Molecular Cell Biology - Membrane Trafficking & Regulation PDF

Molecular Cell Biology- Bolino Lezione 3 15th october 2024 Membrane trafficking and its regulation Vesicular transport Today we will focus on vesicular transport that occurs through topologically similar compartmen...

Molecular Cell Biology- Bolino Lezione 3 15th october 2024 Membrane trafficking and its regulation Vesicular transport Today we will focus on vesicular transport that occurs through topologically similar compartments. This transport happens from the plasma membrane to the outside and from the outside into the inside. This is the way the cell communicates with the environment. We will then focus on membrane contact sites, lysosomal function and autophagosome in autophagy. Topologically similar means that they are compatible to fuse as vesicular trafficking membranes, but the compartments do this from a donor organelle or a vesicle to an acceptor compartment in a very polarised, which means that everything that is inside the vesicle will be inside the new one as well, and regulated way. Polarised means that everything that is inside or outside, in case of delivery out of the cell, will be in the new organelle as well, also everything that is inserted with a polarity, so with a direction, will be maintained in the new organelle. There are different kinds of vesicular transport (figure 1): Exocytic transport (in red): the vesicles come from the endoplasmic reticulum, go to the Golgi and then to the plasma membrane in a very regulated way. The vesicle needs a continuative stimulus to fuse with the membrane. Exocytosis occurs also when something is released from the trans-Golgi to the early endosome or from the Golgi to the late endosome and lysosome. This means that exocytosis occurs not only when things go out of the plasma membrane into the extracellular environment, but also within the cell. Figure 1 Endocytic transport (in green): molecules are ingested in endocytic vesicles derived from the plasma membrane and delivered to the early endosome. They follow the endocytic pathway from the early endosome to the multi-vesicular bodies, late endosome and lysosome. Retrieval transport (in blue): in this type of transport there is a recycling of proteins and receptors. The recycling pathway in the plasma membrane communicates to a portion of the endosome using vesicles that go back to the membrane. Another pathway is from the early endosome, lysosome, and late endosome back to the Golgi, and then back to the ER. All these pathways have different functions. All these processes must be highly regulated to make sure everything goes where their supposed to. Different molecular players have a role in this pathway, and they tell each vesicular what to do and where to go. Each membrane has a coating, so an identity, that is made of phosphoinositide-phospholipids (PIP) and other proteins like signalling proteins, GTPs, and regulators of phosphoinositide that are built like nanodomains on each membrane. Each vesicle (donor) knows where to go because they recognise the target (acceptor) as compatible because they have a similar molecular composition. The vesicles are transported through the cytoskeleton, therefore through microtubules. To restore the identity of the membrane of the donor and the acceptor some proteins regulators are activated every time these movements occur. All the vesicles move almost simultaneal throughout the cell, so there are almost thousands of vesicles that are continually internalised. Phosphoinositide: 1 Molecular Cell Biology- Bolino Lezione 3 15th october 2024 They are signalling molecules, they change in a very dynamic way, and they convert into each other, depending on what they have to do and which membrane identity they have to code. They are bound to the membrane via the fatty acid chain and are exposed on the cytosol, because they have to talk and recognise the acceptor membrane, so they have a hydrophilic side. The PIPs derive from the poliphosphorylation, in different positions (3, 4 or 5), of the phosphatidylinositol, which is the precursor. The lipids can be signals as they change in a very precise and regulated way and the effectors recognise these lipids and bind them through modules, so the domain of the protein can specifically recognise them. The PIP3 is located on the plasma membrane, it is the main signalling lipid of the plasma membrane, it can drive all the signals downstream of tyrosine kinase receptor into the cell and also influence transcription and the cell cycle progression. This lipid is distributed in low concentration only in a small domain of a plasma membrane where the tyrosine kinase receptor is. This happens to focus the downstream signal only in that specific part of the cell. Also, cytoskeleton remodelling is possible with this lipid to be able to do a plasma membrane deformation to be able to accommodate a pathogen. PI(4,5)P2 is also important for endocytosis. This type of regulation occurs also thanks to coating, which contributes to this kind of recognition. This recognition is the deformation of the membrane and the selection of what has to be transported. Membranes do not fuse only to increase their surface but also to transport things inside. There are different types of coating proteins, depending on the vesicles that have to be moved (figure 3): COPI (vedi libro) which is in between different cisternae of the Golgi. COPII (vedi libro) which is from the ER to the Golgi. They both help the formation of the vesicle membrane and select the cargo. Clathrine: is a protein formed by a heavy chain and a light chain. The light chain interacts with the actin, and the heavy chain selects the cargo that has to be transported through an adaptor. Clathrine is not only important for clathrin- mediated endocytosis but also to Figure 3 move lysosomal enzymes from the Golgi to the lysosome. Clathrines form a structure called triskelion, which helps actin to deform the membrane and also is needed to select the cargo to be transported. Receptors are needed because molecules cannot pass the membrane, so they have to be selected and brought inside. Receptors are also needed to downregulate a signal because they are internalised with a ligand, this mechanism is faster than decreasing the transcription of the gene that is coding for the receptor itself. Clathrine binds to an adaptor protein (AP2) that co-participates to this mechanism of selection. While there is the clustarisation of the membrane the adaptor helps the clathrine with the deformation of the membrane. The 2 Molecular Cell Biology- Bolino Lezione 3 15th october 2024 membrane then has to be cut so the vesicles are formed and to do so dynamine is needed. The membrane then assumes a round shape because is exposed to water. Figure 4: invagination of the membrane formed by the receptor and the cargo The vesicles are uncoated otherwise they don’t know where to go because the clathrin is masking what is underneath the membrane (phosphoinositide PI(4,5)P2 and proteins). The membrane then has to be deformed (figure 5) which happens with the help of actin, clathrine and bar protein which are alpha helix and positively charged, so they have high affinity to the negatively charged membrane. Figure 5 Arp2/3 is a protein that binds actin to give the direction of the polymerisation of a new filament and the rearrangement of the actin filament already present. Another mechanical force that makes this invagination possible is the actin that is polymerised at the edge of the vesicle, at the point where the vesicle has to be pushed out towards the cytosol. Dynamin is the constrictor, when it dimerises and Figure 6 hydrolyses the GTP, it changes its conformation physically constricts the membrane and fuses the two layers together to release the vesicle. (figure 7) GTPase and GTP hydrolysis are dimerised and form ultrastructures that constrict and force the two layers of the membrane to stay very close and fuse. Figure 7 There are other coating proteins, such as COPI and COPII, the difference is that in the clathrin-mediated endocytosis, there is the phosphoinositide that is the trigger that attracts the adaptor AP2, the receptor and all the machinery necessary, whereas in COPI and II there are other types of GTPases as signalling molecules (e.g. Arf, Sar, Rab GTP). 3 Molecular Cell Biology- Bolino Lezione 3 15th october 2024 COPII is a double layer of coating vesicle (figure 8) that goes from the ER to the Golgi, this is made possible by Sar-1. Sar-1 has an active and inactive state that can be switched by GEF and GAP which promote the GTP or GDP-bound state with hydrolysis. There is also the recruitment of Sec 23, 24, 13, and 31 which are proteins that build the cage in the vesicles that have to move from the ER to the Golgi. Figure 8 Other actors that play a key role in directing the vesicles are the regulators. Phosphoinositide species are produced by phosphorylation and then phosphatases remove the phosphate and convert these into something else. Other regulators are needed as well as Rab GTPase to help the trafficking of vesicles. Rab + PIP + SNARE cooperates to define membrane identity and tell the membrane where to go. Figure 9: Ras is the superfamily. (vedi Alberts) GTP and GDP are converted with the help of GAP and GEF which promote their phosphorylation and dephosphorylation. GDI is a protein that binds in the cytosol in the GDP state helping the protein to stay inactive. GTP is needed for the integration of several Figure 9 signalling events they are located all over the cell, not only on the plasma membrane. Rheb, which is a part of the Ras superfamily, is very important because it converges on the signalling of mTORC. Ras are very important in cancer because they control growth, cell proliferation and transcription of genes. Small Rho GTPases are the ones that help the cytoskeleton to modify itself. Rab and Arf are the main small GTPases that are relevant for membrane trafficking and that cooperate to provide the membrane its identity. Miro is a GTPase that is bound to the outer membrane of the mitochondria. It is a new addition to this family, and it was discovered a few years ago. All these are attached to membranes but not necessarily to the plasma membrane. The only one that is not attached to the membrane is Run because it is cycled in and out of the nuclear core, as a GTP and GDP. GTP interacts with GEF, which bounds chromatin in nuclear lamina. Rab: They govern the fusion of membranes and are constituents of the coat for membrane identity. Rab are GTPases that belong to the Ras superfamily and are involved in membrane trafficking. Rab is composed of several members, that are called with numbers. There are specific Rabs that reach specific locations of organelles. Rab 5 is localised in the early endosome and Rab 7 in the late endosome. Vesicles are reminded by many actors where to go as they are with the acceptor membrane. Rab and SNARE are activated to mediate this fusion. Rab recognises another protein and interacts with it, vSNARE and 4 Molecular Cell Biology- Bolino Lezione 3 15th october 2024 tSNARE recognise each other as a cognate pair of proteins that can interact together and bring the membranes close to each other. This has to be brought in proximity by a motor protein. Figure 10 The nanodomains for membrane identity are composed of PIP, SNARE and Rab and are formed in a specific location thanks to a cascade of events that is initiated by the first player which then recruits the rest of the proteins needed. To build this platform, in Figure 11, you need to have a Rab activated by a GEF, which is then converted to GTP, the active version, and it promotes the action of a kinase that converts phosphatidylinositol in P3P. They then recruit other GEF to bind Rab, which is already active, Figure 11: and this stimulates more molecules to be 1. Rab is activated though GEF activated. This starts a cascade event which 2. Rab-GTP is recognised 3. Rab-GTP binds a motor protein to bring the vesicle to the target ends in the formation of microdomains, starting membrane from a single molecule. 4. Rab-GTP+effector bind to SNARE à vSNARE +tSNARE is formed 5. Fusion and Rab binds GDI PIPs, SNAREs and Rab make up the identity coat of the membrane, thanks to which they can fuse. They also have to acquire a new identity as they mature, which is a completely different event. The early endosome is formed because there are vesicles that fuse from the plasma membrane by endocytosis, it then becomes something else by maturing. For example, early endosomes may become the precursor of lysosomes. This switch into a different identity occurs because this early endosome that is destined to become late endosome loses the Rab 5 identity and becomes Rab 7 positive. This happens because Rab 5 activates effectors, that this membrane needs to function, and GEF that are specific for Rab 7. In the end, this membrane switches into Rab 7 positive, which is a maker for late endosome, lysosome and other phosphoinositides species. 5 Molecular Cell Biology- Bolino Lezione 3 15th october 2024 Transport from the ER to Golgi: COPII is a double coat that covers the vesicles, this is formed because the macrodomains attract all the regulators, the double coating and the selection of the cargo, which is attracted from the lumen to be selected. The cargo has to be selected and moved outside of the ER, because the Golgi will be modified by glycosylation or because enzymes and adaptors have to be moved as this function is needed in another compartment. To build the other compartments, the enzymes also have to be moved. In the ER only proteins that are correctly folded can travel outside, otherwise they are assisted by chaperons so they can be correctly folded. If these proteins are not correctly folded the proteasomal mediated degradation occurs. (Already said last time) These vesicles bud with COPII from the ER and they don’t immediately fuse with the Golgi but they mature in a Golgi compartment and fuse to become intermediates of the vesicular tubular Figure 12 clusters and then go to the Golgi. Maturing means that they acquire functions that are typically from another compartment, so they switch their identity. This homotypic fusion of the vesicles is possible because of the Rabs and the tSNARE and vSNARE which are both on the Figure 13: homotypic fusion vesicles to bring the membranes close so they can fuse and form clusters. (figure 13) They always have to be attached to the cytoskeleton of the organel’s vesicles. Several of these then exchange material through COPI to recycle this like receptor that will be needed again. KDEL is a stretch of four aminoacids of proteins that are resident in the ER and are needed by the ER to be transported back. They have a receptor that leaves the ER and goes to the Golgi, then it does another cycle of transport through a vesicle and is recycled back to a retrieval mechanism to select this receptor through the interaction of it with KDEL, which is the C-terminal of the protein that needs to be recycled back. (figure 14) (vedi alberts per una spiegazione più dettagliata). Figure 14 There are different types of glycosylations in the cell: N-glycosylation: the conjugation with the asparagine, which is needed as a marker to sense the correct folding of the protein. O-glycosylation: there is a covalent bond with an oxygen that is exposed to the lateral chain of another aminoacid through which the glycosylation is attached. In the ER the proteins are subjected to multiple steps of glycosylation after already being glycosylated during the N- glycosylation. The first one happens in the ER and it is the mannose-6-phosphate attachment of the marker for lysosomal Figure 15 enzymes, which marks lysosomal enzymes. This is the first post- translational modification that happens to the Golgi, but only to proteins that are destined to become lysosomal enzymes. There are then several modifications, including the removal of mannose, and the addition of GlcNAc and N-acetylglucosamine. These modified proteins leave the Golgi to go into the lysosome, which is an exocytosis, or to go to the plasma membrane in a regulated or not regulated way. 6 Molecular Cell Biology- Bolino Lezione 3 15th october 2024 (vedi libro per i tipi di glicosilazione) Glycosylation is multiple and they come from the ER, then the glucose is released to mark the protein folding until only one is remaining, and they leave the ER with high mannose content. They are then modified as mannose is removed and N-glucositilglucosamine is attached. Endo H sensitive or insensitive can recognise the glycosylation groups of a protein so it can be used in an experiment if you want to see if there is a putative defect in glycosylation because of a genetic defect, and you can monitor if a protein is correctly glycosylated or not. If a protein is sensitive to this treatment, it means that it has a high concentration of mannose, so it is still in the middle portion of the Golgi, if the protein is resistant to this chemical attractor, then it means it has less mannose but more sialic acid, so it is in the last part of the maturation process. This is done with a western blot analysis. Glycosylation is not only used to assist proteins to have a mature functional conformation but also as a signal, as many receptors can bind to proteins that are glycosylated. Transport from the trans-Golgi: (figure 16) Exocytosis is not only needed to deliver vesicles to the plasma membrane but also to deliver enzymes to the lysosome. The mannose-6-phosphates tag of the lysosomal enzyme is the first reaction that happens in the cis- Golgi. 70 different enzymes are contained in the lysosome, in multiple copies, which is why the lysosome is small but very dense. 1. Signal-mediated diversion to lysosome: This sorting of lysosome enzymes occurs in the cis- Golgi as exocytosis can deliver to the lysosome by conjugating the proteins with the mannose- 6-phosphate (M6P) (figure 16 in yellow). There is then a selection, with the help of clathrine, of the receptor and the enzymes that are conjugated with the signal mannose-6- phosphate that is brought out of the membrane where these vesicles then lose the clathrine uncoating and then fuse with the lysosome. The Figure 16 lysosome itself is created by this fusion of several vesicles. In the lysosome there is also the disassembly of the M6P receptor as well as its enzyme, this happens because the lysosome has an acidic PH that other compartments within the cell do not have, which helps the dissociation of the receptor. The receptor is then recycled and depending on the witch compartment it is in, they use different retromer complexes that assist the budding and the selection of the receptor without a cargo to be retransported back to the Golgi or the ER. (Figure 17) The lysosome is acidic because it has a pump that imports protons, with an ATP-dependent active transport from a less concentrated location to a more concentrated one. The Figure 17 endosome has a PH that is not acidic but not as much as the cytosol that is 6.5. (Vedi Alberts on how the M6P is conjugated) 2. Regulated exocytosis: The exocytosis can be regulated or not, depending on if a stimulus is needed for the fusion between the vesicle and the plasma membrane to occur or it can be constitutive, so it occurs without stimulus. (Vedi Alberts for examples of exocytosis) A prototypical regulation of exocytosis is the synaptic transmission, which is the fusion of synaptic vesicles with the plasma membrane after endocytosis, it is calcium-dependent so different players interact with each 7 Molecular Cell Biology- Bolino Lezione 3 15th october 2024 other. They are both in the plasma membrane and the vesicles and act as SNARE to attract the vesicles and allow them to be very near to the target membrane to be fused with it. It is regulated because you need calcium which is not released randomly but it is released from the compartments in which it is stored, (lysosome, ER and mitochondria) in a very regulated way. This release happens during the depolarisation of the membrane. The vesicles do not always need to be fused with the membrane to exocytosis cargo, because vesicle exocytosis may be needed also to increase the membrane surface. The plasma membrane surface may need to be increased during cell division when there is the constriction, thanks to the actin-myosin cytoskeleton ring, otherwise, the new cells would be smaller. The vesicles are delivered here through vesicle fusion to the plasma membrane thanks to exocytosis from other compartments, such as the Golgi. In Phagocytosis there is the need for vesicles to fuse to increase the surface and to modify the membrane so the pathogen can be internalised. If the is a break of the membrane, the vesicles come from lysosomes in a calcium-dependent fusion event, there is an increase in the membrane surface and so the wound is repaired. It also happens during cellularisation because several tissues have to be built and increase the number of cells. Transcytosis is a type of exocytosis typical of polarised cells, which are cells that junctions that seal the space between them creating a barrier that impedes the transport of substances in one way so they can only enter from the apical to the basolateral portion of the membrane. Different domains have to be built in these polarised cells (figure in red means proteins that constitute this domain that are not present elsewhere), and to maintain this particular distribution of proteins there is the need pf transcytosis. The delivery of one side and then endocytosis and redistribution on the other side. It is a specialised way of endocytosis and exocytosis that is needed in cells to make different Figure 18 domains of plasma membrane depending if they are apical or basolateral. Endocytosis is the internalisation of material from the plasma membrane or the extracellular compartment into the cell. Vesicular trafficking is highly regulated, the main regulators are the coat that each membrane has as an interplayer between proteins and lipids. Some compartments are fusing, and others are maturing to acquire new identities and become something else. The endo-lysosomal trafficking: ER, Golgi matures from ER intermediates that acquire new identities, Golgi delivers vesicles to the lysosome, and lysosomes are formed ex-novo from vesicles that fuse. Early endosomes form ex-novo In order we have plasma membrane, vesicles that are fusing together and form early endosomes, which mature in multi-vesicular bodies into late endosomes. Late endosome forms by fusing of lysosome and lysosome fuse with autophagosome into an autophagosome. 8