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.