From engine room to executioner mitochondrial functions in cell death

Video Transcript

• 00:00 - 00:07: Hi, welcome to Abcam's webinar today. Today's principal speaker is Dr. Stephen Tate from
• 00:07 - 00:13: Cancer Research UK, Beatson Institute and University of Glasgow. Stephen carried out
• 00:13 - 00:19: his graduate studies at the Institute for Animal Health in Pirbright, UK. This is where
• 00:19 - 00:25: he investigated viral immune evasion strategies. His post-doc training was carried out at the
• 00:25 - 00:31: Netherlands Cancer Institute in Amsterdam and at St. Jude Children's Research Hospital, Memphis.
• 00:32 - 00:39: Since 2012, Stephen has had his own research group based at the Cancer Research UK, Beatson Institute
• 00:39 - 00:44: and the University of Glasgow. His group is supported in part by a fellowship from the
• 00:44 - 00:52: Royal Society. His lab's main research interest is investigating deregulation of mitochondrial
• 00:52 - 00:59: functions in cancer, focusing upon cell death and autophagy. Joining Stephen today will be David
• 00:59 - 01:08: Bruce, Research Area Content Associate from Abcam. David has a BSc in Biomedical Sciences, Pharmacology
• 01:09 - 01:15: from the University of Aberdeen. Before joining Abcam, David studied for his PhD at the University
• 01:15 - 01:22: of Dundee, where he examined the regulation of the TGF-beta signaling pathway by novel protein
• 01:22 - 01:29: phosphatases and ubiquitin E3 ligases. I will now hand over to Stephen who will start this webinar.
• 01:30 - 01:38: Stephen. Thanks very much, Lucy. Okay, and thanks for the introduction. Right, so today I'm really
• 01:38 - 01:44: going to talk about the mitochondrial pathways of cell death and how to investigate these processes.
• 01:45 - 01:51: I'm really going to divide my talk into three separate sections. One is going to be discussing
• 01:51 - 01:57: the mitochondrial or intrinsic pathway of apoptosis, its mechanism, its dysfunction and
• 01:57 - 02:04: disease and really outline a couple of outstanding questions that remain in the field. From that,
• 02:04 - 02:10: I'm going to go on to discuss at least some methods that can be used for detecting mitochondrial
• 02:10 - 02:16: dependent apoptosis in the system that you're investigating. Finally, I'm going to kind of
• 02:16 - 02:22: showcase a new approach that we've developed to determine mitochondrial function in non-apoptotic
• 02:22 - 02:32: cell death. Okay, so first I'm going to talk about apoptosis. Apoptotic cell death requires activation
• 02:32 - 02:38: of proteases called caspases which bring about the rapid demise of a cell. Effectively, there
• 02:38 - 02:45: are two different pathways, two main pathways whereby caspases can be activated. One is called
• 02:45 - 02:51: the extrinsic pathway of apoptosis and this typically is activated by death receptor ligands.
• 02:51 - 02:59: These bind to receptors which in turn cluster and activate caspase 8. Active caspase 8 goes ahead
• 02:59 - 03:05: and then cleaves caspase 3 and 7, bringing about rapid cell death. I'm not really going to talk
• 03:05 - 03:09: much about the extrinsic pathway of apoptosis. Today, we're going to focus on the intrinsic
• 03:09 - 03:13: pathway of apoptosis, also known as the mitochondrial pathway of apoptosis
• 03:14 - 03:19: which is activated by one of numerous stresses such as DNA damage or ER stress.
• 03:20 - 03:27: This leads to activation of BH3-only proteins that in turn activate two proteins in the process
• 03:27 - 03:32: BAX and BAK. We'll discuss this in much more detail, but these proteins, when they're activated
• 03:32 - 03:38: lead to mitochondrial outer membrane permeabilization. This then, in turn, activates
• 03:38 - 03:44: caspases through the release of proteins such as cytochrome c or SMAC/OMI from the mitochondria.
• 03:44 - 03:51: These then drive caspase activation in the case of cytochrome c. SMAC/OMI, on the other hand,
• 03:51 - 03:57: inhibits a caspase inhibitor called XIAP, and the net effect is once mitochondrial
• 03:57 - 04:02: permeabilization has occurred, also known as MOMP or referred to as MOMP through the talk as well,
• 04:03 - 04:06: the cell dies within a matter of minutes to a couple of hours.
• 04:11 - 04:16: So really just emphasizing the mitochondrial pathway of apoptosis. In healthy cells,
• 04:16 - 04:22: mitochondria retain various proteins in their mitochondrial intermembrane space
• 04:22 - 04:27: such as cytochrome c, SMAC/OMI. When these proteins are released, they actively kill the cell.
• 04:28 - 04:33: And so in one sense, mitochondria can be considered similar to suicide capsules.
• 04:34 - 04:40: When a cell receives a trigger to die that activates BH3-only protein, one of these is BID.
• 04:42 - 04:47: These, in turn, activate BAX and BAK, and somehow when BAX and BAK are activated,
• 04:47 - 04:52: they selectively permeabilize the mitochondrial outer membrane. Mitochondrial outer membrane
• 04:52 - 04:58: permeabilization has catastrophic consequences for the cell. As mentioned previously,
• 04:58 - 05:05: cytochrome c binds this adaptor molecule APAF1. That, in turn, activates caspases. They cleave
• 05:05 - 05:11: hundreds of different proteins bringing about rapid apoptotic cell death. And as mentioned,
• 05:11 - 05:17: SMAC/OMI inhibits this caspase inhibitor XIAP, and so the net effect is you get rapid,
• 05:17 - 05:24: robust caspase activation and rapid cell death. Okay, and so we can image this whole process
• 05:25 - 05:29: by confocal microscopy. And this is shown here. And what you're going to see in this movie
• 05:30 - 05:36: are the mitochondria being labeled red with a fluorescent fusion protein SMAC-mCherry.
• 05:36 - 05:42: They’re also expressing YFP-BAX, which is predominantly cytosolic in healthy cells.
• 05:43 - 05:48: Now, as the cell dies, the cell looks perfectly healthy up until the point the mitochondrial
• 05:48 - 05:53: permeabilization occurs. And at that point, SMAC-mCherry is released throughout the cell.
• 05:53 - 05:59: You'll see it go from this punctate pattern to diffuse pattern. And at the same time,
• 05:59 - 06:05: BAX translocates onto the mitochondria. So it moves onto the mitochondria. It goes from this
• 06:05 - 06:11: diffuse pattern to punctate pattern. Once mitochondrial permeabilization has occurred,
• 06:11 - 06:18: this engages rapid and robust caspase activity that leads to the hallmarks of apoptosis. And
• 06:18 - 06:22: you can see that quite nicely in these cells. The cells shrink, the nucleus condenses,
• 06:23 - 06:29: and finally, the cells expose phosphatidylserine. And this can be detected because we've added
• 06:30 - 06:37: an annexin V, which is conjugated to APC. And it binds phosphatidylserine as it's flipped from
• 06:37 - 06:42: the inner to the outer leaflet of the plasma membrane. And so I'll play this movie through
• 06:42 - 06:47: a couple of times just really to emphasize this point, the mitochondrial permeabilization
• 06:47 - 06:50: is the key event in the mitochondrial pathway of apoptosis.
• 06:57 – 07:04: Eventually, these cells, as I say, expose phosphatidylserine. And in our bodies,
• 07:04 - 07:07: these cells are rapidly removed by neighboring phagocytes.
• 07:10 - 07:14: So again, at that point, we get massive caspase activation, the cells shrink,
• 07:14 - 07:19: membranes bleb, the nucleus condenses, and we get phosphatidylserine exposure.
• 07:22 - 07:28: Okay, so that would give you the idea that mitochondrial permeabilization leads to cell
• 07:28 - 07:32: death through caspase activity. Well, actually, even in the absence of caspase
• 07:34 - 07:39: cleavage of substrates, mitochondrial permeabilization often represents a point of no
• 07:39 - 07:45: return. And this is shown in this movie here in which cells have been transfected from the
• 07:46 - 07:450: protein GFP-TBid, which causes mitochondrial permeabilization. These cells have already
• 07:50 - 07:57: released SMAC-mCherry throughout the cell. However, these cells have been incubated with
• 07:57 – 08:02: caspase inhibitor. And whilst the cells don't die in the short term, you'll see quite nicely
• 08:02 - 08:07: over time, the cells undergo massive vacuolarization and undergo what looks like a
• 08:07 - 08:12: necrotic cell death. So even if we prevent caspase activation, providing mitochondrial
• 08:12 - 08:18: permeabilization has occurred, cells die. Usually, this event causes caspase-dependent apoptosis.
• 08:20 - 08:26: But somehow, cells, in the absence of caspase activity, still die. Some studies would argue
• 08:26 - 08:23: that this may be an active role for mitochondria in triggering caspase activation,
• 08:23 - 08:29: still die. Some studies would argue that this may be an active role for mitochondria in triggering
• 08:29 - 08:33: caspase-independent cell death. Alternatively, or even additionally,
• 08:34 - 08:37: death may simply be due to a gradual loss in mitochondrial function.
• 08:41 - 08:48: So mitochondrial apoptosis has many roles in health and disease. It's required for proper
• 08:48 - 08:52: embryonic development, and that's best evidenced in mice that are deficient in mitochondrial
• 08:52 - 08:58: permeabilization. So, for example, ones that lack BAX and BAK. It's often embryonic lethal.
• 08:59 - 09:04: It's also required, amongst many other functions, for things like regulating the immune system.
• 09:04 - 09:07: So deficiency in mitochondrial permeabilization leads to defective
• 09:07 - 09:12: lymphocyte maturation, homeostasis, ultimately triggering autoimmunity.
• 09:12 - 09:18: And so these are just two examples of the role of mitochondrial apoptosis in maintaining health.
• 09:19 - 09:26: It also has many roles in disease, and perhaps the best evidence role for deregulation of
• 09:26 - 09:32: mitochondrial apoptosis is seen in cancer. So one of the hallmarks of cancer is that
• 09:32 - 09:40: during the tumorigenic process, cells must evade apoptosis. And why is this the case?
• 09:40 - 09:46: Well, many tumor suppressor pathways engage apoptosis and kill a cell that's at risk
• 09:46 - 09:50: of becoming malignant. So inhibiting apoptosis promotes transformation.
• 09:51 - 09:56: Moreover, it promotes cell survival under hypoxic or nutrient-poor conditions,
• 09:56 - 09:59: common conditions that a tumor cell faces.
• 10:00 - 10:06: Another aspect whereby apoptosis promotes cancer is many cells, when they detach from
• 10:06 - 10:13: their substratum, undergo a form of apoptosis called anoikis. This prevents cells from moving
• 10:13 - 10:18: around the body and prevents metastasis. So obviously, inhibiting apoptosis, again,
• 10:18 - 10:22: would facilitate this process and the movement of cancer cells around the body.
• 10:23 - 10:30: And finally, it has this kind of secondary effect. Many anticancer therapies can induce apoptosis.
• 10:30 - 10:35: And so by inhibiting apoptotic cell death, a cell can not only become cancerous,
• 10:35 - 10:38: it can also evade chemo or radiotherapy.
• 10:43 - 10:48: So, as mentioned a couple of slides back, BAX and BAK are really two key proteins that are
• 10:49 - 10:53: essential for MOMP, mitochondrial outer membrane permeabilization, and apoptosis.
• 10:53 - 10:59: On a pro-apoptotic trigger, that activates a BH3-only protein leading to BAX and BAK activity,
• 11:00 - 11:03: ultimately triggering MOMP. And that's best evidenced in mice that lack BAX and BAK.
• 11:05 - 11:13: One example, or physiological example, of cell death is the interdigital space in our limbs.
• 11:13 - 11:18: This webbing dies through the process of apoptosis. If we knock out BAX and BAK,
• 11:18 - 11:24: we effectively prevent cell death occurring in these interdigital webs, and you can see the webbing remains.
• 11:24 - 11:29: Moreover, if we take cells from animals that are deficient in BAX and BAK,
• 11:30 - 11:37: we can see quite nicely these DKO cells, all the intrinsic or mitochondrial triggers
• 11:37 - 11:43: of apoptosis, such as staurosporin, etoposide, or UV, are completely, well,
• 11:44 - 11:48: in the absence of BAX and BAK, cells are completely resistant to these stimuli.
• 11:50 - 11:57: So, how do these proteins get activated? Well, BAX, as shown in that movie a couple of slides
• 11:57 - 12:04: ago, predominantly in healthy cells is a cytosolic protein. So, this is a green GFP-BAX shown here.
• 12:04 - 12:09: As a cell undergoes mitochondrial permeabilization, undergoes apoptosis,
• 12:09 - 12:15: BAX translocates from the cytosolic location onto the mitochondria, as you can see quite nicely here,
• 12:15 - 12:21: co-localizing with mitochondria. And upon this translocation, it engages mitochondrial
• 12:21 - 12:25: permeabilization, and you'll see that nicely in this movie here. BAX translocates,
• 12:25 - 12:31: it goes from this diffuse localization to a punctate mitochondrial localization,
• 12:31 - 12:37: and really simultaneously, mitochondrial permeabilization is engaged. SMAC-mCherry, which resides
• 12:37 - 12:43: in the mitochondrial intermembrane space, is then released from the mitochondria throughout the cell,
• 12:43 - 12:55: and ultimately the cell will die. As you can see quite nicely here, BAX will translocate,
• 12:55 - 12:57: and at the same time, mitochondrial permeabilization occurs.
• 13:02 - 13:07: So, how do active BAX and BAK cause mitochondrial permeabilization? Well,
• 13:07 - 13:11: this is really a matter of much debate. One thing that is clear is that during
• 13:12 - 13:18: the process of apoptosis, BAX and BAK form homooligomers, and that's quite evident
• 13:19 - 13:24: in experiments that have been done using chemical crosslinkers. Cells or mitochondria that have been
• 13:24 - 13:29: treated with the active form of TBID to engage mitochondrial permeabilization have been treated
• 13:29 - 13:36: in the presence of crosslinker. When we look at BAX, it goes from this monomeric state.
• 13:36 - 13:41: In the presence, or when it's activated, we can crosslink higher molecular weight species.
• 13:42 - 13:48: Exactly the same thing goes for BAK. So, two schools of thought. One would argue that,
• 13:48 - 13:54: upon activation, BAX and BAK form homodimers dependent, kind of if you like, head-to-head
• 13:54 - 13:59: homodimers. These then form higher weight oligomers, ultimately causing permeabilization
• 13:59 - 14:06: in the mitochondria. Alternatively, active BAX and BAK have been proposed to cause these daisy
• 14:06 - 14:12: chain-like homooligomers that ultimately cause mitochondrial permeabilization.
• 14:15 - 14:22: So, how does the mitochondrial outer membrane permeabilize? Two main models argue that,
• 14:22 - 14:28: upon activation of BAX and BAK, they either form proteinaceous channels, so they form
• 14:28 - 14:33: holes in the mitochondria themselves, allowing the release of intermembrane space proteins.
• 14:34 - 14:37: The alternative model, and there's certainly a lot of controversy
• 14:38 - 14:50: about this, is that rather than directly causing pores in the mitochondrial outer membrane,
• 14:50 - 14:55: activated BAX and BAK serve to bend the mitochondrial outer membrane lipids,
• 14:55 - 14:59: forming lipidic pores, which ultimately allow the release of cytochrome c.
• 15:00 - 15:04: Exactly how this process occurs, as I mentioned, is still highly debated.
• 15:07 - 15:14: So, how is it controlled? Well, simply, what I've discussed so far would give you the idea
• 15:14 - 15:18: that it's just unidirectional. Provided there's an activating signal, BAX and BAK will be
• 15:18 - 15:24: activated. However, that's definitely not the case. There are many proteins that restrain
• 15:24 - 15:31: BAX and BAK activation. So, essentially, BCL2 protein family members regulate mitochondrial
• 15:31 - 15:39: permeabilization. This family can be divided into two main subsets. One is the anti-apoptotic BCL2
• 15:39 - 15:48: protein family, comprised of BCL2, BCL-W, BCL-XL, A1, and MCL1, that have four BCL2 homology domains.
• 15:48 - 15:55: Secondly, there are pro-apoptotic BCL2 proteins, and as I mentioned, BAX and BAK are two of them.
• 15:55 - 16:01: These are effector molecules. BOK shares similarity to these proteins. Whether in itself it's an
• 16:01 - 16:06: effector protein is unclear. And finally, there's the class of, if you like, apoptosis signaling
• 16:06 - 16:14: molecules, the BH3-only proteins, of which there are numerous, BID, BIM, BAD, BIK, BMF, and so on,
• 16:14 - 16:22: that activate apoptosis. And so, anti-apoptotic BCL2 proteins restrain the process of mitochondrial
• 16:22 - 16:29: permeabilization. And they do this, the whole system works through protein-protein interactions.
• 16:30 - 16:36: So, for example, this is a structure shown here of MCL1 binding the BH3 domain of the BH3-only
• 16:36 - 16:45: protein BIM. What happens is the BH3-only domain of BIM binds into the hydrophobic groove of MCL1,
• 16:45 - 16:51: and in doing so, MCL1 can neutralize BIM function. And in this part of the slide here,
• 16:51 - 16:55: you can see quite nicely, these are the anti-apoptotic BCL2 proteins.
• 16:56 - 17:01: They bind to varying degrees, different BH3-only proteins. So, for example,
• 17:01 - 17:06: BIM, PUMA, and TBID appear to be able to bind all of them. BAD and NOXA,
• 17:06 - 17:10: which are other BH3-only proteins, are more restricted in their binding specificity.
• 17:12 - 17:18: Secondly, BCL2 proteins can bind BAX and BAK. And again, there appears to be some
• 17:18 - 17:25: specificity as to which anti-apoptotic BCL2 proteins can bind either BAX or BAK.
• 17:29 - 17:35: So, exactly how BCL2 proteins regulate MOMP is still a matter of controversy. There are two,
• 17:35 - 17:41: or until relatively recently, there were two models suggested to propose this effect.
• 17:42 - 17:49: One is that BAX or BAK, for that matter, are directly activated by BH3-only proteins.
• 17:49 - 17:55: So, for example, BIM activates BAX, and that goes on to drive mitochondrial permeabilization.
• 17:56 - 18:03: BCL2 proteins in this model, the direct model of activation, simply serve to bind and sequester
• 18:03 - 18:09: these activating BH3-only proteins. And in doing so, they can prevent BAX and BAK activity.
• 18:10 - 18:18: The other model, the indirect model of activation, is that rather than these proteins,
• 18:18 - 18:24: such as BAX and BAK, being directly activated, they exist in a constitutively active form and
• 18:24 - 18:30: such that BCL2 family members, anti-apoptotic proteins, serve to block activated BAX and BAK.
• 18:32 - 18:37: The BH3-only proteins in this model neutralize anti-apoptotic BCL2 function,
• 18:38 - 18:42: allowing activated BAX and BAK to drive mitochondrial permeabilization.
• 18:44 - 18:50: More recently, there's been a model called Unified, which is an elaboration of the
• 18:50 - 18:56: Embedded Together model, which really argues that both the direct and indirect models are likely
• 18:56 - 19:04: true. And in this model, what happens is BIM or another BH3-only protein can activate BAX,
• 19:04 - 19:07: leading to BAX activation and mitochondrial permeabilization.
• 19:08 - 19:14: BCL2, anti-apoptotic BCL2 proteins restrain the process, both by binding these activator
• 19:14 - 19:22: BH3-only proteins and by binding activated BAX and BAK. So most likely, aspects of both
• 19:22 - 19:30: of these models hold true. And recently, new drugs have been developed to therapeutically
• 19:30 - 19:37: target mitochondrial apoptosis in cancer. These drugs have been termed BH3 mimetics,
• 19:37 - 19:42: and they work by binding the hydrophobic groove in anti-apoptotic BCL2 family proteins
• 19:43 - 19:45: and neutralizing their protective function.
• 19:48 - 19:53: So by binding in this hydrophobic groove in, say, for example,
• 19:53 - 20:00: BCL-XL, this drug, Navitoclax (ABT-263), prevents BCL-XL from being activated by BAX.
• 20:00 - 20:06: BH3-only proteins from binding into the BCL2 family member, in turn, liberating these BH3-only
• 20:06 - 20:09: proteins to go on and activate BAX and BAK.
• 20:09 - 20:16: And so there's a variety of BH3 mimetics that have been developed that target a panel, for
• 20:16 - 20:24: example, BCL-XL, BCL-W, and BCL-2, of BCL2 family members. So ABT-737263 targets all three
• 20:24 - 20:25: of them.
• 20:25 - 20:32: More recently, newer ones have been developed, WEHI-539, that target BCL-XL. ABT-199 targets
• 20:32 - 20:37: BCL-2. It's important to note, and this is something we'll touch on later as well, that
• 20:37 - 20:42: there are various other anti-apoptotic BCL2 family members for which we can't specifically
• 20:42 - 20:46: target at present.
• 20:46 - 20:54: So how do these drugs work in practice? Well, as I've stated, anti-apoptotic BCL2 proteins
• 20:54 - 21:02: prevent mitochondrial permeabilization. When we add in a BH3 mimetic compound, this targets
• 21:02 - 21:08: and neutralizes anti-apoptotic BCL2 function, and so it removes this break, if you like,
• 21:08 - 21:13: upon the apoptotic process. And now cells are much more sensitive. You can trigger cell
• 21:13 - 21:21: death with a lower drug treatment, for example, leading to release or upregulation of a lower
• 21:21 - 21:26: amount of BH3-only proteins, triggering mitochondrial permeabilization. So in the presence of these
• 21:26 - 21:31: drugs, many cells, and specifically tumor cells, are actually more sensitive to cell
• 21:31 - 21:34: death.
• 21:34 - 21:41: And here's some evidence suggesting that they work, and these drugs, beyond being very useful
• 21:41 - 21:46: as research tools, they appear to be very effective either on their own or in combination
• 21:46 - 21:52: with chemotherapy, inducing tumor cell death. And so as an example, these are xenograft
• 21:52 - 21:57: tumors grown in a nude mouse. They are grown with vehicle, and you can see quite nicely
• 21:57 - 22:02: that the tumor keeps growing, or in the presence of ABT-737.
• 22:02 - 22:07: Secondly, in the clinic, they've also been used. This is a patient that's been suffering
• 22:07 - 22:12: from lymphoma pre-treatment with one of the BH3 mimetics. You can see the lymphoma mass
• 22:12 - 22:18: here, and then post-treatment, much of that lymphoma is regressed. And so as I said, they
• 22:18 - 22:25: look to be very promising drugs, either on their own in certain lymphomas or leukemias,
• 22:25 - 22:31: or together with additional chemotherapies to specifically kill cancer cells.
• 22:31 - 22:36: So to summarize this part, what I've told you so far is that mitochondrial outer membrane
• 22:36 - 22:42: permeabilization is the key event in the mitochondrial pathway of apoptosis. It leads to the release
• 22:42 - 22:48: of proteins such as cytochrome c from the mitochondrial intermembrane space that activate
• 22:48 - 22:55: caspases. However, importantly, even in the absence of caspase activity, MOMP typically
• 22:55 - 23:00: represents a point of no return, so cells die once it's occurred.
• 23:00 - 23:06: BCL2 family proteins are the main regulators of mitochondrial permeabilization. Pro-apoptotic
• 23:06 - 23:12: BH3-only proteins signal the pro-apoptotic signal, or relay the pro-apoptotic signal
• 23:12 - 23:21: to activate BAX and BAK, and block protective anti-apoptotic BCL2 function. BAX and BAK
• 23:21 - 23:27: upon activation permeabilize the mitochondrial outer membrane. Anti-apoptotic BCL2 proteins
• 23:27 - 23:35: bind both classes of pro-apoptotic proteins, thereby preventing apoptosis. And this network
• 23:35 - 23:40: of interactions has been exploited by new drugs that have been designed to specifically
• 23:40 - 23:46: bind and neutralize anti-apoptotic BCL2 proteins.
• 23:46 - 23:51: So I've just outlined three outstanding questions, I think, that remain regarding the mitochondrial
• 23:51 - 23:55: pathway of apoptosis. There are many others, but these are at least three that spring to
• 23:56 - 24:01: my mind, at least. One is, how do activated BAX and BAK permeabilize the mitochondrial
• 24:01 - 24:07: outer membrane? As I've stated, there are two models for this, but it's really unclear
• 24:07 - 24:11: how this process occurs, and it's critical that we do understand it, because it's the
• 24:11 - 24:15: really defining event in whether a cell lives or dies.
• 24:15 - 24:20: Secondly, when we think about targeting this network in disease, can we effectively target
• 24:21 - 24:28: BCL2 family members in cancer? And what I mean by this is, we can clearly develop drugs
• 24:28 - 24:34: to inhibit anti-apoptotic BCL2 proteins, but are there going to be problems with resistance?
• 24:34 - 24:39: For example, upregulation of MCL1, we can't target that just now. And secondly, if we
• 24:39 - 24:46: target many anti-apoptotic BCL2 proteins simultaneously, is that going to lead to toxicity problems?
• 24:46 - 24:53: Moreover, kind of getting away from this idea of sensitizing cells to apoptosis, there are
• 24:53 - 24:58: some diseases in which too much apoptosis is a bad thing, and this, for example, various
• 24:58 - 25:05: neurodegenerative diseases may be contributed to by too much apoptosis.
• 25:05 - 25:11: So on the flip side, can we target BCL2 family members to prevent mitochondrial outer
• 25:11 - 25:17: membrane permeabilization and cell death? And really, I think that's a relatively unexplored area
• 25:17 - 25:19: that deserves more attention.
• 25:19 - 25:26: Okay, so next I'm going to discuss with you some methods, certainly not an exhaustive
• 25:26 - 25:33: list, but some methods for detecting mitochondrial-dependent apoptosis.
• 25:33 - 25:40: So firstly, you have a stimulus of interest that's killing the cells. Does MOMP actually
• 25:41 - 25:46: occur after my stimulus of interest? And most of the assays that revolve around detecting
• 25:46 - 25:52: mitochondrial permeabilization really rely upon this robust release of intermembrane-based
• 25:52 - 25:59: proteins such as cytochrome c, SMAC, and OMI from the mitochondria into the cytosol.
• 25:59 - 26:05: And as I showed you earlier in that movie, it's really a black-and-white event. Most,
• 26:05 - 26:09: if not all, the mitochondria permeabilizing the cell lead to a massive release of these
• 26:09 - 26:15: proteins into the cytosol. So we can use this differential localization to assay for
• 26:15 - 26:18: the presence of MOMP.
• 26:18 - 26:24: We can do this in several ways. One is to use immunofluorescent staining for cytochrome
• 26:24 - 26:30: c, SMAC, or OMI. It's punctate under normal healthy conditions, and it goes to diffuse
• 26:30 - 26:37: cytosolic state when MOMP occurs. And this is shown quite nicely in these figures here
• 26:37 - 26:42: in which cytochrome c has been stained either in healthy cells, and you can see it has this
• 26:42 - 26:47: punctate localization, or cells that have been treated to undergo apoptosis with actinomycin
• 26:47 - 26:52: D. And you can see here the cells round up, but cytochrome c is released throughout the
• 26:52 - 26:57: cell. In the presence of Z-VAD, we see this cytochrome c release from the mitochondria
• 26:57 - 27:03: throughout the cell. Z-VAD is a caspase inhibitor. This prevents the cells rounding up and actually
• 27:03 - 27:05: makes them easier to visualize.
• 27:05 - 27:11: So I think performing these analyses, a key tip is really to add a caspase inhibitor into
• 27:11 - 27:17: the experiment to prevent cell rounding and detachment. The important thing to know is
• 27:17 - 27:23: that's only going to work provided caspases aren't upstream of mitochondrial permeabilization.
• 27:23 - 27:31: So initially, maybe both ways of doing it, either in presence or absence of caspase inhibitor,
• 27:31 - 27:33: would be beneficial.
• 27:33 - 27:38: The pros of it is that it's straightforward. There are very good antibodies to mitochondrial
• 27:38 - 27:43: intermembrane space proteins, and it's very obvious when a cell has undergone MOMP.
• 27:43 - 27:49: It's not very subjective analysis. You can see quite clearly there's a difference between
• 27:49 - 27:54: a healthy cell and a cell that's undergone MOMP, and it can be very quantitative.
• 27:54 - 27:59: I think one of the major cons is that it's quite laborious when quantifying many different
• 27:59 - 28:01: samples.
• 28:01 - 28:05: Alternatively, we can detect the release of proteins from the intermembrane space throughout the
• 28:05 - 28:11: cell using Western blotting of cytosolic and/or mitochondrial membrane fractions.
• 28:11 - 28:15: So this is a case of stimulating cells, fractionating by different methods.
• 28:15 - 28:23: We often use digitonin-based lysis, but equally as good, hypotonic lysis also works.
• 28:23 - 28:27: And by using these selective lysis conditions, you can selectively permeabilize the plasma
• 28:27 - 28:33: membrane, but leave the mitochondrial intermembrane intact.
• 28:33 - 28:37: And again, I think it's beneficial to add QVD, a caspase inhibitor, or Z-VAD into the
• 28:37 - 28:45: experiment to prevent cell lysis and cytochrome c loss, at least from the cytosolic fraction.
• 28:45 - 28:49: And so as an example here, these cells have been treated either with control or with etoposide
• 28:49 - 28:55: or gamma radiation for different lengths of time.
• 28:55 - 28:59: They've been hypotonically lysed, and then the cytosol has been probed for cytochrome
• 28:59 - 29:01: c as a measure of mitochondrial permeabilization.
• 29:01 - 29:06: And you can see quite nicely, these are apoptotic triggers, and we can detect cytochrome c quite
• 29:06 - 29:10: effectively in the cytosolic extract.
• 29:10 - 29:14: So one of the major pros is it's quite straightforward.
• 29:14 - 29:20: However, a con to this approach is that you need a larger sample than doing immunostaining,
• 29:20 - 29:23: for example, and it's not very quantitative.
• 29:23 - 29:27: And with this, obviously, we can't gain a percentage of cells that have undergone
• 29:27 - 29:31: mitochondrial permeabilization, only that it's occurred in at least a subset.
• 29:35 - 29:41: The last way, at least using the antibody immunostaining approach I'm going to discuss
• 29:41 - 29:45: today, is to detect the release of proteins from the intermembrane space throughout the cell
• 29:45 - 29:47: by FACS analysis.
• 29:47 - 29:52: Of cytochrome c, or this can be done also for SMAC or OMI, released from the mitochondrion.
• 29:52 - 29:58:  And so this method was developed by Nigel Waterhouse several years ago now, and the
• 29:58 - 30:00:  rationale behind it is here.
• 30:00 - 30:06:  We can take a healthy cell when you selectively permeabilize the plasma membrane.
• 30:06 - 30:12:  The mitochondria are still intact, so we have no loss of mitochondrial intermembrane space
• 30:12 - 30:18:  protein signal, and we can immunostain these cells and stain them by FACS, and so these
• 30:18 - 30:20:  stain high.
• 30:21 - 30:27:  However, when a cell has undergone apoptosis, the intermembrane space proteins leak throughout
• 30:27 - 30:29:  the cell.
• 30:29 - 30:34:  We can then selectively permeabilize the plasma membrane, and this protein, if you like,
• 30:34 - 30:38:  the cytochrome C, can be released from the cell into the cytosol.
• 30:38 - 30:44:  We stain these cells up, and these stain low.
• 30:44 - 30:46:  And so in essence, we can get two peaks.
• 30:46 - 30:49:  One healthy, no mOMP has occurred.
• 30:49 - 30:54:  The second unhealthy, mOMP has occurred, and we can distinguish this in a very quantitative
• 30:54 - 30:58:  manner, and that's shown quite nicely here from Nigel's paper.
• 30:58 - 31:02:  These are healthy cells here, not undergone mOMP.
• 31:02 - 31:06:  These are the ones that have undergone mOMP, and we can very easily quantitate that, as
• 31:06 - 31:09:  shown here from some work I did a few years ago.
• 31:09 - 31:14:  The pros of this approach is it's straightforward, quantitative, and actually you don't need
• 31:14 - 31:16:  very many cells for it as well.
• 31:16 - 31:20:  You can do many different samples at the same time.
• 31:20 - 31:24:  The cons is, at least my experience of this approach, is sometimes the positive and negative
• 31:24 - 31:30:  peaks overlap somewhat, and that complicates quantification.
• 31:30 - 31:38:  And secondly, digitone and concentration must be optimized on a per-cell type basis.
• 31:38 - 31:43:  The final approach for detecting mOMP that I'm going to discuss with you today is really
• 31:43 - 31:46:  to use live cell imaging.
• 31:46 - 31:53:  And so we can fluorescently label mitochondria with proteins such as cytochrome cGFP, SMAC
• 31:53 - 31:56:  mCherry, SMAC GFP or RONY mCherry.
• 31:56 - 32:00:  And these proteins obviously reside in the mitochondrial intermembrane space in healthy
• 32:00 - 32:06:  cells, but we can image these cells repeatedly over time, and the proteins are then released
• 32:06 - 32:10:  as the cell undergoes mitochondrial permeabilization.
• 32:10 - 32:14:  And we can detect the release of these proteins throughout the cell.
• 32:14 - 32:19:  And I think this is shown quite nicely here in which these are cells, MCF7 cells, that
• 32:19 - 32:22:  have been treated to undergo apoptosis.
• 32:22 - 32:27:  They express mitochondrial matrix-targeted ds-RED, which stays in the mitochondria even
• 32:27 - 32:30:  when they undergo mitochondrial permeabilization.
• 32:30 - 32:35:  However, these cells also express SMAC GFP, and you'll see as the cells undergo
• 32:35 - 32:40:  mitochondrial permeabilization, SMAC GFP is really released in an explosive manner throughout
• 32:40 - 32:43:  the cell very rapidly.
• 32:46 - 32:49:  You can see that quite nicely there.
• 32:49 - 32:56:  So it's a relatively straightforward methodology to look at mitochondrial permeabilization
• 32:56 - 32:59:  in real time.
• 32:59 - 33:04:  One of the huge advantages of it is we can look on a per cell basis, we can image these
• 33:04 - 33:10:  cells, and we can quite easily quantify by gating around individual cells when a cell
• 33:10 - 33:13:  has undergone mitochondrial permeabilization.
• 33:13 - 33:19:  So we apply a so-called punctate diffuse index, and this is averaged over many different cells
• 33:19 - 33:20:  here.
• 33:20 - 33:26:  This is the start of mitochondrial permeabilization, and really within 10 minutes, most cells have
• 33:26 - 33:32:  undergone complete release, complete mitochondrial permeabilization, and then ultimately die.
• 33:32 - 33:37:  So the pros of this approach is it's quantitative, small cell numbers required, can easily multiplex
• 33:37 - 33:38:  with other probes.
• 33:38 - 33:44:  So, for example, we can look at caspase activation, Bax translocation at the same time.
• 33:44 - 33:50:  The cons are that it's relatively low throughput, the analysis time can be considerable, and
• 33:50 - 33:55:  at least for some of these reporter proteins, they may have to be introduced stably into
• 33:55 - 33:56:  cells.
• 33:56 - 34:02:  So transient transfection, especially with things like cytochrome C, it tends to mislocalize
• 34:02 - 34:05:  and so it doesn't localize properly to the mitochondria.
• 34:05 - 34:11:  So in that sense, it may take a while to generate a cell line that's of much use.
• 34:11 - 34:17:  So moving on from that, I want to really ask the question, or give some hints, how can
• 34:17 - 34:21:  I tell if my treatment is inducing mitochondrial-dependent apoptosis?
• 34:21 - 34:27:  Well, first one, you want to address whether your cell is undergoing apoptosis, and commonly
• 34:27 - 34:30:  we do this in the lab by flow cytometry.
• 34:30 - 34:36:  And here we make use of a cell-impermeant dye, such as propidium iodide, which is taken
• 34:36 - 34:41:  up into dying cells, and all dying cells take up propidium iodide, whether they undergo
• 34:41 - 34:42:  apoptosis or not.
• 34:42 - 34:49:  However, we co-label cells with an XN5 together with PI, and this allows us to specifically
• 34:50 - 34:57:  stain the apoptotic population, which is XN5 positive and usually PI negative, although
• 34:57 - 35:02:  ultimately when cells are left long enough, they'll take up PI.
• 35:02 - 35:05:  And so an example of this is shown here.
• 35:05 - 35:08:  These cells have been engaged to undergo apoptosis.
• 35:08 - 35:10:  These are the healthy cells.
• 35:10 - 35:18:  We have PI staining along the bottom here, and XN staining along the side.
• 35:18 - 35:23:  Cells, as they undergo apoptosis, they become XN5 positive, and this is because of this
• 35:23 - 35:29:  phosphatidylserine exposure that I mentioned at the beginning of my talk that occurs during
• 35:29 - 35:30:  apoptosis.
• 35:30 - 35:35:  It's flipped from the inner to the outer leaflet of the plasma membrane, and we can detect
• 35:35 - 35:36:  that with an XN5.
• 35:36 - 35:42:  Ultimately, these cells that are XN5 positive will take up PI as well.
• 35:42 - 35:47:  But we can specifically measure apoptosis by looking at XN5 positive, PI negative
• 35:47 - 35:48:  population.
• 35:51 - 35:58:  Secondly, you can address whether apoptosis is occurring by adding in chemical caspase
• 35:58 - 36:04:  inhibitors, of which there are many, too, that we use often in the lab, or QVD, or ZVAD.
• 36:04 - 36:10:  And these should, if a caspase-dependent process is occurring, caspase-dependent apoptosis,
• 36:10 - 36:12:  it should at least slow cell death.
• 36:12 - 36:17:  With the one caveat that mOMP can also lead to slower caspase-independent cell death,
• 36:17 - 36:20:  which makes it important to do extensive kinetics.
• 36:22 - 36:27:  Moreover, you can ask whether mitochondrial permeabilization is required for cell death
• 36:27 - 36:32:  by generating cell lines that overexpress anti-apoptotic BCL2 proteins.
• 36:32 - 36:40:  So an example here at my lab where we've expressed BCLXL in U2OS cells.
• 36:40 - 36:44:  BCLXL is an anti-apoptotic BCL2 family member.
• 36:44 - 36:48:  Then we've measured cell death following proteasome inhibitor.
• 36:48 - 36:54:  You can see quite nicely you get cell death by looking at XN5 positivity.
• 36:54 - 36:57:  BCLXL inhibits this process.
• 36:57 - 37:04:  Which would argue that the proteasome inhibitor MG132 is triggering cell death
• 37:04 - 37:07:  through a mitochondrial-dependent effect.
• 37:07 - 37:13:  Alternatively, or additionally, in addition to overexpressing anti-apoptotic BCL2 proteins,
• 37:13 - 37:16:  we can also knock down BAX or BAK.
• 37:16 - 37:24:  With the caveat that in most situations, either BAX or BAK is sufficient to drive apoptosis.
• 37:24 - 37:28:  And so in most cases, both will need to be knocked down.
• 37:30 - 37:36:  And as stated just a minute ago, I think it's important to do cell death assays
• 37:36 - 37:40:  in many cases to do it with extensive kinetics.
• 37:40 - 37:43:  And this is just really highlighting this example.
• 37:43 - 37:51:  We can overexpress BCLXL, treat cells with staurosporin, which is an apoptotic trigger.
• 37:51 - 37:55:  Looking at the percentage of mitochondrial permeabilization,
• 37:55 - 38:01:  all these different concentrations of staurosporin and BCLXL effectively block cell death.
• 38:01 - 38:03:  At least in the short term.
• 38:03 - 38:05:  These cells are left longer.
• 38:05 - 38:09:  You can see quite nicely only at the lower concentrations of staurosporin
• 38:09 - 38:12:  does BCLXL allow clonogenic survival.
• 38:12 - 38:17:  And so clearly this compound, staurosporin, and many other triggers are like this as well.
• 38:17 - 38:24:  In a longer-term scenario, these apoptotic stimuli or pro-death stimuli
• 38:24 - 38:26:  can trigger other forms of cell death.
• 38:26 - 38:32:  And so it's important to do, wherever possible, these analyses with extensive kinetics.
• 38:34 - 38:36:  Other aspects that can be investigated.
• 38:36 - 38:38:  One can look at BAX activation.
• 38:38 - 38:42:  For example, by looking at translocation onto mitochondria,
• 38:42 - 38:46:  making use of an active confirmation-specific antibody, 6A7,
• 38:46 - 38:49:  that was developed in Richard Ewell's lab many years ago,
• 38:49 - 38:51:  to look at activated BAX.
• 38:51 - 38:54:  And that can be used by imaging or flow cytometry.
• 38:54 - 39:01:  Equally, BAX activity can be looked at by making use of a confirmation-specific antibody, AB2.
• 39:02 - 39:09:  Moreover, extensive studies have been carried out to MOMP in its role in cell death,
• 39:10 - 39:14:  making use of in vitro mitochondrial permeabilization assays.
• 39:15 - 39:20:  And so these are, in many cases, using mitochondria that are isolated from mouse liver,
• 39:20 - 39:26:  although they can be isolated also from many cell lines as well.
• 39:27 - 39:33:  And mouse liver mitochondria can be incubated in an Eppendorf tube
• 39:33 - 39:35:  along with different BH3-only proteins.
• 39:35 - 39:37:  This is just an example here.
• 39:37 - 39:41:  You incubate it along with these proteins that engage MOMP,
• 39:41 - 39:43:  engage mitochondrial permeabilization.
• 39:44 - 39:48:  Then you can fractionate out quite easily, simply by centrifugation,
• 39:48 - 39:51:  the mitochondrial membrane pellet or the supernatant.
• 39:51 - 39:55:  And you can see quite nicely here that we block for cytochrome C.
• 39:55 - 39:57:  In the controlled situation, there is no MOMP.
• 39:57 - 39:59:  All cytochrome C remains in the pellet.
• 40:01 - 40:04:  In the activated situation, where we have mitochondrial permeabilization,
• 40:04 - 40:07:  cytochrome C is completely released from the mitochondria.
• 40:08 - 40:12:  And so I would refer you to this paper for more detailed methods
• 40:12 - 40:17:  on how to analyze mitochondrial permeabilization in an in vitro manner.
• 40:19 - 40:23:  Okay, so for the last few minutes, I'm going to talk to you or discuss with you
• 40:23 - 40:27:  some new approaches that we've been utilizing to look at mitochondrial importance
• 40:27 - 40:30:  in non-apoptotic forms of cell death.
• 40:33 - 40:37:  And we've been using this approach to look at the role of mitochondria in necroptosis.
• 40:37 - 40:43:  And this is a form of cell death that's been first described several years ago now.
• 40:43 - 40:48:  And it's strange in that it's a form of cell death that's actually inhibited by caspases.
• 40:51 - 40:55:  I'm not going to go into extensive detail about how caspases inhibit the process,
• 40:55 - 41:00:  but this really demonstrates that we can treat cells with tumor necrosis factor.
• 41:00 - 41:04:  Many cell types are fine in the presence of tumor necrosis factor.
• 41:04 - 41:10:  QVD, caspase inhibitor, is co-added, and then we see massive extent of cell death.
• 41:10 - 41:13:  And this is detected by uptake of Cytotox screen.
• 41:13 - 41:16:  It's one of these membrane impermeant dyes.
• 41:16 - 41:18:  When cells die, we take it up, and they light up green.
• 41:19 - 41:24:  So the textbook view of cell death is that it can really be divided into two forms.
• 41:24 - 41:29:  One is necrotic, which is a passive, unprogrammed form of cell death.
• 41:29 - 41:32:  It's not regulated by any set of proteins.
• 41:32 - 41:35:  Apoptosis is clearly an active form of cell death.
• 41:35 - 41:37:  It's programmed.
• 41:37 - 41:41:  And there are certainly some discussions as to whether this is true,
• 41:41 - 41:48:  but traditionally apoptosis is thought of as a non-inflammatory form of cell death,
• 41:48 - 41:50  whereas necrosis is considered inflammatory.
• 41:51 - 41:53:  Necroptosis falls somewhere in between,
• 41:53 - 41:57:  in that it's a programmed form of cell death with necrotic-like features.
• 41:58 - 42:01:  And it's thought that this form of cell death may actually promote inflammation.
• 42:04 - 42:08:  So it's a non-apoptotic, programmed form of cell death that's elicited by various stimuli,
• 42:10 - 42:15:  such as viruses, tumor necrosis factor, pathogen recognition receptors,
• 42:16 - 42:19:  and undoubtedly many other stimuli can also activate necroptosis.
• 42:20 - 42:24:  So far, they all require activation of this kinase called RIPK3.
• 42:25 - 42:28:  This, in turn, activates another protein in the cascade called MLKL,
• 42:29 - 42:32:  which leads to cell death with necrosis-like features.
• 42:34 - 42:37:  We were interested in how cell death is executed.
• 42:39 - 42:45:  Many papers over the years have implicated the central role for mitochondria in necroptosis execution.
• 42:45 - 42:51:  So, for example, during necroptosis ROS, reactive oxygen species have been shown to be increased,
• 42:51 - 42:55:  which may come from the mitochondria.
• 42:55 - 42:59:  There's a loss in ATP, and obviously mitochondria being the powerhouse of the cell,
• 42:59 - 43:03:  loss of mitochondrial function would lead to loss of ATP,
• 43:03 - 43:09:  again implicating mitochondrial dysfunction in necroptosis.
• 43:09 - 43:16:  More recently, mitochondrial proteins such as pGAM5 or DRP1 have been implicated in necroptotic cell death.
• 43:16 - 43:20:  And clearly, based on previous form in apoptotic signaling,
• 43:20 - 43:26:  there's some precedence that mitochondria may be involved in actively causing cell death.
• 43:27 - 43:35:  So we got interested in addressing whether mitochondria were involved in necroptotic cell death,
• 43:35 - 43:42:  really from initial movies that we've carried out looking at mitochondrial function as cells undergo necroptosis.
• 43:42 - 43:46:  And so these cells have been stained up with a potentiometric dye called TMRE,
• 43:46 - 43:51:  which stains up healthy mitochondria, stains them up red.
• 43:51 - 43:55:  And we image these cells as they undergo necroptotic cell death.
• 43:55 - 43:58:  And so over time, what you'll see is these cells eventually burst.
• 43:58 - 44:06:  They then take up an XM5488, which goes into the cell and lights up the plasma membrane from the inside.
• 44:06 - 44:10:  And I just want to emphasize what you would—I'll play it a couple of times—
• 44:10 - 44:17:  is that as the cells undergo death, at that point, they lose their TMRE, they lose their membrane potential,
• 44:17 - 44:22:  really arguing that loss of mitochondrial function is a late event in necroptosis.
• 44:25 - 44:27:  As you can see there.
• 44:40 - 44:43:  Okay, and so that's just the still taken from that movie,
• 44:43 - 44:48:  really showing at the point of lysis these mitochondria still retain at least some membrane potential.
• 44:48 - 44:57:  But this movie at least doesn't implicate or discount the role that mitochondria may have in necroptosis.
• 44:57 - 45:04:  So we thought it would be really cool and really important if we could take cells and ask,
• 45:04 - 45:11:  if we deplete mitochondria from them, does it impart any resistance to necroptosis?
• 45:12 - 45:19:  And so the way we've done that is make use of enforced Parkin-mediated mitophagy.
• 45:19 - 45:27:  And so Parkin is an E3-ubiquitin ligase that was described back in 2008 by Richard Youle in mammalian cells
• 45:27 - 45:35:  to cause mitophagy, which is selective removal of damaged mitochondria through the process of autophagy.
• 45:36 - 45:43:  And so if you disrupt the transmembrane potential of mitochondria by an uncoupler such as CCCP or FCCP,
• 45:43 - 45:52:  Parkin is recruited onto these mitochondria and then ubiquitinates them, and this drives the removal from the cell.
• 45:52 - 46:00:  And so we've taken this approach to stably generate cell lines or cells that stably express YFP-Parkin
• 46:00 - 46:05:  and then treated them with an uncoupler and then looked at mitochondrial content.
• 46:05 - 46:09:  And you can see quite nicely here the mitochondria are in red here.
• 46:09 - 46:13:  Cells express YFP-Parkin, which is in green.
• 46:13 - 46:20:  Once they're treated with an uncoupler, in most cases, we fail to detect mitochondria left.
• 46:20 - 46:26:  By confocal analysis, we've extended this also to do EM analysis.
• 46:26 - 46:29:  And you can see the mitochondria are highlighted in red here.
• 46:29 - 46:32:  These are YFP-Parkin control cells.
• 46:32 - 46:35:  You can see they're full of mitochondria.
• 46:35 - 46:43:  Once these cells are treated with an uncoupler for a couple of days, we completely deplete mitochondria for most of the cells.
• 46:43 - 46:54:  So we can generate cell lines that really homogeneously deplete mitochondria in a really effective manner.
• 46:54 - 46:56:  You can confirm that in various other ways.
• 46:56 - 47:00:  One is to do an immunoblot for different mitochondrial proteins.
• 47:00 - 47:07:  Only in the presence of Parkin and only after an uncoupler is added, we see depletion of different mitochondrial proteins.
• 47:07 - 47:13:  I think it's important to note that if you're carrying out this sort of approach in the lab,
• 47:13 - 47:17:  it's really beneficial to blot against different mitochondrial proteins.
• 47:17 - 47:25:  For example, TOM20 has been shown to be ubiquitinated by Parkin and removed from mitochondria in the absence of mitophagy.
• 47:25 - 47:29:  So it's important to look at matrix proteins and intermembrane space proteins.
• 47:29 - 47:31:  You have effective mitochondrial depletion.
• 47:31 - 47:36:  These should also be removed from the cells.
• 47:36 - 47:43:  So importantly, at least some cell lines can actually survive short-term following mitochondrial depletion.
• 47:43 - 47:44:  And you see that quite nicely.
• 47:44 - 47:49:  Here we've taken cells that express Parkin, uncoupled the mitochondria.
• 47:49 - 47:57:  We get effective mitochondrial depletion and usually analyze these cells at 48 hours post addition of CCCP.
• 47:57 - 47:59:  But the cells actually stick around.
• 47:59 - 48:01:  We look at cell viability here.
• 48:01 - 48:04:  They stick around for many days after depletion.
• 48:04 - 48:07:  Ultimately, they all go on and die.
• 48:07 - 48:13:  So we've not generated a cell line that can survive long-term in the absence of mitochondria.
• 48:13 - 48:16:  We do see a robust inhibition of clonogenic survival.
• 48:16 - 48:20:  But I think the important thing to note is we have this window of opportunity.
• 48:20 - 48:25:  We will have cells that have mitochondria or not, and then we can ask different questions.
• 48:25 - 48:32:  And so we've used this approach to ask, if we deplete mitochondria, do we have an effect on necroptosis?
• 48:32 - 48:40:  And so here we've used the system that was developed by Andrew Oberst that's based over at the University of Washington in Seattle,
• 48:40 - 48:46:  where he's developed really nice systems to cleanly activate caspase-8 and trigger apoptosis,
• 48:46 - 48:51:  or cleanly activate RIPK3 and trigger programmed necrosis or necroptosis.
• 48:51 - 48:57:  So here he's fused FKBP domains to either caspase-8 or RIPK3.
• 48:57 - 49:04:  In the presence of a dimerizer drug, this brings caspase-8 together, causing its activation, which then allows apoptosis.
• 49:04 - 49:06:  The same goes for RIPK3.
• 49:07 - 49:11:  It actually causes oligomerization in RIPK3 and can trigger cell death.
• 49:11 - 49:18:  And so using this approach, we've either engaged apoptosis or necrosis and depleted mitochondria to ask,
• 49:18 - 49:23:  if we get rid of mitochondria, does it affect the kinetics or the extent of cell death?
• 49:23 - 49:28:  You can see quite nicely here, these are caspase-8 dimerizer cells.
• 49:28 - 49:32:  You add the chemical dimerizer, you trigger rapid apoptotic cell death.
• 49:33 - 49:38:  However, if you deplete mitochondria from the cells and then trigger caspase-8 activation,
• 49:38 - 49:44:  you completely block the ability of caspase-8 to trigger apoptosis in these cells,
• 49:44 - 49:51:  which argues, at least in these cells, that caspase-8 requires mitochondrial involvement to trigger apoptotic cell death.
• 49:54 - 49:59:  On the other hand, when we do this with RIPK3, we dimerize or oligomerize it.
• 49:59 - 50:02:  We get rapid levels of necroptosis.
• 50:02 - 50:06:  You do the same thing when cells have been depleted of mitochondria.
• 50:06 - 50:09:  We see exactly the same extent of cell death.
• 50:09 - 50:12:  And we can quantitate that very nicely.
• 50:12 - 50:17:  These are cells that have been depleted of mitochondria and triggered to undergo necroptosis.
• 50:17 - 50:22:  These are the ones that are mitochondrial replete and undergo necroptosis.
• 50:22 - 50:25:  And effectively, you can overlay the two graphs.
• 50:26 - 50:33:  Really arguing that mitochondrial elimination does not affect cell death triggered by direct activation of RIPK3.
• 50:35 - 50:41:  So, what I want to just highlight in this last part is that mitochondrial elimination does not prevent necroptosis,
• 50:41 - 50:48:  either driven by direct activation of RIPK3, or we've also done it after TNF-induced necroptosis.
• 50:48 - 50:56:  Beyond this, I think Parkin-driven mitophagy is a powerful tool to define mitochondrial importance in any given biological process,
• 50:56 - 50:58:  not solely cell death.
• 50:58 - 51:04:  And so, RIPK3 triggers necroptosis, we believe, independently of the mitochondria.
• 51:04 - 51:09:  And actually, subsequent to our work, there's evidence that RIPK3, through MLKL,
• 51:09 - 51:14:  can trigger necroptosis by directly causing plasma membrane permeabilization.
• 51:15 - 51:23:  So, I'd just like to finish up by acknowledging the guys in my lab that are certainly helping with this work and continuation of it.
• 51:23 - 51:33:  Moreover, I've mentioned during this presentation, Andrew Oberst developed these clean ways of killing cells either via apoptosis or necroptosis.
• 51:33 - 51:37:  The initial mitophagy work was started in Doug Green's lab.
• 51:37 - 51:40:  These are the guys that fund my lab.
• 51:41 - 51:45:  With that, I'd like to thank you for listening to me.
• 51:45 - 51:51:  And I'll hand you back to David, who's going to talk to you about some seminar-related products.
• 51:51 - 51:52:  Thanks very much.
• 51:54 - 51:56:  Thank you, Stephen, for a really great talk.
• 51:56 - 51:59:  I'm sure you'll have plenty of questions from our listeners.
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• 53:11 - 53:14:  So why not try a RabMab from Abcam?
• 53:15 - 53:24:  We have a broad range of secondary antibodies to complement our primary antibodies, including our new range of Alexa Fluor conjugated secondaries.
• 53:24 - 53:34:  We produce these in-house, and they are available pre-absorbed to different species and in a range of Alexa Fluor-fluorochrome conjugates.
• 53:35 - 53:44:  We also offer cell staining reagents, including our Cytopainter range of reagents, which are ideal for multicolor staining.
• 53:44 - 53:54:  These allow you to visualize subcellular components, such as the mitochondria or lysosome, and can be used on live and fixed cells.
• 53:54 - 53:58:  Abcam offers a number of dyes for fluorescence imaging.
• 53:58 - 54:01:  These include DRAQ5 and DRAQ7.
• 54:01 - 54:08:  These are small molecules which label double-stranded DNA or permeabilized cells.
• 54:08 - 54:12:  CyTraq Orange, which stains both the nucleus and cytoplasm.
• 54:12 - 54:18:  Nuclear Yellow and Nuclear Green, which are both DNA dyes. and you can find more information about these on our website.
• 54:19 - 54:25:  Abcam also has an extensive offering of products to support your immunohistochemistry protocols.
• 54:26 - 54:33:  These include various types of detection systems, such as X-POSE-IHC kits for high-sensitivity staining,
• 54:33 - 54:37:  as well as multicolor detection systems or chromogens for staining multiple markers
• 54:40 - 54:46:  You can find a wide range of reagents to support your IHC, including blocking reagents and sera,
• 54:46 - 54:56:  antigen-retrieval buffers, as well as histology stains to assist you in identifying tissue morphology.
• 54:59 - 55:03:  We have a number of Western blotting antibody cocktails, including our apoptosis cocktail.
• 55:03 - 55:09:  These reagents allow you to detect multiple proteins in one immunoblot.
• 55:11 - 55:17:  We also have apoptosis detection kits that monitor the flipping of phosphatidylserine, or PS,
• 55:17 - 55:23:  from the interface of the plasma membrane to the cell surface after apoptosis has been detected.
• 55:23 - 55:29:  Last year, we launched a new range of ELISA kits, or simple-step ELISA kits.
• 55:29 - 55:35:  The time taken to develop our simple-step ELISA kits is reduced when compared to traditional ELISAs.
• 55:35 - 55:40:  This is because our new kits only require one washing step.
• 55:40 - 55:46:  This reduced washing also has the benefit of improving their sensitivity as well as their specificity.
• 55:46 - 55:50:  Abcam also offers a wide range of pharmacological tools.
• 55:51 - 55:57:  These include over 4,000 enzyme, receptor, ion channel inhibitors, and activators.
• 56:00 - 56:10:  Our range of pharmacological tools includes some of the compounds Stephen spoke about today, including the caspase substrate inhibitors and SBCBP.
• 57:10 - 57:16:  I'll now pass you back over to Stephen, who is ready to answer your questions we've received during the webinar.
• 57:16 - 57:19:  Thank you for your attention.
• 57:19 - 57:21:  Thanks very much, David.
• 57:21 - 57:25:  Yeah, so thanks for guys that sent in the questions.
• 57:25 - 57:30:  I'll try my best to answer some of them.
• 57:30 - 57:41:  Okay, so one was from Victoria, and that was what concentration of CCCP was I using for these experiments?
• 57:41 - 57:46:  Typically, we've used in the lab 12.5 micromolar.
• 57:46 - 57:56:  Whilst we've been using CCCP, we've moved over a little bit more now to using antimycin A and oligomycin.
• 57:56 - 58:01:  The reason being is that CCCP also affects lysosomal function,
• 58:01 - 58:08:  so confirming our results using other methods has been really beneficial.
• 58:08 - 58:11:  So this is another one.
• 58:11 - 58:14:  Does necroptosis induce inflammation?
• 58:14 - 58:22:  I think there's certainly a few mouse models that lack RIPK3, for example,
• 58:22 - 58:31:  that would argue that necroptosis doesn't induce inflammation, so it reduces inflammatory bowel disease.
• 58:31 - 58:35:  Well, at least I guess that argues that RIPK3 is required for it.
• 58:35 - 58:43:  I think there's still a lot of debate as to whether these mouse models that are implying that necroptosis has a role in inflammation,
• 58:43 - 58:50:  whether it's really to do with necroptosis or the ability of RIPK3 to actually induce cytokine production.
• 58:50 - 58:55:  So I think that's still very much a matter of debate.
• 58:55 - 59:03:  Moreover, some labs would argue that necroptosis serves to actually inhibit inflammation by killing the cell.
• 59:03 - 59:11:  So I think it's still, as I said, still an active area of debate whether necroptosis is inflammatory per se.
• 59:11 - 59:14:  Okay, so another question.
• 59:14 - 59:18:  During multiplexing, which probe is suitable?
• 59:18 - 59:25:  And I think that's obviously relative to the imaging approaches that I was discussing earlier,
• 59:25 - 59:29:  whereby we're looking at mitochondrial permeabilization.
• 59:29 - 59:40:  So the Annexin V conjugates that I use for PS or to look at phosphatidylserine exposure, they come in a range of colors.
• 59:40 - 59:48:  And so if you image looking at, say, mCherry, SMAC mCherry release from the mitochondria,
• 59:48 - 59:57:  you can certainly use an Annexin V coupled to, say, an Alexa 488 to look at phosphatidylserine exposure.
• 59:57 - 60:10:  Moreover, there are various caspase activity probes that can be used. Many of these are based on FRET or loss of FRET, so fluorescence resonance
• 60:10 - 60:16:  energy transfer. Upon caspase cleavage, you get a loss of signal. These can typically
• 60:16 - 60:25:  be combined with SMAC mCherry. There's a few of those reporters out there. To look at plasma
• 60:25 - 60:34:  membrane integrity, simply PI is a very good compound to look at that. It's taken up very
• 60:34 - 60:39:  quickly, very robustly when cells undergo apoptotic cell death, ultimately at the end of
• 60:39 - 60:43:  the whole process, they take up PI. And that can be, again, multiplexed with many of these
• 60:43 - 60:49:  SMAC GFP or SMAC mCherry type live cell imaging approaches.
• 60:50 - 60:56:  Right. I'm just seeing if there are other questions here. Can it affect the other functions
• 60:56 - 61:01:  of cells if a cell doesn't have mitochondria? Undoubtedly, that's the case. This is something
• 61:01 - 61:08:  we're investigating now. The role of mitochondria in, for example, protein secretion, vesicular
• 61:08 - 61:14:  trafficking, we're quite interested in addressing these questions in the absence of mitochondria.
• 61:14 - 61:20:  How are these functions actually affected? Not simply through the role of mitochondria
• 61:20 - 61:26:  in providing energy, but do mitochondria have a signaling function related to organelle
• 61:26 - 61:31:  transport, vesicular trafficking in cells? I think, undoubtedly, that's going to be the
• 61:31 - 61:35:  case. You remove mitochondria from a cell, it's going to have many different effects
• 61:35 - 61:37:  on that cell.
• 61:38 - 61:48:  Okay. And I'll just finish up with a couple other questions. One is, do all cancer therapies
• 61:48 - 61:54:  kill via apoptosis? This is certainly a matter of debate as well. As I mentioned in one of
• 61:54 - 62:04:  my slides, apoptotic cell death is often the primary way in which cells die. But clearly,
• 62:05 - 62:14:  during cancer therapy, cells often undergo necrotic cell death as well. So if cells are
• 62:14 - 62:20:  exposed to high enough levels of drugs or radiation, whilst apoptosis may be the primary
• 62:20 - 62:26:  way in which they undergo cell death, ultimately the cells will die by other means as well.
• 62:26 - 62:30:  Another question is, how does mitochondrial permeabilization kill the cell in the absence
• 62:30 - 62:35:  of caspase activity? Really, we don't know how this occurs. I think the best evidence
• 62:35 - 62:40:  in the literature would simply be that there's a progressive loss in mitochondrial function,
• 62:40 - 62:43:  which ultimately leads to cell death.
• 62:43 - 62:49:  Okay. So with that, I'd like to thank you for your questions, and thanks for tuning
• 62:49 - 62:57:  into the webinar. And I'd like to hand myself back to David. Thanks very much.
• 62:57 - 63:06:  Hi, Stephen. Thank you for that. I'll now pass you over to Lucy, who's going to just
• 63:06 - 63:08:  tie up the end of the seminar.
• 63:08 - 63:16:  Okay. Thanks, David. So we would like to thank everyone who attended the webinar today. If
• 63:16 - 63:21:  you have any questions about anything that has been discussed today or have any technical
• 63:21 - 63:28:  inquiries, the Abcam support team would be very happy to help you. They can be emailed
• 63:28 - 63:35:  at technical@abcam.com. And more details about relevant phone numbers can also be found
• 63:35 - 63:38:  on the Abcam website.
• 63:38 - 63:43:  We hope you found this webinar informative and useful to your work, and we hope we can
• 63:43 - 63:48:  welcome you to another Abcam webinar in the future. Thank you again for attending, and
• 63:48 - 63:50:  good luck with your research.