Release of insulin granules by simultaneous, high-speed correlative SICM-FCM.

J Bednarska,A.I Tarasov,P Novak,Andrew Shevchuk,P Rorsman,Y Korchev

doi:10.1111/jmi.12972

Abstract

Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic β-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.

Highlights

Examples of over 50 biologically active peptides/low molecular-weight substances released by exocytosis include body’s only glucose-lowering hormone insulin, adrenaline regulating the ‘flight-orfight’ response as well as dopamine, serotonin and large neurotransmitter molecules(Golan et al, 2011)
Scanning Ion Conductance Microscopy (SICM) topographical images of control uninfected INS1-E cells revealed the cell surface decorated with numerous microvilli and dorsal ruffles that could be seen as membrane protrusions (Fig. 2A)
Superimposing fluorescence and topographical images revealed smooth membrane bulges spatially correlating with NPY-Venus fluorescence spots, which could be interpreted as membrane-docked insulin-containing vesicles (Fig. 2B, red arrows)

Summary

Introduction

Examples of over 50 biologically active peptides/low molecular-weight substances released by exocytosis include body’s only glucose-lowering hormone insulin, adrenaline regulating the ‘flight-orfight’ response as well as dopamine, serotonin and large neurotransmitter molecules(Golan et al, 2011). Biophysical alternatives to fluorescence microscopy of single-vesicle exocytosis are nominally free from the label problem; at the same time, they are less informative than imaging and, again, do not measure insulin release directly. Monitoring of single-vesicle exocytosis using patch-clamp electrophysiology(Neher & Marty, 1982) or electrochemical techniques(Huang et al, 1995; Zong et al, 2010) provides excellent signal-to-noise ratio but largely lacks spatial information. This problem may be resolved by using scanning probe microscopy (SPM) techniques. Scanning Ion Conductance Microscopy (SICM) has been successfully utilised for the detection of the exocytotic sites by the scanning probe in chromaffin cells, fixed before and after the stimulation(Shin & Gillis, 2006)

Methods

Results

Discussion

Conclusion