Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Elena Levi-D&#8217;Ancona; Vaibhav Sidarala; Scott A. Soleimanpour

doi:10.3791/65789

Journal

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Biology

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Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells

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Biology

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JoVE Journal Biology

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells

1,400 Views

•

07:04 min

•

September 15, 2023

DOI:

10.3791/65789-v

DOI:

10.3791/65789-v

•

07:04 min

September 15, 2023

•

1313 Views

Elena Levi-D’Ancona^1,2, Vaibhav Sidarala¹, Scott A. Soleimanpour^1,3,4

¹Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes,University of Michigan, Ann Arbor ²Graduate Program in Immunology,University of Michigan Medical School ³Department of Molecular and Integrative Physiology,University of Michigan, Ann Arbor ⁴VA Ann Arbor Healthcare System

Transcript

Our protocol describes two complementary methods to study beta-cell mitophagy flux at the single-cell resolution via flow cytometry. The mt-Keima protocol utilizes a genetic mitophagy reporter, whereas the MtPhagy protocol combines three fluorescent dyes, which makes it easy to extrapolate to difficult-to-transfect cells in human samples. Assessments of mitophagy using traditional biochemical approaches and imaging approaches are both time consuming and challenging.

Thus, it’s important to develop new, robust, and effective methods to study mitophagy flux in beta-cells. Both the mt-Keima and MtPhagy method are quantitative assessments that are efficient for studying mitophagy flux in pancreatic beta cells. So, the Soleimanpour lab focuses on the different mechanisms that drive pancreatic beta-cell failure and diabetes.

And we specifically focus on the different aspects of mitochondrial lifecycle, including mitophagy, to understand how defects in mitochondrial function drive the development of diabetes.

Summary

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic β-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.