Plasma Membrane Repair: Norma Andrews

Part 1: Mechanisms of Plasma Membrane Repair

00:00:07.14 Hi.
00:00:08.14 My name is Norma Andrews.
00:00:09.18 I'm a professor at the University of Maryland.
00:00:13.04 And I'm delighted to be here to talk to you about a fascinating process, which is the
00:00:18.11 mechanism by which cells repair wounds on their plasma membrane.
00:00:23.16 So, what most people think when we talk about wound repair is this different, but also remarkable,
00:00:31.15 ability that all of us multicellular organisms have of repairing injury to our tissues.
00:00:37.17 So, this is a complex, multi-stage process that starts with formation of a clot and then
00:00:43.23 this is followed by infiltration of inflammatory cells that directly attack microbes that may
00:00:49.14 have invaded, followed by extensive remodeling and cell proliferation that ultimately completely
00:00:57.06 heals the injury.
00:00:58.10 So, what I want to talk about today is something quite different -- it is the ability that
00:01:04.03 single cells have, without the participation of any other cells in tissues, to repair injury
00:01:12.00 to their plasma membrane.
00:01:14.08 We can demonstrate this process easily in the lab with several experimental settings,
00:01:18.24 and I'll show you one here.
00:01:20.11 What we have here is a monolayer of cells that are shown by scanning electron microscopy.
00:01:28.24 And a simple way to induce wounds on the membrane of these cells is to drop glass beads over
00:01:34.18 this culture.
00:01:35.18 So, the impact of this bead really causes a lot of damage, as illustrated here.
00:01:40.19 We used a DNA dye to stain all the cells that remain attached to the surface and you can
00:01:46.20 see here that, at the site where the bead hit, the cells were removed.
00:01:52.17 And we followed this rapidly with a membrane-impermeant dye, in this case a red dextran.
00:02:00.10 And this dye cannot cross intact membranes, so what this experiment immediately tells
00:02:05.07 us is that the cells on the periphery of this wound got wounded
00:02:09.07 and allowed entry of the dye.
00:02:12.05 We can then come with a second membrane-impermeant dye, in this case a green dextran, and we
00:02:17.22 can change the timing in which we add the second dye to see the process of repair.
00:02:24.03 So, for example, in this first example, here, we waited 20 seconds between adding the red
00:02:30.07 dye and then adding the green dye.
00:02:32.06 And this monolayer, here, was hit in two places by the beads and we see here all the cells
00:02:38.14 that got injured, and then in just 20 seconds we see that several of the cells were able
00:02:44.04 to block entry of the second dye, indicating that they were able to repair.
00:02:49.01 And then, if we wait a little longer, one minute for example, we can see that more cells
00:02:54.04 in this culture were able to completely reseal their membrane.
00:02:57.22 So, this kind of experiment really tells us a very important property of plasma membrane
00:03:03.11 repair, which was also observed in several different experiments in different labs, which
00:03:09.14 is that repair occurs in a few seconds.
00:03:12.07 And this is really quite important because cells, to survive, they have to control the
00:03:17.07 influx of calcium -- that is much more abundant outside cells than inside -- and also they
00:03:22.18 cannot afford to keep losing critical factors from the cytosol.
00:03:27.21 This also happens in vivo and one of the tissues in which it is easier to demonstrate this
00:03:32.18 is in skeletal muscle.
00:03:34.03 So, these images here are from a study of Richard Lovering
00:03:37.21 at the University of Maryland Medical School.
00:03:40.16 And what they did here was to use a green dye to stain the sarcolemma, which is the
00:03:47.04 plasma membrane of muscle fibers, and these are sections across this muscle.
00:03:54.01 So, we are seeing the outline of the fibers and they injected into these rats
00:03:59.12 a red membrane-impermeant dye.
00:04:02.01 And the... what is demonstrated here is that when the animals are just kept in their normal
00:04:06.23 resting condition, you don't see any entry of the dye, indicating that these fibers remain
00:04:13.10 intact, but when animals are made to exercise, specifically to run downhill on a treadmill,
00:04:22.04 you can see clearly that several fibers got injured, and this was caused by these lengthening
00:04:27.10 contractions, which are known to cause injury in muscle.
00:04:34.08 Although mechanism of plasma bring repair has not been by far as widely studied as the
00:04:42.02 mechanism of tissue repair, we have already, in the last decade, several very interesting
00:04:47.12 examples in which specific proteins were identified.
00:04:51.12 And what is a common feature between these studies is that all these proteins are necessary
00:04:59.08 for plasma membrane repair, and mouse models in which mutations were introduced into these
00:05:04.15 proteins, these animals developed muscle pathology.
00:05:07.16 So, I have here images from a study by Joe Roche, also at the University of Maryland
00:05:13.10 Medical School, showing that wild-type animals, when they are subjected to this kind of contraction,
00:05:21.11 they have very limited injury, and they restored this very effectively with time.
00:05:27.18 When they did the same experiment with mice deficient in... that carried mutations in
00:05:35.11 this protein, dysferlin, you can see a dramatic entry of the red dye, which indicates initial
00:05:41.05 injury, and then... which I can show closer in this other slide, that... while in wild-type
00:05:49.09 animals, we see the fibers normally intact, we see that in the dysferlin-deficient mice,
00:05:59.19 after injury, there is a massive infiltration, shown in blue, of inflammatory cells.
00:06:05.21 That can be seen clearly, also, up to 72 hours after the injury.
00:06:10.13 So, this established an important concept, because for some time there was some discussion
00:06:14.24 that it might be the inflammation of the muscle that was causing the injury.
00:06:19.22 And in this study Joe Roche showed that, actually, the injury precedes inflammation.
00:06:26.12 And this is very interesting because, in recent years, exciting findings have been happening
00:06:31.19 in the field of immunology which revealed the existence of a machinery inside cells
00:06:37.17 that detects wounds on the plasma membrane.
00:06:41.20 So, this injury to the membrane can happen physiologically many different ways, with
00:06:47.01 toxins inserted by microbes or directly with mechanical or chemical injury.
00:06:56.03 And what has emerged is that there is a machinery that senses changes like, for example, efflux
00:07:03.02 of potassium, which is an ion that is more abundant inside cells, as opposed to sodium
00:07:08.17 that is present inside... outside of cells.
00:07:13.12 So, we have here an example of how cells really have adapted the innate immune response to
00:07:23.04 recognize injury and to trigger inflammation.
00:07:27.18 So, how does this happen?
00:07:30.14 So, how do cells really restore this remarkable structure that is composed not only of a lipid
00:07:37.24 bilayer, but also of transmembrane proteins, and also important links with the cytoskeleton
00:07:43.21 inside the cells and the extracellular matrix outside?
00:07:47.10 So, how cells do this is what we are interested in.
00:07:52.06 And we followed on the steps of really very interesting, creative experiments that were
00:07:59.14 done in the past.
00:08:01.16 The modern history of plasma repair really started with the landmark studies of how Heilbrunn
00:08:07.17 and Chambers, in which they were looking at sea urchin eggs in seawater.
00:08:14.12 And they noticed that, when they injured the membrane of these eggs, they saw a surface
00:08:19.03 reaction in which they saw a transient blebbing and rearrangement of the surface, and then
00:08:24.12 these eggs went back completely to normal,
00:08:27.15 and they were able even to be fertilized and develop.
00:08:31.19 But, just by removing calcium from the water, they saw that this repair process did not
00:08:38.03 happen, and these eggs just leaked and died.
00:08:42.03 So, with these studies, a very important gold standard of plasma membrane repair was established.
00:08:48.12 Up to now, this is an absolute requirement that the presence of calcium outside of cells
00:08:55.07 is required as a trigger for membrane repair.
00:08:59.06 Then, another important breakthrough happened decades later, in the 90s, with the studies
00:09:04.11 of Paul McNeil and Richard Steinhardt, in which they were directly looking at also sea
00:09:10.12 urchin eggs and other cell types, in which they made very localized wounds on the membrane
00:09:16.15 and they saw that intracellular vesicles migrated to the wound site and fused with the plasma
00:09:23.01 membrane, and this process was... occurred within a few seconds that repair happens in.
00:09:30.08 And they then proposed this very important concept that exocytosis, that delivery of
00:09:35.19 the internal membranes to the cell surface, is a very important step for the resealing
00:09:43.09 of the plasma membrane.
00:09:45.15 This is... illustrates, here, a very elegant study that Paul McNeil did, in which he compared
00:09:50.23 directly red blood cells, which are really filled with hemoglobin and they don't have
00:09:56.11 the internal membrane structures that nucleated cells have, which is indicated here in this
00:10:02.07 beautiful image by George Palade.
00:10:04.16 So, he compared side by side the ability of
00:10:08.08 these two different cell types to respond to injury.
00:10:11.17 And he showed that in... in red blood cells, there is a very slow resealing.
00:10:16.14 So, what we are seeing here in this green line is the sequence... is the entry of a
00:10:23.24 membrane-impermeant dye into the cells that gets stabilized only after a significant period
00:10:30.07 of time.
00:10:31.07 So, these cells remain leaky and this is something that is known by people who have been working
00:10:35.09 with preparing erythrocyte ghosts, that they do reseal partially but it takes time.
00:10:41.13 And then, when in parallel he did the same experiment, looking at cells that have this
00:10:48.14 more complex structure, like fibroblasts or epithelial cells, he saw that there was a
00:10:53.12 much faster resealing -- you can see that the scale is different here.
00:10:57.15 In a few seconds, these cells, in the presence of calcium, in the green line, were able to
00:11:02.12 stop the influx of the dye, and this did not happen when calcium was not present.
00:11:08.02 So, this study really settled this very interesting concept that endomembranes are required for
00:11:14.09 repairing the plasma membrane.
00:11:16.24 So, this is when we really got very interested in this process, because we were interested
00:11:24.02 in the question, what is the nature of these vesicles that are present in most cell types
00:11:29.13 and respond to calcium by fusing with the plasma membrane?
00:11:32.17 So, these vesicles would have to be present in every cell type and they would have to
00:11:37.05 rapidly respond to calcium influx.
00:11:40.10 So, this happened quite unexpectedly in my lab when we were studying something quite
00:11:46.11 different, which was cell invasion by this parasite Trypanosoma cruzi.
00:11:50.23 So, T. cruzi, shown here, is... this is the infective stage attached to the surface of
00:11:56.23 a host cell, and this is a parasite that invades cells, as you can see in this remarkable movie
00:12:02.15 made by Hertha Meyer back in the '40s.
00:12:05.10 And this causes a serious disease in Latin America.
00:12:08.03 So, this parasite can invade many different cell types, and it transforms intracellularly,
00:12:13.20 after replicating, back into the infective stage, which we are seeing here, this highly
00:12:19.02 motile form that fills up the cells and then escapes from these cells to reinfect others.
00:12:24.05 This is a serious disease and we were interested in understanding, what is the mechanism by
00:12:31.02 which these infective stages enter host cells?
00:12:35.02 So, through these studies, we saw that images made by Edith Robins of the invasion process
00:12:42.01 really don't look like phagocytosis; it looks like the parasite is diving into the cell.
00:12:46.16 And the really surprising finding was when we stained these cells for markers for lysosomes.
00:12:51.05 Here, antibodies against an abundant glycoprotein of lysosomes Lamp-1.
00:12:55.23 We saw that the parasites, even when they were only partially into the cells, they were
00:13:01.14 already stained.
00:13:02.14 There was a tight vacuole formed around the parasites that were stained with markers for
00:13:06.22 lysosomes.
00:13:08.01 So, this told us something very important, and we also found through these studies an
00:13:14.12 important link with calcium.
00:13:16.02 So, what is shown up here is cells loaded with a calcium-sensitive dye in which we are
00:13:21.07 perfusing parasites through the culture, and the infective stage of T. cruzi is able to
00:13:27.05 trigger a very strong elevation in intracellular calcium, which then promotes fusion of lysosomes
00:13:36.00 with the plasma membrane, and formation of a vacuole, derived from lysosomal membranes,
00:13:42.06 that the parasites use to invade and then later on they escape into the cytosol.
00:13:48.07 So, these studies really allowed us to arrive at a very novel and interesting concept, and
00:13:57.04 this is really a great example of how you really should keep your eyes open, because
00:14:02.06 this is not at all what we were planning to do, but when we saw lysosomes behaving this
00:14:07.02 way we thought that it should be possible, then, if we thought calcium was the trigger
00:14:13.14 for this fusion of lysosomes with the plasma membrane, we should be able to remove the
00:14:17.23 parasites from the picture and just manipulate the intracellular calcium concentration, and
00:14:23.13 see if we could make lysosomes, which were normally viewed as these terminal compartments
00:14:29.01 of the endocytic pathway, to fuse with the plasma membrane.
00:14:33.18 And this is exactly what happened.
00:14:35.09 Through these studies, we had the help of some great collaborators -- one of them was
00:14:39.08 Sandy Simon.
00:14:40.08 This was the beginning of the era of the total internal reflection microscopy, which is really
00:14:45.13 a very useful technique in which what is illuminated in these cells is just a very small field
00:14:51.18 at the bottom of the cells, close to the coverslip.
00:14:54.04 And intracellular vesicles labeled with a fluorescent dye, when they enter this evanescent
00:15:00.00 field, they become progressively brighter and it's possible to catch them in the moment
00:15:05.19 of exocytosis.
00:15:06.22 So, in this movie here, ly... lysosomes were first loaded by endocytosis with a fluorescent
00:15:14.17 dextran, and these puffs that you are seeing reflect the release of the dextran, when these
00:15:19.19 cells were stimulated with a calcium ionophore.
00:15:21.14 If you focus on this lysosome, here, you can see that, at the moment in which it fuses
00:15:26.17 with the plasma membrane, showing directly that lysosomes can respond to elevation in
00:15:32.23 cytosolic calcium with exocytosis.
00:15:34.20 Conventional lysosomes, and this is a normal mouse fibroblast.
00:15:39.15 So, another important piece of information that came out of these studies was the presence
00:15:47.07 of a peripheral population of lysosomes because normally, right, when you look at cells and
00:15:52.12 stain them for lysosomal markers and look by epifluorescence, what we see is a cluster
00:15:57.23 of lysosomes in the central part of the cell, and this is really very well-known due to
00:16:03.21 movement of lysosomes back into the center of the cell on microtubules.
00:16:10.24 But what this TIRF microscopy technique allowed us to see is that cells also have a resident
00:16:19.13 peripheral population of lysosomes that is already sitting close to the plasma membrane,
00:16:24.01 ideally positioned to respond to elevations in calcium with exocytosis.
00:16:30.06 And Jyoti Jaiswal in Sandy Simon's lab quantified these fusion events and we can see, here,
00:16:36.16 using three different ways to elevate calcium in cells -- a calcium ionophore or two physiological
00:16:42.17 agonists, thrombin and bombesin -- in the vast majority of cases, right, comparing the
00:16:47.05 green with blue columns... that the vast majority of fusion events detected happened from that
00:16:54.07 peripheral population.
00:16:55.07 So, there were very few examples of lysosomes that were in the dark, deeper into the cell,
00:17:02.15 and enter this population at the periphery.
00:17:05.20 So, this told us that there is a population of peripheral lysosomes dedicated for exocytosis,
00:17:14.05 or perhaps for other functions, and it's really quite gratifying that recently there have
00:17:18.24 been several labs that have been working out, what is the molecular machinery that is retaining
00:17:24.04 these lysosomes at the periphery?
00:17:27.09 We then moved, of course... that we were interested in seeing if this phenomenon of lysosomal
00:17:34.03 exocytosis was related to those vesicles that Paul McNeil and Richard Steinhardt had shown
00:17:41.22 to promote plasma membrane repair.
00:17:45.07 And we found that this was the case.
00:17:47.20 In this case, here, what we have is an experiment in which this monolayer was scratched and
00:17:56.00 we see, again, using a red dextran as a way to label the injured cells, we went... then
00:18:04.03 came with antibodies against the... this lysosomal glycoprotein, and this was done with antibodies
00:18:10.01 against the luminal domains, so that normally is not exposed on the surface, so these antibodies
00:18:14.21 can only reach this region of the protein if the lysosome first fused and exposed its
00:18:21.08 luminal side outside.
00:18:23.06 And we see that there's extensive labeling with these antibodies against the luminal
00:18:27.16 domain of Lamp-1, only on the cells that were wounded.
00:18:30.19 So, these cells that... with the blue nuclei... we showed that they were not injured in the
00:18:37.02 process and they did not undergo lysosomal exocytosis.
00:18:40.24 So, over the years, we really investigated this process in detail, and we found that
00:18:53.09 we could manipulate the machinery.
00:18:55.22 We have the molecular machinery responsible for lysosomal exocytosis, and when we blocked
00:19:02.12 this process of lysosomal exocytosis triggered by a wound on the membrane we could also inhibit
00:19:09.24 membrane repair.
00:19:12.01 And one of the tools that we used for this process was a calcium sensor that we found
00:19:18.17 on the membrane of lysosomes.
00:19:20.24 I'll talk more about this in my second talk, but I would just like to mention here that
00:19:26.00 what has emerged recently is that lysosomes themselves are also an important source of
00:19:31.21 calcium.
00:19:32.21 So, there have been very interesting studies looking at the channels that mediate calcium
00:19:37.10 release from lysosomes, like mucolipin-1, and mice with mutations in these channels
00:19:44.02 also develop muscle pathology, a form of muscular dystrophy.
00:19:48.18 So, again, this is what we would predict, that if you have abnormal response of lysosome
00:19:56.03 to calcium release you end up with muscle pathology, and probably the calcium inside
00:20:03.02 of lysosomes is also very important for this process.
00:20:07.22 So, what has really come together recently... so, there was in the beginning quite a bit
00:20:14.18 of skepticism, which is quite normal, because lysosomes were supposed to be just these terminal
00:20:21.12 compartments of the endocytic pathway, but as you can see here from this figure from
00:20:26.01 a recent review by Andrea Ballabio, that lysosomes really recently have emerged as this really
00:20:32.22 important hub, that are not only responsible for the degradation of material coming in
00:20:37.24 by endocytosis, but also for degradation of material taken up by autophagy and also for
00:20:45.16 communication with the nucleus -- you know, very important functions of sensing nutrients
00:20:50.05 and regulating metabolism.
00:20:52.11 And plasma membrane repair, it seems also to be one of the functions that maybe this
00:21:00.21 peripheral population of lysosomes, in particular, is involved in.
00:21:06.06 [So, a question that I get very often when I talk about this is, why lysosomes, right?
00:21:12.00 So, why use such a dangerous compartment, full of these degradative enzymes, for plasma
00:21:17.05 membrane repair?
00:21:18.12 So, thinking about this, and we actually had uhh.. real... real experiments that we did
00:21:24.05 to test this hypothesis, is that we have to think that nucleated cells co-evolved with
00:21:31.07 bacteria for millions of years, and bacteria produce an arsenal of agents that wound the
00:21:37.00 plasma membrane, for example, the type III secretion needle or several other secretion
00:21:42.20 systems the bacteria have, that have been shown directly to puncture the membrane of
00:21:48.19 host cells and cause calcium influx.
00:21:52.00 And, of course, there are many pore-forming toxins that I'm going to talk more about in
00:21:56.03 my second talk.
00:21:57.11 So, this process, we know that when you have influx of calcium there is a response of lysosomal
00:22:04.21 exocytosis, so what we say is that we believe that using lysosomes for this response to
00:22:11.03 injury, since the injury in the early days of the evolution of complex cells was probably
00:22:16.21 caused by bacteria, that this provided a selective advantage.
00:22:21.18 Because, at the same time that the cell was receiving its membrane, it might be destroying
00:22:27.06 the microbes that was... were causing the injury.
00:22:30.06 And this concept we find very interesting, because our immune system has a very important
00:22:36.09 cell type, the cytotoxic lymphocyte, that's shown here in this beautiful image by Gillian
00:22:41.15 Griffiths, in which in red are the secretory lysosomes, which are these lysosomes that
00:22:47.02 have been modified in these cells to contain specialized proteins that are secreted in
00:22:52.23 a polarized fashion into target cells, and are used to kill infected cells and tumor
00:23:00.04 cells, extensively, in our immune responses.
00:23:02.24 So, we really like this idea that using lysosomes for plasma membrane repair is coupled to the
00:23:10.10 defense function of lysosomes that has been maintained in our immune system.
00:23:16.24 So, what I'm gonna do in my second part is to discuss how lysosomal exocytosis would
00:23:24.13 repair the plasma membrane.
00:23:25.21 I have discussed with you why we think this was a good idea, but in my second lecture
00:23:31.08 I will tell you about another series of very surprising findings we made along the way,
00:23:36.17 in which we found that the exocytosis of lysosomes is followed by an endocytic response that
00:23:43.12 the enzymes released by lysosomes can trigger a process of membrane invagination that can
00:23:50.02 carry lesions into the cells, and that this would be a way that the cells remove the lesions.
00:23:56.02 So, quite an unexpected way that I would be happy to discuss with you.
00:24:00.18 So, I would just like to stop here, but, before that, I must acknowledge some very talented
00:24:07.00 people in my lab and also our collaborators that I mentioned the work they did.
00:24:13.03 Thank you.

Part 2: Ca2+-Dependent Lysosomal Exocytosis Mediates Endocytosis and Wound Healing

00:00:07.15 Hi there.
00:00:08.15 I'm Norma Andrews.
00:00:09.15 I'm a professor at the University of Maryland, and in the second part of my seminar I will
00:00:15.15 be discussing with you a series of surprising findings we made over the years, and these
00:00:21.13 findings gave us unexpected insights over the mechanism by which cells reseal wounds
00:00:29.01 on their plasma membrane.
00:00:31.24 As I mentioned in my first talk, a major breakthrough in the field of plasma membrane repair came
00:00:36.24 from these early studies of Heilbrunn and Chambers, who demonstrated that the presence
00:00:41.22 of calcium in the extracellular medium was essential for the membrane resealing.
00:00:48.11 And what they suggested at the time was that this process might occur through aggregation
00:00:54.10 of proteins that might form a clot-like structure, like in blood clotting, and that this would
00:01:00.00 be responsible for preventing loss of the intracellular components.
00:01:05.13 Very interestingly, decades later, there is quite a bit of evidence that they were right,
00:01:13.00 that there is a role for proteins that are present in the cytosol and that respond to
00:01:17.20 calcium influx by associating with membranes.
00:01:21.03 So, one class of proteins that does this are the annexins, which undergo conformational
00:01:27.22 changes when they are exposed to calcium, and they associate with phospholipids on the
00:01:33.22 plasma membrane.
00:01:34.22 And this image here is from a study... one of these studies... showing that this can
00:01:42.20 be directly visualized after injuring cells, the association of an annexin V with membranes
00:01:48.21 around wounds.
00:01:50.06 And what these authors proposed is that these annexins would form a two-dimensional array
00:01:56.04 that would then not only prevent further expansion of the wound but perhaps facilitate the subsequent
00:02:04.08 steps that reseal the membrane.
00:02:07.22 It is very possible, actually, that additional calcium-responsive proteins in the cytosol,
00:02:14.14 such as transglutaminases, for example, that have the capacity of cross-linking on the
00:02:18.11 proteins when exposed to calcium, play a role in forming a temporary clot.
00:02:23.17 But what we also know, and this is from the landmark studies of Paul McNeil and
00:02:28.15 Richard Steinhardt, is that the membrane is capable of really completely resealing the bilayer;
00:02:35.05 it's reconstituted.
00:02:36.10 And what they showed is that this process involves exocytosis, fusion of intracellular
00:02:43.11 compartments that donate membrane to promote the resealing.
00:02:48.04 So, this is when my lab entered the field, because, as I explained in our first lecture,
00:02:54.10 we were doing something completely different, studying invasion of an intracellular parasite,
00:02:59.03 and we stumbled into this unexpected behavior of lysosomes.
00:03:03.20 The lysosomes actually have a molecular machinery on their surface that allows them to bind
00:03:10.02 to the plasma membrane and fuse, releasing their contents.
00:03:14.06 And, using these molecules that we found on the surface of lysosomes, we could manipulate
00:03:22.21 the process, and really determine that lysosomes were very good candidates for the vesicles
00:03:28.16 initially identified as important for exocytosis and plasma membrane repair.
00:03:36.03 One molecule that was extremely useful for us in these studies was synaptotagmin VII,
00:03:41.04 which is a member of a family of calcium sensors that are anchored on membranes, and they are
00:03:48.24 not part of the SNARE complex that promotes membrane resealing, but they have been shown
00:03:54.05 extensively by several labs that they convert calcium sensitivity to membrane fusion reactions.
00:03:59.16 So, what we found is that there is a very broadly expressed isoform of this family,
00:04:07.08 synaptotagmin VII, that is present on the membrane of lysosomes and that this molecule
00:04:14.24 facilitates the response of lysosomes to calcium influx during plasma membrane wounding.
00:04:19.11 So, there was quite a bit of skepticism in the beginning when we started proposing that
00:04:24.08 we had found the lysosomal isoform of synaptotagmin, because these proteins were mostly studied
00:04:31.09 before in synaptic vesicles of neurons, where they mediate... they are very important players
00:04:37.06 on the release of neurotransmitters.
00:04:41.12 So, it was not expected since, at that time, lysosomes were not viewed as compartments
00:04:48.24 that could undergo calcium-regulated exocytosis.
00:04:53.03 So, what you do in these cases -- this is very common in science -- you just have to
00:04:57.06 persist and you have to think of independent ways to demonstrate something that you think
00:05:03.08 is going on.
00:05:04.08 So, for us, one of the ways in which we could settle this issue of a lysosomal isoform of
00:05:11.14 synaptotagmin was by figuring out how synaptotagmin VII is targeted to lysosomes.
00:05:18.04 So, when this protein is synthesized in the endoplasmic reticulum and then travels through
00:05:23.19 the Golgi, it was not clear how we could reach lysosomes, because the cytosolic domain that
00:05:30.20 contains these calcium-sensing domains doesn't have a classical targeting motif that can
00:05:37.01 take proteins to lysosomes.
00:05:38.18 So, what we found in the lab was that synaptotagmin VII is modified by lipids -- it's palmitoylated.
00:05:46.13 And this palmitoylation allows it to associate with a lysosomal resident protein, CD63, which
00:05:52.16 is a classical, very well-known lysosomal protein that has a very well-defined targeting
00:05:58.11 motif in its cytosolic domain.
00:06:00.14 So, basically synaptotagmin VII catches a ride with CD63 to get to lysosomes.
00:06:05.24 And, as you can see in this experiment, here, when we just tag these different proteins
00:06:11.09 with different colors, we can see that... in blue, CD63, in greens, synaptotagmin VII,
00:06:19.12 and in red, Lamp1, which is an independent marker for lysosomes that does not associate
00:06:24.17 with synaptotagmin VII.
00:06:26.18 And you can see that... in this overlay image, that we can see a very good colocalization
00:06:32.20 between these three proteins, really indicating Syt VII on lysosomes.
00:06:36.18 We just have to mutate one amino acid on the cytoplasmic tail, on this targeting motif
00:06:41.24 of CD63, to completely change this picture.
00:06:45.00 And you see, here, that this mutation is changing the distribution of CD63 from lysosomes to
00:06:52.06 the plasma membrane, and that exactly the same happens with synaptotagmin VII with this
00:06:57.05 mutation that was done on CD63.
00:07:01.11 And this manipulation did not alter at all the lysosomal localization of Lamp1, really
00:07:08.08 demonstrating that this is the process by which this isoform of synaptotagmin gets targeted
00:07:14.12 to lysosomes and then functions there as a calcium sensor.
00:07:20.20 And this was really important for us because, at this time, we already had quite a bit of
00:07:24.20 data indicating that synaptotagmin VII depletion in cells or in animals interferes with plasma
00:07:32.19 membrane repair.
00:07:33.19 And what is shown, here, is knockout mice deficient in synaptotagmin VII, and a cross
00:07:39.15 section of their skeletal muscle -- again, as in the other examples that I showed you
00:07:44.01 before of other proteins involved in plasma membrane repair, there is an inflammatory
00:07:48.09 infiltrate that develops over time in these mice, really consistent with other evidence
00:07:56.11 we have of consequences of defective plasma membrane repair.
00:08:00.10 We can see by electron microscopy in these fibers that some of the muscle fibers of these
00:08:05.01 mice are completely infiltrated by macrophages.
00:08:08.11 And we have also additionally inflammatory cells that really characterize this response
00:08:14.10 in these mice as an inflammatory myositis.
00:08:17.17 So, what was really interesting about this is that we were studying synaptotagmins that
00:08:23.18 have these two independently folding C2 domains on their cytosolic domains, and at the same
00:08:31.09 time -- the same year, actually, that we published a muscle defect in the synaptotagmin VII-deficient
00:08:38.07 mice -- a paper came out from Kevin Campbell's lab, showing that a human form of muscular
00:08:44.09 dystrophy that was known to be caused by mutations in dysferlin was also caused by defects in
00:08:52.10 plasma membrane repair.
00:08:53.10 It was the first time that this type of muscular dystrophy
00:08:56.16 was associated with plasma membrane repair.
00:08:59.11 And what's very interesting is that dysferlin is a much larger protein and it doesn't have
00:09:04.06 only two C2 calcium-binding domains, but it has up to seven.
00:09:10.11 And this protein is actually associated with the membrane of muscle fibers, with the sarcolemma.
00:09:16.09 So, with all these calcium binding domains, really in direct contact with the cytosol.
00:09:22.00 And this is one of several other types of indications we have that muscle cells have
00:09:27.22 a specialized machinery, since dysferlin is expressed mostly in muscle cells, that this
00:09:33.11 additional machinery might be necessary for cells that are
00:09:37.15 exposed frequently to membrane damage.
00:09:40.12 So, this brings us to, how is this working?
00:09:43.20 Alright?
00:09:44.20 So, the first models that were proposed in this field were by Paul McNeil, who suggested,
00:09:51.00 as I mentioned already, that he saw this exocytic response happening during exocytosis, and
00:09:57.07 then on an electron microscopy studies he kept consistently seeing an aggregation of
00:10:02.06 vesicles that was very close to the wound site.
00:10:05.03 So, what he proposed that calcium was doing is that by flowing through the wound it was
00:10:09.15 causing homotypic fusion of these compartments, and then this would form a large patch of
00:10:15.04 membrane that would somehow be applied to the wound and reseal the membrane.
00:10:21.01 Richard Steinhardt proposed a different model, in which also the major player would be exocytosis
00:10:28.01 of intracellular compartments, but in this case what he proposed is that the reduction
00:10:33.06 in tension of the plasma membrane, with exocytosis happening in the vicinity of the site where
00:10:39.05 calcium flows into the cells, this would release tension on the membrane, and he actually could
00:10:43.19 directly measure this with laser tweezers, and it would be this releasing tension that
00:10:48.17 would favor spontaneous resealing.
00:10:51.03 So, what got really interesting was, later on, when papers started coming out that cells
00:10:57.15 can also repair a very different type of wound on their membranes in a calcium-dependent
00:11:02.22 manner, which is what is shown here with this bacterial, very well-studied bacterial toxin,
00:11:08.16 streptolysin O, and this is the structure of this protein.
00:11:14.22 Monomers of this toxin bind to cholesterol on plasma membranes then oligomerize, and
00:11:20.20 this complex then surface... then suffers a vertical collapse in which a stable protein-lined
00:11:27.09 pore is formed on the membrane.
00:11:29.02 So, this is a very stable pore and it would be very hard to imagine how this kind of membrane
00:11:35.15 wound could be repaired by a membrane patch coming from the inside or by just releasing
00:11:42.17 tension in the membrane.
00:11:44.01 So, we started working with these toxins in the lab and what this video, here, is showing
00:11:49.12 is that we have a calcium-sensitive dye loaded into the cells and we really can directly
00:11:54.20 visualize how transient is the influx of calcium, that instead of keeping the intracellular
00:12:01.03 calcium concentration high, which is when the fluorescence gets brighter, the cells
00:12:07.01 could rapidly control this process.
00:12:09.10 And we showed this in many different ways and this really showed us that something else
00:12:13.18 was going on, that there was a mechanism to remove pores from the membrane.
00:12:18.05 And so this is another way that is very useful to look at membrane wounding and repair.
00:12:24.20 So, when we are looking at cells that don't have the toxin, we just see this dye a lipophilic
00:12:31.00 dye, FM1-43, intercalates on the membrane, gets brighter, but cannot cross the membranes.
00:12:36.11 So, we see it only on the outside of the cells.
00:12:41.11 So, what you see looping here, in conditions without calcium that are not permissive to
00:12:48.04 repair, we added a toxin and this dye immediately flows into the cells, showing that, really,
00:12:54.24 they cannot contain the dye because they have gaps on their membrane.
00:12:59.15 So, very interestingly, when we do this in the presence of calcium, the conditions permissive
00:13:05.04 for repair, if you look there is an initial flow of the dye into the cells, and the movie
00:13:11.18 is looping here, but you see that the cells rapidly control this, and as they are doing
00:13:16.11 this you see that the membrane is blebbing.
00:13:19.16 And this observation of blebbing on the membrane of cells that are undergoing wounding and
00:13:26.03 repair is actually a common observation that has been seen in many systems, and this has
00:13:31.12 influenced a lot the thinking the investigators, because it's quite tempting, when you see
00:13:36.07 extensive blebbing of the membrane, if you're dealing with a pore-forming toxin, to conclude,
00:13:41.03 well, this is the answer, that the cells are shedding these pores by blebbing.
00:13:46.03 But, if you pay attention in what this movie here is showing is that this blebbing is very
00:13:51.04 transient.
00:13:52.06 And we looked very carefully into this and we found
00:13:55.01 no evidence that these blebs are shed.
00:13:58.04 They are extended and then they retract and we believe this is happening because of a
00:14:03.03 cortical cytoskeleton that is temporarily disrupted by the calcium influx -- when it's
00:14:08.01 restored, the bleb retracts.
00:14:11.04 And we did experiments to really directly demonstrate this point.
00:14:15.23 What is shown here is that we treated the cells which cytochalasin D, which is a drug
00:14:20.24 that disrupts the actin cytoskeleton.
00:14:22.17 So, again, when we add the toxin in calcium-free conditions, the dye flows in, indicating that
00:14:29.23 they can't repair.
00:14:30.23 But, very interestingly, you see here that this cytochalasin D disruption of the actin
00:14:36.10 cytoskeleton completely abolished the blebbing, but these cells were very capable of blocking
00:14:43.16 the influx of the dye -- they actually do it better than that cells with an intact cytoskeleton.
00:14:50.09 This and other experiments persuaded us that we don't believe, at least in conditions that
00:14:55.18 we have worked, that blebbing is a major factor in the repair of pore-forming toxins.
00:15:03.16 So, what we learned, which was also very informative... so, this is measuring the fluorescence intensity
00:15:09.18 inside these cells, in these experiments of intracellular flow of FM1-43, that cells reseal...
00:15:16.08 so, they... they block entry of the dye that is seen here in cells, and in the absence
00:15:22.13 of calcium or cells depleted in ATP... this control of the calcium influx happens within
00:15:29.12 30 seconds.
00:15:31.00 And this first was very interesting, because at that point we didn't know, well, the cells
00:15:34.14 have different mechanisms to repair different types of wounds -- maybe the... the pores,
00:15:39.22 these stable pores, are dealt with in a different way.
00:15:44.05 But it is a big coincidence that the cells reseal within the same kinetics that was described
00:15:49.06 before for mechanical wounds by several different labs, so it's also a fast repair, within around
00:15:55.09 30 seconds.
00:15:57.07 So, this is when the big surprise came, we were not... and we were not expecting this
00:16:02.01 at all, but, after we had ruled out the blebbing out of the pores, we started noticing that
00:16:09.02 when we did these experiments with the membrane-impermeant dyes, instead of adding it after the injury
00:16:14.18 event -- we left the dye since the beginning of the experiment -- we saw what we expected,
00:16:20.01 that you see this massive influx of the dye into the cytoplasm if you don't have calcium
00:16:26.01 to promote the resealing, but when you have the cells in the presence of calcium that
00:16:30.13 resealed we noticed that there was accumulation of this dye that had been added extracellularly
00:16:36.10 in small vesicles inside the cells.
00:16:38.10 So, the dye did not enter the cytosol but appeared inside these small vesicles.
00:16:42.12 So, in this experiment here, done with the help of Derek Toomre at Yale, we used a fluorescent
00:16:48.24 marker for the plasma membrane and then analyzed the fluorescence intensity -- it's what we
00:16:53.14 call kymographs -- along these white lines just underneath the plasma membrane.
00:16:58.09 And you can see that, in cells not exposed to the toxin, in this short period of time
00:17:04.01 of just 180 seconds there is no significant endocytosis; we don't see signs of plasma
00:17:10.23 membrane moving into the cell.
00:17:12.16 But, after adding the toxin, which was adding at this red dotted line, here, we saw a massive
00:17:19.00 appearance of intracellular vesicles, membrane from the plasma membrane being carried in
00:17:25.00 the cell.
00:17:26.00 And this started will happen as soon as 12 seconds after the cells were injured.
00:17:30.11 So, this is really what put us on the track of really, this... it was quite surprising
00:17:36.08 that nobody had seen this before, because when we looked at these cells a few minutes
00:17:41.24 after they had been exposed to the toxins the cells looked like Swiss cheese.
00:17:46.15 They are full of these vesicles that have really this very irregular morphology -- I
00:17:52.06 have here a clathrin... a classical clathrin-coated endosome just for comparison, and we saw that
00:17:58.03 these vesicles did not have a clathrin coat, but they contain markers, which is what these
00:18:03.07 arrows here are pointing to... that...
00:18:06.18 BSA albumin coupled to gold particles that we could see on electron microscopy.
00:18:12.06 So, the presence of these particles inside these vesicles told us that they originated
00:18:17.24 by endocytosis -- they were not pre-existing vesicles.
00:18:21.16 And this really was an important realization because it helps us understand and interpret
00:18:27.15 other experiments.
00:18:29.13 And this can be quantified on electron microscopy and you can see here that, if we chelate calcium
00:18:35.22 with a calcium chelator in these cultures, there's a big inhibition of the number of
00:18:41.09 these vesicles formed by endocytosis.
00:18:44.07 So, this is the point that I was making before, that studies... the model proposed by Paul
00:18:52.08 McNeil, was really in a big way guided by the observation of endocy... of large compartments
00:19:04.16 close to the wounds in cells, and also the fact that these compartments became gradually
00:19:10.19 larger.
00:19:12.00 And this is what was proposed as the patch that would be applied to the membrane.
00:19:16.06 But, just by adding an endocytic tracer on the outside and doing very similar experiments
00:19:21.15 and wounding these cells, we saw that these vesicles that form close to the wound site,
00:19:27.14 we believe they are endocytic.
00:19:29.07 So, they are moving in by pinching off from the plasma membrane and they carry extracellular
00:19:34.22 markers with them.
00:19:36.10 And these compartments really get large and we studied this over time, so you can see
00:19:42.10 here that, starting really soon after adding the toxin to the cells, and then fixing and
00:19:49.13 examining these cells by electron microscopy at different periods of time, we saw that,
00:19:54.06 initially, small vesicles are formed inside the cells
00:19:57.10 and they gradually merge with each other.
00:20:00.05 We can see these figures that are very suggestive of homotypic fusion, and then they join these
00:20:05.14 structures that are quite well-known with the morphology of late endosomes, that have
00:20:11.14 these internal vesicles -- they are called multivesicular bodies -- which then mature
00:20:16.21 into lysosomes.
00:20:18.05 So, we could show in a series of biochemical experiments that the toxin, because we could
00:20:23.03 follow the toxin that left the plasma membrane and moved all the way into lysosomes, and
00:20:29.04 we could show that this toxin was degraded inside lysosomes.
00:20:34.00 So, what is the link with lysosomal exocytosis?
00:20:37.05 Right?
00:20:38.05 I spent a lot of time telling you that there's a direct correlation between the ability of
00:20:42.11 cells to fuse lysosomes with the plasma membrane and their ability to reseal the membrane.
00:20:49.13 So, the very interesting link that emerged from our studies was a lysosomal enzyme, acid
00:20:56.05 sphingomyelinase, which, as shown here in this graph just showing levels of activity
00:21:02.19 of the enzyme using a specific substrate... and we saw that, under conditions in which
00:21:07.07 we trigger lysosomal exocytosis, which is permeabilizing the membrane in the presence
00:21:11.17 of calcium, there is a very clearly detectable release of this enzyme into the supernatant.
00:21:19.00 And if we don't have calcium this does not happen, and this also does not happen spontaneously
00:21:24.11 if the cells are not permeabilized with a toxin.
00:21:28.19 So, what is really interesting about.... about acid sphingomyelinase is that this enzyme
00:21:33.20 is linked to a serious human disease, which is Niemann-Pick type A and B. So, this is
00:21:39.23 an autosomal recessive disease that, in the type A of the disease, as you can see here,
00:21:44.15 again, in this activity plot of cells isolated from these patients, that the enzyme levels
00:21:51.21 that are normally seen in normal human controls are almost absent in these patients.
00:21:58.07 And this causes a very severe neurological disease and these patients don't live long
00:22:02.04 -- they die before two years of age.
00:22:05.06 So, we are now investigating this with a mouse model.
00:22:08.20 We are excited by the possibility that there is also more to be learned about Niemann-Pick
00:22:15.16 type A disease relative to plasma membrane repair, because, since these patients really
00:22:21.05 have such a short life, there is not much... many studies done with their dishes.
00:22:28.02 But there is also a mild form of the disease, which is Niemann-Pick type B, in which there
00:22:33.11 are varied levels of expression of this enzyme, and the pathology that appears in these patients
00:22:40.18 is extremely variable.
00:22:42.14 So, it could eventually be very useful to understand and to help these patients with
00:22:48.14 the milder form of the disease if we find out if there are
00:22:52.02 also defects in plasma membrane repair.
00:22:54.22 So, we think, at least in isolated cells in culture, that this is the case, because what
00:23:00.08 is indicated here is a different type of membrane-impermanent dye.
00:23:04.20 We again wounded cells with a pore-forming toxin.
00:23:07.20 In the absence of calcium, we see that the dye enters most of the cells and we see the
00:23:14.01 nuclei stained with this dye.
00:23:16.21 But, when we have calcium, the cells effectively stop this dye influx.
00:23:22.11 In cells from the patients, we have a much less efficient ability to block influx of
00:23:30.12 propidium iodide, really indicating a defect that we can measure using an instrument called
00:23:36.10 a flow cytometer, in which, if we run cells that are not permeabilized, that were exposed
00:23:42.15 to this dye, you see that most of the cells are in the low fluorescence end of the spectrum,
00:23:47.21 but by adding the toxin in the absence of calcium we can see that both cell types -- from
00:23:52.19 normal human controls or from the Niemann-Pick type A patients -- they are equally injured
00:23:58.16 and most of the cells move to the high fluorescence end.
00:24:02.18 And this graph, here, in the presence of calcium, really illustrates how the cells from these
00:24:08.16 patients are less eff... effective in repairing these pore wounds, because more cells actually
00:24:18.00 have a difference between 62% of the cells repairing in controls under these conditions
00:24:23.16 to just 30% in these patients.
00:24:26.13 We could also do experiments in the lab using RNAi-mediated silencing to really reduce the
00:24:35.11 levels of these enzymes in HeLa cells, and what you can see looping here is, again, without
00:24:42.12 calcium you see the FM1-43 dye flowing into the cells in response to the pore-forming
00:24:47.13 toxin, the cells can control this very well, which is what is indicated in this panel,
00:24:52.20 here, that you don't see fluorescence, but when we reduce expression levels of this enzyme
00:24:59.05 we see that the defect appears.
00:25:01.20 Some of these cells are really not able to stop the dye from flowing in and, remarkably,
00:25:08.02 just by adding acid sphingomyelinase to the supernatant outside of these cells, we can
00:25:14.22 really repair these defects.
00:25:16.23 So, this really indicated for us something very exciting, that this enzyme is working
00:25:22.09 on the outside of cells to remodel the plasma membrane and promote this process of repair.
00:25:28.03 So, around this time, we were very happy to see this completely independent study coming
00:25:32.18 out of Don Hilgemann's lab, in which, using completely different techniques -- electrophysiology
00:25:38.11 and measuring membrane capacitance -- he detected what he called massive calcium-activated endocytosis,
00:25:45.08 and he showed that this form of endocytosis followed exocytosis.
00:25:50.08 And this endocytosis, interestingly, could be triggered by elevation in intracellular
00:25:55.15 calcium, like we see, or by adding to the cells extracellular sphingomyelinase, also
00:26:03.10 very similar to the results that we see with acid sphingomyelinase from lysosomes.
00:26:08.13 So, how would this be working?
00:26:11.08 You have... there's several studies emerging recently in which we are understanding really
00:26:17.16 much better how sphingomyelinase works on membranes, and the substrate of sphingomyelinase
00:26:23.14 is this very abundant lipid on the outer leaflet of the plasma membrane of cells, sphingomyelin,
00:26:31.02 and when the head group of sphingomyelin is cleaved it generates ceramide, which is a
00:26:36.18 lipid known to coalesce and to form these microdomains that
00:26:41.11 have the tendency to invaginate in cells.
00:26:43.20 You can see this directly in this experiment, shown here, that was done with giant liposomes
00:26:49.04 that contain sphingomyelin, and, just by adding with a micropipette, sphingomyelinase at this
00:26:56.05 spot on this giant liposome, you can see the formation of an invagination that is being
00:27:03.20 formed by the accumulation of ceramide on this membrane.
00:27:07.02 So, this really brings us to something quite interesting, and we think this is really going
00:27:13.03 to help us understand many different aspects of membrane biology, is that it has been known
00:27:19.04 for quite some time, and there's a lot of interest in this, that they are microdomains
00:27:23.12 on the plasma membrane that are called lipid rafts that are enriched in
00:27:28.13 sphingolipids and cholesterol.
00:27:30.12 And these microdomains segregate from other lipids and they have specific functions that
00:27:36.05 are still being investigated.
00:27:38.01 And there is a very interesting structure, caveolae, which involves these lipid rafts
00:27:44.06 domains, and they have formed these very defined morphological structures with the help of
00:27:49.03 coat proteins called caveolins and additional proteins called cavins that stabilize these
00:27:54.14 domains in an invaginated form on the membrane.
00:27:57.18 And there has been a lot of discussion whether these are stable structures on the membrane,
00:28:02.18 if their function is to flatten out and release membrane tension, or if they also function
00:28:09.07 as endocytic vesicles.
00:28:11.01 So, what we saw, and I showed you before that the initial vesicles that we saw just looking
00:28:17.04 30 seconds after we permeabilized cells with a pore-forming toxin, when we looked first
00:28:23.22 at control cells that we don't...
00:28:25.24 do not add any toxin, we see occasional caveolae, as expected, along the plasma membrane.
00:28:33.19 Many different cells express this, but very interestingly caveolae are more abundant in
00:28:38.16 cells that are more frequently under mechanical stress, such as muscle and endothelial cells.
00:28:45.05 But in this case, here, we're just looking at a normal epithelial cell in which we saw
00:28:52.02 that, after we added the toxin, there was a really very significant increase in the
00:28:58.00 number of these small vesicles that had the size and the morphology very consistent with
00:29:03.12 vesicles that would have been generated from caveolae on the plasma membrane.
00:29:07.16 And, very interestingly, without injuring this membrane at all, just by treating the
00:29:12.18 cells with sphingomyelinase on the outside, we could also increase the number of these
00:29:17.22 caveolae-like structures.
00:29:20.03 We could show... using several important controls in which we can distinguish what is inside
00:29:26.00 the cells from what is outside, we could show that a labelled toxin colocalizes inside cells
00:29:33.24 in compartments that have the marker for caveolae...
00:29:37.14 Cav1... caveolin-1.
00:29:39.03 And, you can see here in the overlay, there was pretty good colocalization with several
00:29:44.13 of these compartments, indicating that the toxin does travel into cells.
00:29:49.02 And we did electron microscopy as well, using gold of different sizes -- the small gold
00:29:55.16 indicates the toxin and the large particles indicates the marker for caveolae.
00:30:00.18 So, you can see here, in these vesicles, at the size expected for caveolae, there is,
00:30:07.24 clearly detectable, the presence of the toxin, consistent with our hypothesis that the toxin
00:30:13.10 is being removed from the cell surface and trafficking into cells by endocytosis of these
00:30:19.16 caveolar structures.
00:30:21.19 If we inhibit the expression of this protein required for the formation of caveolae, using
00:30:26.23 the same assay of this intracellular flow of FM1-43, we can see, in the control cells,
00:30:35.08 exactly the fact that I showed you several times, that when you don't have calcium you
00:30:39.17 have the dye flowing, when you have calcium the cells control this, and by using RNAi
00:30:46.23 to deplete cells of this protein that is necessary for the formation of caveolae you see that
00:30:53.08 the dye starts to flow in, which is quantified in this purple line, here, indicating a partial
00:30:59.15 defect that is not as strong as when we don't have any calcium, but it's a clearly detectable
00:31:05.09 defect in repair... in removal of this pore from the membrane and the blockage of entry
00:31:14.03 of the dye.
00:31:15.13 So, what is really interesting in all this is, as it... one of the most gratifying things
00:31:22.10 in science that sometimes, after many years, we find these unexpected links with literature
00:31:28.20 that already exists, so there are many papers for many years that the proteins necessary
00:31:34.22 to form caveolae, caveolin and cavin, have been studied and have been linked in humans
00:31:41.08 to serious pathology, both in skeletal muscle and in cardiomyocytes.
00:31:45.07 And, of course, this could be related to additional functions of caveolae, but, since it is so
00:31:52.08 common to see muscle pathology in mutations associated with proteins required for repair,
00:31:59.10 we were very excited to see that this is evidence that already exists for caveolins, these proteins
00:32:07.00 necessary to form these special structures, caveolae.
00:32:11.18 So, what about cells that don't express caveolins, that don't contain caveolae because they don't
00:32:17.04 express the proteins, like caveolins, necessary to form these structures?
00:32:21.18 So, we investigated this in collaboration with a wonderful colleague at Maryland, Wenxia
00:32:27.08 Song, and what we learned is that, very interestingly, B lymphocytes that do not have caveolae, they
00:32:34.16 also respond to injury to their membrane with exocytosis of lysosomes, and then rapidly
00:32:41.20 with a form of endocytosis, but in the absence of caveolin that normally forms... forms these
00:32:47.11 very defined structures of a very defined size, we saw these tubules -- longer invaginations
00:32:54.14 of the membrane -- that were also associated with the ability of these lymphocytes to reseal
00:33:00.14 their membrane.
00:33:01.14 So, what we are excited about is that these protocols of membrane injury and repair may
00:33:07.01 provide a long-sought trigger for this form of endocytosis.
00:33:11.19 Several groups are very interested in what we call clathrin-independent and caveolae-independent
00:33:18.03 forms of lipid raft endocytosis.
00:33:21.16 And there has been a lot of variation between labs and we are speculating that, maybe, these
00:33:28.14 variations come from unintended injury of cells, because we know that cells... the plasma
00:33:36.22 membrane of cells can get injured just when cells are crawling through cells or interacting
00:33:41.21 with different surfaces.
00:33:43.03 So, we are now investigating if we can use membrane wounding to see if this is a process
00:33:50.03 that can be triggered, particularly in cells that don't express caveolae, and if we can
00:33:55.16 learn more about this process.
00:33:57.18 So, I want to leave you with this provocative concept, that what we are thinking is that
00:34:05.05 a very important element of the plasma membrane repair is actually remodeling of the plasma
00:34:12.07 membrane, it is a process that happens as a consequence of the release of lysosomal
00:34:17.22 hydrolases on the cell surface.
00:34:19.21 And we have direct evidence for... for a role for acid sphingomyelinase, but we also think
00:34:25.11 that other hydrolases in lysosomes, like proteases, could be involved in facilitating the generation
00:34:31.13 of ceramide, which may be the trigger for the formation of these membrane invaginations
00:34:38.05 and endocytosis that can carry lesions into cells for degradation.
00:34:43.15 So, this has really been a great adventure for us in the lab and this is really what...
00:34:50.17 I think it's a great example of how you should keep your eyes open in the lab, because sometimes
00:34:57.19 you can be studying something completely different like we were, studying invasion of a parasite,
00:35:02.12 and you stumble into something that gradually, although very unexpected, little by little,
00:35:09.12 starts making sense.
00:35:10.19 And this is an extremely gratifying part of science because nature, once in a while, gives
00:35:15.20 us these little glimpses of really beautiful complexity of biological systems, and we have
00:35:24.07 to be humble and we have to be aware that what we are seeing
00:35:28.03 is just the tip of the iceberg.
00:35:31.24 But, in many cases, following these processes can really be very useful in several important
00:35:37.11 interventions including disease.
00:35:39.24 So, I'll end here by acknowledging wonderful people, again, the present and past members
00:35:47.06 of my lab, and also collaborators that I mentioned their work in this part of my talk.
00:35:53.15 Thank you.

Videos in this Talk

Part 1: Mechanisms of Plasma Membrane Repair
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: Ca2+-Dependent Lysosomal Exocytosis Mediates Endocytosis and Wound Healing
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Norma Andrews

Total Duration: 1:01:04

Recorded: March 2017

All Talks in Cell Biology

Talk Overview

Dr. Norma Andrews overviews the mechanisms of cellular plasma membrane repair. As she describes, a lesion is followed by a Ca2+-dependent movement of vesicles to the plasma membrane. By studying how the Trypanosoma cruzi parasite enters the cell, Andrews’ laboratory discovered that an increase of intracellular calcium was triggering lysosomal fusion to the plasma membrane. This unexpected observation allowed them to conclude that upon an injury to the plasma membrane, a Ca2+ influx induces lysosomal exocytosis mediated repair. This research demonstrates that lysosomes are not only responsible for the degradation of material that comes inside the cell through endocytosis, but also have an important role in plasma membrane repair.

In her second talk, Andrews further explains how Ca2+-dependent exocytosis of lysosomes aids membrane repair. Her laboratory showed that after lysosomal exocytosis, an injury to the plasma membrane would also trigger a Ca2+-dependent endocytosis that is required for the repair mechanism. Andrews laboratory showed that lysosomes release the enzyme acid sphingomyelinase (ASM) which induce the endocytosis required for plasma membrane repair.

Speaker Bio

Norma Andrews

Norma Andrews is currently a Professor and Chair of the Department of Cell Biology and Molecular Genetics at the University of Maryland. She received a B.S. degree in biology (1977) and a Ph.D. degree in biochemistry (1983) from the University of São Paulo, Brazil. In 1990, after completing postdoctoral studies in the laboratory of Victor… Continue Reading