Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 161, Issue 3, Pages 298-313Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2007.09.015
Keywords
cellular tomography; 3D reconstruction; mammalian; insulin secretion; beta cell; islets of Langerhans; montaging; segmentation; automation; transforms; insulin; electron microscope tomography; Golgi; pancreas; secretory granules; trans-Golgi; endocrine; exocytosis; cytoskeleton
Funding
- NCRR NIH HHS [P41 RR004050, P41 RR008605, RR08605, RR04050] Funding Source: Medline
- NIDA NIH HHS [R01 DA016602, DA016602] Funding Source: Medline
- NIDDK NIH HHS [R21 DK071236-02, R21 DK071236, R21 DK071236-01] Funding Source: Medline
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We have developed a simplified, efficient approach for the 3D reconstruction and analysis of mammalian cells in toto by electron microscope tomography (ET), to provide quantitative information regarding 'global' cellular organization at similar to 15-20 nm resolution. Two insulin-secreting beta cells-deemed 'functionally equivalent' by virtue of their location at the periphery of the same pancreatic islet-were reconstructed in their entirety in 3D after fast-freezing/freeze-substitution/plastic embedment in situ within a glucose-stimulated islet of Langerhans isolated intact from mouse pancreata. These cellular reconstructions have afforded several unique insights into fundamental structure-function relationships among key organelles involved in the biosynthesis and release of the crucial metabolic hormone, insulin, that could not be provided by other methods. The Golgi ribbon, mitochondria and insulin secretory granules in each cell were segmented for comparative analysis. We propose that relative differences between the two cells in terms of the number, dimensions and spatial distribution (and for mitochondria, also the extent of branching) of these organelles per cubic micron of cellular volume reflects differences in the two cells' individual capacity (and/or readiness) to respond to secretagogue stimulation, reflected by an apparent inverse relationship between the number/size of insulin secretory granules versus the number/size of mitochondria and the Golgi ribbon. We discuss the advantages of this approach for quantitative cellular ET of mammalian cells, briefly discuss its application relevant to other complementary techniques, and summarize future strategies for overcoming some of its current limitations. (C) 2007 Elsevier Inc. All rights reserved.
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