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doi: 10.1242/10.1242/jcs.00326

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Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22

¹ Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Biologia, Universita' di Udine, P.le Kolbe 4, 33100 Udine, Italy
² Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie AREA Science Park, Padriciano 99, 34142 Trieste, Italy
³ Dipartimento di Biologia, Universita' di Trieste, v. Giorgieri 5, 34100 Trieste, Italy
⁴ Dipartimento di Scienze Neurologiche e della Visione Universita' di Genova, v. dei Toni 5, 16138 Genova, Italy
⁵ MATI Center of Excellence, Universita' di Udine, P.le Kolbe 4, 33100 Udine, Italy

View larger version (66K): [in a new window]   Fig. 1. Expression of the Gas3/PMP22-GFP. (A) Immunodetection of Gas3/PMP22-GFP in transfected U20S cells. U2OS cells were transfected with pEGFP-N1-Gas3/PMP22 and cellular extracts were treated with or without PNGase-F or Endo-H as indicated. Immunodetection was performed using the anti-GFP antibody. (B) Subcellular localization of Gas3/PMP22-GFP and Gas3/PMP22-L16P-GFP. Gas3/PMP22-GFP and Gas3/PMP22-L16PGFP were overexpressed in NIH3T3 by nuclear microinjection. 6 hours after microinjection, cells were fixed and double-stained to visualize GFP and ER, using biotinylated concanavalin A. Bar, 18 µm. (C) Time-lapse images of Schwann cells overexpressing Gas3/PMP22-GFP. Frames at selected hours after microinjection, as indicated, showing Schwann cells overexpressing Gas3/PMP22-GFP. Arrowheads underline a reduction in spreading; asterisks indicate floating dead cells. (D)Time-lapse images of Schwann cells overexpressing GFP. Frames at selected hours after microinjection showing Schwann cells overexpressing GFP. Bar, 30 µm.

View larger version (96K): [in a new window]   Fig. 2. Accumulation of membrane vesicles in cells expressing Gas3/PMP22. (A) Time-lapse images of SCs overexpressing Gas3/PMP22-GFP. Frames at selected hours after microinjection, as indicated, showing Schwann cells overexpressing Gas3/PMP22-GFP. Arrowhead indicates cluster of vesicles in the perinuclear region and asterisks indicate vacuoles. Bar, 18 µm. (B) Time-lapse images of NIH-3T3 cells overexpressing Gas3/PMP22-GFP. Frames at selected hours after microinjection, as indicated, of a representative cell injected with pEGFP-N1-Gas3/PMP22 (10 ng/µl) are shown. Arrowhead indicates cluster of vesicles in the perinuclear region; arrows indicate vacuoles; asterisks indicate a vacuole close to the area retracted from the adhesion substrate. Bar, 14 µm. (C) Electron micrograph of a SC from sciatic nerve of homozygous PMP22tg rat, 100,000x. Intracellular myelin figures not associated with an axon are present (arrow). Moreover, empty vacuoles made up by redundant layers of SC membrane (asterisk) and plasma membrane invagination (arrowhead) can be observed. (D) Electron micrograph of a homozygous PMP22tg SC in culture (5 days), 60,000x. Concentric sheaths of membrane are present in the cytoplasm (arrows). Vacuoles can be observed either in the centre of the layer, or related to the SC membrane (asterisk).

View larger version (35K): [in a new window]   Fig. 3. ROK and Bcl-2 are unable to suppress vacuoles accumulation in cells expressing Gas3/PMP22. (A) The indicated ROK constructs were co-expressed with gas3/PMP22-GFP in NIH3T3 cells. After 24 hours from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22-GFP and ROK as described in Materials and Methods. Cells were scored for reduced spreading as previously described (Brancolini et al., 1999). Data represent arithmetic means±s.d. for four independent experiments. (B) Immunofluorescence analysis of NIH3T3 cells co-expressing gas3/PMP22-GFP and ROK1–1271 or Gas3/PMP22-GFP and ROK K112A. NIH3T3 cells 24 hours after seeding were microinjected with pEGFP-N1-gas3/PMP22 (20 ng/µl) and pXJ40- ROK1–1271 (80 ng/µl) or with pXJ40- ROK K112A (80 ng/µl). After 24 hours cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22 and HA-tagged ROK. Bar, 18 µm. (C) Gas3/PMP22-GFP and hPLAP, as controls, were overexpressed in REF 52 cells. After 21 hours from microinjection 10 µM of Y-27632 (final concentration) was added to the culture medium and cells were fixed 1 hour later and processed for immunofluorescence to score the Gas3/PMP22 phenotype. Data represent arithmetic means±s.d. for four independent experiments. (D) Immunofluorescence analysis of REF 52 cells expressing Gas3/PMP22-GFP or hPLAP treated or not with Y-27632. REF 52 cells 24 hours after seeding were microinjected with pEGFP-N1-gas3/PMP22 (20 ng/µl) or with pGDSV7S-hPLAP (80 ng/µl). Bar, 18 µm. (E) Gas3/PMP22-GFP and the indicated genes were co-expressed in NIH3T3 cells. After 18 hours from microinjection cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22-GFP and the co-expressed protein. Cells were scored for accumulation of vacuoles. Data represent arithmetic means±s.d. for four independent experiments.

View larger version (34K): [in a new window]   Fig. 4. Characterization of the Gas3/PMP22-positive vacuoles. (A) NIH3T3 or IMR90-E1A cells were microinjected with Gas3/PMP22-GFP and 12 hours later were fixed and probed with antibodies to annexin II, Tfn-R, and P0 or stained with WGA as indicated. Endocytosis of biotinylated Tfn was performed as described in Materials and Methods. The arrowhead indicates vacuoles negative for annexin II staining, whereas arrows point to vacuoles negative for Tfn-R and Tfn staining. Bar, 8 µm. (B) Dynamin1 wt and the point mutated derivative K44A defective in GTP binding and hydrolysis were co-expressed with Gas3/PMP22-GFP. After 18 hours from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22-GFP and dynamin as described in Materials and Methods. Cells were scored for accumulation of vacuoles. Data represent arithmetic means±s.d. for four independent experiments.

View larger version (73K): [in a new window]   Fig. 5. Gas3/PMP22 co-localizes with Arf6 and Arf6-Q67L in vacuoles. NIH3T3 cells were co-injected with Gas3/PMP22-GFP and with HA-tagged Arf6, Arf6-Q67L, Arf6-T27N or FLAG-tagged p95-C3. 18 hours later cells were fixed and probed with antibody to HA to visualize Arf6 and the different mutants or with antibody to FLAG to visualize p95-C3 as shown. Bar, 8 µm.

View larger version (98K): [in a new window]   Fig. 6. Gas3/PMP22-induced vacuoles are PIP2-positive and trap MHC I. (A) U2OS cells were transfected with Gas3/PMP22-VSV and PHGFP or with Gas3/PMP22-L16P-VSV and PH-GFP. 40 hours later cells were fixed and probed with an antibody to VSV. Bar, 8 µm. (B) NIH3T3 cells were co-injected with Gas3/PMP22-VSV and PH-GFP, with Gas3/PMP22-L16P-VSV and PH-GFP or with Gas3/PMP22-MG-VSV and PH-GFP. 18 hours later cells were fixed and probed with antibody to VSV to visualize Gas3/PMP22. Bar, 8 µm. (C) IMR90-E1A cells were microinjected with Gas3/PMP22-GFP. 15 hours later cells were fixed and probed with an antibody to MHC I. Bar, 4 µm. (D) IMR90-E1A cells were microinjected with Gas3/PMP22-GFP. 15 hours later they were allowed to internalize MHC I antibody for 60 minutes and then were fixed and processed for visualizing internalized antibody. Bar, 5 µm. (E) U2OS cells were microinjected with Gas3/PMP22-GFP. 18 hours later they were allowed to internalize MHC I antibody for 60 minutes and then were fixed and processed for visualizing internalized antibody. Bar, 5 µm.

View larger version (75K): [in a new window]   Fig. 7. A subset of vacuoles induced by Gas3/PMP22 are coated with actin. NIH3T3 cells were microinjected with HA-Arf6-Q67L, with Gas3/PMP22-GFP or with PH-GFP and Gas3/PMP22-VSV. 15 hours later cells were fixed and probed with an antibody to HA and phalloidin (top), with phalloidin (middle), or with an antibody to VSV and phalloidin (bottom) as shown. (Inset) Enlargement of box showing differences in actin coating and PIP2 accumulation in Gas3/PMP22 vacuoles. Bar, 3 µm.

View larger version (133K): [in a new window]   Fig. 8. Overexpressed Gas3/PMP22 was route to the late endosomes. (A) NIH3T3 cells were microinjected with Gas3/PMP22-GFP. 15 hours later cells were fixed and probed with an antibody to LBPA. Arrowheads indicate vacuoles in the perinuclear region double positive for Gas3/PMP22 and LBPA. (B) NIH3T3 cells were microinjected with Gas3/PMP22-GFP. 15 hours later cells were fixed and probed with an antibody to LBPA. The arrow indicates large peripheral vacuoles negative for LBPA staining. (C) NIH3T3 cells were microinjected with Gas3/PMP22-GFP. 15 hours later cells were fixed and probed with an antibody to LBPA. Arrowheads indicate clusters of aggregated vacuoles in the perinuclear region double positive for Gas3/PMP22 and LBPA. (D) NIH3T3 cells were microinjected with Claudin-15-GFP. 15 hours later cells were fixed and probed with an antibody to LBPA. (E) NIH3T3 cells were microinjected with Claudin-15-GFP. 15 hours later cells were fixed and probed with an antibody to annexin II. (F) NIH3T3 cells were microinjected with Gas3/PMP22-GFP. 15 hours later cells were fixed and probed with an antibody to annexin II. Arrows indicate large peripheral vacuoles positive for annexin II staining. (G) NIH3T3 cells were microinjected with Gas3/PMP22-GFP. 15 hours later cells were fixed and probed with an antibody to LBPA and phalloidin TRITC. Arrows indicate large peripheral vacuoles coated with actin and arrowheads point to clusters of aggregated vacuoles in the perinuclear region double positive for Gas3/PMP22 and LBPA. Images were obtained using a Leica TCS confocal microscopy and in the overlay are displayed in pseudocolors: green (GFP fused proteins), red (LBPA, annexin II and actin) blue (LBPA). Bar, 5 µm.

View larger version (20K): [in a new window]   Fig. 9. Gas3/PMP22 requires Arf6 to induce membrane internalization. (A) Arf6, Arf6-Q67L and Arf6-T27N were co-expressed with Gas3/PMP22-GFP in NIH3T3 cells. After 18 hours from microinjection cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22-GFP and Arf6. Cells were scored for accumulation of vacuoles double positive for Arf6 and Gas3/PMP22. Data represent arithmetic means±s.d. for four independent experiments. (B) Arf6, Arf6-Q67L and Arf6-T27N were co-expressed with PH-GFP in NIH3T3 cells. After 18 hours from microinjection cells were fixed and processed for immunofluorescence to visualize PH-GFP and Arf6. Cells were scored for accumulation of vacuoles. Data represent arithmetic means±s.d. for four independent experiments. (C) Arf6 and Arf6-T27N were co-expressed with Gas3/PMP22-GFP in NIH3T3 cells. After 18 hours from microinjection cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22-GFP and LBPA. Cells were scored for accumulation vesicles double positive for Gas3/PMP22 and LBPA. Data represent arithmetic means ± s.d. for four independent experiments.

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