Biology subjects

Barrow, H. G.

Publications and source records attributed to Barrow, H. G..

7 recordsLinked to original sources

bioRxiv2026

Human iPSC Models of Ganglioside Deficiency Reveal a Sialylated Lipid Requirement for Plasma-Membrane Organization and Neuronal Activity

Gangliosides are abundant neuronal glycosphingolipids, yet their roles in organizing the plasma membrane and supporting neuronal function remain poorly defined. Mutations in the biosynthetic enzymes ST3GAL5 or B4GALNT1 cause severe neurodevelopmental disorders, yet their cellular consequences are unclear. Using isogenic human iPSC-derived cortical neurons, we show that loss of these enzymes eliminates major neuronal gangliosides but produces strikingly divergent outcomes. ST3GAL5 deficiency reprograms the glycosphingolipid repertoire toward non-neuronal species and abolishes network-level electrical activity. In contrast, B4GALNT1-deficient neurons retain near-normal excitability, supported by accumulation of simple sialylated precursors (GM3/GD3). Quantitative proteomics reveals a profound loss of plasma membrane proteins, including ion channels and synaptic organizers, only in ST3GAL5-deficient neurons. These findings identify sialylated glycosphingolipids as essential scaffolds for plasma membrane organization and neuronal excitability, providing a mechanistic basis for the severe phenotype caused by loss of GM3 synthase in humans. TEASERHuman neuronal models reveal why loss of GM3 synthase causes severe neurodevelopmental disease

cell biology↗

bioRxiv2025

Optimisation of lytic herpes simplex virus infection in human induced pluripotent stem cell derived cortical neurones

Herpes simplex virus (HSV)-1 infection of cortical neurones is a leading cause of encephalitis. While we have substantial knowledge about the molecular virology of HSV-1 lytic infection in cells of the periphery, like keratinocytes or fibroblasts, we know much less about infection of human neurones owing to the challenges of working with neuronal cell-based models. Here we demonstrate the use of a human induced pluripotent stem cell (iPSC)-derived cortical neurone model (i3Neurones) for HSV-1 infection. i3Neurones are highly scalable and can be rapidly and efficiently differentiated into an isogenic population of cortical glutamatergic neurones. We show that i3Neurones support the full HSV-1 lytic replication cycle. We present an optimised protocol for the infection of i3Neurones with HSV-1 that allows their synchronous infection at near-100% efficiency, and optimised fixation methods that preserves organelle and neurite structure for immunocytochemistry analysis. Our study highlights i3Neurones as a robust, scalable platform for microscopy and biochemical studies of HSV-1 and other neurotropic pathogens. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2025

Conformational Dynamics and Membrane Insertion Mechanism of B4GALNT1 in Ganglioside Synthesis

Glycosphingolipids (GSLs) are crucial membrane components involved in essential cellular pathways. Complex GSLs, known as gangliosides, are synthesised in the ER/Golgi by a series of glycosyltransferase enzymes. Imbalances in GSL metabolism can lead to severe diseases, often affecting the nervous system. B4GALNT1 is a key enzyme in the ganglioside synthetic pathway, synthesising complex gangliosides including GM2 and GD2 from GM3 and GD3. These products are precursors to the major brain gangliosides. Loss of B4GALNT1 function causes hereditary spastic paraplegia 26 (HSP26), while its overexpression is linked to cancers including childhood neuroblastoma. Here, we present crystal structures of the homodimeric B4GALNT1 enzyme demonstrating conformational changes upon binding of donor substrate ligands and product. These structures support a catalytic mechanism that involves dynamic remodelling of the substrate binding site during catalysis. We also demonstrate that processing of lipid substrates by B4GALNT1 is severely compromised when surface loops flanking the active site are mutated from hydrophobic residues to polar. Molecular dynamics simulations support that these loops can insert into the lipid bilayer explaining how B4GALNT1 accesses and processes lipid substrates. By combining structure prediction and molecular simulations we propose that this mechanism of dynamic membrane insertion is exploited by other, structurally distinct GSL synthesising enzymes.

biochemistry↗

bioRxiv2024

Molecular Insights into Neuronal Dysfunction in GM2 Gangliosidoses

Glycosphingolipids (GSL) are important bioactive components of cellular membranes. Complex GSLs, containing sialic acid residues are known as gangliosides and are highly abundant in the brain. Diseases of ganglioside metabolism often result in severe, early-onset neurodegeneration. The ganglioside GM2 is the substrate of the hydrolytic lysosomal {beta}- hexosaminidase A (HexA) enzyme and when subunits of this enzyme are non-functional, GM2 lipid accumulates in cells leading to the GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases. We have developed high-quality i3Neuron-based models of Tay-Sachs and Sandhoff diseases, that demonstrate storage of GM2, formation of membrane whorls and accumulation of endolysosomal proteins consistent with disease phenotypes. Importantly, in addition to lysosomal dysfunction, the composition of the plasma membrane (PM) is significantly impacted in these diseases with changes in the abundance of both lipids and proteins. The changes to the PM proteome are driven in part by exocytosis of lysosomal material resulting in the aberrant accumulation of lysosomal proteins and lipids on the cell surface. The altered abundance of GM2 at the PM was striking, bringing the abundance of this precursor lipid up to that of the common neuronal gangliosides. Furthermore, the PM profiling identifies significant changes in synaptic protein abundances with direct functional impact on neuronal activity including rapid electrical firing consistent with neuronal hyperactivity. This work provides mechanistic insights into neuronal dysfunction in the GM2 gangliosidoses and highlights that these are also severe PM disorders. This work has broad implications for other lysosomal storage disorders and late-onset neurodegenerative diseases involving sphingolipid dysregulation.

neuroscience↗

bioRxiv2024

Encephalomyocarditis virus protein 2B* antagonises innate immune signalling by interacting with 14-3-3 protein family members

Encephalomyocarditis virus (EMCV) has for decades served as an important model RNA virus. Although most of the EMCV proteins are obtained via proteolytic cleavage of a long polyprotein, 2B* is expressed from a short overlapping open reading frame via an unusual protein-stimulated temporally dependent ribosomal frameshifting mechanism. The function of 2B* has not yet been characterised, though mutant viruses that are unable to express 2B* have a small plaque phenotype. Here we show that 2B* binds all seven members of the 14-3-3 protein family during virus infection. Binding is dependent on the 2B* C-terminal sequence RRNSS. IFN-{beta} and IL-6 signalling are impeded following overexpression of 2B* but not a truncated version lacking the RRNSS residues, thus suggesting a 14-3-3-dependent role for 2B* in inhibiting MAVS signalling. We also find that this function is distinct from the effect of 2B* on plaque size, as a virus in which 2B* was similarly truncated exhibited near-wildtype plaque size, thus indicating that 2B* also harbours additional functions. This work provides the first identification of a role of 2B* in innate immune antagonism and expands our knowledge of the protein complement of this important model virus. IMPORTANCEEncephalomyocarditis virus (EMCV) infects a range of species, causing economically important reproductive disorders in pigs and encephalitis and myocarditis in rodents. Due to its wide host range, it is an important model pathogen for investigating virus-host interactions. EMCV expresses an accessory protein, 2B*, from an overlapping open reading frame via an unusual ribosomal frameshifting mechanism. Although the frameshifting mechanism has been established, the function of the 2B* protein had not previously been explored. Here, we determined the host proteins to which 2B* binds and found that it specifically binds to the entire 14-3-3 protein family which, among other roles, contribute to the innate immune response to viral infection in mammalian cells. This interaction requires a specific stretch of amino acids at the end of 2B*. By interacting with the 14-3-3 proteins, 2B* blocks immune response activation. Thus, 2B* is a novel antagonist of innate immunity.

microbiology↗

bioRxiv2024

Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis

Numerous viral genes are involved in assembly of herpes simplex virus-1 (HSV-1), but their relative importance and function remain poorly characterised. Transmission electron microscopy has been used to study viral protein function in cells infected with HSV-1 mutants; however, these studies were usually conducted without correlative light microscopy to identify specific viral components. In this study, fluorescent capsid (eYFP-VP26) and envelope (gM-mCherry) proteins were imaged by structured illumination microscopy under cryogenic conditions (cryoSIM) and cellular ultrastructure was captured from the same infected cells using cryo-soft-X-ray tomography (cryoSXT). Nine fluorescent HSV-1 mutants, each lacking a different viral protein, were compared to assess the importance of viral proteins in different stages of HSV-1 morphogenesis. The relative importance of five viral proteins to nuclear egress were ranked (pUL34 > pUL21 > VP16 > pUL16 > pUS3) according to the levels of attenuation observed for each virus. Correlative imaging also revealed the roles of five viral proteins in cytoplasmic envelopment. VP16 was found to be important in capsid delivery to envelopment compartments, while cytoplasmic clusters of virus particles plus features of stalled envelopment not previously described were observed in the absence of pUL11, pUL51, gK, and gE. Finally, this 3D imaging approach was used to capture different assembly stages during cytoplasmic envelopment and to determine that envelopment occurs by particle budding rather than wrapping. The findings demonstrate that tomographic 3D correlative imaging is an emerging technology that sheds new light on viral protein functions and virion morphogenesis. ImportanceTo date, the characterisation of HSV-1 mutants in the study of virus assembly has been limited to transmission electron microscopy (TEM) without the addition of correlative light microscopy to identify fluorescently labelled viral proteins. In addition, only a small number of mutants are typically used in each study. Herein, a comparative analysis of nine HSV-1 mutants lacking specific structural proteins was performed using correlative fluorescence microscopy and X-ray tomography for the first time, revealing the relative roles of each viral protein in virus assembly. pUL11, pUL51, gK, and gE were shown to have important roles in cytoplasmic envelopment, with the loss of their functions leading to various stalled cytoplasmic envelopment phenotypes involving polarised arrays of capsids at one side of cytoplasmic vesicles that to our knowledge have never been seen with TEM. This correlative imaging approach enabled the study of cytoplasmic envelopment in 3D, revealing an envelopment mechanism driven by capsid budding rather than membrane wrapping. Further cryoSIM experiments revealed that pUL16 and pUL21 are important in nuclear egress of HSV-1 and that VP16 promotes nuclear egress and delivery of capsids to cytoplasmic envelopment compartments. By providing novel and comparative insights into the roles of different viral proteins in various stages of HSV-1 assembly, these findings highlight the utility of correlative cryo-fluorescence plus cryo-soft-X-ray tomography for probing trajectories of intracellular pathogen assembly.

microbiology↗

bioRxiv2019

Herpes simplex virus-1 pUL56 degrades GOPC to alter the plasma membrane proteome

Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over a whole time-course of herpes simplex virus (HSV)-1 infection was used to characterize changes in the host-cell proteome and to probe the kinetics of viral protein production. Several host-cell proteins were targeted for rapid degradation by HSV-1, including the cellular trafficking factor GOPC. We identify that the poorly-characterized HSV-1 protein pUL56 binds directly to GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling revealed that pUL56 mediates specific changes to the surface proteome of infected cells, including loss of IL18 receptor and Toll-like receptor 2, and delivery of Toll-like receptor 2 to the cell-surface requires GOPC. Our study highlights an unanticipated and efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the abundance of multiple signaling molecules at the surface of infected cells.

microbiology↗