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doi: 10.1242/10.1242/jcs.00326
Research Article |
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
* Author for correspondence (e-mail: cbrancolini{at}makek.dstb.uniud.it)
Accepted 17 December 2002
Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate (PIP2)-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles, and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP2-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane–endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.
Key words: Tetraspan proteins, Peripheral neuropathies, Schwann cells, Myelin, PIP2
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