OECs is mediated by Nogo-A. (A,B) OEC migration is not affected by oligodendrocyte-derived matrix and diffusible factors. Coverslip fragments containing OECs or OECs-DiI were inverted on laminin and cultured with culture medium (Laminin), the condition-defined medium (CM), the oligodendrocyte-conditioned medium (OLs-CM), the oligodendrocyte-derived matrix with culture medium (OLs-GM), or on a monolayer of oligodendrocytes with culture medium (OLs-MON). The migration index and distribution of the number of migrated cells were examined. Migration Index (%)=Maximum distance travelled by OECs as a percentage of the maximum distance traveled by OECs on laminin. **P<0.01 versus laminin. (C) Cos-7 cells were transfected with Nogo-A and immunostained with antibody against Nogo-A without being permeabilized with Triton X-100. (D) DiI-labelled OECs were placed over a mock-transfected Cos-7 cell monolayer (Cos-7-Mock), and a Nogo-A-transfected Cos-7 cell monolayer (Cos-7-NogoA). The adherent cells were counted and normalized against the Cos-7-Mock group. (E,F) The cell motility of OECs on a monolayer of Cos-7-NogoA was assessed using inverted coverslip migration assay. **P<0.01. (G,H) Attenuation of OEC migration by OLs-MON is NgR-dependent. OECs were pre-treated with or without PI-PLC, NgR antibody or goat IgG and applied to inverted migration assay in which OECs migrate on laminin or OLs-MON. Bars, 50 µm.

 

Figure 8
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  		Fig. 8. The migration of implanted OECs is facilitated by neutralizing NgR in a spinal cord hemisection injury model. (A) The animals implanted with DiI-labelled OECs were treated with N.S., anti-NgR antibody or goat IgG every day through a pipe embedded in the subarachnoid space. After 10 days, the length, width and area of the region invaded by DiI-postive cells were measured by using MetaMorph Imaging System analysis software. The three right panels are locally magnified images of the three left panels. (B) Quantitative assessment of cell migration in vivo. Migration Index (%)=Maximum length, width or area traveled by DiI-positive cells in the presence of anti-NgR antibody or irrelevant IgG as a percentage of the maximum length, width or area traveled by DiI-positive cells in the presence of N.S. **P<0.01 versus N.S. Bar, 50 µm.

 

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© The Company of Biologists Ltd 2007