| Abstract |
Small GTPase Rabs are evolutionarily conserved regulators of intracellular membrane traffic and regulate multiple steps in membrane trafficking. Although various approaches have been used to identify the function(s) of individual Rabs, no simple tool that reversibly inhibits the function of Rab has ever been reported. Here, we developed a novel tool, named AID-2×RBD27 (auxin-inducible degron-tagged tandem Rab27-binding domain) that reversibly inhibits the function of Rab27 and then evaluated its usefulness by using Rab27A-mediated melanosome transport as a model. We showed that expression and degradation of AID-2×RBD27 in melanocytes caused reversible changes in melanosome distribution between perinuclear melanosome aggregation and peripheral melanosome dispersion. By performing 3D live-cell imaging in combination, we found that two types of anterograde melanosome transport are involved in peripheral melanosome dispersion, i.e., fast, long-range melanosome transport in the microtubule-enriched inner cellular region, especially in the dendrite, and slow, intermittent melanosome transport along the cortical actin filaments. Our new concept of an AID-RBD system would apply to all other Rabs as a means of investigating various Rab-mediated membrane trafficking events by reversibly inhibiting them.
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