Randy Schekman (HHMI & UCB) 1: Secretory Pathway: How cells package & traffic proteins for export

Summary

In this insightful video presentation, Randy Schekman, a molecular biologist from UC Berkeley, discusses the cellular secretory pathway, which enables cells to package and deliver proteins outside the cell. He examines the historical context of biological membranes, focusing on their role in protein encapsulation and transportation. Schekman delves into the molecular level using examples from simple organisms like Saccharomyces cerevisiae (baker's yeast), explaining the genetic and biochemical methodologies that furthered our understanding of this complex biological process. He concludes by highlighting recent experiments related to small RNA molecule packaging within vesicles and their intercellular communication roles.

Highlights

• Randy Schekman's lecture details how cells export internally made proteins 🧬.
• The presentation covers the functionality of biological membranes in organizing cell operations like protein transport 📜.
• Saccharomyces cerevisiae serves as a model organism for understanding genetic and biochemical processes in protein trafficking 🚚.
• Schekman describes innovations in visualizing cellular structures and functions using electron microscopy 📷.
• Recent studies on RNA packaging into vesicles and their communicative role between cells add a new dimension to cellular biology 📡.

Key Takeaways

• The secretory pathway is vital for cells to export proteins made inside to the outside environment 🌍.
• Biological membranes, particularly the endoplasmic reticulum and Golgi apparatus, play crucial roles in protein packaging and transport 📦.
• Using baker's yeast, scientists can study genes involved in cellular processes due to their simpler structure 🧫.
• Techniques like pulse-chase radiolabeling and electron microscopy were instrumental in understanding the secretory pathway 🔬.
• Randy Schekman used genetics in yeast to dissect mechanisms of the pathway, earning him significant accolades in the field 🏅.

Overview

Randy Schekman's engaging lecture guides us through the secretory pathway — an essential cellular process. By illustrating the journey of proteins from inside the cell to the external environment, Schekman provides an in-depth look at how cells maintain functionality through dynamic processes like protein folding and transport.

He artfully describes groundbreaking techniques developed over the decades, from electron microscopy enhancements to genetic manipulation in simple organisms like yeast, which illuminate our understanding of complex cellular mechanisms. These histories not only celebrate scientific milestones but demonstrate the intricate dance of biological components working in unison.

The talk culminates in a modern exploration of RNA vesicle packaging, highlighting how cutting-edge research continues to reveal new layers of cellular complexity. This evidence-based journey is both educational and inspiring, showcasing the relentless pursuit of knowledge that defines molecular biology.

Randy Schekman (HHMI & UCB) 1: Secretory Pathway: How cells package & traffic proteins for export Transcription

• 00:00 - 00:30 hello my name is Randy Schekman I'm at the University of California at Berkeley in the department of molecular and cell biology today I'll be giving three presentations on the cellular process that is used to package protein molecules that are made inside the cell but have to be shipped outside of the
• 00:30 - 01:00 cell in my first presentation I'll discuss some of the historical aspects of how we learned about biological membranes and how they are deployed to encapsulate protein molecules as they are made inside the cell in my next lecture I'll describe how this process was understood at the molecular level using a simple nucleated organism a simple you carry out called
• 01:00 - 01:30 Saccharomyces cerevisiae or baker's yeast and i'll tell you about both genetic and biochemical approaches to understanding this process and finally in my last lecture I'll discuss some very recent experiments on how cells package small RNA molecules that are encapsulated into vesicles that are discharged at the cell exterior and may communicate between cells in our body but let's begin with a discussion of how
• 01:30 - 02:00 cells are organized the basic principle of organization of cells comes with an understanding of the structure of a biological membrane that's a depicted on my first slide so here you see a cartoon of a biological membrane consisting of two leaflets of molecules called lipids or phospholipids they are shown here with
• 02:00 - 02:30 red balls that are the water-loving or hydrophilic head groups of a phospholipid molecule connected to these thin tails they are the water-hating or hydrophobic fatty acid side chains that constitute the core of the membrane bilayer two leaflets of lipids come together to form this bilayer embedded in a biological
• 02:30 - 03:00 membrane are these green structures they are protein they depict protein molecules some of them go clear through the bilayer as you see here this example is maybe for instance a channel in the membrane through which small molecules may come and go or this example may be of a protein that is a receptor that sits on the outside of the cell and recognizes hormones that may interact with a cell to convey information to the
• 03:00 - 03:30 cell interior now all membranes in cells have this basic structure but each membrane in the cell has a different kind of personality and they go from very simple organizations to very complex organizations here for instance is the simplest cell this is a red blood cell coursing through your bloodstream it consists at least in a human of a single membrane surrounding an internal
• 03:30 - 04:00 cytoplasm that is filled with hemoglobin the protein molecule in your red cells that carries oxygen to your peripheral tissues so just a simple cell with a single membrane but in contrast for example a much more complex cell this is a very important cell in your pancreas it's called the beta cell in the islets of langerhans it's responsible for
• 04:00 - 04:30 manufacturing insulin that is discharged outside of the cell carried within the cell by these little granules these these look like little i-ight spots but they're granules that how is insulin and convey it through the cytoplasm to the cell surface where it is discharged by a process called membrane fusion that we'll discuss in a few minutes so enormous difference in
• 04:30 - 05:00 complexity between in a cell that has many functions such as in the pancreas or a simple cell such as the red blood cell now a cartoon of the various membrane organelles that are found inside of the cell is depicted here this is a cell from an epithelium surrounding a tissue that is responsible for making many protein molecules that are shipped to different places in and
• 05:00 - 05:30 out of the cell at the base of the cell you see the nucleus housing the chromosomes surrounding that nucleus our membranes that constitute a network called the endoplasmic reticulum that are dotted with these little particles their ribosomes ribosomes are the machines that stitch amino acids one next to another to make protein molecules that are often transmitted
• 05:30 - 06:00 across a membrane into this clear space of the endoplasmic reticulum and we'll talk about that in a few minutes there are many other membrane organelles nacelle the powerhouse organelle the mitochondrion a structure called the Golgi apparatus through which protein molecules are conveyed and other membranes that have specialized functions like the peroxisome or the endosome these all all have biological
• 06:00 - 06:30 membranes surrounding them and each has different protein molecules that execute the unique functions of these organelles now much of what we know about the organization of an animal cell came from the pioneering work of cell biologists in the middle part of the 20th century prominent among them was a brilliant cell biologist by the name of George Pilate dr. Pilate was an émigré from
• 06:30 - 07:00 Romania he came to New York where he established his laboratory at the Rockefeller University in the mid 1950s it was pilate who discovered the ribosome he did as with much of the rest of the of his work by perfecting an instrument called the electron microscope which you see you see here him seated behind on he and Keith Porter and other scientists at the
• 07:00 - 07:30 Rockefeller devised procedures to fix cells and tissues and to preserve them so that they could be sectioned in a diamond knife and then visualized under an intense electron beam where the electrons were scattered by structures within the cell and all of the beautiful pictures some of which I'll show you were interpreted by him to understand many of the functions of membranes that
• 07:30 - 08:00 communicate with one another by the process of protein secretion now let's go through step by step each of the organelles that Pilate and his students were able to appreciate both by visualizing them in the electron microscope but also by isolating them and studying them as biochemical entities the first organelle that he was able to understand is the endoplasmic reticulum here you see a section through a cell of
• 08:00 - 08:30 the pancreas these cells in the pancreas are differentiated they're already developed to their full potential their major role is in the production packaging and secretion of proteins that go in eventually into the gutter into the bloodstream and as a result the network responsible for the manufacture of these proteins is highly elaborate in
• 08:30 - 09:00 a Cell of the pancreas that is differentiated to make proteins for export the endoplasmic reticulum this network of membranes can have a surface area that is 25 fold greater than the surface area of the membrane that surrounds the cell so it's an enormous and will end a quite elaborate platform and you'll note that these platforms are studded with ribosomes each of which
• 09:00 - 09:30 is acting to produce a protein molecule which will eventually find its way across the membrane of the endoplasmic reticulum to rest in the clear luminal space this luminal space then represents a kind of a a canal system within the cell a large fluid volume collecting molecules that have passed the barrier of the endoplasmic reticulum membrane and are poised to be shipped along this
• 09:30 - 10:00 canal network through the cell by steps that I will elaborate over the next few minutes now you can get a better sense of the dimensional arrangement of this endoplasmic reticulum in the cartoon shown on my next slide you see here that it is not just a set of tubules but it's actually a set of sheets of leaflets that are envelopes that spread throughout the cytoplasm and can occupy
• 10:00 - 10:30 a great fraction of the cell most of this membrane has the ribosomes studying its surface but there are also parts of it that are smooth that are free of ribosomes that may represent transitional zones from which molecules become packaged into vesicles that convey this material downstream in the pathway as you'll see now what clotty did to pursue this understanding of the
• 10:30 - 11:00 function of the endoplasmic reticulum what to devise techniques to break cells open to take tissue homogenized to break cells open and then to obtain partially and eventually highly purified fractions of membranes that could be studied for their molecular composition and their bio synthetic potential here is a very simple first step that Pilate and his colleagues Christians Adu and Albert
• 11:00 - 11:30 claude devised to begin to fractionate membrane organelles so one starts with a tissue a pancreatic tissue for instance and this tissue can be disrupted by a physical agitation to break the cells open but to preserve membranes relatively intact in a Cell homogenate or cell lysate now in this lysate if the cells have been gently broken membranes retain different sizes and shapes and they can
• 11:30 - 12:00 begin to be separated from one another by a series of steps of centrifugation steps where the homogenate is placed in a centrifuge tube and sediment it at different speeds at very low speed of sedimentation large membranes for example the nucleus sediment out of suspension to form a pellet at the bottom of the tube at medium speeds of centrifugation other somewhat smaller organelles like the mitochondrion the
• 12:00 - 12:30 lysosome or the peroxisome can be sedimented and obtained in a slightly enriched form and then at higher speeds of sedimentation very small membranes small vesicles eventually sediment out of suspension and form a pellet at the bottom of the tube and so these distinct pellet fractions can be examined for their biochemical composition and for their structure as seen in the microscope now another principle that
• 12:30 - 13:00 Pilate perfected too specifically to isolate those membranes that have ribosomes bound to them is shown on the next slide this is a procedure where membranes are separated according not to their size but to their buoyant density the membranes have distinctive buoyant density membranes that are free of ribosomes tend to be more buoyant less
• 13:00 - 13:30 dense and they can be separated from membranes that retain ribosomes and which are have a higher buoyant density so in a homogenate the sample having both membrane bound and unbound structures can be applied to the top of a gradient typically a gradient of sucrose from low to high and then the sample can be sedimented for a bear long time so that the membranes achieve
• 13:30 - 14:00 an equilibrium buoyant density and the smooth membranes lacking ribosomes are sediment to a position of low buoyancy whereas those membranes that have ribosomes sediment to position of high buoyancy of high buoyant density cleanly separating these two membranes this high buoyant density fraction is a relatively enriched source of membranes that have ribosomes and as you'll see have the
• 14:00 - 14:30 ability to take protein molecules that are destined for secretion and pass them across the membrane into the clear interior space of the organelle now I'm going to summarize work not only of dr. Pilate but a principally of his protege another very famous cell biologist by the name of Gunter blobel who was able to pursue Pilates original
• 14:30 - 15:00 pioneering work using biochemical cell biology to understand the precise mechanism that proteins views as they pass from a ribosome across the membrane of the endoplasmic reticulum into the clear interior space the first step and a long sequence of events that alleged eventually will leave the protein molecules secreted outside of the cell so here is then a summary of a great deal of work that dr. global achieved
• 15:00 - 15:30 and for which he won the Nobel Prize we start with ribosomes that assemble together a large subunit of the ribosome and a small subunit of the ribosome they come together along with a messenger RNA in this case a messenger RNA that encodes a protein that is going to be secreted oh what doctor global discovered is that proteins that are
• 15:30 - 16:00 destined for secretion have a special sequence at the very end terminus the beginning of the protein that tends to be somewhat a polar or hydrophobic and that sequence draws called a signal peptide draws the ribosome messenger RNA nascent protein chain eventually to a channel in the ER membrane through which the polypeptide
• 16:00 - 16:30 is inserted and progresses into the clear interior space the luminal states of the ER in the course of the biosynthesis of this protein global discovered that the hydrophobic the a polar signal peptide is clipped by a special protease in the ER membrane that produces the mature n terminal domain of the secretory protein that is now free to fold into a functional tertiary
• 16:30 - 17:00 structure in the lumen of the ER folded properly and ready to progress through the pathway so this call eventually called the signal hypothesis predicted the existence of a channel and in my next lecture I'll tell you about how my laboratory was able to use genetics to discover the genes that encode this channel now after molecules have folded
• 17:00 - 17:30 and are ready to go they are ready to perform their function eventually outside of the cell they are recognized and conveyed in vesicles that I'll describe in my next lecture to the next station in the secretory pathway a structure called the Golgi apparatus here is a depiction of the Golgi apparatus it's kind of looks like a stack of pancakes although in three
• 17:30 - 18:00 dimensions it's a rather more complex organelle where the membranes are interrelated not only stacked one on top of the other but have tubular connections this was a structure that was first described in the 19th century by an Italian psychologist by the name of Camillo Golgi who whose discovery was based on his finding of a dye a chemical dye that highlighted this
• 18:00 - 18:30 membrane in nerve cells it highlighted this membrane at the expense of other membranes we now know that this dye that Golgi devised recognizes carbohydrate and carbohydrate is rich on glycoproteins that are packaged and conveyed through the Golgi apparatus but after this discovery in the late 19th century very few investigators were able to make progress for nearly 60 years
• 18:30 - 19:00 this organelle was considered a cellular curiosity with no obvious function and it was not until the 1960s and 70s when dr. Pilate focused his vision on this structure were we able to deduce that it is a station on route between the endoplasmic reticulum and the cell surface through which molecules are conveyed much as passengers would be conveyed through a bus station they are conveyed through the Golgi apparatus and
• 19:00 - 19:30 shipped to different destinations in the cell and outside of the cell I'll have more to say about this Golgi structure as time goes on now once molecules progress through this station they are ready they are mature they are ready to be encapsulated within granules that eventually convey them to the cell surface and there's a simple diagram that I'd like to share with you that describes what happens next after the
• 19:30 - 20:00 Golgi apparatus so here is a very simple depiction of the fate of secrets secreted molecules as they are packaged into granules and eventually delivered to the cell surface so here you see such a cartoon of a granule that's got a membrane and the red dots on the end on the inside represent molecules like insulin that are being manufactured inside of a beta cell of a pancreas at a certain time this mature granule finds
• 20:00 - 20:30 its way to the cell surface and the membrane of the granule merges with the membrane of the cell surface to form a continuous bilayer that results in the interior content of this granule being discharged to the cell exterior and crucially this happens without breaking the cell without breaching the permeability barrier of the membrane that surrounds the cell or of the cell with lice so you can then
• 20:30 - 21:00 affect secretion of water-soluble molecules like insulin and hormones and antibody molecules by this process of membrane fusion and the final product then is seen outside of the cell now let's look at a real example from a cell that Pilate visualized showing virtually the same thing that I've depicted in my cartoon at a certain crucial moment the content of this granule condensed in its
• 21:00 - 21:30 interior and surrounded by a biological membrane migrates to the cell perimeter where the two membranes the membrane of the granule and the membrane surrounding the cell come to very close opposition so close that the cytoplasmic content between these two membranes is squeezed out the membranes come so close that they can approach each other within angstroms and then at a key moment the cell receives a signal that causes the
• 21:30 - 22:00 membranes to merge by this process of membrane fusion and as you saw a moment ago the interior of the granule is ejected to the cell exterior in this case this granule is condensed in somewhat crystalline but it dissolves when it leaves the cells and eventually protein molecules such as insulin are distributed into the bloodstream so this is a crucial step that occurs not only in the pancreatic beta-cell but in all
• 22:00 - 22:30 cells and virtually all cell that are manufacturing protein let me give you a couple of examples here's a cell that contains a huge supply of proteins that are to be secreted enormous numbers of granules are built up in this cell and eventually when the cell is triggered by some stimulant to engage in protein secretion the granules all reach the cell perimeter and then look what happens the cell is almost appears as though it's exploding
• 22:30 - 23:00 but the cell in this case still remains intact but all of the material has been secreted and the cell surface membrane is distorted by having accumulated a lot of this membrane that was in granules that now is at least temporarily merged and fused at the cell perimeter the cell restores itself some of the excess membranes taken back into the cell it feels these granules are filled up in the process can be repeated now in the brain this process takes shape in the
• 23:00 - 23:30 secretion of chemicals not necessarily proteins but chemicals particularly chemical neurotransmitters and here's an example this is not a human brain but this is actually the connection between a nerve cell and a muscle cell at a structure called the neuromuscular Junction this sample happens to be taken from a frog but the same is true in all metazoan cells so this is a nerve cell this is a nerve terminal the membrane
• 23:30 - 24:00 that surrounds the nerve terminal is up is a traditional plasma membrane but as you'll see inside in the cytoplasm of the nerve terminal there are many small granules in this case they're called vesicles or synaptic vesicles and these synaptic vesicles house the chemical transmitters that mediate communication between a nerve cell in a muscle cell for instance these synaptic vesicles
• 24:00 - 24:30 house molecules like serotonin that affects mood and mood disorders in humans or these synaptic vesicles may house dopamine the chemical neurotransmitter that is responsible for much of our movement and also affects cognition and which is drastically reduced in patients suffering from Parkinson's disease
• 24:30 - 25:00 another very important in center called acetylcholine responsible for much of the communication between nerve cells and which is very tragically lost in patients that succumb to Alzheimer's disease so these granules then are manufactured they collect very high chemical concentrations of neurotransmitters and they come up right up to the cytoplasmic side of the membrane surrounding the
• 25:00 - 25:30 nerve terminal and you can actually visualize the process of fusion of these vesicles at the presynaptic membrane by very clever experiment that was first devised by John Heiser in st. Louis some years ago that allowed him to stimulate a nerve terminal and then very quickly within milliseconds capture images in frozen samples that allow one to
• 25:30 - 26:00 actually see the membranes begin to merge with the plasma membrane of the nerve cell here is a time sequence a resting nerve cell followed by stimulation and rapid processing within five milliseconds you can begin to see events where the vesicle has just started to merge and the interior of the vesicle becomes secreted to the space in this case between a nerve cell and a muscle cell the chemicals that diffuse
• 26:00 - 26:30 into this cleft the synapse then bind on the muscle side to receptors that allow a muscle cell eventually to contract so all movement is based on this rapid communication of neurotransmitters mediated by vesicles that share much of the same process of secretion that we see in cells such as the beta cells of the pancreas
• 26:30 - 27:00 now Pilate dr. Pilate didn't just take lots of pretty pictures he did an amazing experiment that allowed him to in a way visualize the stages in this process step by step using a combination of pulse chase radio labeling Auto radiography and thin section electron microscopy that gave us the picture that we now have now 50 years later of how
• 27:00 - 27:30 this process is organized in eukaryotic cells and this has been a simple cartoon that this displays Pilates final pioneering work for which you won the Nobel Prize in 1974 we know from his work that proteins originated on ribosomes bound to the endoplasmic reticulum they are allowed to fold in this clear interior space they are then packaged into little vesicles that
• 27:30 - 28:00 convey material to the Golgi apparatus material then flows through the Golgi apparatus some is diverted from the Golgi apparatus to an intracellular organelles such as the lysosome which is the kind of digestive organ of a cell where protein molecules may be broken down or other granules are formed by budding at the Golgi apparatus to produce mature secretory granules that
• 28:00 - 28:30 move and by a process of membrane fusion discharge their contents of the cell surface now I had the privilege of meeting dr. Pilate when I was a graduate student and then an important event in my career came when I was a postdoctoral fellow at UC San Diego and I heard dr. Pilate described his pioneering work to an audience of the American Society for cell biology this
• 28:30 - 29:00 was in 1974 just as he had returned from Stockholm having received his Nobel Prize and I was trained as a biochemist not as a cell biologist it was clear how brilliant the work that Pilate had done was and how revolutionary it was for the field of cell biology but as a biochemist what struck me was that this beautiful image summarizing decades of work describing an obviously essential
• 29:00 - 29:30 cellular process was nonetheless devoid of any molecular mechanistic understanding that is in 1974 when Pilate was recognized for his work we didn't know about any gene or protein molecule involved in organizing this pathway nothing literally nothing was known and so I resolved when I began my career at the University of California at Berkeley to study this process in an organism that would allow
• 29:30 - 30:00 a molecular dissection of the mechanism of this pathway everyone until then had studied mammalian cells or animals where at least in the mid-1970s the techniques of genetics and biochemistry were not well developed and so what I decided to do at the outset of my independent career was to explore this process in a simple organism baker's yeast baker's
• 30:00 - 30:30 yeast can be grown in large quantities here is an image of a scanning eeehm picture of yeast cells such as you might see growing on the surface of a grape yeast cells grow by a process of asymmetric budding where a small bud emerges from the surface of a mother cell and grows in preference to the mother cell during the first 90 minutes or so of the growth of the cell until the daughter cell achieves the size of
• 30:30 - 31:00 the mother cell at which point they divide and if the nutritional conditions are correct the cell can continue through yet another cycle of division now yeast was particularly important organism in the history of molecular biology because of the use of traditional classical genetic approaches that allow one to understand genes and thus proteins that are involved in any
• 31:00 - 31:30 cellular process even essential cellular processes and I'd like to highlight as a recognition of this application of genetics the work of a pioneering geneticist Easton the name of Leyland Hartwell who is able using very simple visual techniques to identify genes that are required for the progression of cells through the cell division cycle he did this by
• 31:30 - 32:00 introducing using a chemical mutagen mutations into the East genome and then looking among these mutants for those that affect a particular step in the ability of the cell to complete its division cycle these are called CDC or cell division cycle mutants and each represents a mutation in a gene that is essential for cell viability the way you can study these genes is by obtaining mutations that are conditional in their
• 32:00 - 32:30 effect that is that allow the cell to grow at for instance room temperature but till the cell when the cell is warm to human body temperature 37 degrees and so he obtained dozens of such mutations each of which defines a gene required for protein for for progression through the cell division cycle so I'm going to conclude this first lecture with a simple image of a normal yeast cell that
• 32:30 - 33:00 gave my laboratory some inkling that yeast cells might be a good test system to have used the logic of Heartwell to discover the genes involved in protein export here is a thin slice through a normal a wild type yeast cell obviously very different than a pancreatic cell it's dominated the cytoplasm is dominated by a very high granular content of ribosomes but as you can see
• 33:00 - 33:30 there are some organelles these are membrane organelles this large structure is the yeast vacuole it is the equivalent of a mammalian lysosome in other sections of this cell you can see that yeast cells have a nucleus you can also see that there are tubular membranes that are similar to the endoplasmic reticulum but my laboratory was particularly intrigued by the appearance of a cluster of small vesicles that
• 33:30 - 34:00 congregate under the bud portion of the dividing cell these vesicles seem likely to be responsible for conveying proteins for secretion into the growing bud surface of a cell this is the bud of the cell but further we imagine that the membrane of the vesicle would contain the building blocks for the assembly of the plasma membrane and thus by this process of membrane fusion the vesicle
• 34:00 - 34:30 would not only discharge proteins into the cell wall that surrounds the yeast cell but that the membrane of the vesicle would be in a sense the building block of the plasma membrane so the fundamental prediction that I'll leave you with in this part in which I will elaborate on in my next lecture is that the genes involved in the production of these vesicles we predicted would be required for cell growth and secretion
• 34:30 - 35:00 and therefore the genes could only be studied by obtaining conditional or temperature sensitive lethal mutations so we'll leave it there and pick it up in my next lecture thank you you