How are proteins transported across the stacked cisternae of the Golgi apparatus? Do they stay within the cisterna while the latter matures and progresses in an anterograde manner, or do they navigate between the cisternae via vesicles? Using synthetic biology, we engineered new tools designed to stabilize intercisternal adhesion such that Golgi cisternae are literally glued together, thus preventing any possible cisternal progression. Using bulk secretory assays and single-cell live imaging, we observed that small cargoes (but not large aggregated cargoes including collagen) still transited through glued Golgi, although the rate of transport was moderately reduced. ARF1, whose membrane recruitment is required for budding COPI vesicles, continues to cycle on and off glued Golgi. Numerous COPI-size vesicles were intercalated among the glued Golgi cisternae. These results suggest that cisternal progression is not required for anterograde transport, but do not address the possibility of cisternal maturation in situ
