Automated detection of whole‐cell mitochondrial motility and its dependence on cytoarchitectural integrity

Abstract

Current methodologies used for mitochondrial motility analysis tend to either overlook individual mitochondrial tracks or analyze only peripheral mitochondria instead of mitochondria in all regions of the cell. Furthermore, motility analysis of an individual mitochondrion is usually quantified by establishing an arbitrary threshold for “directed” motion. In this work, we created a custom, publicly available computational algorithm based on a previously published approach (Giedt et al., 2012) in order to characterize the distribution of mitochondrial movements at the whole-cell level, while still preserving information about single mitochondria. Our technique is easy to use, robust and computationally inexpensive. Images are first pre-processed for increased resolution, and then individual mitochondria are tracked based on object connectivity in space and time. When our method is applied to microscopy fields encompassing entire cells, we reveal that the mitochondrial net distances in fibroblasts follow a lognormal distribution within a given cell or group of cells. The ability to model whole-cell mitochondrial motility as a lognormal distribution provides a new quantitative paradigm by which to compare mitochondrial motility in naïve and treated cells. We further demonstrate that microtubule and microfilament depolymerization shift the lognormal distribution in directions which indicate decreased and increased mitochondrial movement, respectively. These findings advance earlier work on neuronal axons (Morris and Hollenbeck, 1993) by relating them to a different cell type, applying them on a global scale, and automating measurement of mitochondrial motility in general.

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins

“…Consistent with the ‘tip-tracking’ model described above, a number of research studies showed that mitochondrial motility was impaired upon microtubule depolymerization (Kandel et al, 2015; Ligon and Steward, 2000; Morris and Hollenbeck, 1995; Muller et al, 2005; Yi et al, 2004). While a consensus has emerged regarding the role of the microtubular cytoskeleton, conflicting results have been published on the relationships between the actin cytoskeleton and mitochondrial movements.…”

“…While a consensus has emerged regarding the role of the microtubular cytoskeleton, conflicting results have been published on the relationships between the actin cytoskeleton and mitochondrial movements. Actin filament depolarization was shown to influence mitochondrial motility either negatively (Muller et al, 2005; Quintero et al, 2006) or positively (Ligon and Steward, 2000; Morris and Hollenbeck, 1995), partly depending on the cell type analyzed (Kandel et al, 2015). Interestingly, depolymerizing the actin cytoskeleton was recently suggested to blunt mitochondrial motility only in neuronal processes, and not in the cell body (Narayanareddy et al, 2014), providing further support to the idea that distinct control mechanisms may operate in different spatially-restricted cytoplasmic regions.…”

Mitochondrial respiration is sensitive to cytoarchitectural breakdown

“…Adult human dermal fibroblasts between passages 1 and 5 (Lifeline Cell Technology, Walkersville, MD) were cultured in FibroLife cell culture media (Lifeline Cell Technology) as previously described [16]. Cells were seeded approximately 48 hours prior to experiments.…”

“…Changes in mitochondrial inner membrane potential and morphology in single cells were assessed using wide-field fluorescence microscopy as previously described [16]. Cell viability was also measured using this setup.…”

“…Here we examine the mitochondrial consequences of cytostructural impairment. This work builds upon our previous research characterizing the effects of cytoskeletal toxins on mitochondrial motility in particular [16]. …”