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Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria.

Zhdanov AV, Dmitriev RI, Papkovsky DB - Cell. Mol. Life Sci. (2010)

Bottom Line: This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm.Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm.Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Republic of Ireland. a.zhdanov@ucc.ie

ABSTRACT
Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca(2+) and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 ((d)PC12), (d)SH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm. The response to Baf is regulated by cytosolic Ca(2+) fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.

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Baf induces flickering depolarization of the ΔΨm in dPC12 cells. a Colocalization of the TMRM and mitoCase12 probes in cells treated with Baf for 45 min decreases from 90–95% to 50–80%. b, c Large fluctuations of TMRM signals in individual mitochondria located in cell neurites and costained with mitoCase12 probe (binset) reveal a flickering pattern of ΔΨm depolarization. The dashed line (c) shows a cross-section of representative mitochondria selected for analysis. d, e In individual mitochondria localized in neurites (darrows), TMRM intensity periodically drops by 90 ± 5% and then returns to the basal level, while the mitoCase12 fluorescence shows 15–25% fluctuations. f Inhibition of PTP opening by CsA (2 μM) strongly increases the effect of Baf on ΔΨm depolarization. In contrast, thapsigargin (10 μM, Thaps) does not change the effect of Baf on the TMRM signal. However, the morphology of the mitochondria changes dramatically: they become large round compartments with an average diameter of 1–2 μm. g 15 min after the addition of Baf to the cells preincubated with CsA, TMRM intensity decreases to 15–50%, which is significantly different to the cells treated with Baf alone. Inhibition of F0F1 ATPase with oligomycin (10 μM, Olig) has a similar effect. Antimycin A completely depolarizes the mitochondria. Thapsigargin strongly increases variability of the response to Baf. All preincubations were done for 30 min. DMSO was used as a negative control. Bar 20 μm; asterisks significant differences
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Fig5: Baf induces flickering depolarization of the ΔΨm in dPC12 cells. a Colocalization of the TMRM and mitoCase12 probes in cells treated with Baf for 45 min decreases from 90–95% to 50–80%. b, c Large fluctuations of TMRM signals in individual mitochondria located in cell neurites and costained with mitoCase12 probe (binset) reveal a flickering pattern of ΔΨm depolarization. The dashed line (c) shows a cross-section of representative mitochondria selected for analysis. d, e In individual mitochondria localized in neurites (darrows), TMRM intensity periodically drops by 90 ± 5% and then returns to the basal level, while the mitoCase12 fluorescence shows 15–25% fluctuations. f Inhibition of PTP opening by CsA (2 μM) strongly increases the effect of Baf on ΔΨm depolarization. In contrast, thapsigargin (10 μM, Thaps) does not change the effect of Baf on the TMRM signal. However, the morphology of the mitochondria changes dramatically: they become large round compartments with an average diameter of 1–2 μm. g 15 min after the addition of Baf to the cells preincubated with CsA, TMRM intensity decreases to 15–50%, which is significantly different to the cells treated with Baf alone. Inhibition of F0F1 ATPase with oligomycin (10 μM, Olig) has a similar effect. Antimycin A completely depolarizes the mitochondria. Thapsigargin strongly increases variability of the response to Baf. All preincubations were done for 30 min. DMSO was used as a negative control. Bar 20 μm; asterisks significant differences

Mentions: The marked decrease in cellular TMRM signal upon Baf treatment (Fig. 1e) can be interpreted as partial depolarization of the whole mitochondrial pool of the cell. However, when analysing confocal images for spatial distribution of TMRM (ΔΨm) and mitoCase12 (mitochondrial Ca2+) within individual cells, we found a significant (25 ± 10%) decrease in their colocalization 45 min after Baf addition (Fig. 5a). Since the mitoCase12 probe has no tendency to leak (being a mitochondria-targeted protein), this result pointed to a decrease in the proportion of polarized mitochondria, rather than a more general partial depolarization. Indeed, further analysis of the TMRM signal in individual mitochondria in dPC12 cell neurites visualized by mitoCase12, revealed random, flickering depolarization, which can be seen 10 min after Baf application and lasts for several hours (Fig. 5b, c; Supplementary movie). In galactose(+) medium the flickering developed even more rapidly (within 5 min in individual cells). Although such flickering had rather high interexperiment variability complicating its detailed statistical analysis, it was clearly visible after 45-min incubation with Baf in about 15–55% of cells, and progressed to about 100% of the cell population in 1–3 h. Detailed time-lapse image analysis of cells after 1 h of Baf treatment (images taken every 0.5 s) revealed that ΔΨm flickering between the polarized and depolarized states occurred with an average period of 20 ± 10 s (Supplementary Fig. S6). Marked fluctuations in TMRM fluorescence (90 ± 5%) showed that most individual mitochondria underwent full depolarization and restoration of the ΔΨm. In control cells, transient fluctuations in TMRM signal without prominent flickering were observed in <3% of cells. Confocal images recorded at different sampling frequencies (every 5–20 s) showed that ΔΨm fluctuations were independent of illumination of the TMRM probe (which can also depolarize mitochondria upon photoactivation [46]).Fig. 5


Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria.

Zhdanov AV, Dmitriev RI, Papkovsky DB - Cell. Mol. Life Sci. (2010)

Baf induces flickering depolarization of the ΔΨm in dPC12 cells. a Colocalization of the TMRM and mitoCase12 probes in cells treated with Baf for 45 min decreases from 90–95% to 50–80%. b, c Large fluctuations of TMRM signals in individual mitochondria located in cell neurites and costained with mitoCase12 probe (binset) reveal a flickering pattern of ΔΨm depolarization. The dashed line (c) shows a cross-section of representative mitochondria selected for analysis. d, e In individual mitochondria localized in neurites (darrows), TMRM intensity periodically drops by 90 ± 5% and then returns to the basal level, while the mitoCase12 fluorescence shows 15–25% fluctuations. f Inhibition of PTP opening by CsA (2 μM) strongly increases the effect of Baf on ΔΨm depolarization. In contrast, thapsigargin (10 μM, Thaps) does not change the effect of Baf on the TMRM signal. However, the morphology of the mitochondria changes dramatically: they become large round compartments with an average diameter of 1–2 μm. g 15 min after the addition of Baf to the cells preincubated with CsA, TMRM intensity decreases to 15–50%, which is significantly different to the cells treated with Baf alone. Inhibition of F0F1 ATPase with oligomycin (10 μM, Olig) has a similar effect. Antimycin A completely depolarizes the mitochondria. Thapsigargin strongly increases variability of the response to Baf. All preincubations were done for 30 min. DMSO was used as a negative control. Bar 20 μm; asterisks significant differences
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Related In: Results  -  Collection

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Fig5: Baf induces flickering depolarization of the ΔΨm in dPC12 cells. a Colocalization of the TMRM and mitoCase12 probes in cells treated with Baf for 45 min decreases from 90–95% to 50–80%. b, c Large fluctuations of TMRM signals in individual mitochondria located in cell neurites and costained with mitoCase12 probe (binset) reveal a flickering pattern of ΔΨm depolarization. The dashed line (c) shows a cross-section of representative mitochondria selected for analysis. d, e In individual mitochondria localized in neurites (darrows), TMRM intensity periodically drops by 90 ± 5% and then returns to the basal level, while the mitoCase12 fluorescence shows 15–25% fluctuations. f Inhibition of PTP opening by CsA (2 μM) strongly increases the effect of Baf on ΔΨm depolarization. In contrast, thapsigargin (10 μM, Thaps) does not change the effect of Baf on the TMRM signal. However, the morphology of the mitochondria changes dramatically: they become large round compartments with an average diameter of 1–2 μm. g 15 min after the addition of Baf to the cells preincubated with CsA, TMRM intensity decreases to 15–50%, which is significantly different to the cells treated with Baf alone. Inhibition of F0F1 ATPase with oligomycin (10 μM, Olig) has a similar effect. Antimycin A completely depolarizes the mitochondria. Thapsigargin strongly increases variability of the response to Baf. All preincubations were done for 30 min. DMSO was used as a negative control. Bar 20 μm; asterisks significant differences
Mentions: The marked decrease in cellular TMRM signal upon Baf treatment (Fig. 1e) can be interpreted as partial depolarization of the whole mitochondrial pool of the cell. However, when analysing confocal images for spatial distribution of TMRM (ΔΨm) and mitoCase12 (mitochondrial Ca2+) within individual cells, we found a significant (25 ± 10%) decrease in their colocalization 45 min after Baf addition (Fig. 5a). Since the mitoCase12 probe has no tendency to leak (being a mitochondria-targeted protein), this result pointed to a decrease in the proportion of polarized mitochondria, rather than a more general partial depolarization. Indeed, further analysis of the TMRM signal in individual mitochondria in dPC12 cell neurites visualized by mitoCase12, revealed random, flickering depolarization, which can be seen 10 min after Baf application and lasts for several hours (Fig. 5b, c; Supplementary movie). In galactose(+) medium the flickering developed even more rapidly (within 5 min in individual cells). Although such flickering had rather high interexperiment variability complicating its detailed statistical analysis, it was clearly visible after 45-min incubation with Baf in about 15–55% of cells, and progressed to about 100% of the cell population in 1–3 h. Detailed time-lapse image analysis of cells after 1 h of Baf treatment (images taken every 0.5 s) revealed that ΔΨm flickering between the polarized and depolarized states occurred with an average period of 20 ± 10 s (Supplementary Fig. S6). Marked fluctuations in TMRM fluorescence (90 ± 5%) showed that most individual mitochondria underwent full depolarization and restoration of the ΔΨm. In control cells, transient fluctuations in TMRM signal without prominent flickering were observed in <3% of cells. Confocal images recorded at different sampling frequencies (every 5–20 s) showed that ΔΨm fluctuations were independent of illumination of the TMRM probe (which can also depolarize mitochondria upon photoactivation [46]).Fig. 5

Bottom Line: This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm.Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm.Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Republic of Ireland. a.zhdanov@ucc.ie

ABSTRACT
Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca(2+) and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 ((d)PC12), (d)SH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm. The response to Baf is regulated by cytosolic Ca(2+) fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.

Show MeSH
Related in: MedlinePlus