Biology subjects

Stacey, M.

Publications and source records attributed to Stacey, M..

6 recordsLinked to original sources

bioRxiv2026

Host cell potassium ion channels KCNJ2 (KIR2.1), KCNJ13 (KIR7.1) and KCNMA1 (BKCa) mediate escape of Bunyamwera virus from late endosomal compartments

The Orthobunyavirus genus within the Peribunyaviridae family of enveloped arthropod-borne negative-sense RNA viruses includes species associated with serious or fatal disease in both animals and humans such as Schmallenberg, La Crosse and Oropouche viruses. Orthobunyaviruses (OBVs) are internalised into cells by endocytosis and release their genomes following fusion with late endosomal (LE) membranes, triggered by low pH of the luminal milieu. There is mounting evidence to suggest OBV endosomal escape is also influenced by potassium ions (K+), which increase in concentration as endosomes mature. Endosomal K+ flux is controlled by cellular K+ channels, and we previously showed that K+ channel blockade using broad spectrum pharmacological inhibition abrogates OBV infection, with virions trapped within the endosomes. However, the K+ channels regulating this process are unknown. Herein, to identify the K+ channels involved, we studied Bunyamwera virus (BUNV), the prototypical OBV, and screened a siRNA library targeting 342 human ion channels. We identified 19 K+ channels whose knockdown inhibited BUNV gene expression by over 50%, with at least seven channels affecting BUNV at the entry stage, suggesting they exert a combinatorial influence. Of these seven channels, we used both pharmacological inhibition and genetic channel manipulation to show that OBV escape from CD63- and Rab7-positive LEs is controlled by KCNJ2 (KIR2.1), KCNJ13 (KIR7.1) and KCNMA1 (BKCa). These studies add to the understanding of host factors influencing OBV entry, regulation of endosomal ion flux, and could aid in the design or repurposing of therapeutics for the treatment of OBV-associated disease.

cell biology↗

bioRxiv2025

Electrical Stimulation Directs Articular Chondrocyte and Chondrosarcoma Migration in a 3D Collagen Matrix

Electrical signals are fundamental regulators of cell migration and growing numbers of studies have demonstrated electrically guided cancer cell migration. Chondrosarcomas, cartilage forming tumors, are highly metastatic and resistant to chemo and radiation therapies. To measure cellular migration in a three-dimensional (3D) environment a device was 3D-printed to house a collagen gel with embedded cells while enabling direct current electric field (DC-EF) application. Articular chondrocytes and chondrosarcoma cells were exposed to a 1 V/cm electric field for a duration of 12 hours while tracking their migration behavior. We observed that both cell types migrated towards the anode while chondrosarcoma cells showed a stronger directional response. We observed an EF-induced shift from diffusive migration trajectories towards ballistic migration behavior in articular chondrocytes and directed wobble migration in chondrosarcoma. In articular chondrocytes we observed significant increases in the length of protrusions directed towards the anode (p < 0.05), as opposed to cathode directed protrusions after EF-exposure. Notably chondrosarcoma cells exhibited tiny protrusions of less than a few microns in length which sporadically extruded and retracted. Chondrosarcoma cells were loaded with FluoVolt to track real-time changes in membrane potential. Cells exposed to a 1V/cm electric field for 30 seconds showed a dynamic cell membrane hyperpolarization and repolarization during EF-exposure with a maximum hyperpolarization approximated to be on the order of -5 mV. To our knowledge, these are the first descriptions of the effects of electrical fields on directional cell migration in a 3D environment.

bioengineering↗

bioRxiv2024

Single cell variation and rapid emergence of phenotypic heterogeneity in cell lines- a cautionary tale for devotees of CRISPR-Cas9.

The use and publication of research utilising CRISPR/cas9 in gene editing and knock outs (KOs) within cell lines is now widespread and has proved extremely powerful in the interrogation of gene function. However, the potential of experimental artefacts resulting from the need to generate single cell clones post gene-manipulation has been somewhat overlooked. In this study, we show that the commonly used pulmonary cell line A549 displays significant heterogeneity in terms of their gene expression, and that individual cells from a population exhibit dramatically different susceptibility to viral infection from a range of viruses including respiratory syncytial virus (RSV), influenza A virus (IAV), Hazara virus (HAZV), lymphocytic choriomeningitis virus (LCMV) and Bunyamwera virus (BUNV). Moreover, we demonstrate the rapid re-emergence of phenotypic heterogeneity even after cloning. These results demonstrate the need for caution in interpreting results from CRISPR screens and CRISPR-KO validation studies, especially in the study of viral infection.

microbiology↗

bioRxiv2023

Lymphocytic choriomeningitis arenavirus utilises tunnelling nanotube-like intercellular connections for cell-to-cell spread

The Arenaviridae family within the Bunyavirales order of segmented RNA viruses contains over 50 species grouped into four genera, Antennavirus, Hartmanivirus, Mammarenavirus and Reptarenavirus. Several mammarenaviruses are associated with fatal hemorrhagic fevers, including Lassa, Lujo and Junin viruses. The mammarenavirus member lymphocytic choriomeningitis virus (LCMV) is largely non-pathogenic to humans and represents a tractable model system for studying arenavirus molecular and cellular biology. During infection of cells in culture, a high proportion of LCMV spread is between directly neighbouring cells. Consistent with this observation LCMV-infected cells extrude multiple tunnelling nanotube (TNT)-like structures forming intercellular connections that could provide a route of cell-to-cell spread. To investigate this, we used recombinant LCMV with engineered epitope tags in glycoprotein spike (GP-1) and matrix (Z) proteins, alongside nucleoprotein (NP) antisera, to reveal that all three major structural proteins co-localised within TNT-like connections. Furthermore, utilising fluorescent in situ hybridisation (FISH) we showed NP also co-localised with LCMV genomic sense RNA. Taken together, these observations suggested LCMV virions pass between cells through intercellular connections to infect new cells. Consistent with this, addition of a potent LCMV neutralising antibody to supernatants during infection failed to block LCMV spread through cultures, revealing that cell-to-cell connectivity plays a major role in LCMV transmission. This is the first report of cell-cell infection via TNT-like connections for any species of the 14 families within the Bunyavirales order. This study furthers our understanding of how arenaviruses manipulate the host to establish infection, which may aid in the development of effective anti-viral therapeutics. IMPORTANCEArenaviruses include some of the most serious human pathogens in existence, although no clinically approved vaccines or therapies are currently available to prevent their associated disease. As with most pathogens, transmission of arenaviruses from one cell to another is a critical aspect of infection and resulting pathogenicity. Here, we showed that model arenavirus lymphocytic choriomeningitis virus (LCMV) can spread between cells without exposure to the extracellular space. We visualized the three major LCMV structural proteins, namely nucleoprotein, glycoprotein spike and matrix co-localized along with genomic RNA within tubular structures connecting adjacent cells. The use of a potent neutralizing antibody to block the extracellular route of LCMV transmission reduced spread within cultured cells to approximately half that of untreated cultures. Taken together, these results suggest intercellular connections represent important conduits for arenavirus spread. This information will aid in the development of antiviral strategies that prevent both intra- and extracellular transmission routes.

microbiology↗

bioRxiv2022

VelcroVax: a bolt-on vaccine platform technology improves antibody titres against a viral glycoprotein in mice

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and to newly emerging pathogens. Virus-like particle (VLP) vaccines form the basis of two of the most successful licensed vaccines (against hepatitis B virus (HBV) and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which self-assemble into immunogenic nanoparticles. VLPs can also be modified to present unrelated antigens, and here we describe a universal bolt-on vaccine platform (termed VelcroVax) where the capturing VLP and the target antigen (hapten) are produced separately. We utilise a modified HBV core (HBcAg) VLP, with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus, Junin virus (JUNV). Using this model system, we have solved high-resolution structures of VelcroVax VLPs, and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the non-complexed viral protein. We propose that this system could be modified to present a range of haptens and therefore form the foundation of future rapid-response vaccination strategies.

immunology↗

bioRxiv2021

Structural insight into Pichia pastoris fatty acid synthase

Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 [A] resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.

microbiology↗