RESUMO
Transmigration of circulating monocytes from the bloodstream to tissues represents an early hallmark of inflammation. This process plays a pivotal role during viral neuroinvasion, encephalitis, and HIV-associated neurocognitive disorders. How monocytes locally unzip endothelial tight junction-associated proteins (TJAPs), without perturbing impermeability, to reach the central nervous system remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through endothelial cells. We found that human monocytic OCLN (hmOCLN) clusters at monocyte-endothelium interface, while modulation of hmOCLN expression significantly impacts monocyte transmigration. Furthermore, we designed OCLN-derived peptides targeting its extracellular loops (EL) and show that transmigration of treated monocytes is inhibited in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and we found that the OCLN-derived peptides significantly inhibit HIV dissemination to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent monocyte infiltration and viral neuroinvasion.
Assuntos
Células Endoteliais , Monócitos , Ocludina , Migração Transendotelial e Transepitelial , Peixe-Zebra , Ocludina/metabolismo , Ocludina/genética , Humanos , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/virologia , Animais , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Células Endoteliais/efeitos dos fármacos , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , HIV-1/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/metabolismo , Encéfalo/metabolismo , Encéfalo/virologiaRESUMO
The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. Here, we employed the retention using selective hooks (RUSH) system to show that newly-synthesised SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, we utilised the Drosophila wing imaginal disc model to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, our work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesised SHH secretion with potential impact on embryonic development.
Assuntos
Proteínas Hedgehog , Transdução de Sinais , Feminino , Animais , Proteínas Hedgehog/genética , Membrana Celular , Drosophila , Via Secretória , MamíferosRESUMO
BACKGROUND AND AIMS: Numerous HCV entry factors have been identified, and yet information regarding their spatiotemporal dynamics is still limited. Specifically, one of the main entry factors of HCV is occludin (OCLN), a protein clustered at tight junctions (TJs), away from the HCV landing site. Thus, whether HCV particles slide toward TJs or, conversely, OCLN is recruited away from TJs remain debated. APPROACH AND RESULTS: Here, we generated CRISPR/CRISPR-associated protein 9 edited Huh7.5.1 cells expressing endogenous levels of enhanced green fluorescent protein/OCLN and showed that incoming HCV particles recruit OCLN outside TJs, independently of claudin 1 (CLDN1) expression, another important HCV entry factor located at TJs. Using ex vivo organotypic culture of hepatic slices obtained from human liver explants, a physiologically relevant model that preserves the overall tissue architecture, we confirmed that HCV associates with OCLN away from TJs. Furthermore, we showed, by live cell imaging, that increased OCLN recruitment beneath HCV particles correlated with lower HCV motility. To decipher the mechanism underlying virus slow-down upon OCLN recruitment, we performed CRISPR knockout (KO) of CLDN1, an HCV entry factor proposed to act upstream of OCLN. Although CLDN1 KO potently inhibits HCV infection, OCLN kept accumulating underneath the particle, indicating that OCLN recruitment is CLDN1 independent. Moreover, inhibition of the phosphorylation of Ezrin, a protein involved in HCV entry that links receptors to the actin cytoskeleton, increased OCLN accumulation and correlated with more efficient HCV internalization. CONCLUSIONS: Together, our data provide robust evidence that HCV particles interact with OCLN away from TJs and shed mechanistic insights regarding the manipulation of transmembrane receptor localization by extracellular virus particles.
Assuntos
Hepatite C , Junções Íntimas , Proteína 9 Associada à CRISPR/metabolismo , Claudina-1/genética , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Humanos , Ocludina , Vírion , Internalização do VírusRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.
Assuntos
Encéfalo , COVID-19 , Homeostase , Organoides , SARS-CoV-2 , Sinapses , Humanos , SARS-CoV-2/fisiologia , COVID-19/virologia , COVID-19/metabolismo , COVID-19/patologia , Encéfalo/virologia , Sinapses/virologia , Sinapses/metabolismo , Organoides/virologia , Vírion/metabolismo , Neurônios/virologia , Neurônios/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genéticaRESUMO
Slac2-b, also known as exophilin-5, is a Rab27b effector protein with a role in exosome transport and is encoded by the EXPH5 gene. We previously described biallelic loss-of-function mutations in EXPH5 in an autosomal recessive form of epidermolysis bullosa simplex. However, how the loss of Slac2-b expression leads to skin fragility and erosions is unknown. In this study, we demonstrate that keratinocytes (KCs) isolated from two different individuals with mutations in EXPH5 have significant defects in cellâmatrix adhesion. EXPH5-mutant KCs also showed increased perinuclear accumulation and significantly reduced trafficking of CD63+ vesicles. These phenotypes were also seen in Slac2-bâdeficient KCs. This was coincident with a reduction in Rab27a protein expression in Slac2-bâmutant KCs as well as reduced secretion of extracellular vesicles containing extracellular matrix proteins. Live imaging analysis revealed a strong correlation between CD63+ vesicle trafficking to the plasma membrane and focal adhesion dynamics. These findings support a role for Slac2-b in regulating local focal adhesion dynamics to support effective KC adhesion and provide insight into the underlying pathophysiology of inherited skin blistering.