Abstract
Mitochondrial DNA is tightly packed into nucleoprotein complexes, called nucleoids, formed by mtDNA and mitochondrial transcription factor TFAM. Nucleoids inhabit the organelle, associated to inner mitochondrial membrane. The dynamics of mitochondria involves constant reshaping, and the relative dynamic localization of nucleoids and mitochondrial membranes, remains unexplored.We applied a machine-learning semi-automated method to study the simultaneous dynamic re-distribution of nucleoids and mitochondria. MEF cells were transfected with cDNA codifying mCherry-OMP25 (targeting the outer mitochondrial membrane, OMM) and Tfam-GFP (to visualize the nucleoids), and imaged the cells by high-resolution confocal microscopy at a Zeiss 880 Airyscan confocal microscope. Three-dimensional segmentation of Z-stacks series by means of trainable Weka protocol, allowed us to determine mitochondrial and nucleoid dimensions, shape descriptors, distribution and relative positioning over time, including speed and directionality. Our data show that the semi-automated method, trainable Weka, is a powerful tool to study mitochondrial nucleoids dynamics in the context of permanent mitochondria reshaping.
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