A high content screening microscopy approach to dissect the mechanism of Golgi-to-ER retrograde traffic

The Golgi complex is the central sorting and processing station of the secretory or anterograde pathway. Nascent proteins leaving the endoplasmic reticulum (ER) are first received and collected into an ER-Golgi intermediate compartment, and then if destined for secretion are subsequently transported through the Golgi cisternae to the trans-Golgi network (TGN), during which time a wide range of post-translational modifications occur. The proteins received at the TGN, are selectively sorted and packaged into distinct carrier vesicles that are sorted and transported to either the plasma membrane or endosomal/lysosomal compartments. For certain proteins and lipids however, they may need to be selectively retained in specific Golgi cisternae, or in some cases returned to the ER by active transport. In the face of an incessant flow of material passing through it, the Golgi must maintain its structural and functional integrity. Although much is known about the machinery regulating the anterograde pathway, comparatively little is known about the retrograde route back to the ER, and how the Golgi regulates its structure.In this work, two novel quantitative imaging approaches were devised to systematically identify proteins with a role in regulation of Golgi morphology and retrograde traffic from the Golgi to the ER. Utilising RNA interference (RNAi) technology and fluorescence microscopy coupled to the high throughput capabilities of high content screening a protocol to systematically probe gene function on a large scale and extract single cell multi-parametric data was developed. HeLa cells stably expressing a GFP-tagged Golgi enzyme GalNAc-T2 were used as a model to investigate the proteins regulating Golgi structure, and the metabolite brefeldin A was used as method to induce retrograde traffic from the Golgi to the ER.The newly established approaches were first validated with a test set of targets. The first experiments targeted 70 proteins, composed primarily of the Rab GTPases and several Rab-accessory proteins, while a second larger test set of targets comprised 352 proteins associated with cytoskeleton structure, function and regulation. Systematic depletion of the GTPases confirmed the previously established role for Rab6a in this pathway, and provided clear data for involvement of Rab1a, Rab1b and Rab2a as regulators of Golgi-to-ER transport, in addition to their known role in anterograde traffic. Rab10 and Rab11a were identified as potential novel regulators of this transport step and localisation studies showed their physical presence on a proportion of the Golgi-to-ER tubular intermediates. In addition, combinatorial depletions of Rab proteins also revealed previously undescribed functional co-operation and physical co-occurrence between several Rabs on the transport intermediates. An intricate interplay between the actin and microtubule cytoskeletal network in controlling Golgi structure and Golgi-to-ER trafficking was determined from the screen of cytoskeletal components. Not only did it confirm previously published roles for cytoplasmic dynein complex 1 as a regulator of both structure and trafficking, but it also identified several novel regulators of the two processes, for example Cdc42, Myo18a and Myh9.A genome-wide RNAi screen was then performed, allowing the interrogation of a total of 21,585 proteins, and thereby providing a global analysis of gene function associated with the two processes. This screen revealed that more than 10% of the genome encodes proteins that can be linked to the function of the Golgi complex and confirmed previously described organelle structure regulatory proteins along with multiple novel regulatory proteins and trafficking associated complexes. Network analysis revealed that the regulation of the structure of the Golgi complex occurs at multiple levels, which together with the well-established core machinery, involves cross-talk between small GTP-binding protein regulation, actin and microtubule cytoskeleton organisation and membrane proteins of the ER. Approximately 14% of the genome could be linked to retrograde trafficking. Consistent with the literature, the crucial role of COPI-dependent and COPI-independent trafficking machinery was observed. Roles for the TRAPP and COG tethering complex as regulators of Golgi-to-ER transport were also identified. The results obtained further suggest that ER structural organisation plays a role in the delivery of the Golgi transport carriers to the ER. In addition to the core machinery associated with Golgi-to-ER redistribution, the screen also revealed roles for multiple GTPases, phosphatases and kinases in controlling the dynamics of this trafficking step.Finally, combined analysis of the two screens revealed an intricate interplay between Golgi structure organisation and retrograde traffic. Network analysis revealed key links between Golgi structure organisation, retrograde trafficking, small GTP-binding protein regulation, protein tethering complexes and cytoskeletal organisation. These results provide the most comprehensive assessment of genes associated with Golgi structure, organisation and retrograde pathway function in mammalian cells to date.