Dynamics of GFP-SGLT1 in COS-7 Cells

We recently showed that in Caco-2 cells the high affinity Na+-/Dglucose cotransporter (SGLTl) resides in intracellular endosomes which are attached to microtubules and therefore proposed that the activity of SGLTl is regulated by intracellular trafficking (Kipp et al., 2003). To visualize the dynamics of the transporter, we expressed a GFP-SGLT1 fusion in COS-7 cells. Using fluorescence microscopy, we discovered the GFP-SGLT1 protein in vesicular stmctures resembling those of endogenous SGLTl in Caco-2 cells. We were able to show the SGLT1-containing vesicles moving rapidly within COS-7 cells transiently expressing GFP-SGLT1 by means of time-lapse fluorescence microscopy. This movement was completely abolished by the disruption of the microtubule network with nocodazole. Next, we compared the features of the SGLTl vesicles and vesicles containing the transferrin receptor (TR). Therefore we incubated COS-7 cells expressing GFP-SGLT1 with Alexa Fluor 546-transferrin to compare the localization and the trafficking of SGLTl and the TR. Fluorescence microscopy of these specimens revealed a localization of the TR in vesicular structures that do not colocalize with SGLT1-vesicles and move with a higher speed. Our presented results confirm a vesicular localization of SGLTl and display the microtubule-dependent transport of these vesicles in hing cells. The mechanism of SGLTl endo-/exocytosis is clearly distinguishable from the transport of the transferrin recepto