Expression of the Longin domain of TI-VAMP impairs lysosomal secretion and epithelial cell migration.
BACKGROUND
TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein; also called VAMP7) belongs to the Longin subfamily of v-SNAREs (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors). The regulatory N-terminal extension, called the Longin domain, of TI-VAMP has been shown previously to have a dual biochemical function: it inhibits the capacity of TI-VAMP to form SNARE complexes and it binds to the delta subunit of the AP-3 (adaptor protein 3) complex in early endosomes, thereby targeting TI-VAMP to late endosomes.


RESULTS
We have generated MDCK (Madin-Darby canine kidney) cell lines expressing the Longin domain of TI-VAMP coupled to GFP (green fluorescent protein) in a doxycycline-dependent manner. As expected, AP-3delta (AP-3 delta subunit) is not properly localized in Longin-expressing cells. We have shown that the expression of the Longin domain impairs lysosomal secretion, as determined by the release of a pre-internalized fluorescent fluid-phase marker and by electron microscopy of the membrane-associated released particles. Membrane repair following mechanical wounding, a process requiring lysosomal secretion, is also impaired in cells expressing the Longin domain. Furthermore, cell migration, assessed by wound healing of MDCK monolayers, is also inhibited.


CONCLUSIONS
The results of the present study suggest that the expression of the Longin domain of TI-VAMP regulates lysosomal secretion of epithelial cells and provide molecular evidence for a role of the late endocytic system in cell migration.
Proux-Gillardeaux
Véronique
V
Membrane Traffic in Neuronal and Epithelial Morphogenesis, Inserm Avenir Team, Paris, France.
Raposo
Graça
G
Irinopoulou
Theano
T
Galli
Thierry
T
D000818:Animals; D002460:Cell Line; D021961:Cell Membrane Structures / Q000378:metabolism; D002465:Cell Movement; D004285:Dogs; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology*; D005089:Exocytosis; D015870:Gene Expression; D008247:Lysosomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D050683:R-SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D021122:Protein Subunits; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins