Biology subjects

Regan, P.

Publications and source records attributed to Regan, P..

4 recordsLinked to original sources

bioRxiv2025

A Comprehensive Workflow for Imaging Live Insulin Secretion Events and Granules in Intact Islets

Accurate detection of insulin secretion from pancreatic beta cells is crucial for understanding normal physiological insulin secretion and its pathophysiological counterpart in diabetic states. Traditional methods using fluorescently labeled insulin granules or dye labeling often struggle to distinguish secretion from insulin granule dynamics. We present an optimized protocol using the cell-impermeable Zn2+-binding dye FluoZin-3, which fluoresces upon Zn2+ co-secretion with insulin outside of the islet, more accurately representing secretion. FluoZin-3 combined with intact islet attachment to vascular extracellular matrix and TIRF microscopy offers high spatial and temporal resolution as well as a high signal-to-noise ratio in a minimally perturbed system. Additionally, by integrating the cell-permeable Zn2+-binding dye ZIGIR, we can track insulin granule dynamics alongside secretion events. Our approach generates large datasets, which we efficiently analyze using ilastik machine learning software, enabling fast, accurate, and optionally supervised analysis. This technique builds on our groups previous protocols, detailing a streamlined workflow adaptable to high-resolution, live-cell microscopy for not just insulin but other secretory/granule systems as well. With this method, we investigated secretion behavior of different IG pools in real time during the first phase of insulin secretion: predocked, which appear before high glucose stimulation and are docked at the membrane; docked, which appear upon high glucose stimulation and dock at the membrane; and newcomer, which appear upon high glucose stimulation but dont dock at the membrane. The predocked and newcomer insulin granules are equally secreted and newcomer insulin granules dwell less than one second before secretion upon high glucose stimulation. The predocked and docked insulin granules, however, stay longer at the membrane before secretion. This method is useful for the investigation of functional beta cell heterogeneity of insulin granule secretion in space and time.

cell biology↗

bioRxiv2024

Directed insulin secretion occurs at precise cortical regions with optimal ELKS content that are devoid of microtubules

To maintain normal blood glucose levels, pancreatic beta cells secrete insulin into the bloodstream at specialized regions at the cell periphery, often called secretion hot spots. While many secretory machinery components are located all over the cell membrane, directed secretion relies on distinct cortical patches of the scaffolding protein ELKS and the microtubule (MT)-anchoring protein LL5{beta}. However, using TIRF microscopy of intact mouse islets to precisely localize secretion events within ELKS/LL5{beta} patches, we now show that secretion is restricted to only 5% of ELKS/LL5{beta} patch area. Moreover, the majority of secretion occurs at the margins of ELKS patches. This suggests that additional factor(s) must be responsible for hot spot definition. Because the MT cytoskeleton plays a regulatory role in the insulin secretion process via both delivery and removal of secretory granules from the secretion sites, we test whether local MT organization defines secretory activity at hot spots. We find that the majority of secretion events occur at regions devoid of MTs. Based on our findings, we present a model in which local MT disassembly and optimal ELKS content are strong predictors of directed insulin secretion. Significance StatementO_LIInsulin has to be secreted directly into the bloodstream for efficient regulation of glucose metabolism. Molecular requirements for precise secretion location and microtubule-mediated regulation in directed secretion are uncharacterized. C_LIO_LIUsing intact mouse islets and a live insulin secretion assay, we demonstrate that cortical patches containing ELKS and LL5{beta} display secretory heterogeneity and secretion hot spots are precisely localized to patch edges that are devoid of microtubules. C_LIO_LIThese findings suggest that microtubule absence is critical for secretion and secretion occurs away from presumed sites of secretory granule delivery. This expands the current knowledge of regulation and spatial characteristics of insulin secretion. C_LI

cell biology↗

bioRxiv2023

Epigallocatechin Gallate Modulates Microglia Phenotype to Suppress Pro-Inflammatory Signalling Cues and Inhibit Phagocytosis

Microglia are crucial players in the pathogenesis of late onset Alzheimers Disease (AD), with evidence for both deleterious and beneficial effects. Identifying interventions to modulate microglial responsiveness, to promote Amyloid {beta} (A{beta}) clearance, disrupt plaque formation or to dampen excessive inflammation has therapeutic potential. Bioavailable flavonoids such as the flavan 3-ols are of interest due to their antioxidant, metal chelating, signalling and anti-inflammatory potential. Primary microglia were treated with a series of structurally related flavanol 3-ols to assess effects on phagocytosis, cytokine release and transcriptional responses by RNA sequencing. Data indicated that the extent of hydroxylation and the presence of the galloyl moiety were strong determinants of flavan 3-ol activity. Epigallocatechin gallate (EGCG) was the most effective flavan-3-ol tested and strongly inhibited phagocytosis of A{beta} independent of any metal chelating properties suggesting a more direct modulation of microglia responsiveness. EGCG was broadly anti-inflammatory, reducing cytokine release and downregulating transcription particularly of components of the microglia extracellular matrix such as MMP3 and SerpinB2. Collectively this brings new insight into the actions of flavonoids on microglial responsiveness with potential implications for the therapeutic use of EGCG and structurally related flavanol-3-ols in AD.

neuroscience↗

bioRxiv2019

DAF-16 and SMK-1 contribute to innate immunity during adulthood in Caenorhabditis elegans

Aging is accompanied by a progressive decline in immune function termed \"immunosenescence\". Deficient surveillance coupled with the impaired function of immune cells compromises host defense in older animals. The dynamic activity of regulatory modules that control immunity appears to underlie age-dependent modifications to the immune system. In the roundworm Caenorhabditis elegans levels of the PMK-1 p38 MAP kinase diminish over time, reducing the expression of immune effectors that clear bacterial pathogens. Along with the PMK-1 pathway, innate immunity in C. elegans is regulated by the insulin signaling pathway. Here we asked whether DAF-16, a Forkhead box (FOXO) transcription factor whose activity is inhibited by insulin signaling, plays a role in host defense later in life. While in younger worms DAF-16 remains in an inactive state unless stimulated by environmental insults, we found that even in the absence of acute stress the transcriptional activity of DAF-16 increases in an age-dependent manner. Beginning in the reproductive phase of adulthood, DAF-16 upregulates a subset of its transcriptional targets, including genes required to kill ingested microbes. Accordingly, DAF-16 has little to no role in larval immunity, but functions specifically during adulthood to confer resistance to bacterial pathogens. We found that DAF-16-mediated immunity in adults requires SMK-1, a regulatory subunit of the PP4 protein phosphatase complex. Our data suggest that as the function of one branch of the innate immune system of C. elegans (PMK-1) declines over time, DAF-16-mediated immunity ramps up to become the predominant means of protecting adults from infection, thus reconfiguring immunity later in life.

immunology↗