Abstract
Characterizing relationships between cell structures and functions requires mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Here, we report a unique application of soft x-ray tomography to generate three-dimensional reconstructions of whole pancreatic β cells at different time points following glucose-stimulated insulin secretion. Reconstructions following stimulation showed distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they travel through the secretory pathway. Our results show that glucose stimulation caused rapid changes in biochemical composition and/or density of insulin packing, increased mitochondrial volume, and closer proximity of insulin vesicles to mitochondria. Costimulation with exendin-4 (a glucagon-like peptide-1 receptor agonist) prolonged these effects and increased insulin packaging efficiency and vesicle maturation. This study provides unique perspectives on the coordinated structural reorganization and interactions of organelles that dictate cell responses.
Highlights
The mesoscale architecture of a cell encompasses the organization of all materials ranging in scale from the whole single cell (~10 m diameter) to objects just larger than molecular machines (~50 nm) [1]
We demonstrate that our soft x-ray tomography (SXT) topology mapping pipeline can capture the cellular topology changes induced by both drug treatments and different stimulus time points
Our results are consistent with many previous studies on insulin vesicle diameter, observation of insulin vesicles docked to the plasma membrane (PM) [3, 11, 17], and close association of insulin vesicles and mitochondria [22]
Summary
The mesoscale architecture of a cell encompasses the organization of all materials ranging in scale from the whole single cell (~10 m diameter) to objects just larger than molecular machines (~50 nm) [1]. To more accurately characterize relationships between the substructures and functions of a cell, we must consider how its organelles interact, traffic, and recycle, as well as the related topological rearrangements that occur during the life span of the cell At the mesoscale, these processes include changes in chromatin organization, organelle volumes and distributions, and locations and structural features of different cellular neighborhoods that retain specific functions. New applications and approaches of fluorescence imaging have opened up possibilities for examining 3D cellular organization at higher resolution, including details on organelle and protein localization [7,8,9,10] These fluorescent microscopy approaches require the use of fluorescent probes, which limit the number and type of molecules that can be imaged at one time. With traditional methods alone, it is challenging to reconstruct 3D volumes of multiple cells under a variety of conditions, as is required for mapping the dynamic processes involved in subcellular reorganization
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