Mitochondrial dynamics and quantification of mitochondria-derived vesicles in cardiomyoblasts using structured illumination microscopy.

Ida S Opstad,Krishna Agarwal,Balpreet Singh Ahluwalia,Truls Myrmel,Gustav Godtliebsen,Åsa Birna Birgisdottir

doi:10.1002/jbio.202100305

Abstract

Mitochondria are essential energy-providing organelles of particular importance in energy-demanding tissue such as the heart. The production of mitochondria-derived vesicles (MDVs) is a cellular mechanism by which cells ensure a healthy pool of mitochondria. These vesicles are small and fast-moving objects not easily captured by imaging. In this work, we have tested the ability of the optical super-resolution technique 3DSIM to capture high-resolution images of MDVs. We optimized the imaging conditions both for high-speed video microscopy and fixed-cell imaging and analysis. From the 3DSIM videos, we observed an abundance of MDVs and many dynamic mitochondrial tubules. The density of MDVs in cells was compared for cells under normal growth conditions and cells during metabolic perturbation. Our results indicate a higher abundance of MDVs in H9c2 cells during glucose deprivation compared with cells under normal growth conditions. Furthermore, the results reveal a large untapped potential of 3DSIM in MDV research.

Highlights

Mitochondria are the energy providing organelles in cells and produce energy in the form of adenosine triphosphate (ATP)
Mitochondria are arranged in highly dynamic networks controlled by frequent mitochondrial fusion and fission events driven by mitochondria movements on the cytoskeleton [1]
We investigated the capabilities of threedimensional structured illumination microscopy (3DSIM) for visualizing and quantifying mitochondriaderived vesicles (MDVs) in live and fixed H9c2 cardiomyoblasts with a stable expression of a fluorescent transmembrane domain of the outer mitochondria membrane protein 25 (OMP25) [9]

Summary

| INTRODUCTION

Mitochondria are the energy providing organelles in cells and produce energy in the form of adenosine triphosphate (ATP). The MDVs' size and dynamic nature pose a challenge for conducting imaging studies ( for live imaging) of their formation and trafficking. Elucidation of their significance for mitochondria homeostasis as well as cell function in general is important, especially in high-energy demanding cardiac cells. The acquisition of images at high enough resolution and contrast to allow MDVs to be visualizable by eye is a significant challenge, but not the only hurdle in gathering knowledge about MDVs. Other significant challenges are the appropriate labeling for super-resolution microscopy (e.g., bright, photostable and specific fluorescent markers) and the quantification of these small and (in living cells) dynamic structures. Replacing glucose in the growth media with galactose forces cells in culture to become more oxidative and has been shown to facilitate stress-induced MDV production [10, 11]

| METHODS

| RESULTS AND DISCUSSION

| CONCLUSIONS