mice, the authors found that the BH3-only molecule, tBID, overcomes this problem
by activating two distinct steps; a BAX/BAK-independent structural rearrangement
of the IMM that mobilizes Cyt.c stores out of cristae junctions, followed by a BH3-
domain-dependent oligomerization of BAK and BAX into predicted pores that may
allow transport across the OMM. Interestingly, CsA strongly inhibited tBid-induced
mitochondrial reorganization of cristae, implicating the PTP in this step. Given the
dependence of PTP opening on [Ca2+]m, IP 3 R/RyR Ca2+ spikes from the ER may
cooperate in physiologic settings with BH3-only molecules to allow transient
openings of the PTP, facilitating cristae remodeling and mobilization of Cyt.c in the
absence of large-scale mitochondrial swelling. In vivo, suboptimal activation of ER
Ca2+-release channels, causing transient openings of the PTP, may lead to small
decreases ∆m that are very difficult to detect (Szalai et al., 1999). Perhaps this
explains why many groups do not observe mitochondrial depolarization prior to Cyt.c
release. Large and sustained openings of the PTP, causing a complete loss of ∆m
and mitochondrial swelling, may follow Cyt.c-dependent activation of caspases and
may represent a late stage in apoptosis that ensures the demise of the cell by crippling
its energy production.
3.4
Coordination of Cyt.c release by ER-derived Ca2+ wavesAfter a population of cells is challenged with an apoptotic agent, the time at which
any given cell will undergo Cyt.c release is highly variable and asynchronous.
However, once initiated within a given cell, Cyt.c release is rapid and complete, with
all mitochondria releasing Cyt.c stores within minutes (Goldstein et al., 2000).
Therefore, the decision to execute Cyt.c release is highly coordinated between
mitochondria. The work of Pacher and Hajnoczky (2001) demonstrated that ER-
borne Ca2+ spikes may organize this cross-talk within the mitochondrial population
by eliciting Ca2+ uptake in proximal mitochondria, which then communicate to other
mitochondria by propagating a secondary Ca2+ wave. The authors found that
permeabilized or intact cardiac myotubes exposed to caffeine, a RyR agonist, initiate
ER-derived, low-amplitude, high-propagation-rate [Ca2+]c waves that are coupled to
increases in [Ca2+]m following the same spatiotemporal pattern as the [Ca2+]c wave.
If the cells are briefly pretreated with apoptotic stimuli, such as C2 ceramide, a second
high-amplitude, low-velocity [Ca2+]c wave spreads throughout the cell, and is
accompanied by an ‘apoptotic wave’ of mitochondrial depolarization, Cyt.c release,
and caspase activation, all following the same spatiotemporal pattern as the delayed
[Ca2+]c wave. Generation of the delayed [Ca2+]c wave, and the accompanying
apoptotic wave, was inhibited by CsA, Bcl-xL, and an inhibitor of mitochondrial Ca^2
- uptake sites, but not by caspase inhibitors. In addition, both of these events could
be initiated by exogenous CaCl 2 pulses after the ER Ca2+ store was depleted in
permeabilized cells pretreated with ceramide. Taken together, these results suggest
that the delayed Ca2+ wave originates at mitochondria through Ca2+-induced opening
of the PTP. Interestingly, the authors found that mitochondria located at the
THE ROLE OF THE ENDOPLASMIC RETICULUM IN APOPTOSIS 111