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

Višnjar, Tanja; Chesi, Giancarlo; Iacobacci, Simona; Polishchuk, Elena; Resnik, Nataša; Robenek, Horst; Kreft, Marko; Romih, Rok; Polishchuk, Roman; Kreft, Mateja Erdani

doi:10.1038/s41598-017-13103-x

Cited by 18 publications

(23 citation statements)

References 69 publications

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“…Cultures of normal urothelial cells were established from normal porcine urinary bladders as described previously 17 , 32 . The experiments were approved by the Veterinary Administration of the Slovenian Ministry of Agriculture and Forestry in compliance with the Animal Health Protection Act and the Instructions for Granting Permits for Animal Experimentation for Scientific Purposes.…”

Section: Methodsmentioning

confidence: 99%

Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

Resnik

Prezelj

Luca

et al. 2018

Sci Rep

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Tunneling membrane nanotubes (TnTs) are membrane protrusions connecting nearby or distant cells in vitro and in vivo. Functions of TnTs in cellular processes are various and rely on TnT structure, which also depends on cytoskeletal composition. In the present study, we focused on the organization of microtubules (MTs) and intermediate filaments (IFs) in TnTs of urothelial cells. We analysed TnTs of normal porcine urothelial cells, which morphologically and physiologically closely resemble normal human urothelial cells, and of cancer cells derived from invasive human urothelial neoplasm. Wide-field fluorescence, confocal and super-resolution microscopy techniques, together with image analyses and 3D reconstructions enlightened specific MT-IF organization in TnTs, and for the first time revealed that MTs and IFs co-occur in the majority of normal and cancer urothelial cell TnTs. Our findings show that in the initiation segment of TnTs, MTs are cross-linked with each other into filamentous network, however in the middle and the attaching segment of TnT, MTs can helically enwrap IFs, the phenomenon that has not been shown before within the TnTs. In this study, we assess MT-IF co-occurrence in TnTs and present evidence that such helical organization of MTs enwrapping IFs is only occurring in a minority of the TnTs. We also discuss the possible cell-biological and physiological reasons for helical organization of MTs in TnTs.

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Section: Methodsmentioning

confidence: 99%

Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

Resnik

Prezelj

Luca

et al. 2018

Sci Rep

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“…The further analysis of co-localisation between UPs and lectins in the papillary carcinomas was focused on the apical PM, since biosynthesis of UPs and all other glycoproteins follows the same pathway from the endoplasmic reticulum to the Golgi apparatus and finally culminates in the apical PM. 29 …”

Section: Resultsmentioning

confidence: 99%

Combined lectin- and immuno-histochemistry (CLIH) for applications in cell biology and cancer diagnosis: Analysis of human urothelial carcinomas

et al. 2020

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Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named combined lectin- and immuno- histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.

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“…In this context it will be interesting to investigate further whether the CPV-II that are technically remodeled Golgi cisterna still retain its secretory functions. Remodeling of the GA to create secretory Golgi outposts as result of changes in secretory demands of cells has been reported (Hanus et al, 2008; Kreft et al, 2010; Višnjar et al, 2017). Thus, mobilized Golgi cisternae in the form of CPV-II that clusters near the PM could be an example of viral subversion of this program to induce secretory outposts that function to meet the elevated level of structural protein transport to PM during budding.…”

Section: Discussionmentioning

confidence: 99%

Contribution of the Golgi apparatus in morphogenesis of a virus induced cytopathic vacuolar system

Sengupta

Mihelc

Angel

et al. 2022

Preprint

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The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of peroxidase tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here we propose a model for the conversion of GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our result has implications for alphavirus virus induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.

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

Cited by 18 publications

References 69 publications

Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

Combined lectin- and immuno-histochemistry (CLIH) for applications in cell biology and cancer diagnosis: Analysis of human urothelial carcinomas

Contribution of the Golgi apparatus in morphogenesis of a virus induced cytopathic vacuolar system

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