Session 4: Vesicle Trafficking
Transcript of Part 1: The Secretory Pathway: How Cells Package and Traffic Proteins for Export
00:00:07.20 Hello, my name is Randy Schekman. 00:00:10.01 I'm at the University of California at Berkeley in the Department of Molecular and Cell Biology. 00:00:16.22 Today, I'll be giving three presentations on the cellular process that is used to 00:00:24.05 package protein molecules that are made inside the cell, 00:00:28.08 but have to be shipped outside of the cell. 00:00:31.09 In my first presentation, I'll discuss some of the historical aspects of how we learned 00:00:37.02 about biological membranes, and how they are deployed to encapsulate protein molecules 00:00:44.28 as they are made inside the cell. 00:00:47.19 In my next lecture, I'll describe how this process was understood at the molecular level 00:00:55.10 using a simple nucleated organism, a simple eukaryote called Saccharomyces cerevisiae 00:01:02.11 or baker's yeast, and I'll tell you about both genetic and biochemical approaches 00:01:08.12 to understanding this process. 00:01:10.24 And finally, in my last lecture, I'll discuss some very recent experiments on how cells 00:01:17.14 package small RNA molecules that are encapsulated into vesicles that are discharged at the cell exterior, 00:01:25.15 and may communicate between cells in our body. 00:01:30.23 But let's begin with a discussion of how cells are organized. 00:01:36.02 The basic principle of organization of cells comes with an understanding of the structure 00:01:43.27 of a biological membrane. 00:01:45.22 And that's depicted on my first slide. 00:01:48.22 So, here you see a cartoon of a biological membrane consisting of two leaflets of molecules 00:01:56.25 called lipids or phospholipids. 00:01:59.08 They are shown here with red balls that are the water-loving or hydrophilic head groups 00:02:08.00 of a phospholipid molecule, connected to these thin tails. 00:02:12.16 They are the water-hating or hydrophobic fatty acid side chains that constitute 00:02:19.21 the inner core of the membrane bilayer. 00:02:22.27 Two leaflets of lipids come together to form this bilayer. 00:02:28.15 Embedded in a biological membrane are these green structures. 00:02:33.02 They are protein. 00:02:34.12 They depict protein molecules. 00:02:37.02 Some of them go clear through the bilayer. 00:02:39.12 As you see here, this example is maybe, for instance, a channel in the membrane 00:02:45.15 through which small molecules may come and go. 00:02:48.12 Or this example may be of a protein that is a receptor, that sits on the outside of the cell 00:02:55.04 and recognizes hormones that may interact with a cell to convey information to the cell interior. 00:03:02.20 Now, all membranes in cells have this basic structure. 00:03:09.02 But each membrane in a cell has a different kind of personality. 00:03:13.20 And they go from very simple organizations to very complex organizations. 00:03:19.01 Here, for instance, is the simplest cell. 00:03:22.17 This is a red blood cell, coursing through your bloodstream. 00:03:26.27 It consists, at least in a human, of a single membrane surrounding an internal cytoplasm 00:03:34.18 that is filled with hemoglobin, the protein molecule in your red cells that carries oxygen 00:03:41.20 to your peripheral tissues. 00:03:43.09 So, just a simple cell with a single membrane. 00:03:47.20 But in contrast, for example, a much more complex cell. 00:03:52.26 This is a very important cell in your pancreas. 00:03:56.07 It's called the beta cell in the islets of Langerhans. 00:04:00.17 It's responsible for manufacturing insulin that is discharged outside of the cell, 00:04:09.15 carried within the cell by these little granules. 00:04:11.16 These... these look like little eye... eyespots, but they're granules that house insulin 00:04:19.13 and convey it through the cytoplasm to the cell surface, where it is discharged by a process 00:04:25.27 called membrane fusion, that we'll discuss in a few minutes. 00:04:30.06 So, enormous difference in complexity between a cell that has many functions, such as 00:04:36.21 in the pancreas, or a simple cell, such as the red blood cell. 00:04:40.14 Now, a cartoon of the various membrane organelles that are found inside of the cell is depicted here. 00:04:49.02 This is a cell from an epithelium surrounding a tissue that is responsible for making 00:04:56.01 many protein molecules that are shipped to different places, in and out of the cell. 00:05:02.26 At the base of the cell, you see the nucleus, housing the chromosomes. 00:05:08.19 Surrounding that nucleus are membranes that constitute a network called the endoplasmic reticulum, 00:05:15.24 that are dotted with these little particles. 00:05:19.03 They're ribosomes. 00:05:20.12 Ribosomes are the machines that stitch amino acids, one next to another, to make protein molecules 00:05:28.13 that are often transmitted across a membrane 00:05:33.04 into this clear space of the endoplasmic reticulum. 00:05:37.01 And we'll talk about that in a few minutes. 00:05:38.18 There are many other membrane organelles in the cell. 00:05:42.20 The powerhouse organelle, the mitochondrion. 00:05:45.13 A structure called the Golgi apparatus, through which protein molecules are conveyed. 00:05:51.12 And other membranes that have specialized functions, like the peroxisome or the endosome. 00:05:59.09 These all... all have biological membranes surrounding them and each has different protein molecules 00:06:06.23 that execute the unique functions of these organelles. 00:06:11.14 Now, much of what we know about the organization of an animal cell came from the pioneering work 00:06:18.26 of cell biologists in the middle part of the 20th century. 00:06:24.08 Prominent among them was a brilliant cell biologist by the name of George Palade. 00:06:29.11 Dr. Palade was an emigre from Romania. 00:06:34.02 He came to New York, where he established his laboratory at The Rockefeller University. 00:06:40.25 In the mid-1950s, it was Palade who discovered the ribosome. 00:06:46.15 He did this, as with much of the rest of the... of his work, by perfecting an instrument called 00:06:52.25 the electron microscope, which you see here... you see here him seated behind. 00:06:58.25 He and Keith Porter, and other scientists at the Rockefeller, devised procedures to 00:07:05.12 fix cells and tissues, and to preserve them so that they could be sectioned with a diamond knife, 00:07:13.09 and then visualized under an intense electron beam, where the electrons 00:07:19.16 were scattered by structures within the cell. 00:07:22.01 And all of the beautiful pictures, some of which I'll show you, were interpreted by him 00:07:27.12 to understand many of the functions of membranes that communicate with one another by the process 00:07:34.01 of protein secretion. 00:07:35.24 Now, let's go through, step by step, each of the organelles that Palade and his students 00:07:42.18 were able to appreciate, both by visualizing them in the electron microscope, but also 00:07:49.04 by isolating them and studying them as biochemical entities. 00:07:53.19 The first organelle that he was able to understand is the endoplasmic reticulum. 00:07:59.17 Here you see a section through a cell of the pancreas. 00:08:04.20 These cells in the pancreas are differentiated. 00:08:08.04 They are already developed to their full potential. 00:08:11.25 Their major role is in the production, packaging, and secretion of proteins that go in... eventually, 00:08:21.03 into the gut or into the bloodstream, and as a result the network responsible for 00:08:27.14 the manufacture of these proteins is highly elaborate. 00:08:30.25 In a cell of the pancreas that is differentiated to make proteins for export, the endoplasmic reticulum, 00:08:38.09 this network of membranes, can have a surface area that is 25-fold greater 00:08:45.14 than the surface area of the membrane that surrounds the cell. 00:08:49.02 So, it's an enormous and ela... and quite elaborate platform. 00:08:54.00 And you'll note that these platforms are studded with ribosomes, each of which is acting to 00:09:02.20 produce a protein molecule, 00:09:05.11 which will eventually find its way across the membrane of the endoplasmic reticulum 00:09:10.20 to rest in the clear luminal space. 00:09:14.05 This luminal space then represents a kind of a canal system within the cell, a large 00:09:22.27 fluid volume, collecting molecules that have passed the barrier of the endoplasmic reticulum membrane, 00:09:29.23 and are poised to be shipped along this canal network through the cell, by steps 00:09:36.10 that I will elaborate over the next few minutes. 00:09:38.28 Now, you can get a better sense of the dimensional arrangement of this endoplasmic reticulum 00:09:46.04 in the cartoon shown on my next slide. 00:09:48.00 You see here that it is not just a set of tubules, but it's actually a set of sheets 00:09:54.23 of leaflets that... envelopes that spread throughout the cytoplasm, and can occupy 00:10:01.18 a great fraction of the cell. 00:10:04.10 Most of this membrane has the ribosomes studding its surface, but there are also parts of it 00:10:09.23 that are smooth, that are free of ribosomes, that may represent transitional zones 00:10:16.17 from which molecules become packaged into vesicles that convey this material downstream in the pathway, 00:10:23.28 as you'll see. 00:10:26.06 Now, what Palade did to pursue this understanding of the function of the endoplasmic reticulum 00:10:34.02 was to devise techniques to break cells open, to take tissue, to homogenize, to break cells open, 00:10:41.04 and then to obtain partially and eventually highly purified fractions of membranes 00:10:48.06 that could be studied for their molecular composition and their biosynthetic potential. 00:10:53.25 Here is a very simple first step that Palade and his colleagues, Christian de Duve and 00:11:01.24 Albert Claude, devised to begin to fractionate membrane organelles. 00:11:06.06 So, one starts with a tissue, a pancreatic tissue, for instance, and this tissue 00:11:12.08 can be disrupted by a physical agitation to break the cells open, but to preserve membranes 00:11:18.20 relatively intact in a cell homogenate or cell lysate. 00:11:23.22 Now, in this lysate, if the cells have been gently broken, membranes retain 00:11:30.16 different sizes and shapes. 00:11:32.09 And they can begin to be separated from one another by a series of steps, of centrifugation steps, 00:11:38.18 where the homogenate is placed in a centrifuge tube and sedimented at different speeds. 00:11:44.24 At very low speed of sedimentation, large membranes, for example the nucleus, 00:11:51.16 sediment out of suspension to form a pellet at the bottom of the tube. 00:11:56.02 At medium speeds of centrifugation, other somewhat smaller organelles, like the mitochondrion, 00:12:01.24 the lysosome, or the peroxisome, can be sedimented and obtained in a slightly enriched form. 00:12:08.26 And then, at higher speeds of sedimentation, very small membranes, small vesicles, 00:12:15.15 eventually sediment out of suspension and form a pellet at the bottom of the tube. 00:12:19.24 And so these distinct pellet fractions can be examined for their biochemical composition 00:12:26.23 and for their structure, as seen in the microscope. 00:12:30.13 Now, another principle that Palade perfected to... specifically to isolate those membranes 00:12:37.16 that have ribosomes bound to them is shown on the next slide. 00:12:42.04 And this is a procedure where membranes are separated according not to their size 00:12:48.24 but to their buoyant density. 00:12:51.02 The membranes have distinctive buoyant density. 00:12:54.25 Membranes that are free of ribosomes tend to be more buoyant, less dense. 00:13:01.10 And they can be separated from membranes that retain ribosomes and... which are... 00:13:08.00 have a higher buoyant density. 00:13:09.19 So, in a homogenate, the sample having both membrane-bound and unbound structures 00:13:17.13 can be applied to the top of a gradient, typically a gradient of sucrose, from low to high. 00:13:24.28 And then the sample can be sedimented for a very long time so that the membranes 00:13:30.15 achieve an equilibrium buoyant density. 00:13:33.17 And the smooth membranes, lacking ribosomes, are... sediment to a position of low buoyancy, 00:13:41.13 whereas those membranes that have ribosomes sediment to a position of high buoyancy, 00:13:46.20 of high buoyant density. 00:13:48.22 Cleanly separating these two membranes. 00:13:51.08 This high buoyant density fraction is a relatively enriched source of membranes that have ribosomes, 00:13:59.14 and, as you'll see, have the ability to take protein molecules that are destined for secretion 00:14:08.01 and pass them across the membrane into the clear interior space of the organelle. 00:14:14.24 Now, I'm going to summarize work not only of Dr. Palade but principally of his protege, 00:14:24.04 another very famous cell biologist by the name of Gunter Blobel, who was able to 00:14:29.24 pursue Palade's original pioneering work using biochemical cell biology 00:14:35.26 to understand the precise mechanism 00:14:38.11 that proteins use as they pass from a ribosome across the membrane of the endoplasmic reticulum 00:14:45.14 into the clear interior space, the first step in a long sequence of events that eventually... 00:14:51.23 eventually will leave the protein molecules secreted outside of the cell. 00:14:56.05 So, here is, then, a summary of a great deal of work that Dr. Blobel achieved, 00:15:03.20 and for which he won the Nobel Prize. 00:15:07.21 We start with ribosomes that assemble together, a large subunit of the ribosome and a 00:15:13.28 small subunit of the ribosome. 00:15:15.18 They come together along with a messenger RNA, in this case, a messenger RNA that encodes 00:15:22.00 a protein that is going to be secreted. 00:15:26.22 What Dr. Blobel discovered is that proteins that are destined for secretion have a 00:15:34.22 special sequence at the very N-terminus, the beginning of the protein, that tends to be 00:15:40.04 somewhat apolar or hydrophobic. 00:15:43.12 And that sequence draws... called a signal peptide, draws the ribosome/messenger RNA/nascent 00:15:53.23 protein chain eventually to a channel in the ER membrane, through which the polypeptide 00:16:01.05 is inserted and progresses into the clear interior space, the luminal space of the ER. 00:16:08.24 In the course of the biosynthesis of this protein, Blobel discovered that the hydrophobic, 00:16:15.17 the apolar signal peptide, is clipped by a special protease in the ER membrane. 00:16:22.28 That produces the mature N-terminal domain of the secretory protein, that is now 00:16:29.16 free to fold into a functional tertiary structure in the lumen of the ER. 00:16:37.28 Folded properly and ready to progress through the pathway. 00:16:41.09 So, this call... eventually called the signal hypothesis, predicted the existence of a channel. 00:16:49.26 And in my next lecture, I'll tell you about how my laboratory was able to use genetics 00:16:56.01 to discover the genes that encode this channel. 00:17:00.23 Now, after molecules have folded and are ready to go, they are ready to perform their function, 00:17:10.19 eventually outside of the cell, they are recognized and conveyed in vesicles, that I'll describe 00:17:15.27 in my next lecture, to the next station in the secretory pathway, a structure called 00:17:22.11 the Golgi apparatus. 00:17:23.21 Here is a depiction of the Golgi apparatus. 00:17:26.03 It kind of looks like a stack of pancakes, although in three dimensions it's a rather 00:17:34.01 more complex organelle, where the membranes are interrelated, not only stacked 00:17:40.15 one on top of the other, but have tubular connections. 00:17:43.28 This was a structure that was first described in the 19th century by an Italian cytologist 00:17:48.26 by the name of Camillo Golgi, who... 00:17:51.26 whose discovery was based on his finding of a dye, a chemical dye, that highlighted 00:18:01.28 this membrane in nerve cells. 00:18:04.01 It highlighted this membrane at the expense of other membranes. 00:18:06.23 We now know that this dye that Golgi devised recognizes carbohydrate. 00:18:13.09 And carbohydrate is rich on glycoproteins that are packaged and conveyed through 00:18:19.00 the Golgi apparatus. 00:18:20.23 But, after this discovery in the late 19th century, very few investigators were able 00:18:28.04 to make progress. 00:18:29.17 For nearly 60 years, this organelle was considered a cellular curiosity with no obvious function. 00:18:36.24 And it was not until the 1960s and 70s, when Dr. Palade focused his vision on this structure, 00:18:43.22 were we able to deduce that it is a station, en route, between the endoplasmic reticulum 00:18:50.22 and the cell surface, through which molecules are conveyed. 00:18:54.06 Much as passengers would be conveyed through a bus station, they are conveyed through 00:18:58.25 the Golgi apparatus. 00:19:00.12 And shipped to different destinations in the cell and outside of the cell. 00:19:04.27 And I'll have more to say about this Golgi structure as time goes on. 00:19:09.24 Now, once molecules progress through this station, they are ready... they are mature, 00:19:17.06 they are ready to be encapsulated within granules that eventually convey them to the cell surface. 00:19:24.25 And there's a simple diagram that I'd like to share with you that describes what happens next, 00:19:29.27 after the Golgi apparatus. 00:19:31.14 So, here is a very simple depiction of the fate of secret... secreted molecules as they are 00:19:39.02 packaged into granules and eventually delivered to the cell surface. 00:19:43.04 So, here you see such a cartoon of a granule, that's got a membrane. 00:19:47.24 And the red dots on the in... on the inside represent molecules like insulin, that are 00:19:51.22 being manufactured inside of a beta cell of the pancreas. 00:19:56.00 At a certain time, this mature granule finds its way to the cell surface, and the membrane 00:20:02.25 of the granule merges with the membrane of the cell surface to form a continuous bilayer. 00:20:10.27 That results in the interior content of this granule being discharged to the cell exterior. 00:20:18.02 And crucially, this happens without breaking the cell, without breaching the permeability barrier 00:20:24.11 of the membrane that surrounds the cell, or else the cell would lyse. 00:20:28.15 So, you can then affect secretion of water-soluble molecules like insulin and hormones 00:20:36.27 and antibody molecules molecules by this process of membrane fusion. 00:20:42.14 And the final product, then, is seen outside of the cell. 00:20:46.12 Now, let's look at a real example from a cell that Palade visualized, showing virtually 00:20:51.24 the same thing that I've depicted in my cartoon. 00:20:54.26 At a certain crucial moment, the content of this granule, condensed in its interior 00:21:01.12 and surrounded by a biological membrane, migrates to the cell perimeter, where the two membranes, 00:21:08.13 the membrane of the granule and the membrane surrounding the cell, come to very close apposition, 00:21:16.06 so close that the cytoplasmic content between these two membranes is squeezed out. 00:21:22.13 The membranes come so close that they can approach each other within Angstroms. 00:21:27.10 And then, at a key moment, the cell receives a signal that causes the membranes to 00:21:34.05 merge by this process of membrane fusion. 00:21:36.27 And as you saw a moment ago, the interior of the granule is ejected to the cell exterior. 00:21:43.24 In this case, this granule is condensed and somewhat crystalline, but it dissolves 00:21:48.19 when it leaves the cell. 00:21:50.14 And eventually, protein molecules such as insulin are distributed into the bloodstream. 00:21:55.17 So, this is a crucial step that occurs not only in the pancreatic beta cells, but in 00:22:01.19 all cells, and virtually all cells that are manufacturing proteins. 00:22:05.17 Let me give you a couple of examples. 00:22:07.18 Here's a cell that contains a huge supply of proteins that are to be secreted. 00:22:14.04 Enormous numbers of granules are built up in this cell. 00:22:17.00 And eventually, when the cell is triggered by some stimulus... stimulant, to engage in 00:22:23.26 protein secretion, the granules all reach the cell perimeter. 00:22:27.27 And then look what happens, the cell almost appears as though it's exploding. 00:22:32.13 But the cell, in this case, still remains intact, but all of the material has been secreted 00:22:37.10 and the cell surface membrane is distorted by having accumulated a lot of this membrane 00:22:43.03 that was in granules, that now is at least temporarily merged and fused at the cell perimeter. 00:22:48.27 The cell restores itself, some of the excess membrane is taken back into the cell, it fills... 00:22:54.25 these granules are filled up, and the process can be repeated. 00:22:58.00 Now, in the brain, this process takes shape in the secretion of chemicals. 00:23:06.14 Not necessarily proteins, but chemicals, particularly chemical neurotransmitters. 00:23:11.08 And here's an example. 00:23:12.24 This is not a human brain, but this is actually the connection between a nerve cell and 00:23:19.01 a muscle cell at a structure called the neuromuscular junction. 00:23:22.22 This sample happens to be taken from a frog, but the same is true in all metazoan cells. 00:23:28.20 So, this is a nerve cell. 00:23:30.04 This is a nerve terminal. 00:23:32.15 The membrane that surrounds the nerve terminal is a... is a traditional plasma membrane. 00:23:38.00 But as you'll see, inside, in the cytoplasm of the nerve terminal, there are many small granules. 00:23:44.19 In this case, they're called vesicles or synaptic vesicles. 00:23:49.11 And these synaptic vesicles house the chemical transmitters that mediate communication 00:23:58.08 between a nerve cell and a muscle cell. 00:24:00.16 For instance, these synaptic vesicles house molecules like serotonin. 00:24:08.05 That affects mood and mood disorders in humans. 00:24:11.18 Or these synaptic vesicles may house dopamine, the chemical neurotransmitter that is responsible 00:24:19.27 for much of our movement and also affects cognition, and which is drastically reduced 00:24:28.21 in patients suffering from Parkinson's Disease. 00:24:32.13 Another very important neurotransmitter called acetylcholine, responsible for much of 00:24:38.09 the communication between nerve cells, and which is very tragically lost in patients that 00:24:46.28 succumb to Alzheimer's Disease. 00:24:48.17 So, these granules, then, are manufactured, they collect very high chemical concentrations 00:24:55.08 of neurotransmitters, and they come up right up to the cytoplasmic side of the membrane 00:25:01.26 surrounding the nerve terminal. 00:25:05.24 And you can actually visualize the process of fusion of these vesicles at the presynaptic membrane 00:25:13.12 by a very clever experiment that was first devised by John Heuser in St. Louis, 00:25:19.02 some years ago, that allowed him to stimulate a nerve terminal and then very quickly, 00:25:25.00 within milliseconds, capture images in frozen samples that allow one to actually see the membranes 00:25:33.08 begin to merge with the plasma membrane of the nerve cell. 00:25:37.15 Here is a time sequence. 00:25:39.21 A resting nerve cell, followed by stimulation and rapid processing. 00:25:45.11 Within five milliseconds, you can begin to see events where the vesicle has just 00:25:51.24 started to merge and the interior of the vesicle becomes secreted to the space, in this case, 00:25:58.22 between a nerve cell and a muscle cell. 00:26:00.16 The chemicals that diffuse into this cleft, the synapse, then bind on the muscle side 00:26:08.27 to receptors that allow a muscle cell, eventually, to contract. 00:26:13.04 So, all movement is based on this rapid communication of neurotransmitters, mediated by vesicles 00:26:23.02 that share much of the same process of secretion that we see in cells such as the beta cell 00:26:28.20 of the pancreas. 00:26:31.05 Now, Palade... 00:26:32.24 Dr. Palade didn't just take lots of pretty pictures, he did an amazing experiment 00:26:39.10 that allowed him to, in a way, visualize the stages in this process, step by step, using a combination 00:26:48.28 of pulse-chase radiolabeling, autoradiography, and thin-section electron microscopy 00:26:58.01 that gave us the picture that we now have, now 50 years later, of how this process is organized 00:27:05.04 in eukaryotic cells. 00:27:07.01 And this is, then, a simple cartoon that displays Palade's final pioneering work, 00:27:13.13 for which he won the Nobel Prize in 1974. 00:27:17.02 We know from his work that proteins originate on ribosomes bound to the endoplasmic reticulum. 00:27:23.14 They are allowed to fold in this clear interior space. 00:27:27.27 They are then packaged into little vesicles that convey material to the Golgi apparatus. 00:27:34.12 Material then flows through the Golgi apparatus. 00:27:38.23 Some is diverted from the Golgi apparatus to an intracellular organelle, such as the 00:27:44.21 lysosome, which is the... kind of digestive organ of a cell, where protein molecules 00:27:51.27 may be broken down. 00:27:53.02 Or, other granules are formed by budding at the Golgi apparatus to produce mature secretory granules 00:28:00.03 that move and, by a process of membrane fusion, discharge their content to the cell surface. 00:28:07.18 Now, I had the privilege of meeting Dr. Palade when I was a graduate student. 00:28:14.11 And then an important event in my career came when I was a postdoctoral fellow at UC San Diego. 00:28:21.26 And I heard Dr. Palade describe his pioneering work to an audience of 00:28:28.01 the American Society for Cell Biology. 00:28:30.28 This was in 1974, just as he had returned from Stockholm, having received his Nobel Prize. 00:28:37.26 And I was trained as a biochemist, not as a cell biologist. 00:28:41.28 It was clear how brilliant the work that Palade had done was. 00:28:47.02 And how revolutionary it was for the field of cell biology. 00:28:50.10 But, as a biochemist, what struck me was that this beautiful image, summarizing decades of work, 00:28:57.19 describing an obviously essential cellular process, was nonetheless devoid of 00:29:03.15 any molecular mechanistic understanding. 00:29:07.02 That is, in 1974, when Palade was recognized for his work, we didn't know about 00:29:16.08 any gene or protein molecule involved in organizing this pathway 00:29:20.11 -- nothing, literally nothing was known. 00:29:23.14 And so I resolved, when I began my career at the University of California at Berkeley, 00:29:27.16 to study this process in an organism that would allow a molecular dissection of 00:29:34.06 the mechanism of this pathway. 00:29:36.04 Everyone, until then, had studied mammalian cells or animals, where, at least in the mid-1970s, 00:29:45.16 the techniques of genetics and biochemistry were not well developed. 00:29:50.10 And so what I decided to do at the outset of my independent career was to explore this 00:29:55.13 process in a simple organism, baker's yeast. 00:30:00.02 Baker's yeast can be grown in large quantities. 00:30:02.01 Here is an image of a scanning EM picture of yeast cell, such as you might see growing 00:30:08.04 on the surface of a grape. 00:30:09.24 Yeast cells grow by a process of asymmetric budding, where a small bud emerges from the 00:30:18.08 surface of a mother cell and grows, in preference to the mother cell, during the first 90 minutes or so 00:30:25.17 of the growth of the cell, until the daughter cell approaches... achieves the size 00:30:33.23 of the mother cell, at which point they divide. 00:30:36.08 And if the nutritional conditions are correct, the cells can continue through yet another 00:30:41.07 cycle of division. 00:30:43.07 Now, yeast was a particularly important organism in the history of molecular biology because 00:30:50.03 of the use of traditional, classical genetic approaches that allow one to understand genes, 00:30:57.15 and thus proteins, that are involved in any cellular process, even essential cellular processes. 00:31:06.00 And I'd like to highlight, as a recognition of this application of genetics, 00:31:11.16 the work of a pioneering geneticist, a yeast geneticist by the name of Leland Hartwell, who was able, 00:31:19.05 using very simple visual techniques, to identify genes that are required for 00:31:27.05 the progression of cells through the cell division cycle. 00:31:30.11 He did this by introducing, using a chemical mutagen, mutations into the yeast genome. 00:31:37.10 And then looking among these mutants for those that affect a particular step in 00:31:43.11 the ability of the cell to complete its division cycle. 00:31:46.27 These are called cdc, or cell division cycle, mutants. 00:31:50.14 And each represents a mutation in a gene that is essential for cell viability. 00:31:57.02 The way you can study these genes is by obtaining mutations that are conditional in their effect, 00:32:02.05 that is, that allow the cell to grow at, for instance, room temperature, but kill the cell 00:32:07.27 when the cell is warmed to human body temperature, 37 degrees. 00:32:12.02 And so he obtained dozens of such mutations, each of which defines a gene required 00:32:18.05 for protein... for... for progression through the cell division cycle. 00:32:23.26 So, I'm going to conclude this first lecture with a simple image of a normal yeast cell 00:32:31.19 that gave my laboratory some inkling that yeast cells might be a good test system to 00:32:39.22 use the logic of Hartwell to discover the genes involved in protein export. 00:32:44.22 Here is a thin slice through a normal, a wild type, yeast cell, obviously very different 00:32:50.23 than a pancreatic cell. 00:32:53.19 It's dominated... the cytoplasm is dominated by a very high granular content of ribosomes. 00:33:00.02 But, as you can see, there are some organelles. 00:33:03.06 These are membrane organelles. 00:33:04.26 This large structure is the yeast vacuole. 00:33:08.00 It is the equivalent of a mammalian lysosome. 00:33:11.06 In other sections of this cell, you can see that the yeast cells have a nucleus. 00:33:15.21 You can also see that there are tubular membranes that are similar to the endoplasmic reticulum. 00:33:23.11 But my laboratory was particularly intrigued by the appearance of a cluster of small vesicles 00:33:32.07 that congregate under the bud portion of a dividing cell. 00:33:36.28 These vesicles seem likely to be responsible for conveying proteins for secretion into 00:33:43.07 the growing bud surface of the cell. 00:33:45.27 This is the bud of the cell. 00:33:47.25 But further, we imagined that the membrane of the vesicle would contain the building blocks 00:33:54.08 for the assembly of the plasma membrane. 00:33:57.13 And thus, by this process of membrane fusion, the vesicle would not only discharge proteins 00:34:04.28 into the cell wall that surrounds a yeast cell, but that the membrane of the vesicle 00:34:10.14 would be in a sense the building block of the plasma membrane. 00:34:14.17 So, the fundamental prediction that I'll leave you with in this part, and which I will elaborate on 00:34:19.12 in my next lecture, is that the genes involved in the production of these vesicles, we predicted, 00:34:26.28 would be required for cell growth and secretion. 00:34:31.12 And therefore the genes could only be studied by obtaining conditional or temperature-sensitive 00:34:37.10 lethal mutations. 00:34:38.10 So, we'll leave it there and pick it up in my next lecture. 00:34:41.10 Thank you.