Determination of secretory granule maturation times in pancreatic islet β-cells by serial block-face electron microscopy

AbstractIt is shown how serial block-face electron microscopy (SBEM) of insulin-secreting beta cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the beta cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It is found that 7.2±1.2% (±st. dev.) of the beta cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically ≳100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96±12 hours, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of beta cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically ≲30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135±14 minutes, and the immature transforming granules for an average time of 130±17 minutes.

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Enhanced information from biological materials through technological improvements in cryo-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124343 ◽

1992 ◽

Vol 50 (1) ◽

pp. 788-789

Author(s):

Marc J.C. de Jong ◽

Wim M. Busing ◽

Max T. Otten

Keyword(s):

Electron Microscopy ◽

Electron Beam ◽

Biological Materials ◽

Electron Crystallography ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

The Many ◽

Technological Improvements ◽

Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

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