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 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 β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions.
Highlights
The lifespan of insulin secretory granules (ISGs) within β-cells, from their biogenesis at the Trans Golgi Network (TGN) to their exocytosis at the plasma membrane, is the result of a complex sequence of molecular signalling events[1]
ISGs were labelled with fluorescent protein (FP) fused to peptides or proteins in the granule lumen such as Islet Amyloid Poly-Peptide (IAPP)[26], Syncollin[27], and proinsulin, or to a protein embedded in the granule membrane, such as phogrin[28]
ISG average displacement is linear in time (Fig. 1D inset) only at very short spatiotemporal scales while it substantially deviates from linearity for larger scales[13]
Summary
The lifespan of insulin secretory granules (ISGs) within β-cells, from their biogenesis at the Trans Golgi Network (TGN) to their exocytosis at the plasma membrane, is the result of a complex sequence of molecular signalling events[1]. Spatiotemporal fluorescence fluctuation spectroscopy allows quantitative measurement of average structural and dynamic properties for molecules[18,19,20,21] or sub-cellular organelles[22,23,24] This live-cell-imaging approach does not require any preliminary assumptions or knowledge of the system. Three average parameters of ISGs are extracted from the iMSD trace: size, local diffusivity ‘Dmicro’ (Dm), and anomalous coefficient ‘α’ These values represent a unique point in a 3D parametric space. ISGs were labelled with FP fused to peptides or proteins in the granule lumen such as Islet Amyloid Poly-Peptide (IAPP)[26], Syncollin[27], and proinsulin, or to a protein embedded in the granule membrane, such as phogrin[28] Comparing these different labels, we find similar iMSD parameters for all the lumenal markers, but highlight a clear shift in the shape and position of phogrin-FP cluster. Given the speed and robustness of iMSD, this approach will allow further cell-based screening of ISG structure and dynamics under various physiological and pathological conditions
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