RudLOV is an optically synchronized cargo transport method revealing unexpected effects of dynasore.

Abstract

Live imaging of secretory cargoes is a powerful method for understanding the mechanisms of membrane trafficking. Inducing the synchronous release of cargoes from an organelle is key for enhancing microscopic observation. We developed an optical cargo-releasing method, 'retention using dark state of LOV2' (RudLOV), which enables precise spatial, temporal, and quantity control during cargo release. A limited amount of cargo-release using RudLOV is able to visualize cargo cisternal-movement and cargo-specific exit sites on the Golgi/trans-Golgi network. Moreover, by controlling the timing of cargo-release using RudLOV, we reveal the canonical and non-canonical effects of the well-known dynamin inhibitor dynasore, which inhibits early- but not late-Golgi transport and exits from the trans-Golgi network where dynamin-2 is active. Accumulation of COPI vesicles at the cis-side of the Golgi stacks in dynasore-treated cells suggests that dynasore targets COPI-uncoating/tethering/fusion machinery in the early-Golgi cisternae or endoplasmic reticulum but not in the late-Golgi cisternae. These results provide insight into the cisternal maturation of Golgi stacks.