Peripheral myelin protein 22 alters membrane architecture

Abstract

Peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann cells of the peripheral nervous system. PMP22 genetic alterations cause the most common forms of Charcot-Marie-Tooth disease (CMTD), which is characterized by severe dysmyelination in the peripheral nerves. However, the functions of PMP22 in Schwann cell membranes remain unclear. We demonstrate that reconstitution of purified PMP22 into lipid vesicles results in the formation of compressed and cylindrically wrapped protein-lipid vesicles that share common organizational traits with compact myelin of peripheral nerves in vivo. The formation of these myelin-like assemblies depends on the lipid-to-PMP22 ratio, as well as on the PMP22 extracellular loops. Formation of the myelin-like assemblies is disrupted by a CMTD-causing mutation. This study provides both a biochemical assay for PMP22 function and evidence that PMP22 directly contributes to membrane organization in compact myelin.

Keywords: Charcot-Marie-Tooth Disease; Electron Microscopy; Membrane; Myelin; Neuropathy; PNS; Peripheral Myelin Protein 22; Peripheral Nervous System; Reconstitution.

Fig. 1. PMP22 forms ordered assemblies upon reconstitution into lipid vesicles.

(A to C) Examples of protein-lipid MLAs created when PMP22 is reconstituted into 4:1 POPC/ESM vesicles via the dialysis method and visualized by negative stain EM. (D) Representative image of multilamellar vesicles (MLVs) prepared in the absence of protein via the dialysis method [lipid-only control (LOC)]. (E) MLVs prepared by spontaneous bilayer formation through hydration of lipids with water. (F) Control assemblies containing 4:1 POPC/ESM and the tetraspan VSD of KCNQ1 reconstituted via the dialysis method. Scale bars (all panels), 100 nm. (G) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of wild-type (WT) PMP22, LOC, MLVs, and the tetraspan VSD domain of KCNQ1. All individual object counts were converted to percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control, which was set to 1.0. Green, WT; red, LOC; blue, MLV; orange, VSD. (H) Sucrose gradient analysis of PMP22 reconstituted for 10 days without (−; top) or with (+; bottom) lipids. Fractions were collected from top (low density) to bottom (high density) and analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) with silver staining.

Fig. 2. Differences between MLVs and MLAs are visible by cryo-EM.

(A and B) Representative images of vitrified MLVs prepared in the absence of protein via the dialysis method (A) or by spontaneous bilayer formation through hydration of lipids only with water (B). (C and D) Examples of MLAs created when PMP22 is reconstituted into 4:1 POPC/ESM vesicles via the dialysis method and visualized using cryo-EM. Scale bars (all panels), 100 nm.

Fig. 3. MLAs examined by cryo-ET.

(A) Representative tomographic slices (1.47 nm) of two MLAs. *, MLAs in image. (B and C) Two MLAs from (A). Arrowheads indicate the ends of MLA. (D and E) Segmentation view of the corresponding MLA from (B) and (C). Scale bars, 100 nm (A, B, and D) and 50 nm (C and E). (F) Model demonstrating the compressed wrapped membranes of an MLA. (G) Model demonstrating the nesting vesicles of MLVs.

Fig. 4. Altered PMP22 LPRs disrupt MLA formation.

Representative negative stain images of PMP22 reconstitution assays carried out at LPRs (w/w) of 0.5 (A and B), 1.0 (C and D), and 10.0 (E and F). Scale bars (all panels), 100 nm. (G) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of WT PMP22 reconstitutions at LPRs (w/w) of 0.5, 1.0. 2.0, 4.0, and 10.0. All individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of counts represented by MLAs in the LPR 1.0 sample, which was set to 1.0. Red, LPR 0.5; green, LPR 1.0; blue, LPR 2.0; orange, LPR 4.0; purple, LPR 10.0. Error bars represent SEM between biological replicates. *P < 0.05, **P < 0.01. Statistical significance is only indicated for MLAs.

Fig. 5. MLA formation is not dependent on intermolecular disulfide linkage.

(A and B) Representative negative stain images of MLAs formed in a reconstitution assay using a Cys-less PMP22 mutant (C42S, C53S, C85A, and C109A). Scale bars (A and B), 100 nm. (C) Quantification of the percentage of MLAs present in a series of negative stain EM images of WT and Cys-less PMP22 reconstitutions. All individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control, which was set to 1.0. Green, WT control; red, Cys-less PMP22.

Fig. 6. ECL1 and ECL2 are important for MLA formation.

(A) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of PMP22 reconstitutions of WT PMP22 only, WT PMP22 incubated with GST-ECL1, and WT PMP22 incubated with GST-ECL2. Green, WT control; light blue, GST-ECL1 + WT PMP22 (1:1 molar ratio); dark blue, GST-ECL1 + WT PMP22 (4:1 molar ratio); light orange, GST-ECL2 + WT PMP22 (1:1 molar ratio); dark orange, GST-ECL2 + WT PMP22 (4:1 molar ratio). Error bars represent SEM between biological replicates. **P < 0.01. Statistical significance is only indicated for MLAs. (B) Quantification of the relative percentage of MLAs present in a series of negative stain EM images of PMP22 reconstitutions of WT PMP22; ECL1 loop-mutants PMP22 D37K, L38A, or W39A; and ECL2 loop-mutant PMP22 W124A. Green, WT control; red, D37K; blue, L38A; orange, W39A; purple, W124A. For both panels, all individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control. All values were normalized to the percentage of WT control MLAs, which was set to 1.0.

Fig. 7. The L16P PMP22 (TrJ) mutation disrupts MLA formation.

(A) Quantification of the relative percentage of MLAs present in a series of negative stain EM images in both WT and L16P PMP22 reconstituted on the same day. All individual object counts were converted to the percentage of total counts for a particular sample and were normalized to the percentage of total counts represented by MLAs in the WT PMP22 control, which was set to 1.0. Green, WT control; red, L16P. (B and C) Representative negative stain EM images of the disordered MLAs found in L16P PMP22 reconstitutions. Scale bars (B and C), 100 nm.