Secretory-granule dynamics visualized in vivo with a phogrin–green fluorescent protein chimaera

Abstract

To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 beta-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200-1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine beta-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 beta-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5-10 s and mean displacement <1 microm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5-10 microm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin-EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule-cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis.

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

“…Detection of Kir6.2 and SUR in subcellular fractions of MIN6 cells Equal volumes from the gradient fractions were separated on 9% (w/v) polyacrylamide gels then blotted onto Immobilon-P transfer membrane (Millipore, Watford, UK) and probed with organelle-specific antibodies against mGPDH (mitochondria), phogrin (large dense-core insulin-containing vesicle [LDCV] membranes) [30,35], TGN38 (Golgi) [31], insulin receptor (IR, plasma membrane); LAMP-1 (lysosomes) [36]; SREBP precursor (endoplasmic reticulum [ER]) [32]; 14-3-3-β (cytosol) [37]. The protein and insulin contents in each fraction were determined using a Pierce BCA Protein Assay Kit (Rockford, IL, USA) and a Mercodia Ultrasensitive Mouse Insulin ELISA Kit (Uppsala, Sweden), respectively.…”

Visualising insulin secretion. The Minkowski Lecture 2004

“…In addition, "evanescent wave" microscopy (also called total internal reflection of fluorescence or TIRF microscopy) [53,81,82], in combination with fluorescent probes, allows selective analysis of fusion events at the cell surface (see below). Using the phogrin.EGFP chimera, it was possible to show that at low (sub-stimulatory) glucose concentrations vesicles displayed only short oscillatory ("jiggling") movements about a fixed point [75]. However, when the glucose concentration was raised, much longer (several µm) vesicle excursions occurred, frequently towards the plasma membrane.…”

“…If this were the case in beta cells, then translocation would still be required even for vesicles in the readily releasable pool. By labelling the secretory vesicle membrane with a chimeric construct encoding the membrane-resident protein phogrin (phosphatase on the granule of insulinoma, also called IA-2β [74]) fused to enhanced green fluorescent protein (EGFP), we aimed to image and quantify vesicle movement [75] in single living beta cells. Phogrin, a single transmembrane-spanning vesicle protein, is well suited to the task of localising EGFP to secretory granules, since it possesses multiple and partially redundant targeting sequences [76].…”

“…Rutter, unpublished results). The use of a fluorescent marker combined with confocal microscopy [75,78] provides advantages over the use of earlier microscopic techniques such as differential interference contrast [79,80], since the latter may be complicated by interference from other organelles (e.g. mitochondria, lysosomes).…”

“…Mechanisms involved in the regulation of vesicle recruitment to the plasma membrane Our early studies [75] showed that vesicle movement was sensitive to microtubule disruption with nocodazole, but barely affected by the disruption of actin filaments with colchicine. Moreover, simultaneous imaging of vesicles and microtubules suggested that the vast majority of long excursions occurred along microtubules [83].…”