Mysterious Cell Membranes

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00:00:06.28 My name is Randy Schekman. I'm a professor of molecular and cell biology at the University of California-Berkeley
00:00:13.11 and an investigator of the Howard Hughes Medical Institute.
00:00:17.05 I'm also the editor-in-chief of a new, open access online journal called eLife,
00:00:22.11 which covers all of the life sciences. Today I'd like to tell you about the mysterious membranes within a cell
00:00:30.08 and the cells that I'm going to tell you about are all nucleated cells,
00:00:34.23 they're animal cells or yeast cells, they're multicellular or unicellular,
00:00:39.19 but they all contain the same architecture of membranes
00:00:44.16 that organize a lot of the metabolic processes within all of these cells.
00:00:49.19 Here's a great example. This is a cell that you'll know about because after you eat a meal,
00:00:56.01 it's very busy manufacturing insulin for secretion into your bloodstream.
00:01:01.10 So, here you see a thin sliver through a cell called the beta cell of the pancreas
00:01:07.11 that's manufacturing insulin. Now, all of the dots that you see in the cytoplasm of the cell
00:01:13.29 represent granules of condensed insulin stored within a membrane
00:01:21.19 awaiting shipment outside the cell.
00:01:24.29 So, when you have a meal and you digest carbohydrates, there's a signal that is recognized
00:01:32.07 at the surface of this beta cell that triggers the discharge of this content to the cell exterior
00:01:39.00 and eventually insulin is released.
00:01:41.05 But what we really need to understand for you to appreciate what's going on here
00:01:46.24 is to have a closer look at what all these membranes look like, how they relate to one another
00:01:52.24 and how we can study that in a living cell.
00:01:55.29 So, let's formulate a series of questions that I'd like to answer in the next few minutes.
00:02:01.12 What are all these membranes and what do they do?
00:02:06.00 The first question is how are the membranes in a cell organized?
00:02:10.24 The next question is how do the membranes within a cell communicate with one another?
00:02:16.15 Are they related? Do they interact and how so and how do we study that?
00:02:20.28 How can we study what proteins in membranes do in a living cell?
00:02:25.27 Well, first, let's consider what membranes look like.
00:02:29.26 Membranes, in a very simple cartoon, contain protein molecules,
00:02:36.14 these are the green things that you see here, sometimes spanning through a membrane,
00:02:42.28 that consists of lipid molecules, in this case phospholipid molecules
00:02:48.12 that have a water loving surface on one side of the cell, facing the outside of the cell
00:02:54.19 or facing the cytoplasm of the cell, and then buried in the interior of what is called the bilayer,
00:03:01.20 there are long water-hating hydrophobic molecules called fatty acids that create a very different environment
00:03:10.00 that separates this membrane and allows the cell to remain intact and to function
00:03:16.00 without necessarily spilling its contents to the cell exterior.
00:03:21.06 Now, the lipids at normal body temperature are more or less fluid,
00:03:26.28 which means that the proteins and the lipids within this membrane
00:03:31.09 can move around, they can spin and they can move back and forth,
00:03:35.19 giving the appearance of a kind of a sea of let's say icebergs, or buoys that can roam around within the sea of lipids.
00:03:46.18 But of course, membranes can't be that simple, they have to be organized
00:03:51.01 and the best example of that is to have a look at a very simple cell, one that you've all seen before,
00:03:57.09 and that's the red blood cell.
00:03:59.08 The red blood cell is perhaps the simplest animal cell that we can study.
00:04:03.13 It has no membranes in its interior, instead it is bounded by a single bilayer
00:04:11.02 but note that as you've surely seen before, that it has this dimpled shape,
00:04:16.06 this so-called biconcave shape. If the membrane of the red cell were simply a two dimensional fluid,
00:04:22.22 it couldn't possibly maintain this very characteristic architecture.
00:04:27.18 And so let's have another look at another cartoon that illustrates how cells can organize themselves
00:04:33.27 and restrain the otherwise diffusive forces that would make a membrane completely random.
00:04:40.01 So, here is such a cartoon. So, the membrane is as we've seen before,
00:04:44.17 it has proteins that span the lipid bilayer, but many of the proteins in a red cell actually are anchored
00:04:53.08 by being attached to fiber-forming proteins on the interior that line the cytoplasmic face of the membrane
00:05:02.08 and provide a kind of a skeletal structure that can give long range order not only to impose a shape on the cell,
00:05:10.08 but also to prevent the diffusion of molecules beyond the boundaries of these fenced in, or corralled in regions.
00:05:19.00 So, imagine a cell has this kind of an architecture, it can create now all kinds of surface features
00:05:26.04 and yet keep the membrane still largely fluid.
00:05:30.08 Now, ok, so that's a simple consideration about membranes,
00:05:34.03 but now we want to take a closer look and see what membranes look like inside of cells
00:05:38.23 and not just what the cell surface looks like.
00:05:40.19 And for that, the effort of really a pioneer in the field of cell biology
00:05:46.06 I need to tell you about. This is a man by the name of George Palade
00:05:50.17 who did classic work to use and develop an instrument that you see behind him called the electron microscope.
00:05:58.20 In the 1950's and 1960's and 1970's Palade, at Rockefeller University and his many brilliant students
00:06:07.24 pioneered the use of this instrument, which allows a focused beam of electrons
00:06:12.06 to penetrate through a thin slice through an animal cell and allow the visualization of membranes
00:06:20.24 and other organelles within the cell.
00:06:23.10 Among other things, Palade discovered the ribosome, which is the machine within a cell
00:06:28.17 that manufactures proteins. And as you'll see in the next few slides,
00:06:32.22 he was able to discover all of the many organelles that are within cells
00:06:37.12 and to use really brilliant techniques to map the flow of molecules
00:06:42.28 as they move from one station to another within the cell.
00:06:45.28 So, here's a simple cartoon of one kind of a cell that is an epithelial cell
00:06:53.05 that is organized in a particular polarity. Deep within the cell, we see the nucleus of the cell
00:07:00.03 that houses chromosomes and genes. We see surrounding the nucleus a membrane
00:07:05.18 called the nuclear envelope, about which I'll have more to say later,
00:07:09.14 and then attached to the nuclear envelope we see these dark studded membranes.
00:07:15.25 The dark dots are ribosomes and they are bound to a membrane that's called the endoplasmic reticulum.
00:07:24.01 This membrane is very elaborate, particularly in cells that are manufacturing proteins like insulin,
00:07:31.08 but it's certainly not the only membrane within a cell.
00:07:34.03 There are other organelles, there's a membrane called the mitochondrion
00:07:38.14 that's responsible for the energy production in cells, it makes the energy currency of the cell, called ATP.
00:07:46.00 But there's another very interesting smooth membrane that rests on top of the nucleus
00:07:52.18 called the Golgi apparatus. This membrane was first revealed by a 19th century Italian neuroscientist
00:08:01.09 named Camillo Golgi who discovered a dye that very specifically binds to the membrane of the Golgi apparatus
00:08:08.16 and highlights it in preference to anything else within the cell
00:08:11.27 and so in honor of that discovery, we've called this structure the Golgi apparatus.
00:08:16.13 But now it took many years, many decades, before we actually appreciated what it did
00:08:22.14 and I'll tell you how Palade was able to figure this out.
00:08:25.22 There are other organelles in the cell that move beyond the Golgi apparatus,
00:08:30.22 that move secretory proteins to different destinations in the cell and I'll tell you about that in the next few minutes.
00:08:37.29 Now, we know from Palade's work the logic of flow of molecules like insulin from within the cell.
00:08:47.12 We know, for instance, that insulin molecules and other things that are going to be secreted,
00:08:52.21 other hormones, antibody molecules, certain kinds of growth factors,
00:08:59.27 are manufactured on ribosomes, in this case, now, green dots,
00:09:04.13 that are lined up along the face, the cytoplasmic face of the endoplasmic reticulum
00:09:11.09 and we know that messenger RNAs and coding molecules like insulin, are bound to these ribosomes,
00:09:18.20 they are decoded by the ribosome machine, which stitches amino acids one after another
00:09:26.02 to produce the molecule which is then threaded into the interior of this membrane.
00:09:30.26 I'll tell you more about that as time goes on.
00:09:33.25 We also know from Palade that the proteins, after they're manufactured,
00:09:38.27 and deposited into the interior of the endoplasmic reticulum
00:09:42.25 are captured and transported through this structure that Camillo Golgi discovered called the Golgi apparatus.
00:09:49.25 Further, we know from Palade that some proteins serve, are transported outside of the cell
00:09:58.03 in granules, dense granules, that you see here, where they discharge their content by fusion,
00:10:05.09 by a membrane merging reaction at the cell surface that we'll talk about in a little bit.
00:10:10.14 But some other proteins in the Golgi apparatus are sorted away
00:10:15.26 and are transported to a distinct internal compartment called the lysozome
00:10:21.00 which is kind like the digestive tract of a cell.
00:10:24.15 It serves to degrade proteins and carbohydrates and to allow the reuse of amino acids and sugars for cell metabolism.
00:10:34.04 So, the Golgi apparatus, as revealed by Palade's work turns out to be kind of like a bus station
00:10:40.03 where things are shipped in, sorted into one or another destination and then shipped out.
00:10:45.29 Now, let's have a closer look, again, with the electron microscope at what these membranes look like.
00:10:52.16 And the first one is this beautiful structure, the endoplasmic reticulum;
00:10:56.20 an enormously elaborate array, kind of an assembly-line array of membranes
00:11:03.21 in cells that are manufacturing proteins for export, cells like the beta cell of the pancreas.
00:11:10.02 This network of endoplasmic reticulum membranes is enormously large.
00:11:16.03 It occupies a surface area which has been estimated to be twenty-fold, twenty-five fold in excess
00:11:23.16 of the surface area of the membrane that surrounds a cell.
00:11:27.04 So, it's a template that's laid out within the cell onto which ribosomes bind.
00:11:32.16 They manufacture proteins and they're transported into the interior of this organelle.
00:11:38.21 So, this can be revealed by the electron microscope, which has many advantages because of the spatial resolution,
00:11:45.15 you can look inside of a cell and you can see things that are only angstroms away from each other,
00:11:51.18 maybe 100 angstroms away from each other, you can see these differences,
00:11:55.24 which is really difficult to see when you look at cells in light microscope,
00:12:00.08 even a sophisticated light microscope, the resolving power of a light microscope
00:12:04.24 has not yet approached anything near what one can do with an electron microscope.
00:12:09.10 And yet, the disadvantage of looking at cells in the electron microscope
00:12:14.09 is that you're looking at a shadow of what the cell looked like.
00:12:17.22 The cell has to die, it has to be chemically fixed, it has to be dehydrated,
00:12:23.11 put in a plastic resin and cut in very thin sections
00:12:27.14 in order to allow the electron beam to reveal the interior architecture of the cell.
00:12:34.03 So, of course, cell biologists would rather, or would also, like to look at cells that are living,
00:12:39.25 that are moving around on a plate, on a glass slide.
00:12:43.28 And where molecules are moving around within the cell. This can't yet be done with the electron microscope
00:12:49.08 but it can be done with a light microscope and in particular with a revolutionary technique
00:12:54.24 that was developed in the past 15 years or so that allows one to tag a protein or a membrane
00:13:02.16 with a molecule that has the ability to fluoresce. A fluorescent probe is one that absorbs light
00:13:10.03 that's at a high energy and it emits light at a lower energy and if you put a filter into one stage of the microscope,
00:13:20.06 you can filter and only inspect light that's emitted by a fluorescent probe
00:13:24.28 and that can allow you to visualize things that are going on in a cell that's perfectly viable.
00:13:31.15 Now, how do you do that, though, to allow the investigator to study where a, let's say, a membrane protein,
00:13:39.03 is localized in a cell, and how that protein moves around in a living cell.
00:13:43.09 Well, here's the revolutionary technique.
00:13:45.22 This was developed, as I said, about 15 years or so ago,
00:13:50.07 it was the use of a protein called green fluorescent protein.
00:13:54.28 An investigator at Woods Hole in, near Boston, outside of Boston in Massachusetts,
00:14:03.15 found that jellyfish have a protein that he could isolate that has quite an unusual intrinsic fluorescence.
00:14:12.15 Most protein don't fluoresce. That is, most proteins, when you shine high energy light on them,
00:14:17.23 will not emit light at a lower energy. But this protein, green fluorescent protein, did that,
00:14:23.13 and it was because of the way the protein molecule folds.
00:14:27.19 There's nothing other than amino acids in this protein
00:14:30.16 and yet when you shine a high energy light on it, it will emit at a characteristic wavelength.
00:14:35.10 And that was really quite an astonishing observation in itself, but then it became possible
00:14:43.17 to apply this to use as a reporter to inspect proteins as they move about in cells
00:14:49.20 and the way this is done is the gene for this protein, GFP, can be recombinately stitched onto the gene
00:14:59.11 of a protein that you would wish to investigate
00:15:02.02 and when the hybrid gene is introduced into a living cell, the gene then produces a hybrid, or fusion protein,
00:15:11.25 part your protein, part green fluorescent protein, and if that protein can still do its thing,
00:15:18.04 it will be dragging along with it, inside the cell, a reporter that is intrinsically fluorescent
00:15:24.28 and if you then look at these cells carrying this protein, you can see where the protein lives
00:15:30.26 and where it moves, all consistent with cell viability.
00:15:34.28 Now, an amazing additional adaptation of this was developed by a scientist at UC-San Diego
00:15:41.21 named Roger Tsien who found that he could introduce into the gene that encodes green fluorescent protein
00:15:49.23 mutations that altered the emission spectrum of light that's given off when high energy light is shined onto the molecule.
00:15:59.25 And here are a number of different variants that he created.
00:16:02.29 GFP variants, a veritable rainbow of different molecules that have different emission spectra
00:16:12.07 and one can take each of these different mutant forms of GFP
00:16:16.17 and stitch them onto a protein using recombinant techniques
00:16:20.25 and introduce many different examples into a single cell
00:16:25.21 and if you have a fluorescence microscope with filters that filter out light only at a certain wavelength,
00:16:32.00 you can inspect, in one cell, the location and movement of several different proteins at the same time.
00:16:39.19 Really, quite a remarkable development that has revolutionized cell biology.
00:16:44.12 Now, let's take this example just one step further.
00:16:49.04 How would you then want to investigate a protein in a particular membrane?
00:16:55.24 You have to be able to insert GFP or one of these colorful variants of GFP
00:17:02.10 into a particular membrane if you wish to study how that membrane behaves.
00:17:06.26 And what we know from cell biology studies is that every different membrane in the cell
00:17:14.03 has a different kind of direction signal that allows a protein to enter into the membrane.
00:17:22.10 Each protein has a kind of a localizing signal that you can study and isolate and attach to GFP
00:17:33.21 and cause GFP now, to behave as though it was the protein that you were studying in the first place.
00:17:40.05 And here is a simple cartoon that explains the logic.
00:17:43.19 We start with a gene, in this case, this blue gene, we'll use to indicate GFP
00:17:49.21 and we append to the gene for GFP a piece of information that tells GFP now
00:17:56.27 where we want it to go.
00:17:58.26 It will be a localizing piece of information that says,
00:18:02.11 I want GFP to go into the mitochondrion, this is example A,
00:18:07.21 or I want GFP to go into the endoplasmic reticulum membrane.
00:18:12.17 This is, in this case, a red piece of information.
00:18:16.22 So, you stitch the information onto the GFP gene, you express that in a cell,
00:18:22.13 and then you look, you have a look in the fluorescence microscope
00:18:25.17 to see if it's worked out.
00:18:27.29 Here's a really dramatic example that illustrates the importance of this approach.
00:18:32.21 So, this is a living cell in which the piece of information
00:18:38.07 that tells a protein to go into the endoplasmic reticulum has been stitched onto the gene for GFP
00:18:44.15 and now GFP, which is otherwise a soluble protein in the cytoplasm
00:18:49.14 now becomes attached to the endoplasmic reticulum
00:18:52.18 and reveals this amazingly complex organelle, this intricate network
00:18:57.17 that looks almost like a kind of a disorganized spider web.
00:19:01.13 This was never appreciated until we could use this technology to understand how proteins are organized in cells.
00:19:08.16 And this structure is really so ephemeral, so fragile,
00:19:14.21 that it simply cannot be preserved adequately to be studied in this beauty in the electron microscope.
00:19:22.07 So, fluorescence has had a very important application in this example.
00:19:27.00 Now, that's one approach to study membranes and proteins in living cells
00:19:32.24 but there's another very different approach that will allow us to understand how a protein works,
00:19:39.25 what function the protein serves in a living cell
00:19:44.14 and that is through the use of a very classical approach called genetics.
00:19:48.22 And I'll tell you now about an example that we used in my laboratory many years ago
00:19:53.15 to understand the genes and proteins that are required for movement of secretory proteins
00:20:01.22 within in this case a simple cell, baker's yeast.
00:20:04.16 Now, one way to study genes that are required for cell growth and division
00:20:11.00 is to make mutations in these genes. Now, if a gene is required for the cell to grow and divide,
00:20:17.09 and you're able to use a scalpel to delete that gene from the chromosome, from its normal chromosomal position,
00:20:23.20 of course the cell won't grow and divide, so it's dead.
00:20:26.17 And it's hard to work with a dead cell.
00:20:29.08 But, now, very often, it turns out that proteins that are essential for cells
00:20:34.26 can absorb certain kinds of mutations that don't just kill the cell,
00:20:40.15 but confer an alternate thermal stability to the protein.
00:20:45.02 That is, some mutations will allow a protein to function perfectly normally at one temperature,
00:20:53.12 but at a higher temperature, will cause that protein to unfold and lose its function.
00:20:59.28 And a useful range of temperatures for yeast cells is room temperature or something closer to body temperature,
00:21:11.10 36 degrees centigrade or 23 degrees centigrade.
00:21:15.06 So, in this example, if you have yeast cells and you plate them out on a Petri plate
00:21:20.11 you can produce a series of colonies on these plates.
00:21:23.16 If these were of cells that had been exposed to a chemical mutagen
00:21:27.21 these, some of these cells will continue to grow when the plate is incubated at room temperature.
00:21:35.12 Here, for instance, is an interesting example, please focus on that.
00:21:40.18 But if you make a copy by spotting this Petri plate onto a piece of filter paper,
00:21:48.04 and you copy that filter paper onto a fresh Petri plate and then warm the plate
00:21:54.01 at a temperature close to body temperature,
00:21:56.22 some cells will die because they have a mutation that made the essential protein unstable
00:22:03.16 and thus, a colony will not form at this higher temperature.
00:22:08.17 So, these then can be collected and studied, they're called temperature sensitive lethal mutations
00:22:14.07 and a simple way to think about this is to look at a cartoon of what a protein might look like.
00:22:21.00 In a wild-type cell, a protein on the left has a certain characteristic fold
00:22:29.00 that allows it to function as shown by these red arrows.
00:22:32.28 Whereas, if the gene that encodes this protein acquires sometimes a single mutation,
00:22:40.23 that mutation may cause the protein to be quite different, to be unfolded, at body temperature,
00:22:49.13 unfolded and thus not function,
00:22:51.14 whereas the mutation may not interfere with the folding of this protein at room temperature
00:22:57.22 and thus the cell can grow and divide. So, these then, allow one to use a very simple classical genetic approach
00:23:04.29 to understand how cells, how proteins, may function in a living cell.
00:23:10.13 Now, I'm going to delve into this with a little bit more detail to describe a particular application
00:23:16.20 that a very talented graduate student in my lab, Ray Deshaies, developed many years ago
00:23:22.06 to discover the genes that are required to take a secreted protein from the ribosome
00:23:29.21 across the membrane of the endoplasmic reticulum.
00:23:33.16 The logic of his approach relies on some of the very simple tools that I've already told you about.
00:23:41.05 Here is the set-up for what Ray did.
00:23:44.17 He started with what we've already described as, in this case, an enzyme, not GFP, but an enzyme.
00:23:52.19 The enzyme happens to be the enzyme that makes the amino acid histidine.
00:23:57.29 One of the essential amino acids that are stitched together to make all proteins in the cell.
00:24:02.24 Histidine is manufactured in the cell and the last step before histidine is produced
00:24:08.05 is a chemical call histidinol.
00:24:12.03 These reactions, all of the reactions to make histidine, are ordinarily restricted entirely in the cytoplasm
00:24:20.24 and have no relationship whatsoever to the membranes of the secretory pathway.
00:24:25.19 On the other hand, if you take the gene for this enzyme that makes histidine and you stitch at one end
00:24:33.16 a piece of information that allows this, that would allow another protein to be inserted
00:24:41.22 into the lumen of the endoplasmic reticulum,
00:24:44.08 you can cause the enzyme to be dragged through the normal import machinery in the ER membrane,
00:24:53.12 to then fold in the interior of this organelle, which is removed from where it normally functions.
00:25:03.02 The compound histidinol, manufactured in the cytoplasm,
00:25:08.08 does not gain access to the interior of the endoplasmic reticulum
00:25:14.07 and yet, in this recombinant yeast cell, this enzyme has been dragged in there
00:25:19.29 and can no longer work on histidinol to make histidine. Thus, this cell grows on histidiniol,
00:25:28.20 but this cell will not grow on histidinol. It will not form a colony unless one introduces a mutation
00:25:37.22 into one of the genes that's responsible for the import machinery.
00:25:42.24 So, that's what Ray did.
00:25:44.08 He exposed this cell to a chemical mutagen and he plated these cells,
00:25:50.10 he took this cell, that was not able to grow on histidinol,
00:25:56.22 he exposed it to a chemical mutagen and he plated it on a plate with histidinol,
00:26:02.20 and asked for cells that could now grow and form colonies and among the colonies that grew
00:26:08.03 were ones that contained mutations that partially cripple the import machinery
00:26:14.18 and thus left behind, in the cytoplasm, some of this funny, hybrid protein,
00:26:21.04 enough was left behind to allow the cell to convert histidinol to histidine and thus to grow.
00:26:27.16 He then used recombinant DNA techniques to identify the gene
00:26:33.25 and the gene turns out to encode the channel-forming subunit, not only in yeast,
00:26:39.18 but also in all eukaryotic cells, even in human cells.
00:26:42.23 Here is now a very simple cartoon of what we know about this channel and how it works.
00:26:48.21 This is the cycle of biosynthesis and import of a molecule like insulin.
00:26:55.01 Insulin will be encoded by a messenger RNA and read by a ribosome,
00:27:00.28 the insulin chain will begin to be made by the ribosome
00:27:04.14 and threaded through this channel in the ER membrane, that Ray discovered in yeast,
00:27:10.17 the insulin molecule, or all other molecules that eventually are going to be secreted from cells,
00:27:16.07 are threaded through this channel, the protein folds in the interior,
00:27:20.14 and then escapes the ER for transport through the Golgi apparatus.
00:27:25.10 Now, another very closely organized organelle that is really part of the endoplasmic reticulum
00:27:32.18 sustains another crucial aspect of cell organization and that is the nucleus,
00:27:39.00 which is surrounded by a membrane called the nuclear envelope
00:27:42.12 and the nuclear envelope is itself contiguous with the endoplasmic reticulum membrane.
00:27:47.17 Here is the nuclear envelope, the membrane is unique in that it has pore-like structures
00:27:57.10 that serve to organize the flow of molecules that leave the nucleus
00:28:02.12 or that come into the nucleus from the cytoplasm.
00:28:05.06 Here is an example of a sort of a higher magnification cartoon image of the pore complex.
00:28:13.06 These form in dimples in the nuclear envelope membrane,
00:28:17.08 they're very complex, much larger and more complex than a ribosome,
00:28:21.27 but at the core of this machine, of this pore, there are these green subunits that are nuclear pore proteins
00:28:31.01 that have special features that recognize information on proteins that are to be transported
00:28:38.03 from the cytoplasm into the interior of the nucleus
00:28:41.16 and different information that's required for molecules like messenger RNAs,
00:28:47.21 that are manufactured in the nucleus, to be transported out of the nucleus into the cytoplasm.
00:28:53.05 Now, I'd like to focus on one really crucial experiment that allowed an investigator to identify the information
00:29:01.03 on a protein that allows it to go from the cytoplasm into the interior, into the nucleus.
00:29:07.08 The protein in question is a protein that's manufactured by a tumor virus.
00:29:13.20 This protein is called T-antigen and in the normal tumor-virus infected cell,
00:29:19.05 the T-antigen is exclusively localized in the nucleus,
00:29:23.09 which can be seen using fluorescence microscopy techniques.
00:29:28.13 So, this T-antigen, the normal T-antigen, is only in the nucleus,
00:29:32.04 and what you don't see in this image on the left is the rest of the cell,
00:29:35.06 which is the cytoplasm and other membranes, everything is sequestered in the nucleus.
00:29:41.13 Now, importantly, the T-antigen, which is a very large protein, has a series of amino acids,
00:29:49.10 lysines and arginines, shown here, that comprise the information on the T-antigen
00:29:55.14 that is recognized by the nuclear pore for import from the cytoplasm into the nucleus into the interior.
00:30:02.15 And the way that we can show that is by using genetics to introduce mutations
00:30:08.29 into one or another of these residues. In this case, a mutation is introduced into one of the lysine residues
00:30:15.27 so that now, the mutant gene encodes a protein that has threonine, a different amino acid,
00:30:23.16 in place of lysine. And when this mutant protein, with this one simple mutation,
00:30:28.23 is introduced, in this case, into an animal cell, using again fluorescence microscopy,
00:30:35.09 we see that the T-antigen, this mutant T-antigen, now is only in the cytoplasm
00:30:40.20 and is actively excluded from the nucleus, which appears dark here.
00:30:45.01 So, this experiment, very simple experiment, says that this piece of information
00:30:50.28 is responsible, is the nuclear-localizing signal that allows a protein to be transported into the nucleus
00:30:58.23 and then for the thousands of different proteins that follow that route,
00:31:02.29 to organize nuclear transactions including DNA replication and chromosome separation.
00:31:10.28 Alright, now, very close to the nuclear envelope and also very close to the endoplasmic reticulum
00:31:17.15 is this organelle that we've already discussed very briefly called the Golgi apparatus,
00:31:22.23 which I likened to a bus station that organizes the traffic within the cell.
00:31:27.15 What George Palade discovered was that proteins moving through the Golgi apparatus
00:31:34.04 become packaged into granules; in the case of the insulin-producing cells, these granules are largely containing insulin,
00:31:42.27 but there are many, many examples in biology of granules that have different functions.
00:31:48.11 Let me give you two examples.
00:31:50.18 This is a very different kind of a cell, it's called a mast cell
00:31:55.13 and the mast cell is responsible for secreting things like histamine, allergens,
00:32:02.07 so when you have an inflammatory response, the mast cell is activated
00:32:08.14 and all of these very dense granules, which contain histamine are actively moved to the cell surface,
00:32:16.27 whereby a process of membrane fusion, the content of the granule is discharged to the outside of the cell.
00:32:24.13 So, here's a kind of an explosive secretory pathway that is engaged in the release, in this case, of histamine.
00:32:33.19 Now, let's look at a cartoon that illustrates the steps in this path.
00:32:38.08 The granule is manufactured in the cell following the secretory pathway that I've described.
00:32:47.04 It touches, or docks, on the inside surface of the plasma membrane
00:32:52.13 and then some special event triggers the membrane of the granule to merge with the cell surface membrane,
00:32:59.05 which then results in the content, the internal content of the granule being discharged to the cell exterior.
00:33:06.18 One very important final example of this is in the discharge of a very different kind of molecule,
00:33:14.17 a neurotransmitter that is secreted by a nerve cell.
00:33:18.04 So, in this example, we see a thin slice through a nerve cell,
00:33:22.28 in this case, not touching another nerve cell, but actually touching a muscle cell,
00:33:28.01 where the muscle cell will be induced to contract.
00:33:30.25 That is achieved when chemical neurotransmitters contained within the interior of these smaller granules
00:33:38.14 are discharged by fusion at the, what is called the synapse, the synaptic junction.
00:33:45.16 So, all of these processes, movement from the endoplasmic reticulum, the Golgi apparatus, granules,
00:33:52.23 sustains an enormous range of functions in all of biology,
00:33:58.14 in all of biology, particularly in those cells that have a separate nucleus.
00:34:04.10 Well, let's review some of the key lessons of what I've described in the last several minutes.
00:34:10.01 What have we learned?
00:34:10.26 We've learned that membranes are lipid bilayers
00:34:14.14 and that the constituents of these membranes are organized so as to function properly,
00:34:20.10 but with some freedom to move about within a range in a living cell.
00:34:25.15 We've learned from the use of the electron microscope
00:34:29.14 that cells have a myriad of elaborate membranes, a very fine network of membranes,
00:34:37.23 that serve very specific functions in transmitting proteins from one station to the next,
00:34:44.15 all sorted from one another in this interesting membrane called the Golgi apparatus.
00:34:49.28 And finally, we've learned two crucial techniques in modern cell biology
00:34:54.25 that allow us to make statements of the sort that I've just said.
00:35:00.18 We can study how proteins are organized in cells by stitching a fluorescent probe to them,
00:35:08.12 studying them in living cells and we can study the function of genes and thus of proteins
00:35:13.25 by introducing mutations into those genes to see what effects these mutations have
00:35:19.03 on the ability of the cell to grow and divide.
00:35:21.14 Thank you.

This Talk

Speaker: Randy Schekman

Audience:

General Public
Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: January 2013

Talk Overview

In this talk, Dr. Schekman describes the many different cell membranes found inside a cell and how they are organized to facilitate protein secretion. He explains the important contributions of electron microscopy, fluorescent microscopy and temperature-sensitive mutations to understanding the structure, location, and interactions of different cell membranes.