Dielectrophoretic Separation Reveals Distinct Subpopulations of Insulin Secretory Vesicles in Pancreatic Beta Cells

מושגי ליבה

Direct current insulator-based dielectrophoresis (DC-iDEP) can separate distinct subpopulations of insulin secretory vesicles from pancreatic beta cells, with glucose stimulation altering the biophysical properties and distribution patterns of these vesicle subpopulations.

תקציר

The study used direct current insulator-based dielectrophoresis (DC-iDEP) to investigate the heterogeneity of insulin secretory vesicles in pancreatic beta cells. Insulin vesicles were isolated from INS-1E rat insulinoma cells under unstimulated (n-insulin vesicles) and glucose-stimulated (g-insulin vesicles) conditions.
Key highlights:

DC-iDEP was able to separate distinct subpopulations of insulin vesicles based on their biophysical properties, as reflected in their electrokinetic to dielectrophoretic mobility ratio (EKMr) values.
Distinct distribution patterns were observed for n-insulin vesicles and g-insulin vesicles across a range of applied voltages and EKMr values.
Statistical analysis confirmed significant differences in the overall distribution patterns between n-insulin vesicles and g-insulin vesicles, as well as differences in specific EKMr value ranges.
The results suggest that glucose stimulation alters the biophysical properties and composition of insulin vesicle subpopulations, consistent with previous findings on insulin vesicle heterogeneity and functional maturation.
DC-iDEP provides a powerful tool to discover and quantify subtle differences in organelle subpopulations, which can enable further characterization of their biochemical constituents and functional states.

סטטיסטיקה

Insulin secretion in response to increasing glucose concentrations was confirmed by ELISA.
Density gradient centrifugation and western blotting were used to enrich and identify insulin vesicle fractions.
Dynamic light scattering confirmed the expected size distribution of 150-200 nm for the isolated insulin vesicles.

ציטוטים

"DC-iDEP provides a valuable tool for separating subpopulations of bioparticles with high resolution including viruses, bacteria, organelles, and proteins."
"Distinct subpopulations that were observed in both n- and g-insulin vesicles across a wide range of EKMr values indicate that both n- and g-insulin vesicles are made up of complex and heterogenous subpopulations and are statistically different in their overall distributions pattern as well as in several specific EKMr values."

תובנות מפתח מזוקקות מ:

iDEP-assisted isolation of insulin secretory vesicles

by Barekatain,M... ב- www.biorxiv.org 12-01-2021

https://www.biorxiv.org/content/10.1101/2021.12.01.470798v1

שאלות מעמיקות

How can the isolated insulin vesicle subpopulations be further characterized using techniques like mass spectrometry, electron microscopy, or functional assays to elucidate the biochemical and functional differences between them

To further characterize the isolated insulin vesicle subpopulations, techniques like mass spectrometry, electron microscopy, and functional assays can be employed.

Mass Spectrometry: This technique can be used to identify the proteins present in each subpopulation of insulin vesicles. By comparing the protein profiles of different subpopulations, we can elucidate the specific proteins that are unique to each group. This can provide insights into the biochemical composition and potential functional differences between the subpopulations.

Electron Microscopy: Electron microscopy can offer high-resolution imaging of the insulin vesicles, allowing for detailed visualization of their structure and morphology. Differences in vesicle size, shape, and organization can be observed, providing clues about the maturation state and functional characteristics of the vesicle subpopulations.

Functional Assays: Functional assays can assess the activity and behavior of the insulin vesicle subpopulations. For example, assays measuring insulin secretion kinetics, vesicle fusion dynamics, or response to stimuli can help determine the functional differences between the subpopulations. These assays can provide valuable information on the physiological roles of each subpopulation in insulin secretion.

By combining these techniques, we can gain a comprehensive understanding of the biochemical and functional differences between the isolated insulin vesicle subpopulations, shedding light on their roles in cellular processes.

What other organelle systems could benefit from the application of DC-iDEP to discover and quantify subtle subpopulation heterogeneity

Other organelle systems that could benefit from the application of DC-iDEP to discover and quantify subtle subpopulation heterogeneity include:

Mitochondria: Mitochondria are known to exhibit heterogeneity in terms of function, morphology, and protein composition. DC-iDEP could be used to isolate and characterize distinct subpopulations of mitochondria based on their biophysical properties, providing insights into their functional diversity within cells.

Endosomes and Lysosomes: These organelles play crucial roles in intracellular trafficking and degradation processes. By applying DC-iDEP, different subpopulations of endosomes and lysosomes could be separated and analyzed to uncover variations in their composition and functions.

Golgi Apparatus: The Golgi apparatus is involved in protein processing and sorting. DC-iDEP could help identify subpopulations of Golgi vesicles with specific cargo or processing capabilities, contributing to a better understanding of its role in cellular function.

By exploring the heterogeneity within these organelle systems using DC-iDEP, researchers can uncover novel subpopulations and gain insights into their functional significance in cellular processes.

How might the insights from this study on insulin vesicle heterogeneity inform our understanding of beta cell function and dysfunction in the context of diabetes and other metabolic disorders

The insights gained from studying insulin vesicle heterogeneity can significantly inform our understanding of beta cell function and dysfunction in the context of diabetes and other metabolic disorders.

Functional Implications: By identifying and characterizing distinct subpopulations of insulin vesicles, we can elucidate how these subpopulations contribute to the dynamic regulation of insulin secretion. Understanding the functional differences between vesicle subpopulations can provide insights into the mechanisms underlying glucose homeostasis and insulin release.

Disease Mechanisms: Studying insulin vesicle heterogeneity can offer valuable insights into the pathophysiology of diabetes. Differences in vesicle composition and maturation between healthy and diseased states can help identify potential molecular targets for therapeutic interventions and improve our understanding of beta cell dysfunction in diabetes.

Therapeutic Targets: The identification of specific subpopulations of insulin vesicles associated with glucose stimulation or dysfunction can lead to the discovery of novel therapeutic targets for metabolic disorders. Targeting these subpopulations could offer new strategies for modulating insulin secretion and improving glucose regulation in diabetic patients.

Overall, the study of insulin vesicle heterogeneity through techniques like DC-iDEP has the potential to advance our knowledge of beta cell biology and contribute to the development of innovative approaches for treating metabolic disorders.