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Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells
Published on: April 27, 2018
[Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From
Sergej L Mironov1, Maxim V Ivannikov, Mattias Johansson
1Institute for Physiology, Georg August University, Humboldtallee 23, 37073 Göttingen, Germany. slm@ukps.gwdg.de
Abstract:
The positioning and dynamics of organelles depend on membrane-cytoskeleton interactions. Mitochondria relocate along microtubules (MT), but it is not clear whether MT have direct effects on mitochondrial function. Using two-photon microscopy and the mitochondrial fluorescent dyes rhodamine 123 and Rhod-2, we showed that Taxol and nocodazole, which correspondingly stabilize and disrupt MT, decreased potential and Ca(2+) in the mitochondria of brain stem pre-Botzinger complex neurons. Without changing basal cytoplasmic Ca(2+) ([Ca(2+)](i)), Taxol promoted the generation of [Ca(2+)](i) spikes in dendrites. These spikes were abolished after blockade of Ca(2+) influx and after depletion of internal Ca(2+) stores, indicating the involvement of Ca(2+)-induced Ca(2+) release. Nocodazole decreased mitochondrial potential and [Ca(2+)](m) and produced a long lasting increase in [Ca(2+)](i). MT-acting drugs depolarized single immobilized mitochondria and released previously stored Ca(2+). All of these effects were inhibited by pretreatment with blockers of mitochondrial permeability transition pore (mPTP), cyclosporin A, and 2-aminoethoxydiphenyl borate. Induction of mPTP by Taxol and nocodazole was confirmed by using a calcein/Co(2+) imaging technique. Electron and optical microscopy revealed tubulin bound to mitochondria. Mitochondria, MT, and endoplasmic reticulum (ER) showed strong co-localization, the degree of which decreased after MT were disrupted. We propose that changes in the structure of MT by Taxol and nocodazole promote the induction of mPTP. Subsequent Ca(2+) efflux stimulates the Ca(2+) release from the ER that drives spontaneous [Ca(2+)](i) transients. Thus, close positioning of mitochondria to the ER as determined by MT can be essential for the local [Ca](i) signaling in neurons.
Insights
Microtubule (MT) stabilization or disruption impacts mitochondrial function and calcium signaling in neurons. MT alterations induce mitochondrial permeability transition pore opening, affecting neuronal calcium dynamics.
Area of Science:
- Neuroscience
- Cell Biology
- Mitochondrial Biology
Background:
- Organelle positioning relies on cytoskeleton interactions, particularly mitochondria along microtubules (MT).
- The direct impact of MT on mitochondrial function remains unclear.
Purpose of the Study:
- To investigate the direct effects of MT stabilization and disruption on mitochondrial function and calcium signaling in neurons.
- To elucidate the role of MT-cytoskeleton interactions in regulating mitochondrial calcium handling and neuronal excitability.
Main Methods:
- Utilized two-photon microscopy with mitochondrial fluorescent dyes (rhodamine 123, Rhod-2) in brain stem pre-Botzinger complex neurons.
- Administered MT-acting drugs (Taxol, nocodazole) and assessed mitochondrial potential, mitochondrial calcium ([Ca(2+)](m)), and cytoplasmic calcium ([Ca(2+)](i)) dynamics.
- Employed Ca(2+) influx blockers, internal Ca(2+) store depletion, mitochondrial permeability transition pore (mPTP) blockers (cyclosporin A, 2-aminoethoxydiphenyl borate), and calcein/Co(2+) imaging.
Main Results:
- Taxol and nocodazole decreased mitochondrial potential and [Ca(2+)](m).
- Taxol induced dendritic [Ca(2+)](i) spikes via Ca(2+)-induced Ca(2+) release, while nocodazole caused a sustained [Ca(2+)](i) increase.
- MT-disrupting drugs induced mPTP opening, releasing mitochondrial Ca(2+) and stimulating endoplasmic reticulum (ER) Ca(2+) release, leading to spontaneous [Ca(2+)](i) transients.
- Tubulin was observed bound to mitochondria, and MT disruption reduced mitochondria-ER co-localization.
Conclusions:
- MT structure directly influences mitochondrial function and neuronal calcium signaling.
- MT-dependent positioning of mitochondria near the ER is crucial for local intracellular calcium ([Ca(2+)](i)) signaling.
- MT stabilization or disruption can induce mitochondrial permeability transition pore opening, impacting neuronal excitability.
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