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Human endothelial cells size-select their secretory granules for exocytosis to modulate their functional output.

ABSTRACT: BACKGROUND:The secretory granules of endothelial cells, Weibel-Palade bodies, are released in response to numerous extracellular signals. Their cargo is critical to many vascular functions including hemostasis and inflammation. This presents a fundamental problem: how can these cells initiate tailor-made responses from the release of a single type of organelle, each with similar cargo? Each cell contains Weibel-Palade bodies in a wide range of sizes, and we have shown that experimentally shortening these organelles disproportionately reduces their ability to initiate hemostasis in vitro, leaving leukocyte recruitment unaffected. Could the production of this range of sizes underpin differential responses? OBJECTIVES:To determine whether different agonists drive the exocytosis of different sizes of Weibel-Palade bodies. METHODS:We used a high-throughput automated unbiased imaging workflow to analyze the sizes of Weibel-Palade bodies within human umbilical vein endothelial cells (HUVECs) before and after agonist activation to determine changes in organelle size distributions. RESULTS:We found that a subset of agonists differentially evoke the release of the longest, most pro-hemostatic organelles. Inhibiting the release of these longest organelles by just 15% gives a fall of 60% in an assay of secreted von Willebrand factor (vWF) function. CONCLUSIONS:The size-selection of granules for exocytosis represents a novel layer of control, allowing endothelial cells to provide diverse responses to different signals via the release of a single type of organelle.

SUBMITTER: McCormack JJ

PROVIDER: S-EPMC7155122 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Human endothelial cells size-select their secretory granules for exocytosis to modulate their functional output.

McCormack Jessica J JJ Harrison-Lavoie Kimberly J KJ Cutler Daniel F DF

Journal of thrombosis and haemostasis : JTH 20191002 1

<h4>Background</h4>The secretory granules of endothelial cells, Weibel-Palade bodies, are released in response to numerous extracellular signals. Their cargo is critical to many vascular functions including hemostasis and inflammation. This presents a fundamental problem: how can these cells initiate tailor-made responses from the release of a single type of organelle, each with similar cargo? Each cell contains Weibel-Palade bodies in a wide range of sizes, and we have shown that experimentally ...[more]

PMID: 31519030

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Project description:We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.

Dataset Information

Human endothelial cells size-select their secretory granules for exocytosis to modulate their functional output.

Publications

Human endothelial cells size-select their secretory granules for exocytosis to modulate their functional output.

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