Dataset Information

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

ABSTRACT: Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.

SUBMITTER: Visnjar T

PROVIDER: S-EPMC5634464 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

Višnjar Tanja T Chesi Giancarlo G Iacobacci Simona S Polishchuk Elena E Resnik Nataša N Robenek Horst H Kreft Marko M Romih Rok R Polishchuk Roman R Kreft Mateja Erdani ME

Scientific reports 20171009 1

Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking ...[more]

PMID: 28993693

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Project description:The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins.

Project description:Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. A growing number of studies have reported that autophagy participates in infection by a variety of viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe financial losses to the global swine industry. Although much research has shown that PRRSV triggers autophagy for its own benefits, the exact molecular mechanisms involved in PRRSV-triggered autophagy remain to be fully elucidated. In the current study, we demonstrated that PRRSV infection significantly induced Golgi apparatus (GA) fragmentation, which promoted autophagy to facilitate viral self-replication. Mechanistically, PRRSV nonstructural protein 2 was identified to interact with and degrade the Golgi reassembly and stacking protein 65 dependent on its papain-like cysteine protease 2 activity, resulting in GA fragmentation. Upon GA fragmentation, GA-resident Ras-like protein in brain 2 was disassociated from Golgi matrix protein 130 and subsequently bound to unc-51 like autophagy activating kinase 1 (ULK1), which enhanced phosphorylation of ULK1 and promoted autophagy. Taken together, all these results expand the knowledge of PRRSV-triggered autophagy as well as PRRSV pathogenesis to support novel potential avenues for prevention and control of the virus. More importantly, these results provide the detailed mechanism of GA fragmentation-mediated autophagy, deepening the understanding of autophagic processes.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious swine disease affecting pig farming worldwide. Despite that numerous studies have shown that PRRSV triggers autophagy for its self-replication, how PRRSV induces autophagy is incompletely understood. Here, we identify that PRRSV Nsp2 degrades GRASP65 to induce GA fragmentation, which dissociates RAB2 from GM130 and activates RAB2-ULK1-mediated autophagy to enhance viral replication. This work expands our understanding of PRRSV-induced autophagy and PRRSV replication, which is beneficial for anti-viral drug development.

Dataset Information

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

Publications

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

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