Biology subjects

Aji, A. K.

Publications and source records attributed to Aji, A. K..

3 recordsLinked to original sources

bioRxiv2025

Interactions Between Hantavirus Nucleoprotein and Glycoproteins: a quantitative fluorescence microscopy study

Orthohantaviruses (HV) are tri-segmented negative-sense RNA viruses that can occasionally cause severe pathologies in humans. Currently, limited information exists on the molecular interactions driving HV assembly in infected cells. Specifically, it is not clear how its glycoproteins (Gn and Gc) interact with other viral or host molecules. In this study, we use one- and two-color Number & Brightness fluorescence microscopy approaches to quantitatively characterize the interactions between HV glycoproteins and the nucleoprotein NP in transfected cells. Our results indicate that HV NP homo-interactions are strongly affected by the host environment. Furthermore, we report evidence of Gc-NP interactions, based on i) the high fluorescence cross-correlation between these two proteins and ii) the increased Gc-Gc interactions observed in the presence of NP. Finally, experiments on a Gc deletion mutant suggest that the observed protein-protein interactions are mediated by the cytoplasmic tail of Gc. In conclusion, this study provides new insights into the role of the interactions between HV glycoproteins and NP in the context of HV assembly.

biophysics↗

bioRxiv2023

Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells

Protein-protein-interactions play an important role in several cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for Protein-protein-interactions quantification via fluorescence fluctuation-based methods.

biophysics↗

bioRxiv2020

Detection of Envelope Glycoprotein Assembly from Old-World Hantaviruses in the Golgi Apparatus of Living Cells

Hantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human population. While the structure of the viral envelope has been characterized with high precision, the protein-protein interactions leading to the formation of new virions in infected cells are not fully understood yet. In this work, we use quantitative fluorescence microscopy (i.e. Number&Brightness analysis and fluorescence fluctuation spectroscopy) to quantify the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we have analyzed proteins from Puumala and Hantaan orthohantaviruses in several cellular models. For the first time, we quantified the oligomerization state of each protein in relation to its subcellular localization, concentration and the concentration of its interaction partner. Our results indicate that when expressed separately, both glycoproteins can form homo-multimers in a concentration-dependent manner. Fluorescence fluctuation analysis was applied to prove that Gc:Gc contacts observed on virions are also relevant for Gc-Gc interactions in living cells, in the absence of Gn. Furthermore, we proved that the membrane-distal lobes of Gn are not necessary for Gn homo-multimerization. In cells co-expressing both glycoproteins, we observe clear indication of Gn-Gc interactions and the formation of protein complexes with different sizes, while using various labelling schemes to minimize the influence of the fluorescent tags. Our data are compatible with an assembly model according to which hantavirus spikes are formed via the assembly of Gn-Gc hetero-dimers. Furthermore, our results indicate the interconnection of large Gn-Gc hetero-multimers in the Golgi apparatus. Such large glycoprotein multimers may be identified as multiple interacting viral spikes and provide a possible first evidence for the initial assembly steps of the viral envelope, within this organelle, directly in living cells.

biophysics↗