Quantifying CO₂-dependent positional changes of mitochondria in wild-type and miro mutants of Chlamydomonas...

Exposure of Chlamydomonas reinhardtii (Chlamydomonas throughout) to very low levels of CO₂ (VLC) elicits the synthesis of a carbon concentrating mechanism (CCM; concentrates CO₂ around ribulose-1,5-bisphosphate carboxylase, the Calvin-Benson cycle enzyme involved in the initial CO₂ fixation reaction). Additionally, VLC levels cause relocation of the network of mitochondrial membranes from the cell interior, mostly within the chloroplast "cup," to the cell periphery (cortical region between the chloroplast envelope and plasma membrane), with the tubular network of mitochondrial membranes transitioning from an irregular, interconnected network to a parallel array of apicobasal membrane tubules. In this article, we discuss our newly developed method for quantifying the dynamics of the mitochondrial network as environmental conditions change in both wild-type (WT) cells and a miro1 mutant. This method uses confocal fluorescence microscopy and involves approximating the shape of a Chlamydomonas cell to an ellipsoid in 3D space, precisely calculating the cell's dimensions and determining the length of the fluorescently tagged mitochondrial membrane tubules and their specific subcellular locations. The method is flexible and can be adapted for elucidating the distribution and morphology of mitochondrial membrane tubules and other organelles in a range of organisms. In this study, we use it to evaluate the position and structure of mitochondrial membranes in WT Chlamydomonas cells and a mutant null for a gene encoding the microtubule-mitochondrion interacting protein designated MIRO1. The relocation and rearrangement of the mitochondrial network following exposure of WT cells to different CO₂ levels and light intensities demonstrated the presence of a small population of the mitochondrial membranes that remained apposed to the plasma membrane even under high CO₂ conditions, suggesting the potential occurrence of a distinct plasma membrane-associated population of mitochondrial membranes. Furthermore, there is a marked difference in the cell size and the dimensions of these membranes under high versus low CO₂ conditions, suggesting that the size of the cell and the morphology of the mitochondrial membrane vesicles can be strongly impacted by CO₂ availability, which could alter the cell surface to volume ratio and the ability of the cells to efficiently capture inorganic carbon. In the miro mutant the mitochondrial membranes still migrate to the cell periphery although the coverage of the peripherally facing chloroplast envelope by mitochondrial membranes is approximately 50% that of WT cells, and unlike WT cells, the membrane tubules do not assume a clear apicobasal orientation and appear to aggregate. Furthermore, under elevated CO₂ conditions the miro mutant has many fewer mitochondrial membranes at the cell periphery (proximal to the plasma membrane) relative to WT cells, suggesting that the mutant has an aberration in the relocation process. However, we were unable to detect a major impact on CCM function or fitness for the miro mutant relative to WT cells following exposure to very low CO₂ conditions under the specific growth and assay conditions used in this study.