RudLOV—a new optically synchronized cargo transport method reveals unexpected effect of dynasore

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 exceptional spatial, temporal, and
quantity control during cargo release. A limited amount of cargo-release
using RudLOV successfully visualized 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 revealed
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.