Biology subjects

Gould, G. W.

Publications and source records attributed to Gould, G. W..

12 recordsLinked to original sources

bioRxiv2026

ACE2 associates with insulin-responsive GLUT4 dynamics in adipocytes

Angiotensin-converting enzyme 2 (ACE2) is expressed in adipocytes, yet the mechanisms regulating its intracellular trafficking remain unclear. Here, we investigated whether ACE2 trafficking is coordinated with insulin-responsive vesicle dynamics mediated by the glucose transporter GLUT4 in 3T3L1 adipocytes. Subcellular localization analyses revealed that adipocyte differentiation promotes partial incorporation of ACE2 into insulin-responsive GLUT4-associated vesicular compartments, whereas ACE2 displayed a diffuse distribution in fibroblasts lacking a mature GLUT4 trafficking system. Reconstitution of insulin-responsive GLUT4 vesicle formation through exogenous expression of Sortilin and AS160 in fibroblasts was sufficient to partially recruit ACE2 into perinuclear GLUT4-positive compartments, indicating dependence on canonical GLUT4 vesicle machinery. NanoBiT assays demonstrated a regulated association between ACE2 and GLUT4 that was modestly enhanced by acute insulin stimulation but reduced following prolonged insulin exposure. Insulin stimulation also produced a slight increase in ACE2 surface exposure, while association with GLUT4 was accompanied by reduced ACE2 shedding, suggesting that recruitment into distinct trafficking routes may alter ACE2 accessibility to shedding machinery in adipocytes. Structural modeling further suggested that ACE2 and GLUT4 can form a membrane-compatible complex. Together, these findings indicate that ACE2 trafficking is coordinated with insulin-responsive GLUT4 vesicle dynamics, revealing a previously unrecognized association between metabolic signaling and ACE2 cellular dynamics in adipocytes, with potential implications for metabolic dysfunction and ACE2-associated disease processes.

cell biology↗

bioRxiv2026

A rapid, low-cost approach to solid immersion lens fabrication for enhanced resolution in optical microscopy

Solid immersion lenses (SILs) enhance the spatial resolution of an optical microscope by increasing the effective numerical aperture (NA) without physical modification of the objective lens. However, SIL application remains limited by cost, fragility, and accessibility. We present a rapid, single-step fabrication process to create optical quality hemispherical SILs using consumer-grade UV-curable transparent resin which reduces material costs by over five orders of magnitude relative to commercial glass counterparts. Our method produced resin SILs within seconds which can be easily implemented into conventional microscopy setups for increasing the effective NA. Quantitative imaging of USAF resolution targets and histology muscle preparations demonstrated a resolution enhancement approaching theoretical limits and comparable performance to N-BK7 glass SILs. This enabled visualisation of features usually below the diffraction limit of low NA dry objectives at a fraction of the cost of otherwise required high-powered objective lenses. To demonstrate accessibility and translational potential, our workflow was taught in a practical tutorial of an international microscopy course, where non-expert participants successfully fabricated, characterised, and applied SILs within a single session, reporting high confidence in independent implementation. We established ultra-low-cost resin SILs as a practical, scalable option to enhance the spatial resolution of routine optical microscopes and as an accessible and cost-effective platform for optics education.

biophysics↗

bioRxiv2025

A large 3D-printed integrated lens-biprism element enhances contrast in transmission stereomicroscopy

Stereomicroscopes are routinely used across disciplines for material and surface characterisation due to their simplicity of use and minimal specimen preparation requirements. However, the stereomicroscope transillumination design is suboptimal, as a single incident beam at the specimen plane is shared and transmitted via two laterally offset detection axes. This flaw limits life science applications due to the transparent nature of samples which results in poor contrast images. We use a single additional element in the illumination path to correct the illumination uniformity across the field of view and, by doing so, enhance image contrast and facilitate detection of refractive structures in transparent biological specimens. We designed and fabricated an integrated lens-biprism element using low-cost, consumer-grade 3D printing methods and consumables. This 3D printed lens-biprism distributed diverging rays from a single incandescent light source into two parallel beams that converged at the specimen plane and transmitted through the respective left and right detection axes of a stereomicroscope. This improved transillumination setup increased the image contrast by up to 67.62% compared to the conventional stereomicroscope setup. We demonstrated the benefit of the lens-biprism element by visualising dynamic cellular events in live tissue and discerning refractive structures more easily in transparent specimens.

biophysics↗

bioRxiv2025

PerfectlyAverage: a classical open-source software method to determine the optimal averaging parameters in laser scanning fluorescence microscopy

Laser scanning fluorescence microscopy (LSFM) is a widely used imaging method, but image quality is often degraded by noise. Averaging techniques can enhance the signal-to-noise ratio (SNR), but while this can improve image quality, excessive frame accumulation can introduce photobleaching and may lead to unnecessarily long acquisition times. A classical software method called PerfectlyAverage is presented to determine the optimal number of frames for averaging in LSFM using SNR, photobleaching, and power spectral density (PSD) measurements. By assessing temporal intensity variations across frames, PerfectlyAverage identifies the point where additional averaging ceases to provide significant noise reduction. Experiments with fluorescently stained tissue paper and fibroblast cells validated the approach, demonstrating that up to a 4-fold reduction in averaging time may be possible. Data are also presented to suggest that frame and line averaging with the same number of averages may produce different results, and this may influence the optimal number of averages. PerfectlyAverage is open source, compatible with any LSFM data obtained using line or frame averaging during image acquisition, and it is aimed at improving imaging workflows while reducing the reliance on subjective criteria for choosing the number of averages.

cell biology↗

bioRxiv2025

Inverse Agonism of the FFA4 free fatty acid receptor controls both adipogenesis and mature adipocyte function

Adipocyte disfunction is an important component of many metabolic disorders and there is a need for pharmacological approaches that can restore normal adipocyte function. The FFA4 receptor is a G protein coupled receptor (GPCR), activated by long chain free fatty acids (FFAs), that controls adipocyte function. Importantly, adipocytes produce FFAs, which may directly activate FFA4 and there is a need to better understand how FFAs produced by adipocytes interact with FFA4 signalling. In this study we have employed human and mouse adipocyte cell models to determine how pharmacological agonism or antagonism of FFA4 affects adipogenesis, lipolysis and glucose uptake. We show that a commonly used FFA4 antagonist, AH7614, is an inverse agonist and that treating adipocytes with this compound suppressed adipogenesis, inhibits glucose uptake and enhances isoprenaline stimulated lipolysis. In contrast, treatment with a synthetic FFA4 agonist, TUG-891, has only modest effects on adipogenesis and lipolysis, while showing no effect on glucose uptake. To explore the mechanism for why AH7614 but not TUG-891 affects adipocyte function, we demonstrate that during adipogenic differentiation sufficient FFAs are released into the culture medium to activate FFA4, suggesting AH7614 inhibits an autocrine feedback loop to suppress adipogenesis. In contrast, during lipolysis experiments, insufficient FFAs were released to activate the receptor, suggesting that AH7614 must enhance lipolysis by either inhibiting ligand independent FFA4 signalling, or FFA signalling that does not require the FFAs to be released from the cell. This study will help establish how FFA4 targeting therapeutics could be used to treat adipocyte dysfunction.

pharmacology and toxicology↗

bioRxiv2024

Revealing the Ultrastructure of Live Candida albicans using Stimulated Emission Depletion Microscopy

Candida albicans, a commensal fungal pathogen, is a major cause of opportunistic infections in immunocompromised individuals. Understanding its cellular structures and pathogenic mechanisms is critical for developing targeted antifungal therapies. Stimulated emission depletion (STED) microscopy enables nanoscale visualization of cellular components, surpassing the diffraction limit of conventional light microscopy. In this study, we employed STED microscopy to investigate the ultrastructural organization of C. albicans in live specimens. We showed that dyes commonly used in STED microscopy of mammalian cells are ineffective for the study of C. albicans, and we showed the utility of Nile Red staining for visualising the organisation of dynamic cellular components, including tracking of lipid droplets, using time-lapse recording in experiments exceeding 12 hours. STED microscopy offered more than a two-fold improvement in resolution compared to confocal laser scanning microscopy applied to the same specimens with negligible photobleaching. This study demonstrates the utility of STED microscopy in advancing our understanding of C. albicans biology at the nanoscale, providing a platform for future investigations into fungal pathogenicity and antifungal development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/625149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d46837org.highwire.dtl.DTLVardef@105f661org.highwire.dtl.DTLVardef@728d11org.highwire.dtl.DTLVardef@8b65ae_HPS_FORMAT_FIGEXP M_FIG C_FIG We present an optimised fluorescence staining method for super-resolution live-cell imaging of Candida albicans, using Stimulated Emission Depletion (STED) microscopy to resolve and track sub-cellular structures. We compare the performance of conventional confocal laser scanning microscopy (CLSM) to STED imaging, providing a three-fold resolution improvement beyond the diffraction limit. Finally, we perform live cell tracking to visualise and quantify the trajectories of multiple sub-diffraction limit-sized objects over a period of 12 hours, demonstrating the potential for live-cell STED imaging of Candida to visualise key processes involved in pathogenesis, drug resistance and infection.

microbiology↗

bioRxiv2024

Obtaining super-resolved images at the mesoscale through Super-Resolution Radial Fluctuations

Super-resolution microscopy overcomes the diffraction limit of light to achieve higher spatial resolutions than are typically available when using light microscopy techniques. However, these methods are usually restricted to imaging a very small field of view (FOV). Here, we have applied one of these super-resolution techniques, Super-Resolution Radial Fluctuations (SRRF) in conjunction with the Mesolens, which has the unusual combination of a low-magnification and high numerical aperture, to obtain super-resolved images over a FOV of 4.4 mm x 3.0 mm. We assessed the accuracy of these SRRF images through error maps calculated using a secondary analysis method, Super-resolution Quantitative Image Rating and Reporting of Error Locations (SQUIRREL). We demonstrate it is possible to achieve images with a resolution of 446.3 {+/-} 10.9 nm, providing a [~]1.6-fold improvement in spatial resolution over a uniquely large field, with consistent structural agreement between raw data and SRRF processed images. MotivationCurrent super-resolution imaging techniques allow for a greater understanding of cellular structures however they are often complex or only have the ability to image a few cells at once. This small field of view may not represent the behaviour across the entire sample and the manual selection of which restricted ROI to use may introduce bias. Currently, this is often circumvented by stitching and tiling methods which stitch many small ROI together, however this can result in artefacts across an image which poses an issue when analysing data. To combat this, we have used the Mesolens alongside Super-Resolution Radial Fluctuations analysis, to obtain super-resolved images over a field of view of 4.4 mm x 3.0 mm with minimal error.

biophysics↗

bioRxiv2024

A Novel 3D Imaging Approach for Quantification of GLUT4 Levels across the Intact Myocardium

Cellular heterogeneity is a well-accepted feature of tissues, and both transcriptional and metabolic diversity have been revealed by numerous approaches, including optical imaging. However, the high magnification objective lenses needed for high-resolution imaging provides information from only small layers of tissue, which can result in poor cell statistics. There is therefore an unmet need for an imaging modality that can provide detailed molecular and cellular insight within intact tissue samples in 3D. Using GFP-tagged GLUT4 as proof of concept, we present here a novel optical mesoscopy approach that allows precise measurement of the spatial location of GLUT4 within specific anatomical structures across the myocardium in ultrathick sections (5 mm x 5 mm x 3 mm) of intact mouse heart. We reveal distinct GLUT4 distribution patterns across cardiac walls and highlight specific changes in GLUT4 expression levels in response to high fat diet-feeding, and we identify gender-dependent differences in expression patterns. This method is applicable to any target that can be labelled for light microscopy, and to other complex tissues when organ structure needs to be considered simultaneously with cellular detail. SUMMARY STATEMENTHere we present a novel 3D optical mesoscopy approach that allows the study of both GLUT4 protein expression levels and structural distribution within ultrathick sections of intact murine hearts, in response to high fat diet-feeding.

cell biology↗

bioRxiv2022

Benchmarking thiolate driven photoswitching of cyanine dyes

Carbocyanines are among the best performing dyes in singlemolecule localization microscopy (SMLM), but their performance critically relies on optimized photoswitching buffers. As the chemical buffer composition controls the transitions between non-fluorescent off- and fluorescent on-states, it is ultimately linked to the maximum achievable resolution. Here, we describe a workflow for screening optimal buffer conditions for single-molecule photoswitching. This is achieved by creating an intensity gradient within the field of view using a single micro-electromechanical systems (MEMS) mirror in the excitation path of the wide-field setup. Photon budget, on-state and off-state lifetimes are studied for different concentrations of mercatoethylamine (MEA) and buffer pH values. Both of MEA concentration and buffer pH determine the amount of thiolate, which is the main requirement for carbocyanine dye photoswitching. We show that thiolate acts as a concentration bandpass filter for the maximum achievable resolution and determine a minimum thiolate concentration of [~]1 mM is necessary to facilitate SMLM measurements. We also identify a concentration bandwidth of 1-16 mM in which the photoswitching performance can be balanced between high molecular brightness and high off-time to on-time ratios. Furthermore, we monitor the performance of the popular oxygen scavenger system based on glucose and glucose oxidase over time and show simple measures to avoid acidification during prolonged measurements. Finally, the impact of buffer settings is quantitatively tested on the distribution of the glucose transporter protein 4 within the plasma membrane of adipocytes. Our work provides a general strategy for achieving optimal resolution in SMLM with relevance for the development of novel buffers and dyes.

biophysics↗

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↗

bioRxiv2019

GLUT4 expression and glucose transport in human induced pluripotent stem cell-derived cardiomyocytes

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) have the potential to transform regenerative cardiac medicine and the modelling of cardiac disease. This is of particular importance in the context of diabetic cardiomyopathy where diabetic individuals exhibit reduced cardiac diastolic contractile performance in the absence of vascular disease, significantly contributing towards high cardiovascular morbidity. In this study, the capacity of iPSC-CM to act as a novel cellular model of cardiomyocytes was assessed. The diabetic phenotype is characterised by insulin resistance, therefore there was a specific focus upon metabolic parameters. Despite expressing crucial insulin signalling intermediates and relevant trafficking proteins, it was identified that iPSC-CM do not exhibit insulin-stimulated glucose uptake. iPSC-CM are spontaneously contractile however contraction mediated uptake was not found to mask any insulin response. The fundamental limitation identified in these cells was a critical lack of expression of the insulin sensitive glucose transporter GLUT4. Using comparative immunoblot analysis and the GLUT-selective inhibitor BAY-876 to quantify expression of these transporters, we show that iPSC-CM express high levels of GLUT1 and low levels of GLUT4 compared to primary cardiomyocytes and cultured adipocytes. Interventions to overcome this limitation were unsuccessful. We suggest that the utility of iPSC-CMs to study cardiac metabolic disorders may be limited by their apparent foetal-like phenotype.

biochemistry↗

bioRxiv2018

CHC22 Clathrin Diverts GLUT4 from the ER-to-Golgi Intermediate Compartment for Intracellular Sequestration

Glucose Transporter 4 (GLUT4) is sequestered inside muscle and fat, then released by vesicle traffic to the cell surface in response to post-prandial insulin for blood glucose clearance. Here we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively-secreted GLUT1 transporter and localize CHC22 to the endoplasmic-reticulum-to-Golgi-intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4 and sortilin and down-regulation of either p115 or CHC22, but not GM130 or sortilin abrogate insulin-responsive GLUT4 release. This indicates CHC22 traffic initiates human GLUT4 sequestration from the ERGIC, and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.\n\nSummaryBlood glucose clearance relies on insulin-mediated exocytosis of glucose transporter 4 (GLUT4) from sites of intracellular sequestration. We show that in humans, CHC22 clathrin mediates membrane traffic from the ER-to-Golgi Intermediate Compartment, which is needed for GLUT4 sequestration during GLUT4 pathway biogenesis.

cell biology↗