Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging
Martin Neukam,
Pia Sala,
Andreas-David Brunner,
Katharina Ganß,
Alessandra Palladini,
Michal Grzybek,
Oleksandra Topcheva,
Jovana Vasiljević,
Johannes Broichhagen,
Kai Johnsson,
Thomas Kurth,
Matthias Mann,
Ünal Coskun,
Michele Solimena
Affiliations
Martin Neukam
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany; Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Corresponding author
Pia Sala
Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Center of Membrane Biochemistry and Lipid Research, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
Andreas-David Brunner
Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
Katharina Ganß
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany; Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany
Alessandra Palladini
Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Center of Membrane Biochemistry and Lipid Research, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
Michal Grzybek
Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Center of Membrane Biochemistry and Lipid Research, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
Oleksandra Topcheva
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany; Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany
Jovana Vasiljević
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany; Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany
Johannes Broichhagen
Department of Chemical Biology, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
Kai Johnsson
Department of Chemical Biology, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
Thomas Kurth
TU Dresden, Center for Molecular and Cellular Bioengineering (CMCB), Technology Platform, Electron Microscopy and Histology Facility, 01307 Dresden, Saxony, Germany
Matthias Mann
Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
Ünal Coskun
Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Center of Membrane Biochemistry and Lipid Research, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
Michele Solimena
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany; Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, 01307 Dresden, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Corresponding author
Summary: Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.
