“Technologically this is not so difficult,” says Kreitzer. “But the analysis was very labor intensive.” The reward was a direct readout of fusion events. Basolateral cargoes were located in the most apical two thirds of the cytoplasm and fused with the corresponding region of basolateral membrane. Apical cargoes were concentrated in approximately the top 4 μm of cytoplasm. Their fusion was not observed in polarized cells, and thus was presumed to be restricted to the apical membrane. Neither cargo fused with the basal membrane.
Microtubule depolymerization is known to result in mislocalization of apical membrane proteins, and Kreitzer and colleagues saw fusion of apical cargoes with basal membranes of nocodazole-treated cells. This correlated with syntaxin 3—a fusion machinery protein normally restricted to apical membranes—mislocalizing to basolateral membranes. Thus, syntaxin localization may direct delivery of apical and basolateral cargoes. But it is also possible that syntaxin 3 is itself an apical cargo, and that the real regulation is at the level of loading cargoes onto the correct motors or arrays of microtubules. ▪