Visualizing subcellular rearrangements in intact β cells using soft x-ray tomography

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.

iDEP-assisted isolation of insulin secretory vesicles

“…This new view of the forces imparted on the insulin vesicles aligns with a rather simple proposition that the makeup of the particle has changed: something has been added, subtracted, or altered which changed the force on the particle in a measurable and quantifiable way. This is consistent with findings that glucose can affect the maturation of existing vesicles, increasing their molecular density or the concentration of biomolecules within the vesicle lumen 44 . Additionally, the subpopulations we observe could have been altered by a change in surface protein expression or enrichment of unsaturated lipids 57 .…”

“…Additionally, the subpopulations we observe could have been altered by a change in surface protein expression or enrichment of unsaturated lipids 57 . This is also consistent with findings that glucose enhances vesicle-mitochondria association which is hypothesized to contribute to insulin vesicle maturation 44 . Identical particles always have the same EKMr in the same manner that identical proteins always have the same molecular weight 58 .…”

“…Furthermore, we observed different distribution patterns for insulin vesicles isolated from glucose-stimulated versus unstimulated cells, which suggests glucose stimulation alters the insulin vesicle subpopulations. This is consistent with findings from single cell analysis using cryo-electron tomography and soft X-ray tomography, which revealed heterogeneities in the composition of insulin vesicles 53 and their molecular densities 44 depending on the drug treatment and location of insulin vesicles within the cell. Other studies have also observed enrichment of certain subpopulations of vesicles in response to glucose stimulation 54 .…”

“…This method allows discovery of subpopulations with distinct biophysical properties amongst insulin vesicles from untreated cells (n-insulin vesicles) and 25 mM glucose-treated cells (g-insulin vesicles). Our observations are consistent with previous studies where a pronounced shift in the molecular density of the insulin vesicles was noted under the two conditions 44 . This study substantiates the sensitivity of DC-iDEP separation technique in resolving subpopulations of insulin vesicles, among other organelles, and opens the avenue for numerous studies of the biochemical constituents where complex and heterogeneous populations of organelles are of interest.…”

“…1C). We demonstrate that glucose treatment, which has been shown to influence the dynamics of this organelle within the βcell 44 , affects the biophysical characteristics associated with the vesicle subpopulations captured within our DC-iDEP device. This method allows discovery of subpopulations with distinct biophysical properties amongst insulin vesicles from untreated cells (n-insulin vesicles) and 25 mM glucose-treated cells (g-insulin vesicles).…”