Super Resolution Microscopy Reveals that Caveolin-1 Is Required for Spatial Organization of CRFB1 and Subsequent Antiviral Signaling in Zebrafish

Abstract

Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. Caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. This study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. A novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. Viral infection leads to a downregulation in Caveolin-1b (Cav-1b), disrupting clusters of CRFB1, a zebrafish type I interferon receptor (–R) subunit. Super-resolution microscopy has enabled the first single-molecule imaging of CRFB1 association with cav-1b-containing membrane domains. Strikingly, downregulation of Cav-1b, the major protein component of caveolae, caused CRFB1 clusters to disperse. Dispersal of CRFB1 clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. This response strongly suggests that CRFB1 organization within cav-1b-containing membrane domains is critical for IFN-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter IFN signaling and the antiviral immune response.

Single epicardial cell transcriptome sequencing identifies Caveolin-1 as an essential factor in zebrafish heart regeneration

“…Isoform a drives caveolae formation more efficiently than isoform b (Fujimoto et al, 2000). Also, these two isoforms have different interaction partners and effects on signaling pathways (Nohe et al, 2005;Gabor et al, 2013). Through quantitative RT-PCR using primers pairs targeting the same region of both isoforms or isoform aspecific sequences, we found that the mRNA level of cav1a, not total cav1, is slightly reduced in homozygous mutants.…”

A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance

“…SHRV infection (Gabor et al, 2013; Phelan et al, 2005a) was used to determine the role of mda5 in the antiviral response. Zebrafish liver cells (ZFL) overexpressing mda5 were infected with SHRV at an MOI of 0.01, 0.1 and 1.0 for 24 h prior to methylcellulose overlay and observed for cytopathic effects (CPE) 5 days later.…”

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane

“…Caveolae are poorly characterized invaginated membrane structures that exhibit high curvature 118 . The caveolin Cav-1 was imaged in the context of living zebrafish embryos 119 and with respect to cytokine receptor 120 using PALM microscopy, which showed that assembly of caveolae was important for initiation of receptor signaling. Curvature is also involved in the assembly of structures on the membrane, such as in viral budding and virion formation, which requires deformation of the membrane.…”