We next performed detailed secondary analyses of Ca2+ homeostasis in sh1-MICU1 knockdown cells, using multiple techniques ().
Measurement of mitochondrial Ca2+ uptake kinetics in populations of cells, individual cells, and permeabilized cells
First, we measured mitochondrial Ca2+
uptake in populations of cells using two methods to mobilize Ca2+
. As can be seen in , histamine stimulated IP3
-signaling in control mt-AEQ HeLa cells (pLKO.1) resulted in typical kinetics of mitochondrial calcium uptake3
. However, mitochondrial Ca2+
uptake was impaired by silencing MICU1
, confirming the screening result (). We observed the same Ca2+
phenotype when using store-operated Ca2+
entry to raise intracellular free calcium31
. Namely, we pre-treated cells with thapsigargin, an inhibitor of the SERCA pump, in the absence of extracellular Ca2+
to deplete ER stores and activate store-operated Ca2+
channels at the plasma membrane. The readdition of 2 mM Ca2+
resulted in a transient rise of [Ca2+
in control cells while MICU1
-knockdown cells did not respond (), consistent with the result obtained with histamine stimulation ().
Second, we performed quantitative, single cell analyses of basal [Ca2+
and agonist-stimulated rises in [Ca2+
in HeLa cells using a mitochondria-targeted calcium FRET reporter32
. We observed a significant difference in the baseline [Ca2+
in control cells versus sh1-MICU1
knockdown cells (). Moreover, histamine treatment induced an increase in [Ca2+
in control cells (fractional saturation of 0.75 ± 0.16, n=20) that was abrogated in sh1-MICU1
cells (fractional saturation of 0.20 ± 0.10, n=12), (P
<0.0001). Similarly, when thapsigargin was used as calcium agonist, [Ca2+
increased in control cells (fractional saturation of 0.72 ± 0.15, n=15) but did not rise in MICU1
-silenced cells (fractional saturation of 0.10 ± 0.01, n=11), (P
<0.0001). Representative traces from individual cells are shown in .
Intracellular Calcium Measurements
Third, we measured mitochondrial Ca2+
uptake in digitonin-permeabilized cells. We selectively permeabilized the cell plasma membrane with a titrated amount of digitonin and monitored the clearance of exogenously added Ca2+
with Calcium Green-5N33
(see Methods). Clearance of exogenous Ca2+
is due to uptake by energized mitochondria via the classically defined uniporter, as it is fully abrogated by the ruthenium red derivative Ru-360 or uncoupler (Supplementary Fig. 2a,b
), and not influenced by thapsigargin (Supplementary Fig. 2c
). Although mitochondria of permeabilized control cells were capable of buffering multiple pulses of exogenously added Ca2+
silenced cells showed an attenuated response ().
Collectively, our secondary assays of mitochondrial Ca2+ uptake in cell populations and single cells with three different measurement techniques and two different Ca2+ agonists, confirm that MICU1 is required for mitochondrial Ca2+ uptake in HeLa cells, and that the Ca2+ phenotype in MICU1 knockdown cells is independent of the Ca2+ agonist or the sensor used for detection. Moreover, MICU1 appears to have a very specific role in mitochondrial Ca2+ handling, since resting [Ca2+]ER, [Ca2+]c, and store-operated calcium (SOC) entry rates are not significantly different between control and knockdown cells ().