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Lecture 9

BIO241 Lecture 9

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University of Toronto St. George
Jennifer Harris

Tuesday, February 3, 2009 - We continue with vesicle transport, we’re moving away from the Golgi, the trans Golgi network, that is basically the major branching point in vesicle transport and how do vesicles now make it to the outside or the plasma membrane and release their contents into the outside of the cell (extracellular space)? - Just to recap, this slide is what we’ve been looking at in terms of vesicle transport. We looked at vesicle transport last lecture from the trans Golgi network to the lysosome and how there’s a signal that is added on in the cis Golgi onto proteins that are to be directed to the lysosome. These are for example, lysosomal hydrolases that need to end up in the lysosome and provide the functions of the lysosome in terms of degrading macromolecules. - Just to recap, the trans Golgi network is a major branching point for proteins that are to be sorted to different compartments in vesicles. Proteins that move through the Golgi will end up in the trans Golgi network and the vesicles that bud off the trans Golgi network can have different destinations. - We looked at the lysosome, going through either the late endosome or early endosome, these will eventually mature or become the lysosome. - But vesicles can also bud off the TGN and make it to the plasma membrane, this is called exocytosis. There are two major forms of exocytosis that can occur. One is regulated and one is constitutive and basically what that means is that when the vesicle buds off, it either goes to the plasma membrane and fuses automatically, and that is the constitutive version or the vesicle can sit around in the cytoplasm and wait for a signal in order to fuse to the plasma membrane. That is called the regulated secretory pathway. - We’ll look at those two examples so basically, proteins that are going through the secretion pathway enter the ER, go through the Golgi, there are two different ways they can go through the Golgi that we looked at and then they’re sorted out at the major branching point which is the trans Golgi network to either the lysosome via the early endosomes or to the plasma membrane in a process called exocytosis. - We’ll now look at a small animation that goes through the process till where we are. Animation - The signal sequence of a membrane protein directs the ribosome to the rough ER. The growing polypeptide chain enters the lumen of the ER through a protein import channel in the membrane. There the signal sequence is cleaved, sugars are added and the protein folds. Some enzymes or structural proteins remain in the ER. Most proteins that are soluble in the ER are transported in vesicles to the cis Golgi. - In the cisternal maturation model, the cis Golgi matures to become the medial Golgi, a new cis Golgi is formed by fusion of the ER vesicles. The maturation process includes retrograde vesicular transport of resident Golgi proteins. - Proteins destined for secretion after modification in the Golgi are transported from the trans Golgi to the plasma membrane. When the membranes fuse, the proteins are released into the extracellular space. - Soluble proteins on the outside of a cell can reenter by endocytosis and are sorted to lysosomes for degradation. Other proteins, including lysosomal enzymes sort directly to lysosomes from the trans Golgi. - Looking at exocytosis, the fusion of vesicles to the plasma membrane, the uptake or formation of vesicles at the plasma membrane & uptaking extracellular components and maybe we’ll get into cell communication if we have time.  Lipids  Membrane proteins  Secreted soluble proteins - The first process we’ll look at is exocytosis and basically this is the transport from the trans Golgi network to the plasma membrane. You can see it there and there are two major versions of this: one is again the constitutive or the regulated. - Basically exocytosis is the fusion of transport vesicles with the PM. Obviously these vesicles carry something with them & these transport vesicles are carrying with them lipids. Recall that lipids are formed or synthesized in the ER so they need to be transported to the PM if the plasma membrane is to acquire any of these newly synthesized lipids. These vesicles will contain lipids that will fuse and become part of the plasma membrane. - They also contain transmembrane proteins that we saw in the animation, the squiggly lines going through the plasma membranes and those will, once they’re in the vesicle, once the vesicle fuses with the plasma membrane, it will become plasma membrane associated proteins. - They also include secreted soluble proteins. The vesicles will contain these secreted soluble proteins and once they fuse with the PM, they’ll release these proteins into the extracellular space. Unlike the membrane proteins, these will not remain associated with the membranes and then they’re free to move around outside of the cell. - Now he mentioned that there are 2 different types of exocytosis. One of them is called the constitutive secretory pathway & the other is called the regulated secretory pathway. Basically the major difference b/w the two, is that the vesicles will bud off the TGN, then you’ll have a vesicle that contains different proteins either soluble or membrane proteins, as well as lipids synthesized in the ER. This vesicle will right away fuse with the PM in an unregulated fashion, that is why it is called constitutive, b/c this vesicle won’t be regulated in terms of time when it fuses with the PM so it will form and fuse with the PM. - In contrast the regulated secretory pathway is very similar where you have again a vesicle will bud off the trans Golgi network, shown in the slide and this time the secretory vesicle with its contents, usually these are soluble proteins, that will be released outside of the cell. An example of this we’ll cover is insulin so insulin goes into this regulated secretory pathway and then these vesicles await a signal in order to fuse with the PM. This signal might be a hormone or a neurotransmitter that is recognized at the cell surface and induces an intracellular signaling pathway which might include an increase in cytosolic calcium for example. This intracellular signaling pathway regulates the fusion of this vesicle to the PM so the vesicle will form and await a signal before fusing with the PM. That is the regulated secretory pathway. - The constitutive secretory pathway is basically the default pathway, proteins that don’t have a sorting signal will go through this pathway so if you’ll recall, proteins that are destined to remain in the ER have a specific AA sequence called the KDEL sequence or the AAs K D E and L. If you were to remove that KDEL sequence on an ER localized protein, so now you take that same protein, you mutate that KDEL sequence so it no longer has it, that protein without a KDEL sequence, assuming it doesn’t have any other sorting signals, would end up in the constitutive secretory pathway and go outside the cell. It is the KDEL sequence that will retain it in the ER but if it doesn’t have that sequence, it will go through the Golgi into a vesicle and out the cell, it is the default pathway for proteins that enter the ER and don’t have any other sorting sequence. - An example is antibodies by activated B lymphocytes. The antibodies will be produced and released automatically by the B lymphocytes – they aren’t retained or wait for any signal.  Vesicles fuse with plasma membrane  Found as aggregates in TGN (trans-Golgi network) - The constitutive secretory pathway is the default pathway for proteins without a sorting signal. The regulated secretory pathway on the other hand is basically the vesicles coming out of this pathway are stored near the PM so again they’ll bud off the trans Golgi network and they will accumulate next to the PM and then they wait for a signal such as an increase in intracellular calcium. Once this signal is perceived in the cell, the vesicle will fuse with the PM and then release their contents into extracellular space. - How are these proteins sorted to these types of vesicles? The exact signal that leads to proteins ending up in the regulated secretory pathway is not very well characterized in terms of what is the specific sequence that would lead into this pathway, however it has been observed that the proteins that do end up in this pathway are found as aggregates in the trans Golgi network. They’re usually found in large aggregates in the trans Golgi network & these aggregates just keep getting larger and larger as the secretory vesicles form. Vesicles that are budding off the trans Golgi network will actually fuse together. As the trans Golgi vesicles are budding off, you can see the proteins are already aggregating in the trans Golgi network, they will bud off and form an immature secretory vesicle. What happens here is basically vesicles are continuously adding on components into this immature secretory vesicle and at the same time, clathrin coated vesicles are removing membranes and recycling back to the trans Golgi network. - What you’re getting is an increased concentration of proteins into the secretory vesicle that will form very large aggregates and eventually result in this mature secretory vesicle that is usually packed with a specific type of protein such as insulin. - One of the major cues that leads a protein into the regulated secretory pathway is usually they start to aggregate at the trans Golgi network. - This is the example of insulin. You can see here, here is a regulated secretory vesicle and it is a large crystalline structure of insulin. There are lots of insulin molecules aggregating together forming this fuzzy ball we can see under the electron microscope. There is a lot of aggregation of proteins that occur there. - You’ll recall that insulin is secreted from pancreatic beta cells. These are cells of the pancreas & remember the GLUT2 that results when you increase the blood glucose levels, there is substantial increase in the rate of glucose transport into these pancreatic beta cells & that increase is perceived & will lead to release of insulin by the regulated secretory pathway. - The elevation in blood glucose is perceived by pancreatic beta cells and this increase in blood glucose is actually translated into an increase in intracellular calcium. Increase in blood glucose = increase in intracellular calcium concentrations, this is the signal perceived to induce these vesicles to fuse with the plasma membrane and release insulin into the bloodstream. - Then you’ll recall that in fat & muscle cells, there is actually another glucose transporter, the glucose transporter 4 that when these cells perceive insulin, there is also regulated secretion of this glucose transporter 4 to the PM & then fat & muscle cells will increase their uptake of glucose. - This is an example of the regulated secretory pathway. - That is the 2 examples of exocytosis. Now onto the reverse process: endocytosis. Endocytosis is the transport into the cell from the PM. - Here is the PM and vesicles will bud off the PM and in these vesicles will be content from the extracellular space, the process involved in taking up macromolecules from the outside. One of these in example we’ll go into is cholesterol uptake & how cholesterol is taken up into the cell by endocytosis. - Another junction of endocytosis is to down regulate signaling pathways. A way that this is done is that there are receptors at the PM that will often respond to extracellular signals. These may be hormones for example. Once the signal is perceived, that receptor will signal the signaling pathway that is downstream of it & then in order to down regulate that pathway or turn it off, endocytosis can uptake receptors from the PM & bring them to the lysosome for degradation. There is degradation of the receptor that is recognizing a hormone and that can lead to down regulation of the signaling pathway. - Another example of this that we’ll also go into for the cholesterol receptor is the receptor can actually be uptaken by endocytosis and then recycled back to the plasma membrane and not diverted to the lysosome for degradation. The receptor can have 2 fates, either it can go to the lysosome, be degraded or by recycled back to the PM and continue on with its signaling. - Of course when you’re uptaking lipids for forming vesicles from the PM you’re losing lots of lipids because vesicles are forming, taking with them a little chunk of PM as they bud off and go to their intracellular compartments. If this continued indefinitely without replenishing the lipids then the PM would just get smaller and smaller until the cell disappeared. The lipids in the plasma membrane are replenished by exocytosis. You have endocytosis where the membrane is being taken up, the membrane is shrinking but exocytosis is also replenishing these lipids in the plasma membrane. - If you’ll notice in the slide that as things are coming in from the PM, they first go into the early endosome, and like the trans Golgi network can be thought of as a branching point for the secretion pathway, the early endosome can be thought of as a branching point for endocytosis b/c once things get into the early endosome, they have a number of different fates they can go through. One is to go to the lysosome for degradation, we’re going to see how receptors can do that. Another fate is to be recycled back to the PM so once a protein ends up in the early endosome, it can actually be recycled back to the plasma membrane. There are even some proteins that go directly from the early endosome back to the trans Golgi. You can see that the early endosome is a major branching point in endocytosis like the trans Golgi network is a major branching point for the secretion pathway. - VIDEO: So that is an example of phagocytosis by a neutrophil so you can see that it is ingesting large particles that may give you gas like a bacteria. Then the other example is pinocytosis & this is the example we’ll see in more detail. We won’t go into the details of phagocytosis, we will look more at pinocytosis and see what type of uptake is taken up through pinocytosis and basically in contrast to phagocytosis, pinocytosis is the uptake or the formation of small vesicles that ingest fluids and solutes. One of the major differences between the two is that phagocytosis is large vesicles or large particles that are being uptaken & pinocytosis are smaller vesicles ingesting smaller contents so basically proteins, or even solutes that are in the extracellular space. Both are different types of endocytosis though.  Large vesicles that ingest large particles, microorganisms, dead cells  Small vesicles that ingest fluids and solutes - So the 2 major types of endocytosis. The 1 one is phagocytosis & basically phagocytosis is the ingestion of large or the formation of large vesicles that ingest large particles & these large particles can actually be bacteria or even dead cells. These are huge particles ingested by macrophages for example. - In this case we will look at a type of white blood cell called a neutrophil which accumulate at sites of infection or inflammation and can ingest bacteria at these sites. This is an interesting movie to give us an idea of what phagocytosis looks like. Don’t forget that there are vesicles forming to uptake an entire bacterium into this neutrophil cell. - VIDEO: It shows the neutrophil chasing a bacterium around. Neutrophils are white blood cell that hunt and kill bacteria. In this spread, a neutrophil is seen in the midst of red blood cells. Staphylococcus bacteria have been added, this bacteria release a kemoattractant that is sensed by the neutrophil. The neutrophil becomes polarized and starts chasing the bacteria. The bacteria bounced around by thermal energy move in a random path seeming to avoid their predator. Eventually, the neutrophil catches up with the bacteria and engulfs it by phagocytosis. - The two examples are clathrin coated vesicles forming at the PM in what are called clathrin coated pits. The plasma membrane if you look at one, have certain areas that you can see pits forming and these pits are just basically indentations in the plasma membrane and at the bottom of these indentations, you can see molecules of clathrin forming and these clathrin coated pits will eventually form clathrin coated vesicles. These are sites of pi
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