Fluorescence microscopy opens new perspectives for the analysis of insulin secretory granule movement. In this study, we examined whether recently developed photoactivatable/photoconvertible proteins are a useful tool for studying this process at the single granule level in insulin-secreting cells after glucose stimulation.Methods
Plasmids were generated for expression of fusion proteins of the granule membrane phosphatase phogrin or the granule cargo protein neuropeptide Y (NPY) with the photoactivatable green fluorescent protein mutant A206K (PA-GFP-A206K), the photoconvertible protein Dendra2 and the fluorescent protein mCherry. Transfected insulin-secreting MIN6 cells were analysed by fluorescence microscopy.Results
Point-resolved 405 nm light exposure during image acquisition of MIN6 cells transiently transfected with Phogrin-PA-GFP-A206K or NPY-PA-GFP-A206K as well as of stable MIN6-Phogrin-Dendra2 cells resulted in selective visualisation of few granules by green or red fluorescence, respectively. Movement of these granules was analysed by an automated tracking method from confocal 3D image series. The high spatiotemporal resolution facilitated an elongated tracking of single granules. Interestingly, the track speed and track displacement of granules after 1 h starvation and subsequent glucose stimulation was lower in cells pre-cultured for 48 h at 3 mmol/l glucose than in cells pre-cultured at 25 mmol/l glucose.Conclusions/interpretation
Targeting of the granule membrane or its cargo with a photoactivatable/photoconvertible protein allows in-depth visualisation and tracking of single insulin granules in dependence upon glucose. This technique may also open the way to elucidating the regulation of granule movement velocity within the pancreatic beta cell with respect to secretory defects in type 2 diabetes.