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Simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during apoptosis.

ABSTRACT: Cell apoptosis is a biochemical and molecular pathway essential for maintaining cellular homeostasis. It is an integrated process involving in a series of signal transduction cascades. Moreover, the apoptotic pathways may be initiated inside various subcellular organelles. Increasing evidence indicates that hydrogen peroxide (H₂O₂) is closely related to cell apoptosis, particularly in the mitochondria. However, during the apoptotic process, the synergetic variation of H₂O₂ levels in different compartments is seldom explored, particularly in two important organelles: mitochondria and endoplasmic reticulum (ER). To solve this problem, we developed two new organelle-specific fluorescent probes termed MI-H₂O₂ and ER-H₂O₂ that can detect H₂O₂ in mitochondria and ER, respectively or simultaneously. Experimental results demonstrated that MI-H₂O₂ and ER-H₂O₂ display distinguishable excitation and emission spectra, as well as excellent organelle targeting capabilities. Therefore, we used MI-H₂O₂ and ER-H₂O₂ to successfully image exogenous or endogenous hydrogen peroxide in the mitochondria and ER. Interestingly, during diverse apoptotic stimuli, dual-color fluorescence imaging results revealed that the changes of H₂O₂ levels in mitochondria and ER are different. The H₂O₂ levels are enhanced in both the mitochondria and ER during the l-buthionine sulfoximine (BSO)-treated cell apoptosis process. During mitochondria-oriented apoptosis induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or rotenone, H₂O₂ levels prominently and continuously increase in the mitochondria, whereas the ER H₂O₂ levels were found to rise subsequently after a delay. Moreover, during ER-oriented apoptosis induced by tunicamycin, ER is the major site for overproduction of H₂O₂, and delayed elevation of the H₂O₂ levels was found in the mitochondria. Altogether, this dual-probe and multicolor imaging approach may offer a proven methodology for studying molecular communication events on H₂O₂-related apoptosis and also other physiological and pathological processes within different subcellular organelles.

SUBMITTER: Xiao H

PROVIDER: S-EPMC6024174 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during apoptosis.

Xiao Haibin H Li Ping P Hu Xiufen X Shi Xiaohui X Zhang Wen W Tang Bo B

Chemical science 20160601 9

Cell apoptosis is a biochemical and molecular pathway essential for maintaining cellular homeostasis. It is an integrated process involving in a series of signal transduction cascades. Moreover, the apoptotic pathways may be initiated inside various subcellular organelles. Increasing evidence indicates that hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is closely related to cell apoptosis, particularly in the mitochondria. However, during the apoptotic process, the synergetic variation of H<sub ...[more]

PMID: 30034754

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Project description:Subcellular organelles communicate with each other to regulate function and coordinate responses to changing cellular conditions. The physical-functional coupling of the endoplasmic reticulum (ER) with mitochondria allows for the direct transfer of Ca2+ between organelles and is an important avenue for rapidly increasing mitochondrial metabolic activity. As such, increasing ER-mitochondrial coupling can boost the generation of ATP that is needed to restore homeostasis in the face of cellular stress. The mitochondrial unfolded protein response (mtUPR) is activated by the accumulation of unfolded proteins in mitochondria. Retrograde signaling from mitochondria to the nucleus promotes mtUPR transcriptional responses aimed at restoring protein homeostasis. It is currently unknown whether the changes in mitochondrial-ER coupling also play a role during mtUPR stress. We hypothesized that mitochondrial stress favors an expansion of functional contacts between mitochondria and ER, thereby increasing mitochondrial metabolism as part of a protective response. Hela cells were treated with doxycycline, an antibiotic that inhibits the translation of mitochondrial-encoded proteins to create protein disequilibrium. Treatment with doxycycline decreased the abundance of mitochondrial encoded proteins while increasing expression of CHOP, C/EBPβ, ClpP, and mtHsp60, markers of the mtUPR. There was no change in either mitophagic activity or cell viability. Furthermore, ER UPR was not activated, suggesting focused activation of the mtUPR. Within 2 h of doxycycline treatment, there was a significant increase in physical contacts between mitochondria and ER that was distributed throughout the cell, along with an increase in the kinetics of mitochondrial Ca2+ uptake. This was followed by the rise in the rate of oxygen consumption at 4 h, indicating a boost in mitochondrial metabolic activity. In conclusion, an early phase of the response to doxycycline-induced mitochondrial stress is an increase in mitochondrial-ER coupling that potentiates mitochondrial metabolic activity as a means to support subsequent steps in the mtUPR pathway and sustain cellular adaptation.

Dataset Information

Simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during apoptosis.

Publications

Simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during apoptosis.

Similar Datasets

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