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
1   Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
2   Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany
3   German Center for Diabetes Research (DZD), Neuherberg, Germany
,
M Neukam
1   Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
2   Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany
3   German Center for Diabetes Research (DZD), Neuherberg, Germany
,
A Ivanova
1   Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
2   Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany
3   German Center for Diabetes Research (DZD), Neuherberg, Germany
,
A Sönmez
1   Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
2   Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany
3   German Center for Diabetes Research (DZD), Neuherberg, Germany
,
C Münster
1   Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
2   Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany
3   German Center for Diabetes Research (DZD), Neuherberg, Germany
,
S Kretschmar
4   Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany
5   Biotechnology Center of the TU Dresden (BIOTEC), Dresden, Germany
,
Y Kalaidzidis
6   Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany
7   Moscow State University, Moscow, Russian Federation
,
T Kurth
4   Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany
5   Biotechnology Center of the TU Dresden (BIOTEC), Dresden, Germany
,
JM Verbavatz
6   Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany
8   Université Paris Diderot, Paris, France
,
M Solimena
2   Paul Langerhans Institut Dresden, Medizinischen Fakultät, TU Dresden, Dresden, Germany
3   German Center for Diabetes Research (DZD), Neuherberg, Germany
6   Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany
› Author Affiliations
Further Information

Publication History

Publication Date:
05 May 2017 (online)

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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.