biostudies-literaturePirozzi MAIRC, ItalyMinistero dell'Università e della Ricerca (MUR)Ministero dell'Università e della Ricerca4742-4765https://www.ebi.ac.uk/biostudies/studies/S-EPMC12508209biostudies-literatureUnknown26(19)The classical models of intra-Golgi transport envision a movement of cargoes from cis- to trans-Golgi, followed by their sorting at the trans-Golgi network (TGN). During this vectorial transport, the cargoes are processed by sequentially acting glycosylation enzymes. A number of studies challenged the vectorial transport model and proposed alternative transport routes bypassing either directional transport or the TGN. We have re-visited intra-Golgi transport using varied cargo synchronization protocols, employing both imaging and biochemical methods. We find that cargoes move vectorially across the Golgi stack and reach the TGN. Cell type-dependent variations in transport kinetics and the limited resolution of fluorescence microscopy could have influenced earlier discrepant interpretationsEMBO reportsCargoes move from cis to trans-Golgi compartments and concentrate in the TGN before exiting.PMC12508209IG 15767P2022MMPXHFOE-EuroBioimagingIG 207862022MZJR9XP2022YA3LLCN00000041 CN3 RNARusso DQuarto MLuini AParashuraman SAgliarulo IDi Martino RBeznoussenko GVMironov AAPirozzi MMarzullo VMTuracchio GfalseCargoes move from cis to trans-Golgi compartments and concentrate in the TGN before exiting.The classical models of intra-Golgi transport envision a movement of cargoes from cis- to trans-Golgi, followed by their sorting at the trans-Golgi network (TGN). During this vectorial transport, the cargoes are processed by sequentially acting glycosylation enzymes. A number of studies challenged the vectorial transport model and proposed alternative transport routes bypassing either directional transport or the TGN. We have re-visited intra-Golgi transport using varied cargo synchronization protocols, employing both imaging and biochemical methods. We find that cargoes move vectorially across the Golgi stack and reach the TGN. Cell type-dependent variations in transport kinetics and the limited resolution of fluorescence microscopy could have influenced earlier discrepant interpretations2025-01-01T00:00:00Z2025 Oct2026-06-04T09:24:54.261Z2026-05-07T03:13:04.43ZS-EPMC125082094090349410.1038/s44319-025-00548-9