Optogenetic Manipulation Of Mitochondrial Distribution And Function In Living Cells

Mitochondria are very important organelles in eukaryotic cells.The balance of mitochondrial fission and fusion and their distribution in cells play an important role in the maintenance of cell homeostasis.However,due to the lack of effective regulation methods,the relationship between mitochondrial morphology,intracellular localization and functions is unclear.In this thesis,optogenetics tools were used to realize fast optical manipulation of mitochondrial aggregated distribution and localization in living cells,combined with microscopic imaging technology to study the dynamic changes of mitochondrial distribution and localization during the optical manipulation,and the effects of instantaneous and reversible optical manipulation on mitochondrial functions were explored by biochemical methods.The main results of the theis project are summarized below:1.Optogenetic manipulation of mitochondrial aggregated distribution in living cellsBased on the dimerization of the CRY2-CIBN system and the oligomerization of CRY2 high,the optically-controlled protein element Opto-Mito C(Tom20-CIBN-GFP,m Cherry-CRY2high)was used to realize the optical manipulation of mitochondrial aggregated distribution in COS-7 cells,and the real-time tracking microimaging with quantitative analysis of the aggregation degree were carried out.The results showed:(1)Opto-Mito C successfully induced optically-controlled mitochondrial aggregation in COS-7 cells.(2)An image entropy algorithm was established to quantitatively analyze the degree of mitochondrial aggregation.(3)Although Opto-Mito C was reversible and depolymerized after the removal of blue light stimulation,the mitochondrial aggregated distribution caused by Opto-Mito C was irreversible.2.Optogenetic manipulation of rapid mitochondrial transport to cell membraneBy optogenetic manipulation of KIF5A-CIBN-GFP,a plasmid containing Kinesin Family Member 5A,and Miro1-m Cherry-CRY2 which located in the mitochondrial outer membrane,the mitochondria of COS-7 cells were rapidly transported to the cell membrane,and real-time tracking imaging with the quantitative analysis of mitochondrial distribution were performed.The results showed:(1)The optogenetic manipulation of rapid mitochondrial transport to cell membrane could be realized by recruiting kinesin to mitochondrial outer membrane through the light.(2)The change of the ratio of the perinuclear fluorescence area to the whole-cell fluorescence area can be used to quantitatively analyze the degree of mitochondrial localization around the cell membrane.(3)The transport of mitochondria to the cell membrane induced by optically recuiting kinesin changed mitochondrial morphology and was not completely reversible.3.Influences of mitochondrial aggregated distribution and localization around the cell membrane on mitochondrial functionsThe results showed:(1)The mitochondrial aggregation induced by Opto-Mito C could significantly rescue mitochondrial dysfunctions induced by Niclosamide,could up-regulate the cell ATP level,reduce the cell ROS content,reduce the opening degree of mitochondrial permeability transition pore,and the effects were not reversible.(2)Mitochondrial transport to the cell membrane controlled by light could up-regulate ROS content and increase ATP level in COS-7 cells,and the effects were not reversible in the short term.In a word,this thesis established optogenetic methods to manipulate mitochondrial aggregated distribution and localization in living cells,combined with image quantitative analysis techniques and biochemical methods,to explore the changes of mitochondrial morphology and distribution and their effects on cellular mitochondrial functions.The research results can deepen the understanding of the physiological mechanisms of cellular mitochondria and provide a new method for future research on mitochondrial localization and functions.