Bio 2B03 Module 5 Lecture 2 Script PDF

BIO2B03 scripts Module 5, lecture 2 Script Notes Slide 1 Welcome back to Biology 2B03: Cell Biology. Today, we will wrap up Module 5, Leaving the Cell, with Lecture 2, Vesicular trafficking through the e...

BIO2B03 scripts Module 5, lecture 2 Script Notes Slide 1 Welcome back to Biology 2B03: Cell Biology. Today, we will wrap up Module 5, Leaving the Cell, with Lecture 2, Vesicular trafficking through the endomembrane system. So let’s get started! Slide 2 Experimental evidence has revealed the pathway followed by proteins after co-translational transport in the rough ER. In this lecture we will describe the steps in protein transport away from the ER; and in particular, we will look at experimental evidence and look at the model for protein transport through the Golgi complex and; and finally, we will identify the proteins and mechanisms involved in vesicle trafficking. Slide 3 This cartoon summarizes the path that proteins follow from the rough ER to the cell membrane. While rough ER- resident proteins will stay in the ER, many other proteins will leave via vesicles for destinations such as the Golgi complex, the lysosome, the cell membrane, or secretion out of the cell. As proteins move from the rough ER towards the cell membrane they are transported in vesicles and through the Golgi complex. The Golgi complex is comprised of a series of elongated, flat sacs called cisternae. Vesicles transport proteins from the rough ER to the Golgi cis-cisternae. Vesicles also transport proteins away from the Golgi trans- cisternae. At the left of the image, we see that vesicles fuse with the cell membrane to release proteins from the cell in a process called exocytosis. The constitutive secretory pathway is used by proteins that are released immediately after protein synthesis and transport. The secretory vesicles move straight from the trans- Golgi to the cell membrane. I n c o n t r a s t , t h e regulated secretory pathway is used by proteins that are kept in the cell until a signal triggers release. The secretory vesicles held in the cell are called secretory granules. As we see on the right, other proteins are carried away from the Golgi trans- cisternae in vesicles to form the lysosome. These vesicles will fuse with vesicles formed at the cell membrane called endosomes, that capture and transport macromolecules from outside of the cell. Slide 4 The Golgi complex can be seen in these microscopy images. At the top is a Nomarski or DIC image of the cell in which we can see all of the endomembrane system. How can we distinguish the membrane of the Golgi complex from the membrane of other organelles? Rather than an antibody, we can use a fluorescently- labeled wheat germ agglutinin to recognize the Golgi complex. Wheat germ agglutinin is a lectin that recognizes N-linked polysaccharides found in the Golgi cisternae. At the bottom, the fluorescent image of the same cell allows us to distinguish the subset of endomembranes that comprise the Golgi complex. Slide 5 Now the Golgi complex is not a single organelle, but instead a region that contains a series of elongated vesicles sacs, called cisternae. In this transmission EM image of the Golgi complex, we can see the different compartments of the Golgi complex. The cis-Golgi network is a collection of coalescing vesicles emanating from the ER to form the cis-cisternae. The medial cisternae are in the middle of the complex. The trans-cisternae are furthest from the ER and these break out into vesicles to form the trans-Golgi network. In a three-dimensional reconstruction of the Golgi complex, the cisternae are pseudo-coloured to reveal the different regions from cis-cisternae (shown in red) to trans-cisternae (shown in turquoise). An important aspect of the Golgi network revealed in this image is the large number of mobile spherical vesicles (shown in white), that are associated throughout the Golgi complex. The entire complex is very dynamic as vesicles fuse to form new cisternae, the cisternae change shape and move up through the complex, and vesicles form from membranes of the various cisternae. The Golgi cisternae themselves contain resident proteins that are necessary as well, for further post-translational modification (or PTMs) of transported proteins. Slide 6 Movement of proteins from the rough ER to the cell membrane is called anterograde transport, which means moving in the forward direction (away from the nucleus). While vesicles are important for this movement, proteins must move through the Golgi complex before reaching their destinations at the cell surface or elsewhere. B u t how are proteins moving from the cis-to medial- to the trans-cisternae of the Golgi complex? W e l l , there are two models that can describe transport through the Golgi. Slide 7 Model A suggests that vesicles carrying protein cargo (shown here as red dots) move from cis- to medial- cisternae and from medial- to trans-cisternae. The small red arrows represent the anterograde movement of vesicles with cargo from one cisterna to the next. Model B suggests that proteins (shown again as red dots ) stay in cisternae, but that the cisternae themselves are moving forward through the Golgi complex. This model still requires the use of vesicles that move backwards. These are represented by the empty vesicles moving in a retrograde direction (seen with the small purple arrows). So these are two possible models. What experiments might help us differentiate between the two models? Slide 8 By labeling different types of proteins and following their movement in the cell, we can see which pattern of movement is consistent with our models. In these immuno-TEM images, we can visualize a protein specific antibody to one of two proteins. At the top is an antibody to a cell membrane protein. We expect this protein to move in the anterograde direction through the Golgi towards its target destination. If we look at the image, the cell membrane protein is found only in the cisternal sacs, but not in any associated vesicles. This suggests that vesicles are not required for anterograde movement. The alternative is that the protein stays in a cisterna as it moves forward through the complex. Now one imageis not enough to draw this conclusion, but researchers looked at many such images and never saw this cell membrane protein localized to vesicles. At the bottom is an antibody to a resident medial- Golgi protein, a protein that should only be found in the medial-Golgi. If cisternae are moving en masse through the Golgi complex, a medial-Golgi cisterna would eventually move to the position of a trans- Golgi cisterna. Now, while cargo has moved in the anterograde direction, medial-Golgi resident proteins are mis-localized. This requires the resorting of proteins in the retrograde direction in vesicles. We can see the antibodies to the medial-Golgi protein in the medial Golgi cisternae, but we can also identify vesicles containing the protein. This observation is consistent with the model that suggests that the cisternae move forward through the complex. Together, these two observations are consistent with Model B, the cisternal maturation model. This is also known as the cisternal progression model. This model is generally accepted as the mechanism for protein transport through the Golgi. Slide 9 This animation illustrates the cisternal maturation model. Specifically, cisternal sacs plus cargo move in the anterograde fashion through the Golgi complex (that is towards the top in this animation). As a result, cisternae are defined by their location within the complex: cis-Golgi become medial-Golgi, and medial-Golgi become trans Golgi. In this short animation, the movement of the Golgi cisternae can be seen. The different compartments of the Golgi are coloured to allow you to follow them as they move. The implications of this model are that new cis- cisternae will be formed by coalescing vesicles from the ER, that the trans-Golgi network will dissipate into secretory vesicles, and that Golgi-resident proteins will be mislocalized. Finally, vesicles are used to re-sort Golgi proteins in the retrograde direction, as the bulk of the proteins are cargo being transported in the anterograde direction. Slide 10 This animation illustrates the maturation of the Golgi cisternae carrying protein cargo en masse in the anterograde direction. At the trans-Golgi network, vesicles form that carry cargo to their next destination. Slide 11 This movie reveals the movement of vesicles to, around, and away from the Golgi complex during protein transport. Here, the fluorescence is following a secreted protein in the ER, Golgi (which is brightly labeled)and then in secretory vesicles that leave the Golgi. How are these vesicles formed? Slide 12 There are four steps that we need to consider when we talk about vesicular trafficking. Step 1: Vesicles form by a process called budding; where buds arise from the membrane of the ‘donor’ compartment. Step 2: Cargo proteins are loaded into buds via cargo signal sequences and receptors. Step 3: Vesicle formation and release. and finally, Step 4: Vesicle docking and fusion to membranes of the ‘recipient’ compartment. Let’s take a closer look at these vesicles. Slide 13 There are 3 types of vesicles used in protein transport, that are named after the coat protein used in the formation of a vesicle. The three types of vesicles are the clathrin-coated vesicles, the COP I coated vesicles and the COP II coated vesicles. All of these coat proteins are small GTP binding (or G) proteins with GTPase activity. G-proteins bind to nucleotides GTP and GDP. When the G-protein is bound to GTP, the protein is active. G-proteins have an internal GTPase activity that can hydrolyze the GTP to form GDP. When the G protein is bound to GDP, it is inactive. This image represents the cycle  from active to inactive and back. Active to inactive requires GTPase mediated hydrolysis of GTP to GDP. This is assisted by a GTPase accelerating protein (or GAP). Inactive to active requires the displacement of GDP and its replacement by GTP. This is assisted by a guanine exchange factor (GEF). Slide 14 The three types of coated vesicles are required at different points in the vesicular transport process. Clathrin-coated vesicles are required for transport away from the trans-Golgi network to the endosomes and to the cell membrane (through secretory vesicles). They are also used in the process of endocytosis that uses the cell membrane to transport macromolecules into the cell. COP I vesicles are used specifically for retrograde transport, from the Golgi back to the ER. And the COP II vesicles are required for transport from the rough ER to the cis-Golgi network. Slide 15 This image emphasizes the bidirectional transport from the ER to the cis-Golgi network, and back. On the left, COP II vesicles mediate anterograde transport from the ER to the Golgi. On the right, COP I vesicles mediate retrograde transport from the Golgi to the ER. B u t how are such vesicles formed? Slide 16 Well, let’s return to the four steps of vesicular trafficking. Step 1 is the budding of a vesicle on a donor membrane. Though this process happens in the same way for all vesicles, we will look at the formation of COP II vesicles as our model. A GTPase protein called Sar1 is required for vesicle budding. Sar1-GDP is an inactive, cytosolic protein. Sec12 is a transmembrane protein found on the membrane of the donor compartment, in this case, the ER. The Sec12 is a guanine exchange factor, GEF, that will facilitate the exchange of GDP for GTP on Sar1. Now Sar1-GTP is active. From there, activation of Sar1 involves a conformational change that reveals the hydrophobic N-terminus that will anchor Sar1 in the membrane of the ER. How is this activation going to facilitate the formation of vesicles? Slide 17 In the top image, Sar1 proteins (shown as blue circles) on the donor membrane interact with the cytosolic COP II coat proteins (shown in green). The accumulation of coat proteins on the donor membrane compartment is dependent upon the ability to bind to the Sar1. Localization of the coat proteins onto the membrane is a good first step, but more importantly , the coat proteins can then induce a change in the shape of the underlying membrane. Shown below is once again the association of Sar1 with the GEF (Sec12) to induce activation. The Sar1- GTP then binds with a high affinity to the COP II coat proteins. The COPII proteins include Sec23 and Sec24. Sec23 binds directly to the Sar1 protein, while  Sec24 binds indirectly. Next, Sec13 and Sec31 coat proteins accumulate. The COP II complex has an inherent curvature that then forms. As the proteins associate, they induce the underlying membrane to curve. Slide 18 This schematic shows just the Sec23 and the Sec24 proteins (in green and blue), and the Sar1 protein (shown in red).Dimerized Sec23 and Sec24 are curved and their binding to the membrane-anchored Sar1 forces the surface of the membrane to curve. We can see this curvature in an in vitro reaction using ER membrane micelles. When these micelles are incubated with COPII proteins and activated Sar1- GTP, we can see the budding of new vesicles. Antibodies to coat proteins can be seen in this immuno-TEM image as dark dots. As we can see, curvature of the membrane only occurs where COPII proteins are seen. Slide 19 Budding from the Golgi membranes to form COP I or clathrin-coated vesicles (shown here coming from the trans-Golgi) occurs in the same way. An important difference is the use of the ARF G-protein in place of Sar1. This ARF protein is functionally similar to Sar1, and is used in the formation of COPI and clathrin-coated vesicles. Slide 20 Step 2 is loading of the cargo. Within the curved bud on the ER membrane, cargo accumulates preferentially to mediate loading. Cargo receptors accumulate in the bud and pick up soluble proteins. Transmembrane proteins, such as Golgi enzymes, may also be cargo and these accumulate inside the budding membrane as well. Accumulation of these proteins within the bud is accomplished by the interaction of the cytosolic domains of the receptors of transmembrane cargo with the coat proteins. The coat proteins are therefore acting to collect vesicle- specific cargo. We can also see that some cargo is accidentally loaded. ER resident proteins may also diffuse into that vesicle, but they won’t accumulate to a high concentration in the same way our cargo proteins will. Slide 21 Once the cargo is loaded, the vesicle must be released from the donor membrane. GTP hydrolysis by Sar1 converts Sar1-GTP into Sar1-GDP. Sar1 is no longer membrane-anchored and this releases both Sar1 and the coat proteins in a process called uncoating. The uncoated or naked vesicle is loaded with cargo and ready for transport. It is the uncoated vesicle that is then recognized by a motor protein and carried along the microtubules from the donor membrane to the recipient membrane. Slide 22 We can study various steps in the formation of vesicles by disrupting the process and accumulating transition states. For example, by preventing uncoating, we can see a collection of accumulated coated vesicles. Ordinarily, this stage would be short- lived and difficult to image. Under the TEM, the white line is the membrane of the vesicles and the black dots are the coat proteins forming a fuzzy envelope around the membrane. How might we prevent coat disassembly in order to visualize this transition state for a longer period of time? One way is by adding a non-hydrolyzable form of GTP that maintains Sar1 in it's GTP-bound state. Alternatively we can study a mutation in the Sar1 G protein that inhibits GTPase activity. In both cases, the continued presence of Sar1-GTP prevents vesicle uncoating. It turns out that this will also prevent vesicle transport, vesicle docking, and cargo unloading as the coat proteins mask the proteins that are necessary for these later steps. Slide 23 The second part of Step 3: The process of vesicle release, has been studied in the greatest detail for the clathrin-coated vesicles. The clathrin coat forms a polyhedral lattice using a collection of different clathrin proteins: clathrin heavy chains, clathrin light chains and proteins called adaptor proteins. The figure at the right represents the heavy chain clathrins in pink, and the light chains in blue. Together three light chains and three heavy chains form a structure called a tri-scallion. The tri- scallions interact with one another on the surface of the budding membrane to form the clathrin coat. Slide 24 The G-protein, dynamin, is required for the release of the clath rin-coated vesicles from the budding membrane. In its active form, the dynamin protein is bound to GTP and can associate with the neck of the budding vesicle. The GTPase activity of dynamin hydrolyzes GTP to GDP, changing dynamin shape and structure. This leads to vesicle release. Slide 25 Intermediates in the process of vesicle release can be created by adding non-hydrolyzable GTP or by creating mutants in the dynamin’s GTPase activity. In the TEM image below, black dots represent antibodies to dynamin. We can see that dynamin accumulates specifically around the neck of the budding membrane. This transition state is unusual in that dynamin continues to accumulate without vesicle release, thus creating an unusually long neck. On the right we can see another preparation under the EM. This image allows us to see how dynamin winds around the budding vesicle neck in rings. Slide 26 In this EM image, we can see just dynamin in the presence of GDP. This reveals more information about the structure of the dynamin – dynamin has formed polymers that assemble into spiral structures, the shape of corkscrews. Slide 27 In these cartoons, dynamin (shown in blue) is shown wrapped around the underlying membrane in a helical pattern.Researchers have formulated two models for how dynamin might use its structure to induce vesicle release. On the left, the poppase model suggests that dynamin helices elongate and push the vesicle away from the donor membrane. On the right, the pinchase model suggests that dynamin helices constrict and squeeze the membrane to initiate release. Slide 28 This is an animation of the pinchase model. The dynamin polymer is wrapped in a helical fashion around the vesicle neck and narrowing of the internal diameter of the dynamin helix constricts the vesicle neck and releases the vesicle. Slide 29 This is an animation of the poppase model. Once again, the dynamin polymer is wrapped in a helical fashion around the vesicle neck. But here, the dynamin polymer elongates, and pushes the vesicle off. Slide 30 Can we find evidence to support one model or the other? An in vitro experiment using lipid tubes as models for vesicle necks is shown here. On the left, dynamin is associated with the lipid tubules. On the right, the addition of GTP to the medium (and presumably subsequent hydrolysis) allows a conformational shift that narrows the internal diameter of the dynamin spiral. A s this internal diameter narrows, we can see a concomitant constriction of the lipid tubules. This is consistent with the pinchase model Slide 31 In this in vitro experiment from another lab, we are looking at EM images of lipid tubes in three different states. At the left, undecorated lipid tubes carry no dynamin protein. In the middle, we are looking at the dynamin-GTP stage by arresting the protein in this conformation using non-hydrolyzable GTP (GTP gamma-S). At the right, we see dynamin-GDP after hydrolyzation of normal GTP. GTP hydrolysis is accompanied by a conformational change in the dynamin polymer. We can see here how the helical polymer has elongated as seen by the increased space between the ridges of the dynamin rings. This is consistent with the poppase model. So, which model is “right”? W e l l , this is a topic of discussion in the field. In the end, it’s perhaps a combination of the two models (pinchase and poppase) that acts to induce vesicle release. Slide 32 We can study the function of the dynamin protein in another model system, the fruit fly, Drosophilia melanogaster.The vesicle here is actually a vesicle formed during endocytosis at the cell membrane of a presynaptic neural cell, however, the function and structure of dynamin are the same. In a system different from the secretory pathway that we have been examining,neurotransmitters are loaded from the cytosol into these vesicles that are formed from a presynaptic cell membrane. Eventually, vesicle fusion with the cell membrane then releases the neurotransmitters into the synaptic cleft through exocytosis. The neurotransmitters act as signals to the post-synaptic neural cell by interacting with cell surface receptors. In flies, the shibire gene codes for the dynamin that helps to form these vesicles. Slide A temperature sensitive mutation in disrupts this 33 dynamin protein. Specifically, at the permissive temperature, dynamin is expressed, folded and functional, while at the restrictive temperature, the dynamin protein is denatured and non-functional. As a result, no vesicle formation can occur. While this affects every cell at some point, we can examine the effect that a failure in vesicle formation at the point of vesicle release has on neurotransmitter transport. At the permissive temperature (25°C) the flies are fine, at our restrictive temperature (30°C) the flies are paralyzed. Slide 34 Here we can see two sets of flies. On the right, in green, are wildtype flies without any mutation in the shibire gene. On the left is are flies with the temperature sensitive shibire mutation. Here, the dynamin protein can fold at 25°C, but it cannot fold at 30°C. Let’s watch what happens to these flies as the temperature changes (shown in the top left). To start, flies in both vials are moving around at a temperature of 27°C. As the temperature increases, we can see a difference in the vials. At 29°C, on the left, we can see that some of the flies are beginning to fall down. Flies are no longer able to hold themselves at the top of the vial. This is because of the inability to form and transport neurotransmitters in vesicles, which results in paralysis. At 33°C, in the absence of functional dynamin, there is no transport of neurotramitters in vesicles, due to the inability to form synaptic vesicles. Here, the researchers decide not to leave the flies paralyzed, but instead they reduce the temperature. Interestingly, folding of the dynamin protein is reversible from the restrictive temperature, so the phenotype can be reversed. As you can see, most of the flies at 20 to30 minutes have recovered and they are walking around, except for one. After 30 minutes the last fly has recovered. Slide 35 After vesicle release, the fourth step in protein transport is the release of the cargo into the recipient compartment. This requires vesicle docking and release. We will use the model of secretory vesicle docking to illustrate this process, though it occurs in all vesicles. Another small G protein called Rab GTPase is going to control vesicle docking. Rab-GDP is found free in the cytosol, while Rab-GTP is bound to vesicles by a hydrophobic anchor. This is similar to activation of the Sar1 protein. The protein Rab on the vesicle membrane will associate with a receptor, or an effector, on the target, recipient membrane. Rab-GTP is able to interact and bind with high affinity to the Rab effector to facilitate docking. The Rab effector is anchored to the target membrane and recognizes Rab. Docking, however, is not sufficient to deposit the cargo. The two membranes must fuse for cargo to be released into the recipient compartment. Slide 36 Vesicle fusion is mediate by membrane-anchored protein helices called SNARE proteins. Vesicle SNAREs, or v-SNAREs, are anchored on the vesicle membrane. The v-SNARE here is VAMP. Target SNAREs, or t-SNAREs, are anchored to the target membrane. Examples of t-SNAREs include syntaxin and SNAP25. So while docking brings the vesicle and target membranes close together, the interaction between these SNAREs mediates fusion. Four helices, 2 from SNAP25, one from Syntaxin, and one from VAMP, spiral together to form a four helix bundle. This is a very strong complex that pulls the two membranes close together and allows fusion. Slide 37 A model for membrane fusion is shown here. At the top is the edge of a vesicle and it is docked onto a target membrane. The SNARE complex has already formed. As these helices spiral together, they pull the membranes closer together. The transmembrane domains of the SNARES are pulled apart as the cytosolic domains spiral together. Keep in mind that this is a 2-dimensional picture, but we are actually thinking about 3-dimensional structures. The vesicle on top is more like a soccer ball sitting on a sheet of paper. The transition state in the middle represents a point at which a hole is created in the two membranes simultaneously. The aqueous contents of the vesicle and the target compartment flow into each other, preventing resealing of the original membranes. Instead, resealing occurs such that the vesicle and target membranes are continuous. Slide 38 After fusion and the release of cargo contents, the SNARE complex must be disassembled and the proteins can be reused. NSF and the alpha-SNAP protein associate with the end of the SNARE complex andunwind the 4 helices. Once disassociated, the SNAREs can diffuse in the membrane for recycling  and reuse. Slide 39 In this overview, we are looking at transport between the ER and the Golgi where anterograde transport uses the COP II vesicles. Transport then continues in the Golgi complex. Slide 40 We also have the retrograde COP I pathway in which vesicles are budding off the cis-Golgi cisternae, loading cargo, and docking and fusing with the rough ER. COP I vesicles also move in the retrograde direction between the different Golgi cisternae. Why does the cell need this backwards transport though? Slide 41 Well, to start, there are many proteins that are incorrectly in the Golgi complex and need to return to the ER. In particular, there are ER resident proteins that accidentally leave the ER when they enter a COPII vesicle. In addition, we just saw how vesicle SNAREs need to be recycled back to the donor ER membrane, so they can be reused in the formation and function of new COP II vesicles. This is also true of COP II cargo receptors that are brought to the Golgi and must be returned to the ER. Unfolded proteins will also need to be returned to the ER so that they can be folded, modified, or translocated out to the cytosol for degradation. There are signals specific to ER resident proteins that are required for loading into COPI vesicles. For example, a lysine, aspartate, glutamine, leucine sequence, or KDEL sequence, is found on ER resident proteins. A lysine rich sequence is specific to ER membrane proteins and aspartate-X- glutamate is found on the COPII cargo receptor. These signals are needed to load the proteins into the retrograde COPI vesicles. Slide 42 For example, to retrieve ER resident proteins back to the ER, the KDEL sequence on ER resident proteins is recognized by the KDEL receptor found in the Golgi complex. From there, the KDEL receptor is loaded into the COP I vesicles. These vesicles can detect and accumulate ER resident proteins carrying the KDEL signal. Now the COP I vesicle can bring the ER resident proteins back to the ER. Slide 43 This overview summarizes the steps in protein transport. A collection of secretory proteins mediates each step. Think back to the temperature sensitive secretory mutants identified in yeast in Module 5, lecture 1. Into which of Classes A, B, C, D, or E of secretory mutants do the proteins we discussed today fall? For example, a mutation in a clathrin coat protein will produce a class E phenotype in which secreted proteins accumulate in the trans- Golgi. Consider these scenarios and the class mutant  type, for all the proteins we discussed today. Slide 44 And that’s all for Module 5. As we have seen today, we have outlined the steps in protein transport away from the ER. In doing so, we have identified and described experimental evidence to develop a model for transport through the Golgi complex. And finally, we have also outlined the proteins and mechanisms involved in vesicle trafficking and have described experimental data that support models for vesicle function.