Monitoring Intra-Golgi Transport with Acute Spatiotemporal Control of a Synthetic Cargo.

ABSTRACT

We hereby describe a method to image cargo trafficking from the cis- to the trans-face of the Golgi apparatus. Briefly, we combine nocodazole treatment that breaks down the Golgi ribbon, temperature blocks that slow down cargo transport, and a drug-controlled aggregation system that controls the size of the cargo and its retention at different stages of the secretory pathway. Using this method, we first position the cargo within the cis-face of the Golgi. When traffic resumes upon temperature block release, kinetics of transport can be assessed by confocal microscopy through colocalization of the cargo with cis- and trans-Golgi markers. This method allows for testing various modes of intra-Golgi transports and can be adapted to investigate other steps of the secretory pathway.