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Fig. 4. GLT-1 accumulates in the endocytotic compartments of G93A-expressing MDCK cell lines. (A) Morphological endocytosis assay. Forty-eight hours after GLT-1 transfection, the cells were labelled with FITC-WGA for 30 minutes at 0°C, washed to remove unbound WGA, and cultured for 60 minutes in regular medium to allow the internalisation of surface WGA-labelled glycoproteins prior to fixation and staining with GLT-1 antibody. The merged confocal horizontal section shows the colocalisation (yellow staining) of GLT-1 (red) with the internalised labelled glycoproteins (green). (B) Surface redistribution of GLT-1 in G93A-expressing MDCK cell lines after the inhibition of endocytosis by hypertonic media. Forty-eight hours after GLT-1 transfection, the cells were treated with 0.45 M sucrose for 30 minutes, fixed and double-stained with GLT-1 (red) and ß-catenin (green). The horizontal confocal sections taken at the plane of the lateral surface show the colocalisation of GLT-1 with ß-catenin at the cell surface (yellow), and the disappearance of intracellular perinuclear GLT-1 staining. Bar, 5 µm.





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