Ultrastructural analysis of insulin secretory granule biology by super resolution and transmission electron microscopy

A Müller; M Neukam; A Ivanova; A Sönmez; C Münster; S Kretschmar; Y Kalaidzidis; T Kurth; JM Verbavatz; M Solimena

doi:10.1055/s-0037-1601664

Diabetologie und Stoffwechsel, Table of Contents

Diabetologie und Stoffwechsel 2017; 12(S 01): S1-S84
DOI: 10.1055/s-0037-1601664

Poster: *Poster + Kurzpräsentation

Typ-2-Diabetes I – Langerhans-Inseln

Georg Thieme Verlag KG Stuttgart · New York

Ultrastructural analysis of insulin secretory granule biology by super resolution and transmission electron microscopy

A Müller

¹Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

²Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany

³German Center for Diabetes Research (DZD), Neuherberg, Germany

,

M Neukam

¹Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

²Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany

³German Center for Diabetes Research (DZD), Neuherberg, Germany

,

A Ivanova

¹Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

²Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany

³German Center for Diabetes Research (DZD), Neuherberg, Germany

,

A Sönmez

¹Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

²Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany

³German Center for Diabetes Research (DZD), Neuherberg, Germany

,

C Münster

¹Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

²Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany

³German Center for Diabetes Research (DZD), Neuherberg, Germany

,

S Kretschmar

⁴Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany

⁵Biotechnology Center of the TU Dresden (BIOTEC), Dresden, Germany

,

Y Kalaidzidis

⁶Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

⁷Moscow State University, Moscow, Russian Federation

,

T Kurth

⁴Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany

⁵Biotechnology Center of the TU Dresden (BIOTEC), Dresden, Germany

,

JM Verbavatz

⁶Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

⁸Université Paris Diderot, Paris, France

,

M Solimena

²Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany

³German Center for Diabetes Research (DZD), Neuherberg, Germany

⁶Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

› Author Affiliations

Abstract

Full Text

Insulin secretion in pancreatic islets occurs in two phases: a short and rapid first phase followed by a sustained second phase, with younger insulin secretoy granules (SGs) being preferentially poised for exocytosis relative to older SGs. Here we labeled insulin SGs of different age in beta cells of isolated pancreatic islets from SOFIA (Study of insulin aging) mice, in which an insulin2-SNAP allele had been knocked-in in the Ins2 locus. Combining structured illumination microscopy and transmission electron microscopy (TEM) for correlative light-electron microscopy (CLEM) we precisely tracked age-distinct insulin SGs in Tokuyasu sections of beta cells. Ultrastructural analysis followed by mathematical modeling revealed a significant decrease in the number of labeled SGs starting from 2.7 days of age with a reduction of ˜60% at an SG age of 5 days, indicating a half life of 4.6 days. The reduction was accompanied by the detection of SNAP-labeled older SGs been disposed in multigranular bodies. Furthermore, we could show for the first time the preservation of fluorescence directly associated with protein tags in epoxy resin. This allowed for CLEM of age-distinct insulin SGs in SOFIA islets fixed by high pressure freezing (HPF) and embedded in epoxy resin, providing optimal ultrastructural preservation and contrast. Exploitation of this approach for morphometric analysis of labeled and unlabeled SGs will provide quantitative information about the life cycle of insulin SGs in different metabolic conditions. CLEM in Epon sections after HPF will allow for investigating the interaction of insulin SGs with the cytoskeleton at nanometer-resolution.