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

Scientific Reports
Gianmarco FerriFrancesco Cardarelli

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 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 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 phogr...Continue Reading

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Citations

Feb 16, 2021·Frontiers in Cell and Developmental Biology·Yonela NtamoNireshni Chellan
Mar 7, 2021·International Journal of Molecular Sciences·Diti Chatterjee BhowmickDebbie C Thurmond
Jul 16, 2020·Cell Reports·Ebrahim H Ghazvini ZadehWen-Hong Li
May 6, 2021·Metabolites·Nicholas NorrisMelkam Alamerew Kebede

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Methods Mentioned

BETA
Transmission Electron Microscopy
Structured Illumination Microscopy
Fluorescence
transfection
fluorescence microscopy
PCR
ELISA

Software Mentioned

MATLAB
ImageJ
SPT
STICS
Fiji

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