Figure 4. Inhibition of Lck activity slows SC migration on axons and inhibits myelination.

(a,b) GFP-transfected RSCs on DRG axons were treated with Lck inhibitor (500 nM) or DMSO alone (CTL) and the cultures were imaged every 10 min for 24 h. The migration rate is shown as the percentage of highly migrating (>25 μm h⁻¹; cells 1–3 on the upper frames), slow migrating cells (0–10 μm h⁻¹; cell 3 on the lower frames) or stationary cells (cells 1–2 on the lower frames). Lck inhibition significantly reduced the number of actively migrating cells (n=30, P<0.01, error bars represent ±s.d.), while slow or stationary cells were significantly increased (P=0.05, ±s.d.). (c) RSCs were transfected with siRNAs against Lck, paxillin, Crk, Rac or a non-targeting siRNA. Three days post transfection, the cell migration was recorded on an xCELLigence CIM plate. Reduced expression of Lck, paxillin and Rac significantly decreased RSC migration rate during 2–4 h as compared with control (P=0.01, P=0.03 and P=0.02, respectively, n=3 independent experiments, error bars represent ±s.d.). (d) Levels of Lck, paxillin, CrkII, Rac and actin as a loading control in RSCs used in c. SC-neuron cocultures were treated continuously (e) without or (f) with 500 nM Lck inhibitor for 13 days after the induction of myelination. Cultures were immunostained for MBP (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue). (g,h) Myelin internodes were counted from ten × 10 fields per culture. Treatment with Lck inhibitor significantly reduced the number of myelin internodes (h, P=0.05, n=7 from two independent experiments, error bars represent ±s.e.m.), without affecting total cell numbers (g). SC-neuron cocultures were treated as above then fixed and immunostained for MBP, ezrin and DAPI. Ezrin staining was localized to the nodes in both control (i) and inhibitor-treated (j) cultures and each internode is associated with one nucleus, indicating that Lck inhibition results in shorter, but mature internodes. Arrows indicate an internode between two ezrin-positive nodes (arrow heads). (k) The length of myelin internodes (~100 internodes per culture) was measured and the size distribution is shown. The majority of internodes in Lck inhibitor-treated cultures are shorter than in control cultures (n=7 from two experiments). Scale bars represent 50 μm. Statistical significance was determined by two-tailed Student’s t-test.