iDEP-assisted isolation of insulin secretory vesicles

Understanding the inner workings of cells can be quite a challenge due to the incredible complexity and diversity within each cell. One of the fascinating aspects of cellular biology is the presence of various organelles—specialized structures within a cell that perform distinct functions. However, the current methods for separating and studying these organelles often fall short, especially when it comes to identifying different subpopulations of organelles. This is where a new technique, called direct current insulator-based dielectrophoresis (DC-iDEP), comes into play.

What is DC-iDEP?

DC-iDEP stands for direct current insulator-based dielectrophoresis. It’s a technique that uses an electric field to separate vesicles based on their physical and electrical properties. This method is unbiased, meaning it doesn’t favor any particular type of vesicle, making it especially useful for separating complex mixtures like the contents of a cell.

The Study: Separating Insulin Vesicles

Researchers at the University of Southern California applied DC-iDEP to study insulin vesicles—small sacs within pancreatic β-cells that store and release insulin. They used a multiple voltage strategy with DC-iDEP to increase the range and sensitivity of separation, allowing them to detect different subpopulations of vesicles.

By measuring a “differentiation factor,” which is the ratio of electrokinetic to dielectrophoretic mobility, the researchers were able to characterize the distribution patterns of these vesicles. They found a significant difference between insulin vesicles from cells that had been stimulated with glucose and those that hadn’t. This difference likely reflects the functional maturation of the vesicles in response to glucose, demonstrating that DC-iDEP can achieve high-resolution separation of vesicle subpopulations.

Schematic diagram for the formation of heterogenous insulin vesicles in pancreatic β-cells, graphical summation of disparate protein identifications, and processing of insulin vesicles including DC-iDEP device

A, insulin vesicle formation and maturation in a pancreatic β cell. Newly synthesized insulin is packed inside secretory vesicles which mature to store crystalline insulin in vesicles until secretion is stimulated through different signaling pathways. B, four published insulin vesicle proteomics studies aimed to identify the proteome of the heterogenous populations of secretory vesicles in β-cells with only 5 proteins identified consistently. C, separation of insulin vesicles using a DC-iDEP device. Differential and density gradient centrifugation were used to enrich each sample for insulin vesicle populations. Samples were then immunolabeled and introduced into DC-iDEP device for high resolution separation. Fluorescently labeled particles trapped near various gates in the channel are biophysically different subpopulations with varied EKMr (see text) values. The gates were constricted by increasing sizes of paired triangles, forming channel widths of 73 µm to 25 µm from inlet to the outlet. The different gates created Embedded Image and Embedded Image distributions for EKMr values.

Why Does This Matter?

The ability to separate and study different subpopulations of organelles, such as insulin vesicles, is crucial for understanding how cells function and respond to different conditions. For instance, in the case of pancreatic β-cells, understanding how insulin vesicles mature and release insulin in response to glucose can provide insights into diabetes and potential treatments.

DC-iDEP offers a new path for researchers to explore the subtle biochemical differences within organelle subpopulations, opening up possibilities for discoveries in various biological systems. This technique could revolutionize our approach to studying cell biology, allowing us to see the fine details that were previously hidden from view.

Future Implications

As researchers continue to refine and apply DC-iDEP, we can expect to gain a deeper understanding of cellular functions of organelles and vesicles within the cell. This could lead to breakthroughs in medical research, particularly in understanding diseases that involve cellular dysfunction, like diabetes, cancer, and neurodegenerative diseases.

Barekatain M, Liu Y, Wang Z, Cherezov V, Fraser SE, White KL, Hayes MA. (2024) iDEP-assisted isolation of insulin secretory vesicles. bioRXiv [online preprint]. [article]

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