Aquaporin-4 (AQP4) is found on the basolateral plasma membrane of a variety of epithelial cells, and it is widely accepted that microtubules play an important role in protein trafficking to the plasma membrane. In the particular case of polarized trafficking, however, most evidence on the involvement of microtubules has been obtained via biochemistry experiments and single-shot microscopy. These approaches have provided essential information, even though they neglect the dynamical details of microtubule transport. In this work, we present a high-content framework in which time-lapse imaging, and single-particle-tracking algorithms were used to study a large number (∼104) of GFP-AQP4-carrying vesicles on a large number of cells (∼170). By analyzing several descriptors in this large sample of trajectories, we were able to obtain highly statistically significant results. Our results support the hypothesis that AQP4 is transported along microtubules, but to our surprise, this transport is not directed straight to the basolateral plasma membrane. On the contrary, these vesicles move stochastically along microtubules, changing direction repeatedly. We propose that the role of microtubules in the basolateral trafficking of AQP4 is to increase the efficiency, rather than determine the specificity of the target.
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