Department of Chemistry, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, United States.
School of Molecular Sciences, Arizona State University, Tempe, United States.
Elife. 2024 Aug 27;13:e74989. doi: 10.7554/eLife.74989.
Organelle heterogeneity and inter-organelle contacts within a single cell contribute to the limited sensitivity of current organelle separation techniques, thus hindering organelle subpopulation characterization. Here, we use direct current insulator-based dielectrophoresis (DC-iDEP) as an unbiased separation method and demonstrate its capability by identifying distinct distribution patterns of insulin vesicles from INS-1E insulinoma cells. A multiple voltage DC-iDEP strategy with increased range and sensitivity has been applied, and a differentiation factor (ratio of electrokinetic to dielectrophoretic mobility) has been used to characterize features of insulin vesicle distribution patterns. We observed a significant difference in the distribution pattern of insulin vesicles isolated from glucose-stimulated cells relative to unstimulated cells, in accordance with maturation of vesicles upon glucose stimulation. We interpret the difference in distribution pattern to be indicative of high-resolution separation of vesicle subpopulations. DC-iDEP provides a path for future characterization of subtle biochemical differences of organelle subpopulations within any biological system.
细胞器的异质性和细胞内细胞器之间的接触导致了当前细胞器分离技术的灵敏度有限，从而阻碍了细胞器亚群的特征描述。在这里，我们使用基于直流绝缘子的介电泳（DC-iDEP）作为一种无偏的分离方法，并通过鉴定 INS-1E 胰岛素瘤细胞中胰岛素囊泡的不同分布模式来证明其能力。我们应用了具有更大范围和更高灵敏度的多电压 DC-iDEP 策略，并使用分化因子（电泳和介电泳迁移率的比值）来描述胰岛素囊泡分布模式的特征。我们观察到，与未受刺激的细胞相比，从葡萄糖刺激的细胞中分离出的胰岛素囊泡的分布模式有显著差异，这与葡萄糖刺激时囊泡的成熟一致。我们将分布模式的差异解释为囊泡亚群的高分辨率分离。DC-iDEP 为未来在任何生物系统中对细胞器亚群的细微生化差异进行特征描述提供了一种途径。
