Figure 1.
BIM is critical for heat shock-induced apoptosis.
(A-D) Wild-type, Bim−/−, and Bid−/− MEFs (panel B) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO2–95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel D), Δψm (24 h, panel C) and cell death measurements (24 h, panel A). *p<0.05, significantly different from wild-type cells. (E-H) Human Jurkat T cells, stably depleted of BIM or BID by RNAi (panel F; note: black triangles denote crop marks), were exposed to heat shock (44°C for 1 h) in a humidified incubator (5% CO2–95% air). As before, the cells were then transferred to a 37°C incubator and later collected for MOMP, caspase-3 activation, PARP cleavage (24 h, panel H), Δψm (24 h, panel G) and cell death measurements (24 h, panel E). (I) Wild-type, Bim−/−, and Bid−/− MEFs were exposed to heat shock as described above, but were left in culture afterwards for 72 h, after which the plates were stained with crystal violet. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from wild-type cells.
Figure 2.
Caspase-2 induces apoptosis in a BID-dependent but BIM-independent manner.
(A and B) Cartoons depicting wild-type caspase-2 and the FKBP-Δpro-caspase-2 fusion, in which the prodomain of caspase-2 is replaced with an FKBP protein that dimerizes upon the addition of AP20187. (C and D) Wild-type, Bim−/−, and Bid−/− MEFs were infected with a retrovirus expressing the FKBP-Δpro-caspase-2 fusion protein. The cells were then sorted by flow cytometry for GFP (marker)-positive cells, and the cell pools incubated in the presence or absence of AP20187 for 48 h, after which they were assayed for cell death (panel C) as well as activation of caspases-2 and -3 and BID cleavage where relevant (panel D). *p<0.05, cells treated with AP20187 were significantly different from those treated with DMSO.
Figure 3.
Bax−/−Bak−/− cells resist heat shock-induced MOMP and loss of Δψm, but still undergo caspase-3 activation and cell death.
Wild-type and Bax−/−Bak−/− DKO cells were exposed to (A-C) heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO2–95% air), or (D and E) UV irradiation (4 min) on a transilluminator. The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panels C and E), Δψm (24 h, panel B) and cell death measurements (24 h, panels A and D). cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from wild-type cells.
Figure 4.
Inhibition of apoptosome-dependent caspase-9 activity weakly inhibits heat shock-induced apoptosis but does not provide long-term protection.
(A-C) Vector control MEFs and those expressing a DN-caspase-9 (C287A) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO2–95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel C), Δψm (24 h, panel B) and cell death measurements (24 h, panel A). (D) Wild-type, Bim−/−, and DN-caspase-9 MEFs were exposed to heat shock as described above, but were left in culture afterwards for 72 h, after which the plates were stained with crystal violet. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from vector control cells.
Figure 5.
Loss of MCL-1 or inhibition of BCL-2/BCL-XL potentiates heat shock-induced apoptosis.
(A) Cartoon illustrating the interactions of BH3-only proteins with multidomain proapoptotic (BAX and BAK) and antiapoptotic (MCL-1, BCL-2, and BCL-XL) BCL-2 family members. BH3-only proteins can function as BAX/BAK activators (BIM, tBID, PUMA), or as sensitizers (NOXA, BAD, BMF, BIK, and HRK) that displace activators from antiapoptotic family members. (B-D) Wild-type and Mcl-1−/− MEFs (panel B, inset) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO2–95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel D), Δψm (24 h, panel C) and cell death measurements (24 h, panel B). *p<0.05, significantly different from wild-type cells. (E and F) Wild-type MEFs were preincubated with DMSO (control) or ABT-737 (250 nM) for 1 h and subsequently exposed to heat shock (44°C for 1.5 h) in a humidified incubator (5% CO2–95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel F) and cell death measurements (24 h, panel E). *p<0.05, cells treated with ABT-737 were significantly different from those treated with DMSO. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet.
Figure 6.
Model of BIM-induced cell death following heat shock.
According to the canonical pathway, heat shock induces formation of a RAIDD-caspase-2 complex that activates caspase-2. Following cleavage by caspase-2, truncated Bid (tBID) then stimulates BAX/BAK-dependent MOMP, cytochrome c release, and formation of the Apaf-1/caspase-9 apoptosome complex, which in turn activates the effector caspase-3. In our hands, however, loss of BID or inhibition of the apoptosome provides only modest short-term protection at lower exposures (1 h, 44°C), whereas loss of BIM profoundly inhibits cell death and facilitates long-term protection. In the context of heat shock, BIM induces MOMP and loss of Δψm in a BAX/BAK-dependent manner (and may be responsible for triggering caspase-3 activation in the absence of detectable mitochondrial injury). Thus, BIM appears to mediate an alternative (and perhaps dominant) pathway to heat shock-induced apoptosis.