SGLT-GFP localized at the apical plasma membrane is dependent on the formation of the tight junction framework as seen in wild type SGLT1 [13
]. This observation indicates that SGLT-GFP is useful as a fluorescent probe for apical membrane molecules in living MDCK cells. When the cells were cultured in Ca2+
-free medium for 24 hr, cells could not form intercellular junctions and most of SGLT-GFP signals were localized in cytoplasmic vesicles in the perinuclear region. These SGLT-GFP-positive vesicles started to transfer to the whole plasma membrane 1 hr after the Ca2+
-switch. SGLT-GFP vesicles may be launched to the whole plasma membrane by a vesicular transport system that is stimulated by a signal transduction following the construction of adherence junctions by the Ca2+
-switch. SGLT-GFP signals then gradually accumulated in the apical membrane domain accompanying the formation of the tight junctions, and preferentially localized at the apical membrane after subsequent overnight culture. These results indicate that localization of SGLT-GFP to the apical membrane is dependent on the formation of the tight junction framework, and that the localization pattern of SGLT-GFP during the formation of cell polarity is similar to that in wild type SGLT1.
It has been reported that SGLT1 associates with detergent-resistant membrane microdomains, and disruption of these microdomains by MβCD decreased the amount of SGLT1 in detergent-resistant membrane microdomains, a change that was paralleled by a decrease of sodium-dependent glucose transport activity [10
]. These results suggested that cellular cholesterol may be important for the localization of SGLT1. It has been reported that cellular cholesterol was rapidly depleted from 60% to 50% within 30 min to 2 hr after treatment with MβCD [2
], and that apical targeting of membrane protein HA associated with lipid raft was perturbed [4
]. When confluently cultured MDCK cells were treated with MβCD, the localization of SGLT-GFP was perturbed and switched from apical to whole plasma membrane. This response appeared within 30 min, and the transition of SGLT-GFP to whole plasma membrane was completed by 2 hr. Since the immunofluorescence staining patterns of tight junction protein ZO-1 and apical marker protein gp135 did not change during the transition of SGLT-GFP by MβCD treatment, the change of localization is not due to lateral diffusion by the destruction of the tight junction borders. The effect of MβCD for the transition of SGLT-GFP was completely counterbalanced by the addition of cholesterol. This result indicated that cellular cholesterol is essential for apical localization of SGLT-GFP.
Cytoskeletal tracks are essential for the vesicular transport and selective targeting of membrane proteins. Apical targeting of influenza HA is disrupted accompanied with destruction of microtubule filaments by nocodazol treatment [4
]. We examined possible involvement of microtubules in the localization machinery of SGLT-GFP. By 1 hr after the treatment with colcemid, microtubule networks were completely disrupted, whereas apical localization of SGLT-GFP was maintained. Apical localization of SGLT-GFP was gradually perturbed and SGLT-GFP localized to the whole plasma membrane by 4 hr. Microtubule networks were reconstructed and reverted to normal shape within 1 hr after the removal of colcemid. The apical localization of SGLT-GFP was concomitantly restored accompanied with the reversion of the microtubule filaments. Since the immunofluorescence staining patterns of ZO-1 and gp135 did not change during the transition of SGLT-GFP by colcemid treatment, it is considered that the transition is not due to lateral diffusion by the loss of fence function of the tight junctions. When the cells were treated with colcemid and cycloheximide, transition of SGLT-GFP to whole plasma membrane was observed as well. This result indicates that the basolateral SGLT-GFP originates from that at apical plasma membrane rather than from a newly synthesized source. Destruction of microtubules by colcemid treatment may perturb the apical retention system for SGLT-GFP and the subsequent transcytosis-like transport of apical SGLT-GFP to basolateral membrane, resulting in the whole membrane distribution of it.
From these results, we conclude that the apical localization of SGLT-GFP is maintained by cellular cholesterol and microtubules. Localization of SGLT-GFP to the apical membrane may be maintained by recycling with continuous endocytosis and exocytosis, and cholesterol microdomains and microtubules play important roles in the recycling machinery.