Biology subjects

Ratnayake, D.

Publications and source records attributed to Ratnayake, D..

3 recordsLinked to original sources

bioRxiv2025

Pre-assembly of biomolecular condensate seeds drives RSV replication

During infection many RNA viruses, including respiratory syncytial virus (RSV), form specialized biomolecular condensates, inclusion bodies (IBs), where viral transcription and replication occur1-4. Paradoxically, high protein concentrations are typically required for condensate nucleation5, yet attaining sufficient protein levels in infection is thought to require IBs for viral transcription and replication. To uncover how viruses solve this paradox to establish IBs, we visualized early infection of RSV in real-time with single genomic viral ribonucleoprotein (vRNP) resolution. Our results reveal that IBs are nucleated from infecting vRNPs rather than de novo in the cytoplasm. IB nucleation further requires in-virion pre-assembly of viral protein-protein interaction networks on vRNPs to form pre-replication centers (PRCs). PRCs are potent condensate nucleation seeds due to their resistance to disassembly and efficient recruitment of newly-synthesized viral proteins. The high protein affinity of PRCs also results in increased polymerase complex association, allowing efficient viral transcription even in the absence of IBs. Together, these activities create a feed-forward loop that drives rapid IB formation. Intriguingly, PRC assembly depends on in-virion viral protein levels and is highly heterogeneous among virions, explaining cell-to-cell heterogeneity in infection progression, and identifying heterogeneous virions as the origin of infection heterogeneity. Together, our results show that in-virion pre-assembly of PRCs kick-starts viral condensate nucleation upon host-cell entry, and explains cell-to-cell heterogeneity in RSV infection.

microbiology↗

bioRxiv2020

Macrophages provide a transient muscle stem cell niche via NAMPT secretion

Skeletal muscle is paradigmatic of a regenerative tissue that repairs itself via the activation of a resident stem cell1. Termed the satellite cell, these normally quiescent cells are induced to proliferate by ill-defined wound-derived signals2. Identifying the source and nature of these pro-regenerative cues has been hampered by an inability to visualise the complex cellular interactions that occur within the wound environment. We therefore developed a zebrafish muscle injury model to systematically capture satellite cell interactions within the injury site, in real time, throughout the repair process. This analysis identified that a specific subset of macrophages dwells within the injury, establishing a transient but obligate stem cell niche required for stem cell proliferation. Single cell profiling identified specific signals secreted from dwelling macrophages that include the cytokine, Nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF). Here we show that NAMPT secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (CCR5) expressed on muscle stem cells. This analysis reveals that along with their well-described ability to modulate the pro-inflammatory and anti-inflammatory phases of wound repair, specific macrophage populations also provide a transient stem cell-activating niche, directly supplying pro-proliferative cues that govern the timing and rate of muscle stem cell-mediated repair processes.

cell biology↗

bioRxiv2018

Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations

Protein aggregates such as those formed in neurodegenerative diseases can be degraded via autophagy. To assess changes in autophagic flux in zebrafish models of familial Alzheimers disease (fAD) mutations, we first developed a transgene, polyQ80-GFP-v2A-GFP, expressing equimolar amounts of aggregating polyQ80-GFP and a free GFP internal control in zebrafish embryos and larvae. This assay detects changes in autophagic flux by comparing the relative strength of polyQ80-GFP and free GFP moiety signals on western immunoblots probed with an antibody detecting GFP. However, the assays application is limited by the toxicity of polyQ80-GFP, and because aggregation of this protein may, itself, induce autophagy. To overcome these issues, we subsequently developed a similar ratiometric assay where expression of a GFP-Lc3a-GFP transgene generates initially equimolar amounts of GFP-Lc3a (directed to autophagic degradation) and a free GFP internal control. The sensitivity of this latter assay is reduced by a cellular protease activity that separates Lc3a from GFP-Lc3a, thus contributing to the apparent free GFP signal and somewhat masking decreases in autophagic flux. Nevertheless, the assay demonstrates significantly decreased autophagic flux in zebrafish lacking presenilin2 gene activity supporting that the Presenilin2 protein, like human PRESENILIN1, plays a role(s) in autophagy. Zebrafish heterozygous for a typical fAD-like, reading-frame-preserving mutation in psen1 show decreased autophagic flux consistent with observations in mammalian systems. Unexpectedly, a zebrafish model of the only confirmed reading-frame-truncating fAD mutation in a human PRESENILIN gene, the K115Efs mutation of human PSEN2, shows possibly increased autophagic flux in young zebrafish (larvae).

molecular biology↗