Dataset Information

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living ?-cells.

ABSTRACT: The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living ?-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs' properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living ?-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions.

SUBMITTER: Ferri G

PROVIDER: S-EPMC6393586 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells.

Ferri Gianmarco G Digiacomo Luca L Lavagnino Zeno Z Occhipinti Margherita M Bugliani Marco M Cappello Valentina V Caracciolo Giulio G Marchetti Piero P Piston David W DW Cardarelli Francesco F

Scientific reports 20190227 1

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average d ...[more]

PMID: 30814595

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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.

Project description:It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ?2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only its extracellular action but also the characteristics of its presentation to the extracellular milieu.

Dataset Information

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living ?-cells.

Publications

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells.

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