Abstract
Mitochondrial fission is a process that involves cleavage of mitochondria into smaller fragments and is regulated by the GTPase Dynamin-related protein 1 (Drp1). Higher levels of mitochondrial fission are associated with the induction of apoptosis in cancer cells. However, current methods to accurately quantify mitochondrial fission in order to compare therapeutics that target this process are often ambiguous or rely on subjective assessment. Mitochondria are also prone to aggregation, making accurate analysis difficult. Here we describe an improved approach for the quantification of mitochondrial fragmentation involving several differences from currently existing methods. Cells are first subjected to cytological centrifugation, which reduces cellular z-axis height and disperses individual mitochondria for easier observation. Three commercially available fluorescence analysis tools are then applied to disambiguate remaining mitochondrial clusters that require further inspection. Finally, cut-off scoring is applied, which can be tailored to individual cell type. The resultant approach allows for the efficient and objective assessment of mitochondrial fragmentation in response to treatment. We applied this technique to an experimental question involving chemosensitive and chemoresistant ovarian cancer (OVCA) cells. Cisplatin and the phytochemical piperlongumine were found to induce both mitochondrial fission and apoptosis in chemosensitive cells, while only piperlongumine was able to elicit these cellular responses in chemoresistant cells. Piperlongumine-induced apoptosis appeared to be mediated by Drp1-dependent mitochondrial fission since the apoptotic response was attenuated by the presence of the Drp1 inhibitor mDivi-1. Our study provides groundwork for a more objective approach to the quantification of mitochondrial fragmentation, and sheds further light on a potential mechanism of action for piperlongumine in the treatment of chemoresistant OVCA.
Highlights
IntroductionMitochondria are dynamic organelles found in most eukaryotic cells that undergo the processes of fission (dividing into separate structures) and fusion (merging of two or more adjacent structures)
Mitochondria are dynamic organelles found in most eukaryotic cells that undergo the processes of fission and fusion
Another approach requires the subjective judgment of mitochondrial morphology, and requires the operator to show images that are deemed to be representative of cells with tubular or fragmented mitochondria
Summary
Mitochondria are dynamic organelles found in most eukaryotic cells that undergo the processes of fission (dividing into separate structures) and fusion (merging of two or more adjacent structures). It fails to take into account the 3dimensional structure of mitochondria within cells, which does not allow measurement along the z-axis if a single 2-dimensional image is used Another approach requires the subjective judgment of mitochondrial morphology, and requires the operator to show images that are deemed to be representative of cells with tubular or fragmented mitochondria (other terms used to describe morphology include elongated, fused, intermediate, punctuated and ‘grainy’). Quantification using this approach is essentially based on the opinions of the observer, and each cell is categorized according to which representative image it more closely resembles [9,10,11]. Traditional immunocytochemistry in chambered vessels produces images that typically contain at least some mitochondria that are out of focus during fluorescence imaging, and incomplete representation of the entire specimen
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