MARCH5 regulates mitotic apoptosis through MCL1-dependent and independent mechanisms

Abstract

The anti-apoptotic MCL1 is critical for delaying apoptosis during mitotic arrest. MCL1 is degraded progressively during mitotic arrest, removing its anti-apoptotic function. We found that knockout of components of ubiquitin ligases including APC/C, SCF complexes, and the mitochondrial ubiquitin ligase MARCH5 did not prevent mitotic degradation of MCL1. Nevertheless, MARCH5 determined the initial level of MCL1-NOXA network upon mitotic entry and hence the window of time during MCL1 was present during mitotic arrest. Paradoxically, although knockout of MARCH5 elevated mitotic MCL1, mitotic apoptosis was in fact enhanced in a BAK-dependent manner. Mitotic apoptosis was accelerated after MARCH5 was ablated in both the presence and absence of MCL1. Cell death was not altered after disrupting other MARCH5-regulated BCL2 family members including NOXA, BIM, and BID. Disruption of the mitochondrial fission factor DRP1, however, reduced mitotic apoptosis in MARCH5-disrupted cells. These data suggest that MARCH5 regulates mitotic apoptosis through MCL1-independent mechanisms including mitochondrial maintenance that can overcome the stabilization of MCL1.

Fig. 1. Proteasome-dependent degradation of MCL1 during mitotic arrest.

A Stability of MCL1 during mitotic arrest. HeLa cells were synchronized using a double thymidine procedure. G₂ samples were harvested at 8 h after release from the second thymidine block (indicated as t = −4 h). Cells were trapped in mitosis (M) using NOC and isolated by shake off (t = 0) before further incubated with NOC. Cells were harvested at different time points. Lysates were prepared and analyzed with immunoblotting. Actin analysis was included to assess protein loading and transfer. Phosphorylated histone H3^Ser10 is a marker of mitosis. The intensity of the bands of MCL1, cyclin A, and cyclin B1 was quantified (right-hand panel). B Mitotic arrest stabilizes cyclin B1 and destabilizes MCL1. Cells were synchronized as described in A. The expression of MCL1, cyclin A, and cyclin B1 during G₂ and mitosis was quantified from three independent experiments (mean ± SEM). C Mitotic degradation of MCL1 is proteasome-dependent. Cells were synchronized and trapped in mitosis as described above. Mitotic cells were exposed to either buffer or MG132 and harvested after 3 h. The expression of MCL1 was analyzed with immunoblotting. The MCL1 band intensity was quantified (mean ± SEM from three independent experiments). D MCL1 is degraded during mitosis by an APC/C-independent mechanism. APC4^KO cells expressing ^mAIDAPC4 were generated. The cells were synchronized and arrested in mitosis as before. DI were applied to turn off the expression of ^mAIDAPC4 at the time of second thymidine release. Lysates were prepared and analyzed with immunoblotting. E Disruption of APC4 stabilizes cyclin B1 but not MCL1. Synchronization experiments were performed using ^mAIDAPC4-expressing APC4^KO cells as described in D. The MCL1 and cyclin B1 bands were quantified and shown in the right-hand panels (normalized to G₂ expression). Mean ± SEM of three independent experiments.

Fig. 2. MARCH5 determines MCL1 expression at mitotic entry.

A KO of MARCH5 elevates MCL1 during mitotic arrest. Parental HeLa (WT) and MARCH5^KO cells were synchronized and arrested in mitosis as before. Protein expression was analyzed with immunoblotting. Different exposures of the MCL1 blot are shown to provide a better comparison of the degradation kinetics. The MCL1 and cyclin B1 bands were quantified and shown in the right-hand panels (normalized to G₂ expression in HeLa cells). B Mitotic degradation of MCL1 is independent on MARCH5 and APC/C. APC4^KO expressing ^mAIDAPC4 were generated in a MARCH5^KO background. The cells were synchronized and arrested in mitosis as before. DI were applied to turn off the expression of ^mAIDAPC4 at the time of second thymidine release. Lysates were prepared and analyzed with immunoblotting. C Mitotic apoptosis is negatively regulated by MCL1. HeLa and MCL1^KO expressing ^mAIDMCL1 were synchronized and arrested in mitosis as before. ^mAIDMCL1 was turned off with DI at the time of second thymidine release. Protein expression was analyzed with immunoblotting. D Accelerated mitotic apoptosis in the absence of MCL1. HeLa and MCL1^KO expressing ^mAIDMCL1 were transiently transfected with histone H2B-GFP before synchronized and arrested in mitosis as before. The cells were either untreated or incubated with DI at the time of second thymidine release. Individual cells were tracked using live-cell imaging for 24 h (starting at 8 h after second thymidine release) (n = 50). The duration of mitotic arrest is plotted using Kaplan–Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. ****p < 0.0001. The raw data for individual cells are shown in Fig. S6.

Fig. 3. MARCH5 inhibits mitotic apoptosis through MCL1-dependent and -independent mechanisms.

A Depletion of MARCH5 accelerates mitotic apoptosis. WT and MARCH5^KO cells were transfected with histone H2B-GFP before synchronized and arrested in mitosis as before. Individual cells were tracked using live-cell imaging for 24 h (starting at 6 h after second thymidine release) (n = 50). The duration of mitotic arrest is plotted using Kaplan-Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. **p < 0.01. B Ectopic expression of MCL1 can abolish MARCH5^KO-mediated mitotic apoptosis. Cells expressing ^FLAGMCL1 were generated in HeLa or MARCH5^KO backgrounds. The cells were synchronized and arrested in mitosis as before. Protein expression was analyzed with immunoblotting. C Disruption of MCL1 promotes more apoptosis in MARCH5^KO cells. MARCH5 was inactivated with CRISPR-Cas9 in MCL1^KO expressing ^mAIDMCL1. The cells were synchronized and arrested in mitosis as before. ^mAIDMCL1 was turned off with DI. Protein expression was analyzed with immunoblotting. D Additive acceleration of mitotic apoptosis in the absence of MCL1 and MARCH5. MCL1^KO cells expressing ^mAIDMCL1 in either WT or MARCH5^KO background were transiently transfected with histone H2B-GFP before synchronized and arrested in mitosis as before. The cells were incubated with DI (to turn off MCL1) before individual cells were tracked using live-cell imaging for 24 h. The duration of mitotic arrest is plotted using Kaplan–Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. ****p < 0.0001.

Fig. 4. MARCH5 regulates mitotic apoptosis independently on NOXA, BIM, and BID.

A KO of NOXA does not affect MARCH5^KO-mediated mitotic apoptosis. NOXA^KO was generated in HeLa or MARCH5^KO backgrounds. The cells were synchronized and arrested in mitosis as before. Protein expression was analyzed with immunoblotting. B NOXA does not alter the duration of mitotic arrest in MARCH5^KO. Control or NOXA^KO in MARCH5^KO cells (in a mAID-MCL1 and MCL1^KO background) were synchronized and arrested in mitosis as before. Individual cells were tracked using live-cell imaging. The duration of mitotic arrest is plotted using Kaplan–Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. ns p > 0.05. C KO of BIM or BID does not affect MARCH5^KO-mediated mitotic apoptosis. BIM^KO or BID^KO was generated in HeLa or MARCH5^KO backgrounds. The cells were synchronized and arrested in mitosis as before. Protein expression was analyzed with immunoblotting. The positions of splice variants of BIM (BIM_S, BIM_L, and BIM_EL) are indicated.

Fig. 5. MARCH5 regulates mitotic apoptosis through BAK.

A Disruption of MARCH5 elevates BAK expression. WT and MARCH5^KO cells were synchronized and arrested in mitosis as before. The expression of the indicated proteins was analyzed with immunoblotting. Note that several proteins (MCL1, BCL2, and BCL-XL) displayed mitotic gel mobility shifts. B Increased expression of BAK during mitotic arrest in MARCH5^KO. WT or MARCH^KO cells were synchronized and arrested in mitosis as before. The expression of BAK during G₂ and mitosis was quantified from immunoblots of three independent experiments (mean ± SEM). C PARP1 cleavage in MARCH^KO-mediated mitotic apoptosis is reduced in the absence of BAK. BAX or BAK was ablated with CRISPR-Cas9 in MARCH5^KO cells. The cells were synchronized and arrested in mitosis as before. Protein expression was analyzed with immunoblotting. D MARCH^KO-mediated mitotic apoptosis is delayed in the absence of BAK. MARCH5^KO, MARCH5^KOBAX^KO, and MARCH5^KOBAK^KO (all were MCL1^KO cells expressing ^mAIDMCL1) were transiently transfected with histone H2B-GFP before synchronized and arrested in mitosis as before. Individual cells were then tracked using live-cell imaging. The duration of mitotic arrest is plotted using Kaplan–Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. *p < 0.05; ****p < 0.0001.

Fig. 6. BAK-dependent mitotic apoptosis caused by MARCH5^KO does not require MCL1.

A KO of MARCH5 promotes MCL1–BAK complex formation. HeLa, MARCH5^KO, or MARCH5^KO NOXA^KO were synchronized and arrested in mitosis as before. Cell lysates prepared from different time points were subjected to immunoprecipitation using an anti-MCL1 antiserum. Both the total lysates and immunoprecipitates were analyzed with immunoblotting. B KO of BAK inhibited mitotic apoptosis in cells lacking MARCH5 and MCL1. BAX^KO or BAK^KO was generated from MARCH5^KO MCL1^KO cells expressing ^mAIDMCL1. The cells were synchronized and arrested in mitosis as before. ^mAIDMCL1 was turned off with DI at the time of second thymidine release. Protein expression was analyzed with immunoblotting. C Disruption of BAK delayed mitotic apoptosis in MARCH5^KO MCL1^KO cells. Cells were synchronized and treated as in B (except that they were transiently transfected with histone H2B-GFP before synchronization). The cells were incubated with DI to turn off ^mAIDMCL1 before individual cells were tracked using live-cell imaging. The duration of mitotic arrest is plotted using Kaplan–Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. ****p < 0.0001; ns p > 0.05.

Fig. 7. NOXA contributes to mitotic apoptosis by regulating MCL1–BAX.

A Mitotic apoptosis in MARCH5^KO NOXA^KO is both BAX- and BAK-dependent. BAX^KO or BAK^KO was generated from MARCH5^KO NOXA^KO cells. The cells were synchronized and arrested in mitosis as before. Protein expression was analyzed with immunoblotting. B BAX-dependent mitotic apoptosis in MARCH5^KO NOXA^KO is mediated by MCL1. BAX^KO or BAK^KO was generated from MARCH5^KO NOXA^KO MCL1^KO cells expressing ^mAIDMCL1. The cells were synchronized and arrested in mitosis as before. ^mAIDMCL1 was turned off with DI at the time of second thymidine release. Protein expression was analyzed with immunoblotting. C Disruption of BAK delayed mitotic apoptosis in MARCH5^KO NOXA^KO MCL1^KO cells. Cells were synchronized and treated as in B (except that they were transiently transfected with histone H2B-GFP before synchronization). The cells were incubated with DI to turn off ^mAIDMCL1 before individual cells were tracked using live-cell imaging. The duration of mitotic arrest is plotted using Kaplan–Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. **p < 0.01.

Fig. 8. MARCH5^KO-mediated mitotic apoptosis is dependent on DRP1.

A MARCH5^KO increases DPR1 in mitochondrial fraction. Cellular fractionation of WT and MARCH5^KO cells were conducted to obtain lysates from total cell (T), cytosol (C), and mitochondrial-enriched heavy membrane (M) fractions. The expression of DRP1 was analyzed with immunoblotting. B Depletion of DRP1 alleviates mitotic apoptosis in MARCH5^KO cells. DRP1^KO cells were generated from HeLa WT or MARCH5^KO cells. The cells were synchronized and arrested in mitosis as before. Protein expression was analyzed by immunoblotting. C Depletion of DRP1 promotes MCL1–BAK interaction in NOXA-independent manner. NOXA^KO cells were generated from MARCH5^KO and DRP1^KOMARCH5^KO cells. The cells were synchronized and arrested in mitosis as before. Mitotic cell lysates were subjected to immunoprecipitation using MCL1 antiserum. Both total and immunoprecipitates were analyzed by immunoblotting. The band intensities of MCL1 and BAK in the immunoprecipitates were quantified and the ratios of BAK/MCL are indicated.

Fig. 9. A model of the regulation of apoptosis during mitotic arrest by MARCH5.

The ubiquitin ligase MARCH5 controls the stability of MCL1 during interphase. Mitotic degradation of MCL1 is proteasome-dependent but only partially dependent on MARCH5. Accordingly, MCL1 expression is elevated during early mitosis and takes longer to be depleted during mitotic arrest in MARCH5-depleted cells. NOXA is also stabilized in MARCH5-depleted cells in an MCL1-dependent manner. During late mitotic arrest, the destruction of MCL1 (by yet unidentified ubiquitin ligases X) and NOXA facilitates the activation of BAK and BAX. Our data suggest that the increase of mitochondrial DRP1 also plays a critical role in promoting mitotic apoptosis in the absence of MARCH5.