Biology subjects

McKinney, H.

Publications and source records attributed to McKinney, H..

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↗

bioRxiv2020

Microtubules regulate pancreatic beta cell heterogeneity via spatiotemporal control of insulin secretion hot spots

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize whole mouse islets to determine how microtubules affect secretion toward the vascular extracellular matrix. Our data indicate that microtubule stability in the {beta}-cell population is heterogenous, and that cells with more stable microtubules secrete less in response to a stimulus. Consistently, microtubule hyper-stabilization prevents, and microtubule depolymerization promotes {beta}-cell activation. Analysis of spatiotemporal patterns of secretion events shows that microtubule depolymerization activates otherwise dormant {beta}-cells via initiation of secretion clusters (hot spots). Microtubule depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without microtubules, the timing of clustered secretion is dysregulated, extending the first phase of GSIS. Our findings uncover a novel microtubule function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional {beta}-cell heterogeneity.

cell biology↗

bioRxiv2020

Extreme phenotypes define epigenetic and metabolic signatures in cardiometabolic syndrome.

Improving the understanding of cardiometabolic syndrome pathophysiology and its relationship with thrombosis are ongoing healthcare challenges. Using plasma biomarkers analysis coupled with the transcriptional and epigenetic characterisation of cell types involved in thrombosis, obtained from two extreme phenotype groups (obese and lipodystrophy) and comparing these to lean individuals and blood donors, the present study identifies the molecular mechanisms at play, highlighting patterns of abnormal activation in innate immune phagocytic cells and shows that extreme phenotype groups could be distinguished from lean individuals, and from each other, across all data layers. The characterisation of the same obese group, six months after bariatric surgery shows the loss of the patterns of abnormal activation of innate immune cells previously observed. However, rather than reverting to the gene expression landscape of lean individuals, this occurs via the establishment of novel gene expression landscapes. Netosis and its control mechanisms emerge amongst the pathways that show an improvement after surgical intervention. Taken together, by integrating across data layers, the observed molecular and metabolic differences form a disease signature that is able to discriminate, amongst the blood donors, those individuals with a higher likelihood of having cardiometabolic syndrome, even when not presenting with the classic features.

genomics↗

bioRxiv2020

Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

In the current understanding of adult bone marrow hematopoiesis, megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. Here, we use single cell RNA sequencing to characterize HSCs and MKs from human bone marrow, to investigate MK lineage commitment and maturation. We identify two MK primed HSC clusters exhibiting unique differentiation kinetics, at least one of which is used in steady state and stress thrombopoiesis. By analyzing transcriptional signatures we show that human bone marrow MKs originate from MK primed HSC subpopulations, supporting the notion that these display exclusive priming for MK differentiation. We show that transcriptional programs change with increasing MK ploidy, where genes upregulated in high ploidy states may have functional relevance in platelet production. Finally, we highlight the presence of a specific transcriptional signature in MKs from individuals with myocardial infarction, supporting the aberration of MK differentiation in this thrombotic state.

cell biology↗