Techniques for Quantitative Analysis of Mitochondrial Dynamics

Abstract

Alterations of mitochondrial dynamics (fusion/fission and motility) and ER-mitochondria associations have emerged as feature of a number of pathways. Analysis of these aspects has remained largely qualitative and manual, limiting their potential use as biomarkers. Here we demonstrate techniques for unbiased, quantitative and automatized image analysis of mitochondrial dynamics and ER-mitochondrial interactions. Mitochondrial fusion is evaluated in cultured cells expressing photoactivatable GFP and DsRed in the mitochondria by a simple count of green-only pixels following photoactivation. This approach is validated against a manual count of fusion events. We have employed difference image calculation to measure motility for several years; now we have developed a method to automatically calculate the optimal threshold for this assay. Finally, we have developed methods for quantitative evaluation of the interface between ER and mitochondria in electron micrographs. These scripts provide both granular data on individual interactions (distances, surface covered, etc.) and also attempt to create a simple ‘score’ that can easily be used to compare across conditions.