A versatile mitochondria isolation- and analysis-pipeline generates 3D nano-topographies and mechano-physical surface maps of single organelles

Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cells interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM). Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization.

A Simplified Method to Isolate Rice Mitochondria

Background: Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers participate in the studies of plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, traditional plant mitochondria extraction methods are time-consuming and complicated operation of ultra-centrifuge with the expensive reagent. To develop a more rapid and convenient method for isolation of plant mitochondria, in this study we established a simplified method to isolate rice mitochondria efficiently for further study.Results: To isolate rice mitochondria, the cell wall was first dispelled by enzymolysis to obtain the protoplast which is similar to the animal cell. Then the rice mitochondria were isolated with a modified method basing on the animal mitochondria isolation protocol. The extracted mitochondria were next detected on DNA level and protein level to rule out the contamination of nucleus and chloroplasts. Furthermore, we examined the physiological status and characters of the isolated mitochondria, including the integrity of mitochondria, mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria were remained intact for further studies.Conclusion：The combination of plant protoplasts isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability, and can be broadly applied in the researches on plant mitochondria.