Biology subjects

Bryant, N.

Publications and source records attributed to Bryant, N..

7 recordsLinked to original sources

bioRxiv2026

Dynamic diffusion analysis of the yeast plasma membrane using Airyscan based microscopic techniques

The yeast plasma membrane (PM) is highly compartmentalised into distinct nanoscale domains. The mechanisms by which this organisation regulates surface proteins are not fully understood, and it remains unclear how different biophysical modalities capture diffusion kinetics across varying spatial scales. Using confocal microscopy and an Airyscan2 detector, we benchmarked two prominent techniques: Fluorescence Correlation Spectroscopy (FCS) via the Zeiss Dynamics Profiler and Fluorescence Recovery After Photobleaching (FRAP). We quantified the lateral diffusion of three functionally diverse GFP-tagged model proteins: the exocytic t-SNARE Sso2, the lipid-binding protein Pmp3, and the eisosome-associated protein Ycp4. While diffusion coefficients aligned tightly between both modalities for Pmp3 and Ycp4, Sso2 exhibited a stark 14-fold discrepancy, displaying drastically faster local mobility by FCS compared to macroscopic recovery by FRAP. High-resolution 3D Structured Illumination Microscopy (3D-SIM) shows that Sso2 is partitioned into regional subdomains, that occupy less PM area than the network-like localisation of Pmp3. Our findings suggest that FCS captures rapid, localised diffusion within these microenvironments, whereas FRAP measures highly restricted transit across domain boundaries. Ultimately, this work demonstrates that membrane diffusion coefficients cannot be interpreted in isolation and capturing true lateral mobility requires pairing kinetic measurements with super-resolution spatial mapping to decode complex membrane compartmentalisation.

cell biology↗

bioRxiv2026

Parkinson's disease-associated LRRK2 risk variant, G2385R, enhances Rab substrate phosphorylation and impairs neuronal integrity

Mutations in the LRRK2 gene are the most frequent cause of familial Parkinsons disease (PD) whereas common variants are associated with an increased risk for sporadic PD. LRRK2 encodes a multi-domain protein displaying two functional enzymatic activities: GTPase and kinase. Familial LRRK2 mutations have been linked to alterations in its GTPase and kinase activities, elevated substrate phosphorylation, as well as increased neurotoxicity in cell and animal models. In addition to familial mutations, several common LRRK2 coding variants have been associated with PD risk in different ethnic populations. Little is known about how these coding variants modulate the risk of developing PD. In this study, we aim to evaluate whether a collection of LRRK2 coding risk variants (A419V, N551K, R1398H, R1628P, M1646T, S1647T, G2385R) modify the biochemical properties of the LRRK2 protein and LRRK2-mediated neurotoxicity using cell-based assays. With the exception of G2385R, we find that coding risk variants have minimal impact on LRRK2 steady-state protein levels, GTP-binding, phosphorylation at Ser910, Ser935, and Ser1292, and subcellular localization in human cell lines and cultured primary neurons. G2385R reduces the steady-state levels of LRRK2 and exhibits reduced phosphorylation at Ser910/Ser935 and Ser1292, consistent with diminished kinase activity. Notably, however, LRRK2 variants associated with increased PD risk (A419V, R1628P, M1646T, and G2385R) significantly elevate the levels of LRRK2-mediated Rab10 phosphorylation by two-fold in cells. In contrast, LRRK2 variants associated with reduced PD risk (N551K, R1398H, and N551K-R1398H) do not alter normal LRRK2-mediated pRab10 levels. Intriguingly, we find that both PD-risk and PD-protective LRRK2 variants can respond normally to kinase activation by different lysosomal stressors (chloroquine, nigericin and monensin) to robustly induce pRab10 levels in cells. The PD risk variant, G2385R LRRK2, significantly inhibits neurite outgrowth in primary cortical neurons compared to wild-type LRRK2. Combining each coding risk variant with the familial G2019S mutation has minimal impact on the elevated kinase activity of G2019S LRRK2 or its capacity to inhibit neurite outgrowth. Our study indicates that LRRK2-dependent Rab phosphorylation represents a relevant readout of PD risk induced by LRRK2 coding variants and demonstrates that the PD risk variant, G2385R LRRK2, creates a hyperactive kinase that can impair neuronal integrity at comparable levels to the effects of G2019S LRRK2.

neuroscience↗

bioRxiv2024

Rhinovirus as a Driver of Airway T-Cell Dynamics in Children with Severe Asthma

Severe asthma in children is notoriously difficult to treat, and its immunopathogenesis is complex. In particular, the contribution of T cells and relationships to anti-viral immunity, remain enigmatic. Here, we coupled deep phenotyping with machine learning methods to resolve the dynamics of T cells in the diseased lower airways, and examined rhinovirus (RV) as a driver. Our strategy revealed a T-cell landscape dominated by type 1 and type 17 CD8+ signatures. Interrogation of phenotypic relationships coupled with trajectory mapping identified T-cell migratory and differentiation pathways spanning the blood and airways that culminated in tissue residency, and included transitions between type 1 and type 17 tissue-resident types. These T-cell dynamics were reflected in cytokine polyfunctionality in situ. Use of machine learning to cross-compare T-cell populations that were enriched in the airways of RV-positive children with those induced in the blood after RV challenge in an experimental infection model, precisely pinpointed RV-responsive signatures that mapped to T-cell differentiation pathways. Despite their rarity, these signatures were detected in the airways of uninfected children. Together, our results underscore the aberrant nature of type 1 immunity in the airways of children with severe asthma, and implicate an important viral trigger as a driver.

immunology↗

bioRxiv2024

Single molecule array measures of LRRK2 kinase activity in serum link Parkinson's disease severity to peripheral inflammation

BackgroundLRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinsons disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. MethodsCapitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. ResultspT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil activation, antigenic responses, and the suppression of platelet activation. ConclusionsThe extracellular ratio of pT73-Rab10 to total Rab10 in serum is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics to mitigate associated deleterious immunological responses.

neuroscience↗

bioRxiv2024

Distinct Type 1 Immune Networks Underlie the Severity of Restrictive Lung Disease after COVID-19

The variable etiology of persistent breathlessness after COVID-19 have confounded efforts to decipher the immunopathology of lung sequelae. Here, we analyzed hundreds of cellular and molecular features in the context of discrete pulmonary phenotypes to define the systemic immune landscape of post-COVID lung disease. Cluster analysis of lung physiology measures highlighted two phenotypes of restrictive lung disease that differed by their impaired diffusion and severity of fibrosis. Machine learning revealed marked CCR5+CD95+ CD8+ T-cell perturbations in mild-to-moderate lung disease, but attenuated T-cell responses hallmarked by elevated CXCL13 in more severe disease. Distinct sets of cells, mediators, and autoantibodies distinguished each restrictive phenotype, and differed from those of patients without significant lung involvement. These differences were reflected in divergent T-cell-based type 1 networks according to severity of lung disease. Our findings, which provide an immunological basis for active lung injury versus advanced disease after COVID-19, might offer new targets for treatment.

immunology↗

bioRxiv2021

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type III to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type III impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

cell biology↗

bioRxiv2020

Pathologic α-Synuclein Species Activate LRRK2 in Pro-Inflammatory Monocyte and Macrophage Responses

Missense mutations in the LRRK2 gene that lead to LRRK2 kinase hyperactivity can cause Parkinsons disease (PD). The link between LRRK2 and -synuclein aggregation in PD remains enigmatic. Numerous reports suggest critical LRRK2 functions in microglial responses. Herein, we find that LRRK2-positive immune cells in the brain represent CD68-positive pro-inflammatory, monocyte-derived macrophages, distinct from microglia. Rod -synuclein fibrils stimulate LRRK2 kinase activity in monocyte-derived macrophages, and LRRK2 mutations lead to enhanced recruitment of classical monocytes into the midbrain in response to -synuclein. LRRK2 kinase inhibition blocks -synuclein fibril induction of LRRK2 protein in both human and murine macrophages, with human cells demonstrating much higher LRRK2 levels and kinase activity than equivalent murine cells. Further, interferon-{gamma} strongly induces LRRK2 kinase activity in primary human macrophages in comparison to weak effects observed in murine cells. These results highlight peripheral immune responses in LRRK2-linked paradigms that further connect two central proteins in PD.

neuroscience↗