Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.12.18.520514

Single-Molecule Super-Resolution Microscopy Reveals Formation of NS2B3 Protein Clusters on Mitochondrial Network Leading to its Fragmentation during the Onset of Dengue (Denv-2) Viral Infection

Varghese, J. M.·Joshi, P.·S, A.·Mondal, P. P.
View original source ↗Export metadata ↓

Abstract

Understanding viral proteins at single-molecule level and visualizing their dynamics in a cellular environment is challenging. This calls for sophisticated microscopy techniques (such as SMLM) and image-based study that can reveal details with the precision of a single-molecule. Specifically, NS2B3 is recognized as a critical protein complex responsible for proteolytic activity and processing of viral polyprotein during dengue type 2 (Denv - 2) infection. Using single-molecule-based super-resolution imaging, we study the dynamics of NS2B3 protein, its kinetics and its interaction with cell organelles. Two distinct photoactivable recombinant plasmids (mEos-NS2B3 and PAGFP - NS2B3) and a fluorescent recombinant plasmid (eGFP-NS2B3) were constructed to exemplify the role of NS2B3 protein complex. The study was conducted on NIH3T3 cells and optimized transfection protocol was developed. Studies confirmed the formation of NS2B3 clusters on the mitochondrial network. Statistical analysis of super-resolution data (images) helped determine cluster dynamics and facilitated the estimation of critical biophysical parameters (such as, cluster density, number of molecules / cluster and its spread). Results indicate an average NS2B3 cluster area / spread of [~] 0.050 {micro}m2 with a density of 3500mol./ {micro}m2, and an average of [~] 120 NS2B3 molecules per cluster. In addition, regional analysis suggests a direct positive correlation between NS2B3 cluster formation (single-molecule localization microscopy study) and fragmentation of mitochondrial network (confocal microscopy study). To further exemplify, we have carried out time-lapse imaging to visualize formation of NS2B3 clusters and its dynamics. The corresponding cluster parameters (#clusters, #mol/cluster and cluster area) suggests an increase in average #mol/cluster and cluster area. For the first time, single-molecule-based super-resolution imaging study helped reveal the dynamics of NS2B3 clusters in a cellular system. Understanding the underlying dynamics of NS2B3 clustering at the single-molecule level may help to decipher potential drug targets and the ways to disrupt the NS2B3 clusters. The image-based study has immediate implications in the broad field of single molecule imaging, fluorescence microscopy and disease biology. Statement of SignificanceThe arrival of single-molecule super-resolution imaging techniques and image-based study have advanced the field of cell biology, and our understanding of sub-cellular processes with single-molecule precision. Here, we report the first ever application of super-resolution imaging to visualize NS2B3 clusters in a cellular system that directly links to the fragmentation of mitochondrial network. To facilitate the study, two new photoactivable probes (mEos - NS2B3 and P AGF P- NS2B3) with key protein complex, NS2B3 of dengue virus were developed. The study involves cell transfection studies followed by confocal and single-molecule imaging. The correlative biophysical study (comprising of confocal and single-molecule imaging) and estimated cluster parameters corroborates our findings.

Explore related subjects

pathologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Varghese, J. M., Joshi, P., S, A., Mondal, P. P.. 2022-12-19. Single-Molecule Super-Resolution Microscopy Reveals Formation of NS2B3 Protein Clusters on Mitochondrial Network Leading to its Fragmentation during the Onset of Dengue (Denv-2) Viral Infection. https://doi.org/10.1101/2022.12.18.520514

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

NAE1-Dependent Protein Neddylation Preserves Endothelial Identity and Vascular Integrity

Background: Endothelial dysfunction is a central driver of cardiovascular and inflammatory diseases, yet the post-translational mechanisms that preserve endothelial homeostasis remain incompletely understood. Protein neddylation, the covalent conjugation of a ubiquitin-like modifier, regulates diverse cellular processes, yet its physiological role in the vascular endothelium remains unknown. This study investigated whether protein neddylation is required to preserve endothelial identity and vascular homeostasis. Methods: We generated tamoxifen-inducible endothelial-specific Nae1 knockout mice to inhibit neddylation and combined bulk RNA sequencing, single-cell and single-nucleus transcriptomics, quantitative proteomics, biochemical analyses, and gain- and loss-of-function approaches to define the role of endothelial neddylation in vascular homeostasis and inflammatory injury. Results: Endothelial-specific Nae1 deletion caused rapid mortality associated with vascular leakage, platelet accumulation, inflammation, and multi-organ injury. Multi-omics analyses demonstrated profound loss of endothelial identity, characterized by suppression of core endothelial programs and activation of inflammatory, procoagulant, and pyroptotic pathways. Single-cell analyses revealed progressive endothelial dysfunction culminating in depletion of the endothelial population and remodeling of the vascular niche. Mechanistically, endothelial neddylation deficiency activated gasdermin D (GSDMD)- and gasdermin E (GSDME)-dependent pyroptosis, whereas dual inhibition of GSDMD and GSDME markedly attenuated inflammatory transcriptomic remodeling, vascular injury, hepatocyte death, immune cell infiltration, and platelet accumulation. Translational analyses demonstrated reduced endothelial neddylation in experimental endotoxemia and decreased expression of neddylation pathway components in human atherosclerosis and COVID-19 datasets. Conversely, restoration of endothelial neddylation partially reversed inflammatory endothelial transcriptomic reprogramming in vivo. Conclusions: NAE1-dependent protein neddylation is an essential regulator of endothelial identity and vascular integrity. Loss of endothelial neddylation promotes gasdermin-dependent pyroptosis and thrombo-inflammatory vascular injury, whereas restoration of the neddylation pathway mitigates inflammatory endothelial dysfunction. These findings identify endothelial neddylation as a fundamental mechanism maintaining vascular homeostasis and a potential therapeutic target for cardiovascular and inflammatory diseases.

pathology↗
bioRxiv2024

COVID-19 during pregnancy alters circulating extracellular vesicle cargo and their effects on trophoblast

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) causes placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction and alteration of the placental transcriptome. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized maternal circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro. We found that the gestational timing of COVID-19 is a major determinant of circulating EV function and cargo. In vitro trophoblast exposure to EVs isolated from patients with an active infection at the time of delivery, but not EVs isolated from Controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an active infection, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19 and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.

pathology↗
bioRxiv2024

Human iPSC-derived Committed Cardiac Progenitors Generate Cardiac Tissue Grafts in a Swine Ischemic Cardiomyopathy Model without Triggering Ventricular Arrhythmias

BackgroundIntramyocardial injection of human pluripotent stem cell-derived cardiomyocytes following a myocardial infarction (MI) improves cardiac function in large animal models, but associated ventricular arrhythmias are major safety concern. We hypothesized that transendocardial injection of human induced pluripotent stem cell (hiPSC)-derived committed cardiac progenitor cells (CCPs), combined with cardiac fibroblast-derived extracellular matrix (cECM) to enhance cell retention, will generate cardiac tissue grafts improving contractility without triggering ventricular arrhythmias. MethodshiPSCs were differentiated using bioreactors and small molecules to produce committed cardiac progenitor cells (CCPs). MI was created using a coronary artery balloon occlusion and reperfusion model in Yucatan mini pigs. Four weeks later, epicardial needle injections of CCPs+cECM were performed in a small initial feasibility cohort (n=6), and then transendocardial injections of CCPs+cECM (n=14), CCPs alone (n=14), cECM alone (n=4) or vehicle control (n=13) into the peri-infarct region in a randomized cohort. Arrhythmias were evaluated using implanted event recorders. Magnetic resonance imaging (MRI) and invasive pressure-volume assessment were used to evaluate left ventricular anatomic and functional performance. Detailed histology was performed to detect and characterize human grafts. ResultsA scalable biomanufacturing protocol was developed generating CCPs which can efficiently differentiate into cardiomyocytes or endothelial cells in vitro. Intramyocardial delivery of CCPs to post-MI porcine hearts resulted in engraftment and differentiation of CCPs to form ventricular cardiomyocyte rich grafts. There was no significant difference in cardiac MRI-based measured cardiac volumes or function between control, CCP and CCP+cECM groups; however, pressure-volume analysis showed an improvement in dobutamine-stimulated functional reserve in CCP and CCP+cECM groups. Delivery of CCPs did not result in tumors or ventricular arrhythmias. ConclusionsTransendocardial delivery of CCPs with or without cECM into post-MI porcine hearts resulted in comparable human cardiomyocyte grafts which did not improve resting LV function but did improve stress-induced contractile reserve without triggering ventricular arrhythmias.

pathology↗