mitoLUHMES: An Engineered Neuronal Cell Line for the Analysis of the Motility of Mitochondria.

Perturbations in the transport of mitochondria and their quality control in neuronal cells underlie many types of neurological pathologies, whereas systems enabling convenient analysis of mitochondria behavior in cellular models of neurodegenerative diseases are limited. In this study, we present a modified version of lund human mesencephalic cells, mitoLUHMES, expressing GFP and mitochondrially targeted DsRed2 fluorescent proteins, intended for in vitro analysis of mitochondria trafficking by real-time fluorescence microscopy. This cell line can be easily differentiated into neuronal phenotype and allows us to observe movements of single mitochondria in single cells grown in high-density cultures. We quantified the perturbations in mitochondria morphology and dynamics in cells treated with model neurotoxins: carbonyl cyanide m-chlorophenylhydrazone and 6-hydroxydopamine. For the first time we filmed the processes of fission, fusion, pausing, and reversal of mitochondria movement direction in LUHMES cells. We present a detailed analysis of mitochondria length, velocity, and frequency of movement for static, anterograde, and retrograde motile mitochondria. The observed neurotoxin treatment-mediated decreases in morphological and kinetic parameters of mitochondria provide foundation for the future studies exploiting mitoLUHMES as a new model for neurobiology.