Figure 5: Prohibitins participate in the initial axoglial contact.

(a) Silencing of Phb1 or Phb2 in Schwann cells. Protein lysates of Schwann cells after transfection with shRNA for either Phb1 or Phb2 were blotted and probed for Prohibitin-1, Prohibitin-2 and β-Tubulin. (b) Quantification of Prohibitin-1 and Prohibitin-2 protein levels in Schwann cells silenced for either Phb1 or Phb2. (c) The amount of proteins in Schwann cell pseudopod does not change after Prohibitin silencing. (d) Early interactions between Schwann cells (stained with cell tracker, green) and axons (immunostained for NFH, red) are modified by Prohibitin silencing. Schwann cells (100,000) were seeded on neurons for 30 min or 24 h. Ascorbic acid was added after 8 h in the 24-h condition. (e,f) Number of Schwann cells in contact with neurons after 30 min (e) or 24 h (f). (g) Classification of Schwann cell morphology based on their relationship with axons. Axons are pseudocoloured in white. At 30 min, white indicates a Schwann cell contacting axons, light grey indicates a round Schwann without any protrusion, and dark grey indicates a Schwann cell with a large cytoplasm not extending towards an axon. We observed a significant reduction of Schwann cells contacting axons (white) when Prohibitin-1 or Prohibitin-2 are silenced. Large Schwann cell not extending towards axons were only observed after silencing of Prohibitins. At 24 h, white indicates a bipolar Schwann cell extending along an axon, light grey indicates a round Schwann without any protrusion and dark grey indicates a Schwann extending in more than two directions towards several axons. Error bars indicate s.d. n=3 independent experiments in b,c; n= 3 coverslips from three independent experiments in e–g. Statistical analyses were performed using one-way ANOVA. ***P<0.001, ****P<0.0001. Scale bar, 75 μm.