Figure 1.
Cytoplasmic Ca2+ oscillations run down quickly after MCU knockdown.
A, Control recording to LTC4 in 2 mM external Ca2+. B, Response to LTC4 after MCU knockdown. C, Mitochondrial depolarisation with FCCP (5 µM) accelerates run down of oscillations. D, Aggregate data showing the number of oscillations in 200 seconds recording bins from several experiments are compared (each point is the average of between 36 and 51 cells). E, The size of each oscillation is plotted against Ca2+ oscillation number for the conditions shown (each point is the average of between 35 and 50 cells).
Figure 2.
Graded relationship between mitochondrial depolarisation and run down of Ca2+ oscillations.
A, Increasing the concentration of FCCP leads to increased mitochondrial depolarisation. B, The graph compares the effects of different concentrations of FCCP on mitochondrial membrane potential and the number of Ca2+ oscillations evoked by LTC4 over a 600 seconds recording period. C, Control recording to LTC4. D-F, Effects of increasing FCCP concentration of oscillatory Ca2+ signals. Cells from C-F were all from the same cell preparation and were used on the same day.
Figure 3.
Leukotriene receptor stimulation induces oscillatory Ca2+ signals in the mitochondrial matrix.
A, Oscillatory Ca2+ response to LTC4, measured using the ratiometric pericam. B, The number of oscillations per 200 seconds bin are compared for the conditions shown. Each point is the mean of between 16 and 27 cells. C, The amplitude of each oscillation is compared for the conditions shown. D, Mitochondrial depolarisation with FCCP prevents the matrix rise in Ca2+. E, Knockdown of MCU prevents matrix Ca2+ rise to LTC4. F, Aggregate data are summarised for the conditions shown. Each bar is the average of between 12 and 17 cells. G, Single wavelength pericam (488 nm and 430 nm) recordings are shown along with the corresponding ratio.
Figure 4.
Regenerative Ca2+ release in the absence of Ca2+ influx is regulated by mitochondrial Ca2+ uptake.
A, LTC4 evokes repetitive Ca2+ oscillations in the presence of 0 mM Ca2+ external solution supplemented with 1 mM La3+. B, The oscillations run down quickly after mitochondrial depolarisation. C, The Ca2+ oscillations run down quickly after knockdown of MCU. D, Aggregate data comparing the number of oscillations in each 200 seconds bin from several experiments are compared. E, As in panel D, but the amplitude of each oscillation is compared. F, Oscillatory Ca2+ signals are seen in the matrix in response to LTC4 in 0 mM Ca2+/1 mM La3+. G, Matrix Ca2+ response is prevented by FCCP. H, Knockdown of MCU also suppresses the matrix Ca2+ rise in response to LTC4 challenge. I, Aggregate data from several experiments are compared. Each bar is the average of between 11 and 18 cells. J, Ca2+ release evoked by P2Y receptor activation is reduced by pre-exposure to LTC4. K, Aggregate data from several cells are compared. ATP bar represents 27 cells and LTC4/ATP group is 34 cells. L, Ca2+ release to thapsigargin is unaffected by prior stimulation with LTC4. M, Aggregate data measuring the rate of rise of cytoplasmic Ca2+ following thapsigargin application (as in panel L) are compared. Thap bar represents data from 11 cells, and LTC4/thap 14 cells.
Figure 5.
MCU knockdown impairs leukotriene receptor-dependent gene expression.
A, Mitochondrial depolarisation or knockdown of MCU suppresses c-fos transcription. Aggregate data (mean of 3 independent experiments) are shown in lower panel. Cells were stimulated with LTC4 (160 nM; 4 minutes) in 2 mM external Ca2+ and then cells were perfused with Ca2+-free solution (without agonist) for a further 41 minutes before RNA extraction. B, Expression of GFP (under an NFAT promoter) is shown for the various conditions indicated. C, Aggregate data from several experiments are compared. In these experiments, cells were stimulated with LTC4 in medium for 15 minutes and this was then replaced with LTC4-free medium. GFP expression was measured 24 hours later.
Figure 6.
MCU is required for supporting store-operated Ca2+ entry.
A, Store-operated Ca2+ entry, evoked by 2 µM thapsigargin, is inhibited by knockdown of MCU. B, Aggregate data, measuring the rate of rise of cytoplasmic Ca2+ following readmission of external Ca2+ (as in Panel A) are compared. Control denotes 38 cells and MCU KD 29 cells. C, Matrix Ca2+ measurements show that store-operated Ca2+ influx is buffered by mitochondria in an MCU-dependent manner. D, Aggregate data from experiments as in Panel C are compared. Control denotes 21 cells and MCU KD 17 cells. E, MCU knockdown reduces c-fos transcription to thapsigargin (2 µM applied for 4 minutes in external Ca2+, followed by wash in Ca2+-free solution for 41 minutes before RNA extraction). Aggregate data from 3 independent experiments are shown in the lower panel. F, GFP reporter expression (under an NFAT promoter) stimulated by thapsigargin (100 nM) is reduced following MCU knockdown.