Removing the CTS from Bim abrogates pro-apoptotic activity in HEK293 cells (Weber et al., 2007). While this observation has generally been ascribed to loss of binding of Bim to MOM our observation that the CTS is also involved in binding BimEL to Bcl-XL (Liu et al., 2019) suggested that there may be other explanations for the loss of pro-apoptotic activity for Bim when the CTS is removed. To determine the contribution of the Bim CTS to pro-apoptotic activity, a BimL mutant was generated in which the previously characterized membrane binding domain (carboxyl-terminal residues P121- H140) were deleted (BimL-dCTS) (Wilfling et al., 2012; Liu et al., 2019). This mutant was expressed in cells and the effectiveness of induction of cell death was compared to expression of full-length BimL by confocal microscopy. To detect expression of the constructs in live cells, they included an N-terminally fused Venus fluorescent protein (indicated by a superscripted v in the name). Thus, a construct in which Venus was fused to the amino-terminus of BimL is referred to here as ^VBimL while the mutant lacking the CTS is ^VBimL-dCTS. As an inactive control, we used ^VBimL-4E a mutant in which four conserved hydrophobic residues in the BH3-domain of BimL were replaced with glutamate, thereby preventing binding to all other multi-BH domain Bcl-2 family proteins (Chen et al., 2005; Liu et al., 2019).

To assay pro-apoptotic activity, the constructs were expressed in primary cells and cell lines and both expression and cell death were measured using confocal microscopy. Apoptosis was assessed by detecting externalization of phosphatidylserine by Annexin V staining in cells expressing detectable levels of ^VBimL or the ^VBimL mutants as measured by Venus fluorescence. As a positive control for activation of Bax ^VtBid, the activated form of the BH3-protein Bid fused to the C-terminus of the Venus fluorescent protein was also expressed in cells. As expected, expression of ^VBimL induced apoptosis in all cell types tested, while the negative control protein ^VBimL-4E did not (Figure 1A). Similarly, ^VtBid induced apoptosis in all the cell types except HEK293 cells. As reported previously for Bim-dCTS, the fluorescent version (^VBimL-dCTS) failed to induce cell death in HEK293 cells (Weber et al., 2007). In contrast, expression of ^VBimL-dCTS induced apoptosis to levels similar to ^VBimL in HCT116, BMK and MEF cells but may have reduced potency in CAMA-1 cells. Comparing the AnnexinV intensities for individual cells at a variety of equivalent expression levels of the Bim mutants across the different cell types revealed that the CTS of Bim was required for the pro-apoptotic activity of Bim in HEK293 cells (Figure 1—figure supplement 1).

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Source data indicating that BimL requires a CTS to induce MOMP in HEK293 cells.

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Image data for MEF mitochondrial priming experiments.

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Image data for HEK293 mitochondrial priming experiments.

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To determine if this difference in response to ^VBimL-dCTS expression is a function of the extent to which the apoptotic machinery is loaded in MOM, mitochondria were purified from cells resistant (HEK293) and sensitive (MEF) to ^VBimL-dCTS expression and assayed by BH3-profiling (Potter and Letai, 2016). This assay measures loading of anti-apoptotic proteins with BH3-proteins or active Bax/Bak. Unlike BH3-profiling experiments conducted with BH3-peptides, in these experiments purified full-length proteins were used. Thus, purified cBid, BimL, BimL-dCTS, Bad and Noxa proteins were incubated with mitochondria from each of the cell lines and MOMP was measured by separating supernatant and pellet fractions for each reaction, and immunoblotting for cytochrome c released from the intermembrane space as previously described (Pogmore et al., 2016). Immunoblots were quantified and MOMP assessed as % cytochrome c released (Figure 1B). As expected from the data in Figure 1A, addition of recombinant BimL was sufficient to induce cytochrome c release from mitochondria from both HEK293 and MEF cells. However, addition of BimL-dCTS induced cytochrome c release only in the MEF mitochondria confirming that resistance to BimL-dCTS in HEK293 cells is manifest at mitochondria. Mitochondria purified from HEK293 cells were more sensitive to BimL protein than to recombinant cBid, a phenomenon that is also seen when ^VtBid is expressed in these cells (Figure 1A). This result may be due to the inherent differences between Bim and Bid reported previously (Sarosiek et al., 2013). Nevertheless, in HEK293 cells, ^VtBid was more active than BimL-dCTS and functionally equivalent to ^VBimL in every other cell line tested.

One potential explanation for the difference in response to BimL-dCTS and BimL is that the mitochondria in the cell lines have different dependencies on multi-domain anti-apoptotic proteins for survival, a phenomenon known as priming. If BimL-dCTS has lost one of the functions of Bim such as activating Bax or Bak or inhibiting one of the multi-domain anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 it would be expected to have different activities on mitochondria with different priming. Therefore, to better understand why BimL-dCTS can only permeabilize MEF mitochondria and not mitochondria from HEK293 cells, we compared the sensitivity of mitochondria from the two cell types to addition of BH3-proteins Bad or Noxa that inhibits Bcl-2 and Bcl-XL or Mcl-1, respectively, but that do not activate Bax or Bak (Kale and Osterlund, 2017). Incubation of full-length Bad and/or Noxa with mitochondria from HEK293 cells failed to induce cytochrome c release, while the addition of Noxa or Bad was sufficient to permeabilize MEF mitochondria (Figure 1B). This data suggests that HEK293 cells not depend on expression of Bcl-2, Bcl-XL or Mcl-1 sequestering active Bax, Bak or their BH3-activators while mitochondria from MEFs depend on expression of Mcl-1 and Bcl-XL to prevent apoptosis (Lessene et al., 2013). The results further suggest that removal of the CTS from BimL results in a mutant protein that only kills cells dependent on one or more multi-domain anti-apoptotic proteins for survival. That BimL-dCTS does not kill HEK293 cells further suggests that it does not activate sufficient Bax or Bak to overcome the unoccupied anti-apoptotic proteins in this cell line. In this way, BimL-dCTS functions as a sensitizer similar to proteins like Bad and Noxa. However, unlike other relatively specific sensitizer proteins, the BH3-region of BimL-dCTS binds to and inhibits Bcl-2, Bcl-XL and Mcl-1. Indeed, we have shown that in live cells BimEL-dCTS binds to and inhibits Bcl-2 and Bcl-XL but is more easily displaced than BimEL by small molecule BH3 mimetics (Liu et al., 2019).

Our data with cell lines and their respective purified mitochondria suggests that BimL-dCTS does not kill cells that do not depend on anti-apoptotic proteins for survival. To test this in a more biologically relevant system, we cultured primary murine cortical neurons and assayed their response to expression of the BimL mutants. To enable regulated expression in primary cortical neurons the coding regions for ^VBimL, ^VBimL-4E, and ^VBimL-dCTS were cloned into a tetracycline-responsive lentiviral vector, and introduced into primary cortical neuron cultures through lentiviral infection. After culture for 16 days in vitro, BimL expression was induced in the neurons by the addition of doxycycline. Neuronal cell death was assayed using confocal microscopy after staining neurons with TMRE, a dye that only accumulates in active mitochondria. Thus, a lack of TMRE dye accumulation (TMRE negative) indicates loss of mitochondrial transmembrane potential and in response to expression of a BH3-protein is an early indication of commitment to cell death. Quantification of Venus-expressing neuronal cell bodies revealed that as expected ^VtBid and ^VBimL expression killed cultured primary neurons while ^VBimL-4E did not (Figure 2A–B). However, the expression of ^VBimL-dCTS was largely ineffective to induce cell death in cultured primary cortical neurons (Figure 2B). Our data is consistent with previous reports suggesting that primary murine cultures of hippocampal neurons become resistant to induction of apoptosis by external stimuli over time in culture. This resistance has been reported to be due to a difference in Bcl-2 family protein expression that results in decreased mitochondrial ‘priming’, explaining why our cultures of primary cortical neurons are resistant to ^VBimL-dCTS (Sarosiek et al., 2017).

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Source data for experiments demonstrating that ^VBimL-dCTS does not kill mature cultures of mouse neurons.

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Image data for E15 brain mitochondria priming experiments.

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To determine if resistance to induction of cell death by BimL-dCTS is due to differential sensitivity of neuronal mitochondria to induction of MOMP by BimL and BimL-dCTS, mitochondria were isolated from embryonic day 15 (E15) mouse brains, the same age used to culture primary cortical neurons. Brain mitochondria were used instead of isolating mitochondria from neuronal cultures due to the low yield from primary cultured neurons. Untreated mitochondria from day E15 brain released only low levels of cytochrome c. As expected, addition of 0.1 nM recombinant BimL was sufficient to elicit MOMP as measured by cytochrome c release and detection in the supernatant. In contrast, 100 times more BimL-dCTS (10 nM) failed to induce MOMP (Figure 2C).

Taken together our data suggest that BimL-dCTS kills cells in which the mitochondria are sensitive to inhibition of anti-apoptotic proteins by sensitizers such as Bad and Noxa. Thus, BimL-dCTS did not permeabilize mitochondria extracted from HEK293 cells or E15 whole murine brains, and as a result, BimL-dCTS expression did not kill HEK293 cells or primary cultures of cortical neurons. This finding suggests that inhibition of anti-apoptotic proteins is not sufficient to kill these cells. Therefore, BimL-dCTS differs mechanistically from BimL as the latter kills both cell types resistant and sensitive to BimL-dCTS. Compared to BimL, BimL-dCTS is missing the membrane-binding domain and therefore is not expected to localize at mitochondria (Liu et al., 2019); however, the relationship between Bim binding to membranes and Bim-mediated Bax activation has not been extensively studied. To determine how the molecular mechanism of BimL-dCTS differs from BimL the activities of the proteins were analyzed using cell-free assays.

To investigate the pro-apoptotic mechanism of BimL and BimL-dCTS without interference from other cellular components, both were purified as full-length recombinant proteins and assayed using liposomes and/or isolated mitochondria. To measure direct-activation of Bax by Bim, either BimL or BimL-dCTS was incubated with recombinant Bax and liposomes encapsulating the dye and quencher pair: ANTS (8-Aminonaphthalene-1,3,6-Trisulfonic Acid, Disodium Salt) and DPX (p-Xylene-Bis-Pyridinium Bromide). In this well-established assay (Chi et al., 2014), increasing amounts of BimL activated Bax results in membrane permeabilization measured as an increase in fluorescence due to the release and separation of encapsulated dye and quencher (Figure 3A). This result is consistent with previous observations that picomolar concentrations of BimL induce Bax-mediated membrane permeabilization (Sarosiek et al., 2013). In contrast, three orders of magnitude higher concentrations of BimL-dCTS were required to induce Bax-mediated liposome permeabilization (Figure 3A), suggesting that either or both of binding to membranes and the specific CTS of Bim are required for efficient Bax activation. As expected, similar results were obtained for Bax-mediated release of mitochondrial intermembrane space proteins (Figure 3B). For these experiments, MOMP was measured as release of the fluorescent protein mCherry fused to the N-terminal mitochondrial import signal of SMAC (SMAC-mCherry) from the intermembrane space of mitochondria (Shamas-Din et al., 2014). Similar to the results with liposomes (Figure 3A), and mitochondria from cell lines (Figures 1–2) BimL but not BimL-dCTS triggered Bax mediated SMAC-mCherry release from mitochondria isolated from Bax - /- Bak-/-cells (Figure 3B). In experiments with liposomes and mitochondria, very small amounts of Bim were sufficient to trigger membrane permeabilization because once activated, Bax recruits and activates additional Bax molecules (Tan et al., 2006). To assess the impact of the Bim CTS on the interaction between Bim and Bax, binding was measured using Förster resonance energy transfer (FRET). For these experiments, recombinant BimL proteins were labeled with the donor fluorophore Alexa568, while Bax was labeled with the acceptor fluorophore Alexa647. Unexpectedly, and unlike the BH3-only protein tBid (Lovell et al., 2008), BimL bound to Bax even in the absence of membranes (Figure 3C), while BimL-dCTS had no relevant Bax binding in the presence or absence of mitochondrial-like liposomes (Figure 3C–D). Binding of Bim to Bax in solution suggests that the CTS of Bim may be directly involved in Bim-Bax heterodimerization independent of Bim binding to membranes.

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Source data for experiments demonstrating that BimL-dCTS cannot bind to nor activate Bax.

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To confirm in our system that the labeled BimL proteins bind to membranes via the CTS sequence, binding of Alexa568-labeled recombinant BimL and BimL-dCTS to DiD labeled liposomes was measured by FRET (Figure 4A). In these experiments, DiD serves as an acceptor for energy transfer from Alexa568-labeled BimL. The same approach was used to quantify BimL binding to mitochondrial outer membranes with mitochondria isolated from BAK-/-mouse liver (Figure 4B), which lack Bax and Bak (Shamas-Din et al., 2013a). In both cases, BimL spontaneously bound to membranes with picomolar affinity, while stable binding of BimL-dCTS to liposomes and mitochondria was not-detectable (Figure 4A–B). Furthermore, BimL-dCTS again had no relevant binding to Bax even in the presence of purified mitochondria (Figure 4C).

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Source data for experiments demonstrating the Bim CTS is required to bind BimL to membranes.

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Taken together, our data strongly suggest that the CTS of Bim is required for both BimL to bind to membranes in vitro and for binding Bax with or without membranes. Alternatively purified BimL-dCTS may be completely non-functional. To demonstrate that purified BimL-dCTS binds to and inhibits Bcl-XL as shown for ^VBimL-dCTS expressed in cells (Figure 1) and in Liu et al. (2019), inhibition of Bcl-XL was measured using liposomes and mitochondria.

In addition to direct Bax activation, Bim promotes apoptosis by binding to Bcl-XL and displacing either activator BH3-proteins (Mode 1) or activated Bax or Bak (Mode 2) (Llambi et al., 2011). In the ANTS/DPX liposome dye release assay, BimL-dCTS was functionally comparable to the well-established Bcl-XL inhibitory BH3-protein Bad in reversing Bcl-XL-mediated inhibition of cBid (Figure 5A) or Bax (Figure 5B). Consistent with the observation that BimL-dCTS was less resistant to displacement by BH3 mimetics in live cells, in cell-free assays BimL-dCTS was also less effective than BimL at displacing cBid or Bax from Bcl-XL (Liu et al., 2019). Nevertheless, when assayed with mitochondria BimL-dCTS disrupted the interaction between tBid and Bcl-XL resulting in Bax activation and permeabilization of mitochondria as measured by cytochrome c release (Figure 5C, solid black line). This activity is due to inhibition of Bcl-XL function, as in controls without Bcl-XL the same concentration of BimL-dCTS did not directly activate sufficient Bax to mediate MOMP (Figure 5C, dashed black line). Thus, purified BimL-dCTS is functional and can initiate MOMP by displacing direct-activators (Mode 1) or activated Bax (Mode 2) from Bcl-XL (Figure 5A–C). Finally, BimL-dCTS labeled with Alexa568 retained high-affinity binding for Bcl-XL labeled with Alexa647 both in solution (Kd <16 nM) and on membranes (~35 nM apparent Kd on liposomes and on mitochondria) as measured by FRET (Figure 6B).

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Source data demonstrating that recombinant BimL-dCTS indirectly activates Bax through inhibition of Bcl_XL.

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To identify which residues in the Bim CTS mediate binding to membranes and/or Bax we generated a series of point mutations. Sequence analysis using HeliQuest software (Gautier et al., 2008) predicts that the Bim CTS forms an amphipathic α-helix (Figure 6A). Two arginine residues (R130 and 134) are predicted to be on the same hydrophilic side of the helix, whereas hydrophobic residues (e.g. I125, L129, I132) face the other side (Figure 6A). To determine the functional importance of these residues, Bim CTS mutants were created including: BimL-CTS2A in which R130 and R134 were mutated to alanine; and a series of single hydrophobic residue substitutions by glutamate (V124E, I125E, L129E, and I132E) (Figure 6A). To compare the effects of the CTS mutations on BimL-binding interactions and function, we measured by FRET the Kds for the various binding interactions and the activities of the mutants to promote Bax-mediated liposome permeabilization as EC50’s for ANTS release (Figure 6B and Figure 6—figure supplement 1A–E).

Mutation of individual hydrophobic residues on the hydrophobic side of the Bim CTS (BimL-I125E, BimL-L129E or BimL-I132E) abolished binding to membranes (Figure 6B). In contrast, mutations on the other side of the helix including BimL-V124E and BimL-CTS2A had less effect on Bim binding to membranes (Figure 6B). Despite the dramatic changes in affinity for membranes among Bim CTS mutants, the mutations did not abolish binding to Bax both in the presence and absence of membranes (Figure 6B). Indeed most of the mutants had Kd values for binding to Bax of less than 100 nM and to our surprise many of them bound to Bax better in solution compared to when membranes were present. The presented Kds under ‘membranes present’ is an estimation of the effective Kd (combined solution and two-dimensional Kds), as we are not able to precisely determine the quantity of protein-complexes that form in solution or on the membrane. Nevertheless, this data reflects the situation in cells and further confirms that binding to membranes and Bax are independent functions of the Bim CTS . In the case of BimL-I125E, a mutant that activates Bax to permeabilize liposomes, the initial interaction with Bax must occur in solution as neither protein spontaneously binds to membranes (Figure 6B).

Unexpectedly, there was not a good correlation between BimL binding to membranes and Bax activation. For example, while BimL bound to membranes with a Kd of 31 pM, BimL-CTS2A and BimL-I125E bound to membranes with Kds of ~600 and>1000 pM, respectively yet both mutants triggered Bax-mediated membrane permeabilization, demonstrating that specific residues in the CTS rather than binding to membranes enabled BimL to mediate Bax activation. Moreover, BimL binding to Bax was also not sufficient to activate Bax efficiently. BimL-L129E and BimL-I132E are two Bim mutants that do not bind membranes, retain reasonable affinities for Bax in the presence of membranes (Kds ~ 100–200 nM), but were unable to activate Bax (Figure 6B). These results indicate that these two residues play a key function in Bax activation. As expected, the negative control BimL-4E mutant does not bind to nor activate Bax even though its CTS is intact and the protein binds membranes (Figure 6B). This result confirms the essential role of the BH3-domain and suggests that the Bim CTS provides a secondary role in Bax binding rather than providing an independent high affinity binding site that is sufficient to activate Bax.

Both functional and binding assays for the various point mutants suggest that specific residues in the Bim CTS participate in Bim-Bax protein interactions that lead to Bax activation; however, these mutants did not clearly separate the membrane binding function of the CTS of Bim from a potential function in Bax activation. Thus, it remains possible that restoring membrane binding to BimL-dCTS would be sufficient to restore Bax activation function. To address this, we fused the mitochondrial tail-anchor from mono-amine oxidase (MAO residues 490–527, UniProt: P21397-1) to the C-terminus of BimL-dCTS to restore membrane binding with a sequence unlikely to contribute to Bax activation directly. This protein, BimL-dCTS-MAO, and BimL bound to mitochondrial-like liposomes and purified mitochondria (Figure 7A and Figure 7—figure supplement 1A respectively). As expected, a population of these recombinant Bim proteins remains in solution. To directly assess the Bim CTS contribution to the binding and activation of Bax on the membrane surface, we incubated a defined amount of recombinant Alexa568-labeled BimL or BimL-dCTS-MAO proteins with 0.5 mg/mL ANTS/DPX filled liposomes, then isolated the liposomes using size-exclusion chromatography. Using this procedure, we excluded all recombinant Bim that remained in solution, and obtained the membrane-bound BimL or BimL-dCTS-MAO at a concentration of ~5 nM as calculated based on Alexa568 fluorescence intensity (Precise concentrations labeled in Figure 7B). This equates to 0.6 Bim molecules per liposome. Addition of Bax to these liposomes resulted in ANTS/DPX release for roughly 60% of the liposomes in incubations containing BimL-membrane-bound liposomes, but no significant release from the liposomes in incubations containing BimL-dCTS-MAO-bound liposomes (Figure 7B), suggesting that the Bim CTS contributes to the activation of Bax even on the membrane surface.

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Source data demonstrating that BimL-dCTS-MAO binds to liposomes and Bax, but activates Bax poorly.

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To directly measure binding to Bax, Alexa568-labeled BimL and BimL-dCTS-MAO were incubated with increasing concentrations of Alexa647-labeled Bax protein. In the absence of membranes, no detectable FRET was measured between Alexa568-labeled BimL-dCTS-MAO with Alexa647-labeled Bax (Figure 7C), suggesting that the specific sequence of the Bim CTS contributes to the Bim-Bax interaction in solution. However, when Alexa568-labeled BimL-dCTS-MAO bound to the liposome membrane was isolated by size-exclusion chromatography as described above, a FRET signal was detected between it and Alexa647-labeled Bax. We also detected FRET between Alexa568-labeled BimL-dCTS-MAO and Alexa647-labeled Bax in the presence of purified mitochondria (Figure 7—figure supplement 1B). Although these incubations contained both soluble and membrane-bound Bim protein, we conclude the interaction occurred on the MOM as no FRET signal was detected between these proteins in solution (Figure 7C). Despite measureable binding between BimL-dCTS-MAO and Bax on the liposome membrane (Figure 7D), BimL retained a higher affinity than BimL-dCTS-MAO for Bax (Kds of 21 nM and 49.6 nM, respectively). Together these data suggest that the CTS of Bim contributes to both binding to (Figure 7D) and activation of Bax (Figure 7B) on the liposome membrane.

Our binding and mutagenesis data suggest that the Bim CTS binds to and activates Bax in solution and on membranes. To detect this binding interaction, we used a photocrosslinking approach, in which a BimL protein was synthesized with a photoreactive probe attached to a single lysine residue positioned in the CTS using an in vitro translation system containing 5-azido-2-nitrobenzoyl-labled Lys-tRNA^Lys that incorporates the lysine analog (εANB-Lys) into the polypeptide when a lysine codon in the BimL mRNA is encountered by the ribosome. The BimL synthesized in vitro was also labeled by ³⁵S via methionine residues enabling detection of BimL monomers and photoadducts by phosphor-imaging.

The radioactive, photoreactive BimL protein was incubated with a recombinant His6-tagged Bax protein in the presence of mitochondria isolated from BAK^-/- mouse liver lacking endogenous Bax and Bak to prevent competition and increase BimL-Bax protein interactions. Mitochondrial proteins were then separated from the soluble ones by centrifugation. Both soluble and mitochondrial fractions were photolyzed to activate the ANB probe generating a nitrene that can react with atoms in close proximity (<12 Å from the Cα of the lysine residue). Thus, for photoadducts to form, the atoms of the bound Bax molecule are likely to be located in or near the binding site for the Bim CTS. The resulting photoadduct between the BimL and the His₆-tagged Bax was enriched by Ni²⁺-chelating agarose resin and separated from the unreacted BimL and Bax monomers using SDS-PAGE. The ³⁵S-labeled BimL in the photoadduct with His6-tagged Bax and BimL monomer bound to the Ni²⁺-beads specifically via the His₆-tagged Bax or nonspecifically were detected by phosphor-imaging. A BimL-Bax-specific photoadduct was detected when the ANB probe was located at four different positions in the Bim CTS on both hydrophobic and hydrophilic surfaces of the potential α-helix (Figure 8A). These photoadducts have the expected molecular weight for the Bim-Bax dimer, and were not detected or greatly reduced when the ANB probe, the light, or the His6-tagged Bax protein was omitted (Figure 8A). Consistent with the BimL-Bax interaction detected by FRET in both solution and membranes, the BimL-Bax photocrosslinking occurred in both soluble and mitochondrial fractions. Less photocrosslinking occurred in the mitochondrial fraction likely due to the fact that in membranes homo-oligomerization of activated Bax competes with hetero-dimerization between BimL and Bax.

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As expected, when the ANB probe was positioned in the Bim-BH3 domain as a positive control BimL-Bax photocrosslinking was detected in both soluble and mitochondrial fractions (Figure 8B). Crosslinking with the Bim BH3-domain is consistent with the BH3 interaction with the canonical groove or trigger pocket that is well supported by experimental evidence including co-crystal structures and NMR models (Gavathiotis et al., 2008; Robin et al., 2015). Furthermore, loss of photocrosslinking for BimL mutants with the BH3-4E mutation that abolished binding to Bax demonstrates that direct binding between the proteins is required for crosslinking to be detectable (Figure 8C). Therefore, the crosslinking data suggests that similar to the BH3-domain, the Bim CTS binds to Bax. To further demonstrate that the CTS of Bim binds to Bax independent of both membrane binding and Bax activation, the experiment was repeated with BimL-L129E, a mutant that binds Bax without activating it and that does not bind membranes (Figure 6B). As shown in Figure 8C, the L129E mutant photocrosslinked to Bax in both the soluble and mitochondrial fractions. Furthermore, this mutant also photocrosslinked to Bcl-XL (Figure 8C), consistent with data demonstrating that the Bim CTS also binds to this anti-apoptotic protein (Figure 6B and Liu et al., 2019). The qualitative photocrosslinking data (Figure 8) and the quantitative FRET data (Figure 6B) obtained from the BimL proteins with and without the CTS or BH3-domain mutation are consistent, and both support a model in which the CTS interacts with membranes and binds to Bax, thereby enhancing BH3-domain mediated Bax activation.

To identify the binding site for the Bim CTS in Bax, we used a chemical crosslinking approach. Unlike the photocrosslinking approach that does not reveal the location of the binding site, the chemical crosslinker bismaleimidohexane (BMH) contains two sulfhydryl reactive moieties separated by a 13 Å spacer, and thus formation of a BMH-crosslinked Bim-Bax dimer requires a cysteine in Bim that is in close proximity with another cysteine in the interacting Bax. Therefore, a successful crosslink indicates a close proximity between the two cysteines, potentially revealing the Bim-binding site in Bax.

We used a structurally well-defined Bim-Bax interaction to validate this crosslinking approach. According to the crystal structure (PDB ID 4ZIE; Robin et al., 2015), the BH3-domain of Bim binds to the canonical groove of Bax. Our FRET data shows that BimL and Bax bind in solution and the binding is abolished by the 4E mutation in the Bim BH3-domain that eliminates the nonpolar interactions with the Bax groove. It is therefore expected that this Bim-Bax interaction is mediated by the BH3-domain and the groove, and according to the structure, a BMH molecule would be able to link a cysteine replacing Phe¹⁰¹ in the BH3-domain of Bim to a cysteine replacing Trp¹⁰⁷ in the canonical groove of Bax. We thus synthesized the [³⁵S]Met-labeled single-Cys Bim F101C and Bax W107C proteins in vitro, let them interact in solution, and subjected the sample to BMH crosslinking. When the products were analyzed by SDS-PAGE and phosphor-imaging two BMH-crosslinked products with molecular weights close to that of a BimL-Bax heterodimer were detected (Figure 9A, lane 4, indicated by open and closed triangles). The lower molecular weight band indicated by a closed triangle is the BMH-linked BimL-Bax heterodimer since it was absent in the control reactions when either the single-cysteine BimL or Bax was replaced by the respective cysteine-null (C0) protein (Figure 9A, lane 6 or 2). The higher molecular weight band indicated by an open triangle is the BMH-linked Bax homodimer since it was also present in the control reaction containing the single-cysteine Bax and the cysteine-null BimL (Figure 9A, lane 6). These results demonstrate the BMH crosslinking approach can detect the interaction of the BH3-domain of BimL with the canonical groove of Bax, and hence in principle it can be used to reveal the Bim CTS-binding site in Bax.

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Sequence analysis predicts that similar to the BH3-domain the Bim CTS forms an amphipathic α-helix (Figure 6A). Sequence alignment revealed a high similarity between the CTS and the BH3-domain as both have the same hydrophobic residues at the h0, h1, h2 and h3 positions, and the same polar or charged residue at the h1+two or h2+one position (Figure 9D). The Bim BH3 residues at these positions make critical contacts with the Bax canonical groove that are important for Bax activation (Robin et al., 2015; Weber et al., 2007). To determine whether the Bim CTS binds to the same Bax groove as the Bim BH3-domain, we performed BMH crosslinking using a Bim W137C mutant that has a single cysteine near the C-terminus of the CTS. Like the Bim F101C mutant with the cysteine near the C-terminus of the BH3-domain, Bim W137C crosslinked to Bax W107C (Figure 9A, lane 10, indicated by a closed triangle), suggesting that the BH3-binding groove is also a binding site for the Bim CTS. Consistent with this interpretation, this BimL-Bax heterodimer specific crosslinking did not occur in the control reaction with either single-Cys protein substituted by the respective cysteine-null protein (Figure 9A, lane 8 or 12), unlike the Bax homodimer specific crosslinking that also occurred in the control reaction with the single-cysteine Bax and cysteine-null BimL (Figure 9A, lane 6). Reciprocal immunoprecipitation by BimL and Bax specific antibodies further identified the crosslinked BimL-Bax heterodimer from the cysteine in the Bim CTS or BH3-domain to the cysteine in the Bax groove (Figure 9—figure supplement 1).

To further define this noncanonical CTS-groove interaction, we repeated the crosslinking using other cysteine positions in the Bim CTS and additional cysteine mutants in the canonical groove in Bax. The only strong heterodimer specific crosslinking detected was between Bim M123C (a cysteine near the N-terminus of the CTS) and Bax W107C (Figure 9A, lane 14, indicated by a closed triangle). Since this Bax mutant was also crosslinked to the C-terminus of CTS via W137C, binding of the CTS to the groove seems to occur in either orientation. Additional weak but specific BimL to Bax crosslinking was detected from M123C to E69C and W137C to D98C providing further support for this flexible interaction (Figure 9—figure supplement 1).

To determine whether the physical interaction between the Bim CTS or BH3-domain and the Bax groove detected by the crosslinking is functional, we tested the effect of the L129E mutation in the Bim CTS or the 4E mutation in the Bim BH3-domain on the crosslinking because both mutations greatly inhibited the activation of Bax by BimL (Figure 6B). We found that the L129E mutation reduced the Bim CTS to Bax interaction with the binding groove detected by the Bim W137C or W123C to Bax W107C crosslinking (compare the closed triangle-indicated band in Figure 9B, lane 10 or 14 with that in Figure 9A, lane 10 or 14). Since we used Bim F101C to detect the BH3-domain interaction with the Bax groove, and the F101 was changed to E in the 4E mutant, we generated Bim F101C/3E mutant to assess the effect of the BH3 mutation. As expected, the 3E mutation abolished Bim F101C crosslinking to Bax W107C, and hence, the BH3-interaction with the groove (Figure 9C, the closed triangle indicates the band in lane two that disappeared in lane 4 when the 3E mutant was used). Surprisingly, mutation of the CTS also inhibited the BH3-interaction with the groove (Figure 9B, lane four vs. Figure 9A, lane 4), while the BH3 mutation also abolished the CTS interaction with the groove (Figure 9C, lane 12 vs. lane 10), suggesting that the two interactions are not independent. The BH3-domain may contribute more than the CTS to the overall protein-protein interaction based on the severity of the effect of mutations on the crosslinking and FRET (Figure 6B). Together, the crosslinking data from these loss-of-function Bim mutants demonstrate that the CTS and BH3-interactions with the groove detected between the soluble Bim and Bax proteins are functionally important for Bim mediated activation of Bax.

Together, our data suggests that specific residues within the Bim CTS are involved in different aspects of BimL function. Residue I125 is required for Bim to bind to mitochondria but is of lesser importance in activating Bax. In contrast, residues L129 and I132 are not required for BimL to bind Bax but are important for it to efficiently activate Bax. Finally, BimL-dCTS functions only to bind and inhibit Bcl-XL. The defined mechanism(s) of these mutants makes them useful for probing the differential sensitivity of HEK293 and MEF cells to expression of ^VBimL-dCTS as seen in Figure 1. Expression of the mutants in HEK293 cells by transient transfection revealed that similar to ^VBimL-dCTS, expression of either VBimL-L129E or VBimL-I132E was not sufficient to kill HEK293 cells, despite expression of either mutant being sufficient to kill the primed MEF cell line (Figure 10). In contrast, HEK293 cells were killed by expression of ^VBimL-I125E, albeit to a lesser extent than by ^VBimL (Figure 10). This result is consistent with our findings with purified proteins showing that the EC50 for liposome permeabilization by BimL-I125E was 100 nM compared to ~1 nM for BimL (Figure 6B). The activity of ^VBimL-I125E also demonstrates that BimL binding to membranes is not required to kill HEK293 cells as BimL-I125E does not bind membranes (Figure 6B). Together, this data suggests that unlike MEF cells, only mutants of BimL that can efficiently activate Bax kill HEK293 cells.

Figure 10

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Figure 10—source data 1

Source data demonstrating that mutants of BimL that cannot activate Bax do not kill H.EK293 cells.

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Wild type and single cysteine mutants of Bax, Bcl-XL, and cBid were purified as described previously (Kale et al., 2014). cBid mutant 1 (cBidmt1) was purified with the same protocol used for cBid (Kale et al., 2014). Bad was purified as described previously (Lovell et al., 2008). His-tagged Bax and Bcl-XL proteins were purified as described previously (Ding et al., 2014)

His-tagged Noxa was expressed in E. coli strain BL21DE3 (Life Tech, Carlsbad, CA). E. coli cells were lysed by mechanical disruption with a French press. The cell lysate was diluted in lysis buffer (10 mM HEPES (7.2), 500 mM NaCl, 5 mM MgCl2, 0.5% CHAPS, 1 mM DTT, 5% glycerol, 20 mM Imidazole) and Noxa was purified by affinity chromatography on a Nickel-NTA column (Qiagen, Valencia, CA). Noxa was eluted with a buffer containing 10 mM HEPES (7.2), 300 mM NaCl, 0.3% CHAPS, 20% glycerol, 100 mM imidazole, dialyzed against 10 mM HEPES 7.2, 300 mM NaCl, 10% glycerol, flash-frozen and stored at −80°C.

Purification of BimL and single cysteine mutants of BimL was carried out as previously described (Liu et al., 2019). Briefly, cDNA encoding full-length wild-type murine BimL was introduced into pBluescript II KS(+) vector (Stratagene, Santa Clara, CA). Sequences encoding a polyhistidine tag followed by a TEV protease recognition site (MHHHHHHGGSGGTGGSENLYFQGT) were added to create an in frame fusion to the N-terminus of BimL. All the purified BimL proteins used here retained this tag at the amino-terminus. However, control experiments demonstrated equivalent activity of the proteins before and after cleavage with TEV protease (Data not shown). Mutations as specified in the text were introduced into this sequence using site-directed mutagenesis.

BimL was expressed in Arabinose Induced (AI) E. coli strain (Life Tech, Carlsbad, CA). E. coli were lysed by mechanical disruption with a French press. Proteins were purified from the cell lysate by affinity chromatography using a Nickel-NTA column (Qiagen, Valencia, CA), and eluted with a solution containing 20 mM HEPES pH7.2, 10 mM NaCl, 0.3% CHAPS, 300 mM imidazole, 20% Glycerol. The eluate was adjusted to 150 mM NaCl and applied to a High Performance Phenyl Sepharose (HPPS) column. Bim was eluted with a no salt buffer and dialyzed against 10 mM HEPES pH7.0, 20% glycerol, flash-frozen and stored at −80°C.

Single cysteine mutants of Bax, Bcl-XL, cBid and Bad were labeled with the indicated maleimide-linked fluorescent dyes as described previously (Pogmore et al., 2016; Kale et al., 2014; Lovell et al., 2008). Single cysteine mutants of Bim were labeled with the same protocol as cBid with the exception that the labeling buffer also contained 4M urea.

Liposomes (100 nm diameter) with a lipid composition resembling MOM were prepared as described previously (Kale et al., 2014). Mouse liver mitochondria were isolated from Bak-/-mice as previously described (Pogmore et al., 2016). Liposomes and mitochondria were labeled with 0.5% and 2% mass ratios of DiD, respectively (Life Tech, Carlsbad, CA). The single-cysteine mutant of BimL, BimL Q41C, was labeled with Alexa568-maleimide and incubated with the indicated amount of unlabeled or DiD-labeled mitochondria or liposomes at 37°C for 1 hr. Intensities of Alexa568 fluorescence were measured in both samples as F_unlabeled and F_labelled respectively using the Tecan infinite M1000 microplate reader. FRET, indicating protein-membrane interaction, was observed by the decrease of Alexa568 fluorescence when BimL bound to DiD labeled membranes compared to unlabeled membranes. FRET efficiency was calculated as described previously (Shamas-Din et al., 2013a). The data was fit to a binding model as described below. Lines of best fit were calculated using least squares in Graphpad Prism software.

Membrane permeabilization assays with liposomes encapsulating ANTS and DPX were performed as described previously (Kale et al., 2014). To measure permeabilization of BMK mitochondria, the indicated amounts of proteins were incubated with mitochondria (1 mg/mL) purified from BMK cells genetically deficient for Bax and Bak expressing mCherry fluorescent protein fused to the SMAC import peptide responsible for localization in the inter-membrane space. After incubation for 45 min at 37°C samples were centrifuged at 13,000 g for 10 min to separate the pellet and supernatant fractions and membrane permeabilization was calculated based on the mCherry fluorescence in each fraction (Shamas-Din et al., 2014). For mouse liver mitochondria, cytochrome c release was measured by immunoblotting as described previously (Pogmore et al., 2016; Sarosiek et al., 2013).

Heavy membranes enriched in mitochondria were isolated as described previously (Pogmore et al., 2016; Brahmbhatt et al., 2016). Membrane fractions (1 mg/mL) were incubated with 500 nM of the specified BH3-proteins (Bim, Bad and/or Noxa). For E15 brain mitochondria, 0.5 mg/mL of membrane fractions were used and incubated with the indicated amounts of BH3-only proteins for 30 min at 37 °C. Membranes were pelleted by centrifugation at 13,000 g for 10 min and cytochrome c release was analyzed by immunoblotting using a sheep anti-cytochrome c antibody (Capralogics). Mitochondria from embryonic mouse brains for BH3profiling experiments were prepared from ~20 mouse embryos, E15 in age, following the same protocol used for liver mitochondria (Pogmore et al., 2016).

For FRET experiments, single cysteine mutants of cBid (126C), Bcl-XL (152C), Bax (126C), BimL (41C) and BimL mutants were purified and labeled with either Alexa 568-maleimide (donor) or Alexa 647-maleimide (acceptor) as specified. To determine binding a constant amount of donor protein was incubated with the indicated range of acceptor proteins and where specified liposomes or mitochondria. The intensity of Alexa568 fluorescence with unlabeled or Alexa647-labeled Bcl-XL was measured as F_unlabeled or F_labeled, respectively, and FRET was calculated as described in Pogmore et al. (2016). All measurements were collected using the Tecan infinite M1000 microplate reader. Lines of best fit were calculated using least squares in Graphpad Prism software.

For each pair of proteins a dissociation constant (Kd) was measured in solution and with liposomes. Curves were fit to an advanced function taking into account change of the concentration of acceptor ([A]) when [A] is close to Kd:

[D] is the concentration of donor, F indicates the FRET efficiency with the concentration of acceptor as [A], F_max is the maximum FRET efficiency in the curve (Pogmore et al., 2016).

To produce the proteins for crosslinking using in vitro systems, the DNA sequence encoding murine BimL Cys-null and Lys-null mutant without the His tag and TEV protease recognition site was excised from the pBluescript II KS(+) vector by restriction endonucleases NcoI and ClaI and inserted into the pSPUTK vector (Stratagene, Santa Clara, CA). Mutations as specified in the text were introduced into this sequence using site-directed mutagenesis to generate the single-Lys BimL or single-Cys mutants.

The photocrosslinking method for studying interactions among the Bcl-2 family proteins has been described in detail (Lin et al., 2019). Briefly, using the RNAs produced from the single-Lys Bim DNAs in the pSPUTK vector by an in vitro transcription system, [35S]Met-labeled BimL proteins with a single εANB-Lys incorporated at specific locations were synthesized in an in vitro translation system. 10 μL of the resulting BimL proteins were incubated at 37 °C for 1 hr with 1 μM of 6H-Bax or 6H-Bcl-XL protein and Bak-/-mouse liver mitochondria (0.5 mg/ml total protein and were resuspended in AT buffer with 80 mM KCl and supplemented with energy regenerating system as described previously Yamaguchi et al., 2007) in a 21 μL reaction adjusted by buffer A (110 mM KOAc, 1 mM Mg(OAc)2, 25 mM HEPES, pH 7.5). The mitochondrial and soluble fractions were separated by centrifugation at 13,000 g and 4 °C for 5 min, and the mitochondria were resuspended in 21 μL of buffer A. Both mitochondrial and soluble fractions were photolyzed to induce crosslinking via the ANB probe. The resulting samples were adjusted to 250 μL with buffer B (buffer A with 1% Triton X-100 and 10 mM imidazole) and incubated with 10 μL of Ni²⁺-chelating agarose at 4 °C for overnight. After washing the Ni²⁺-beads three times with 350 μL of buffer B and one time with 400 μL of PBS, the photoadducts of the radioactive BimL protein and the 6H-tagged Bax or Bcl-XL protein and other proteins bound to the Ni2+-beads were eluted with reducing SDS sample buffer and analyzed by SDS-PAGE and phosphor-imaging.

For chemical crosslinking, [³⁵S]Met-labeled single-Cys or Cys-null BimL and Bax proteins were synthesized from the respective mutant Bim and Bax DNAs in the pSPUTK vector using a transcription/translation (TNT)-coupled in vitro system (Promega, Madison, WI). The resulting BimL and Bax proteins, 2 μL each, were paired as indicated in Figure 9, and reduced by 11 μM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in a 20 μL reaction adjusted with buffer A at 37 °C for 1 hr. The sample was diluted to 110 μL with buffer A and split evenly to two aliquots. For a ‘60 min’ BMH crosslinking reaction, one aliquot was incubated with 0.1 mM BMH and 6 mM EDTA at 25 °C for 60 min, and the reaction was stopped by incubation with 50 mM 2-mercaptoethanol at 25 °C for 15 min. For a ‘0 min’ control reaction, the other aliquot was incubated with 2-mecaptoethanol and EDTA for 15 min, and then with BMH for 60 min. The resulting samples were precipitated by trichloroacetic acid. The resulting protein pellets were solubilized in reducing SDS sample buffer and analyzed by SDS-PAGE and phosphorimaging.

To obtain the immunoprecipitation data in Figure 9—figure supplement 1, the indicated single-Cys BimL and Bax proteins produced by the TNT system, 4 μL each, were paired and reduced by TCEP. The sample was diluted to 260 μL with buffer A and crosslinked by BMH. The resulting sample was divided to two aliquots. The 85 μL or 170 μL aliquot was immunoprecipitated by Bax or Bim antibody, respectively. Thus, each aliquot was adjusted to 250 μL with IP buffer (100 mM Tris pH 7.5, 100 mM NaCl, 10 mM EDTA, 1 mM PMSF, 1% (v/v) Triton X-100), and received Bax antibody (made in house, 1:1000 dilution) or Bim antibody (Santa Cruz Biotechnology, Dallas, TX, 1:50 dilution). The samples were rotated at 4 °C for overnight, and after receiving 25 μL of Protein G Sepharose (50% suspension in IP buffer), rotated for 2 more hours. After centrifugation at 2000 g for 0.5 min, the beads were washed three times with 400 μL of IP buffer and one time with 400 μL of 100 mM Tris pH 7.5 and 100 mM NaCl. The bound proteins were eluted with reducing SDS sample buffer and analyzed by SDS-PAGE and phosphorimaging.

HEK293, BMK, MEF, and HCT116 cells were maintained at 37°C (5% v/v CO2) in dMEM complete [dMEM, 10% Fetal Bovine Serum, 1% essential amino acids (Gibco, Grand Island, NY)]. CAMA-1 were maintained the same environmental conditions but using dMEM/F12 (Gibco, Grand Island, NY). Cell lines were routinely confirmed to be mycoplasma-free using a PCR-based protocol as described by Hopert et al. (1993), and their authenticity was verified by short-tandem repeat (STR) profiling at The Centre for Applied Genomics (Toronto, ON, Canada) for human cells. Murine cell lines have not been authenticated. Cells were seeded in CellCarrier-Ultra 384-well plates (1000 cells/well for BMK and MEF, 2000 cells/well for HEK293 and HCT116, 3000 cells/well for CAMA-1). One day later, cells were transfected using FugeneHD (Promega, Madison, WI) with plasmids encoding Venus, or Venus-fused BimL constructs in an EGFP-C3 backbone. Cell culture medium was added to each reaction (50 µl/0.05 μg DNA) and the whole mix added to each well (50 µl/well) of a pre-aspirated 384-well plate of cells. After 24 hr, cells were stained with Draq5 and Rhodamine-labeled Annexin V and image acquisition was performed using the Opera QEHS confocal microscope (Perkin Elmer, Woodbridge, ON) with a 20x air objective. Untransfected cells and cells treated with 1 µg/mL staurosporine were used as negative and positive controls for Annexin V staining. Cells were identified automatically using software as described previously (Shamas-Din et al., 2013a). Intensity features were extracted using a script (dwalab.ca) written for Acapella high content imaging and analysis software (Perkin Elmer, Woodbridge, ON). Cells were scored as Venus or Annexin V positive if the Venus or Annexin V intensity was greater than the average intensity plus two standard deviations for the Venus or Annexin V channels in images of non-transfected cells. Cell death ascribed to the ^VBimL fusion proteins was quantified as the percentage of Venus-positive cells that were also Annexin V positive. For neuron cultures, cell segmentation using conventional methods could not be achieved due to complex cellular morphologies. Therefore, nuclei were first identified, then a ring region ~10% of nuclear area was drawn around each nucleus. Venus intensity was calculated for this ring region, representing the neuronal cell body, to determine if the neuron was expressing the Venus fluorescent protein.

Primary cortical neuron cultures were prepared from embryonic day 15, C57BL/6J mouse embryos as previously described (Mergenthaler et al., 2012). All animal breeding and handling were performed in accordance with local regulations and after approval by the Animal Care Committee at Sunnybrook Research Institute, Toronto. Briefly, after separation from hippocampus and subcortical structures, cortices were washed twice with ice-cold PBS, digested with 1x trypsin for 15 min at 37°C, washed twice with ice-cold PBS and then resuspended with a flame-treated glass pipette in N-Medium (DMEM, 10% v/v FBS, 2 mM L-glutamine, 10 mM Hepes, 45 µM glucose). The dissociated cortices were gently pelleted by centrifugation (200 g for 5 min), N-media was removed, and neurons were resuspended and cultured in Neurobasal-Plus medium (ThermoFisher Scientific) supplemented with B27-Plus (ThermoFisher Scientific) and 1x Glutamax (ThermoFisher Scientific). Neurons were seeded at 5000 cells per well in a 384 well plate (Greiner µclear) after coating with poly-d-lysine (Cultrex). The medium was partially replaced on day 5 in culture with Neurobasal-Plus supplemented with B27-Plus and 1x Glutamax.

Lentivirus to express ^VBimL and other BimL mutants were cloned into the pTet-O-Ngn2-Puro construct with the Ngn2 gene cut out. This construct was a kind gift from Dr. Philipp Mergenthaler, Charité Universitätsmedizin Berlin. Primary neuron cultures were infected with both ^VBimL and rtTA lentiviral particles (~3 μL of each concentrated stock) on the day of seeding. 24 hr later, Neurobasal-Plus medium containing lentiviral particles was removed and replaced with fresh Neurobasal-Plus medium.

Doxycyline (ThermoFisher scientific) was added to 16 day in vitro old cultures of neurons at a concentration of 2 μg/mL to induce ^VBimL protein expression. 5 hr later, neurons were stained with 5 µM Draq5 (Thermofisher scientific) and 0.1 μM TMRE (Thermofisher scientific), then incubated for 30 min at 37 °C. Confocal microscopy was performed immediately after.

Each lentivirus was made using the following protocol adhering to biosafety level two procedures. On day 0, lentiviral vectors psPax2 (10 μg) and pMD2.G (1.25 μg) were mixed with 10 μg of desired ^VBimL lentiviral construct in 1000 μL of Opti-MEM media (ThermoFisher Scientific). Next, 42 μL of polyethylenimine (PEI) solution [1 mg/mL] was added, the mixture vortexed, then allowed to settle for 15 min at room temperature. After 15 min, 1.5 × 10⁷ of resuspended HEK293 cells and the transfection solution were mixed and seeded onto a 100 mm culture dish with 10 mL of dMEM complete plus 10 μM of the caspase inhibitor Q-VD-Oph (Selleckchem), and left to incubate at 37 °C (5% v/v CO₂) for 72 hr. On day 3, media containing lentiviral particles was filter sterilized using a 0.45 μm polyethersulfone filter, and mixed with polyethylene glycol (Bioshop) to achieve a final concentration of 10% (w/v). This was left to mix and precipitate the virus overnight at 4 °C. On day 4, the media was centrifuged for 1 hr at 1600 g, supernatant was then removed and the pellet was resuspended with 400 μL of Neurobasal-Plus media (no additives). Resuspended virus was then stored at −80 °C until needed.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This work was funded by CIHR grant FRN 12517 to DWA and BL and CIHR Foundation grant FDN143312 to DWA, US NIH grants R01GM062964, OCAST grant HR16-026 and Presbyterian Health Foundation grant to JL, and by an Institutional Development Award from the National Institute of General Medical Sciences of US NIH under grant number P20GM103640. DWA holds the Tier 1 Canada Research Chair in Membrane Biogenesis. QL held a post-doctoral fellowship from the Canadian Breast Cancer Foundation.

Animal experimentation: All animal breeding and handling was performed in accordance with local regulations and after approval by the Animal Care Committee at Sunnybrook Research Institute, Toronto AUP #18-558.

© 2020, Chi et al.

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