Lecture 2 - Adherence Junctions.pdf

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University of Toronto St. George
Cell and Systems Biology
Maurice Ringuette

Lecture 2 - Adherence Junctions January 7, 2016:45 PM There are two classes of cells. Epithelium and mesynchymal. Think that they decided to get together and form a multicellular organism. That requires amazing genetic engineering coordination. How do they recognize each other and bind to each other. When you think about the evolution of orgnaisms that all create receptors that selectively binds to each other, you are still gping to end up with a blob of cell. So the coincidence of the adhesion moeldules is the extracellular matrix moelcules that gives your body shape. There is no such thing in the body that has a We are starting today from a simple level and looking at the cell-cell single biological function. Most of them have a adhesion. context dependent activity. It might inhibit the With respect to the starts of cell-cell adhesion and transmembrane same event later. If you think about the proteins that are Ca dependent and called cadherin. They start very cadherence it helps binds a population of cells. early in embryogensis. Cadherence in addition to binding cells together communicate with the actin cytoskeleton and What happens when we interfere them? affects the actin dynamics. This has a lot of complex regulations invovling growth factor signaling, cell- cell adhesions and cross talking between cells. If you communicate to the actin cytoskeltin then you are also indirectly communicating with the other side if the ---- elements tubulin and intermediate filaments. You start off by just binding cells together that recognizes each other. And that rapidly translated the inside of the cell into actin dynamics. Whats the major properties of the actin filament in terms of activiites in addition to cell shape? What do they do? They pull. Mechanical forces comes into play. That’s why its called cellular dynamics during development. CSB331 Page 1 EMT vs MET: Epithelial to Mesynchymal transtition. And vice verca. This is a highly polarized cell it has a apical, basal and lateral domain. This cartoon doenst have the apical distribution of the receptors on the surface, so this image is less polarized. But it is polar. CSB331 Page 2 You have a series of junctions. These are called as junctions because they were seen trying to dense stage. As a tremendous aggregation of proteins at that junction. This is a generic organization. Occluding junctions - tight junctions --> This creates a barrier between environments. Allows the diffusion of the nutrients in the proper direction. Cell cell anchoring junctions: creates the major contribution towards the adhesion interaction of the cell. Cell-cell adhesion in a epithelium we also have cell-matrix interactions. What is so special about the adherence junctions? - Adherence junctions are made from cadherins. What kind of instrument are we going to use? From our naked eye we can see things about 200nm. But many cells are smaller than that. So we need a microscope to see them. When you use the light microscopy you can get into a few hundred nm. Light mic is limited. So to look beyond the cell, you use the electron microscopy. There isa differnce in magnification between the electron mic and light mic and naked eye. Don’t memorize them. Just appreciate the difference. CSB331 Page 3 Adherence junction. - One of the transmembrane adherence proteins are Cadherins. Calcium dependent adhesions. That’s how the name comes. - Classical cadherins: are the ones that are abundant, and the ones that were identified first. - Connects actin filaments. - Connecting between the actin filaments and the recptor cell is fine tunesd by lot of adaptor proteins. This is a very dynamic state, and we focus only on some components - These are made from a dozen of different proteins. There are lot of dynamics involved and fine tuning regulations. Desmosomes: - Are linked to intermediate filaments Classical cadherins: The proteins best made by embryos are cadherence. What they call the classical cadherence. CSB331 Page 4 Cadherin family is expanded. In blue shows some of the signature motifs all the cadherin's and these EC domains and immunoglobin like structures. So The questions is when you see something like that , you see the desmosome cadherins and you see that there is a sequence similarity in the molecular design. There is a EC domain ig like structures. there is a sequence similarity more than a 3D orgnaization of ig. Why am I putting planar polarity here? Think about the epithelia having a multiple dimensions. So you have apical lateral and basal. But you also can have the planar polarity. Think about The hair in the back of insects that is pointing in one direction. That is also another kind of level in polarity. So you have some of the cadherins, like the Flamingo plays a central role in regulating another level of polarity. So where do you think the functional specificity comes from? Its going to be in the endo domain. There are regions that are sitting into the cytoplasm Members of the Fat and Dachsous cadherin subfamilies. Drosophilaas two Fat cadherins (Fat and Fat-like) and one Dachsous cadherin. In vertebrates, four Fat cadherins (Fat-J, Fat1, Fat2 and Fat3) and two Dachsous cadherins (Dachsous 1 and Dachsous 2) have been reported. Fat-J is probably the counterpart of Drosophilaat. Fat1 and Fat3 resemble each other in their overall sequences, and they show weak similarity to Drosophilaat-like. The precise length of Dachsous 2 has not been determined. The rectangular boxes depict the intracellular domains of the various Fats. The regions between the last cadherin repeat and the first EGF motif or the laminin A-G motif (for Fat2) of Fat cadherins show weak similarities to the Flamingo box, which is conserved in Flamingo cadherins ( Usui et al., 1999 From So what's so special about the classical cadherins and what about the complexes in the slide we should rememeber? • Not a memorization slide. Only to remember - E cadherin: found in most epithelia - N cadherin: mostly assocaited with neurons - But these extend to other cadherins as well. P cadherin - placenta cadherin. VE : vascular endothelial cadherin. These were named on the place where they were first found. A cell cant express different combination of cadherins depending ont the seqquence. CSB331 Page 5 This suggestion here is that if youa re going to EMT --> you loss of E cadherin. If you reverse is you are bringing in E cadherin. State if been an epithelia is triggered by the E cadherin. Its not clearly on E cadherin..? If I don’t lose that I don’t lsoe the epithlium character. What triggers the intial polarized state is much more complexly distributed. If you selectively knock down E cadherin you will lose epithelium character. But it is deceiving… The signature feature of MOST cadherins is its homophilic interaction. Heterophilic means: 2 different types binds. Classical cadherins have homophilic interactions. And hetero doesn’t happen. E with E. wont form interactions with N. CSB331 Page 6 Video: To give a perspective on dynamics. This is GFP tagged cadherins. If you look closely and follow a cell, you can see blobs, in real life . Any kind of itneraction in cell is dynamic. There are cues to promoite and inhibit interactions. Ther are lot more dynamic stages going on in the cell. Ectodomain: whats sticking out of the cell. Selectivity and binding comes from this domain. Endodomain: what is in the cytoplasm. Functional specificity. Ectodomain: There are 5 EC1-EC5 Ig like domains. There are little linker regions where Ca2+ ions bind. You need Ca2+ for this to work. There is a very special function on the animals, The N terminal domain is the more globular like domain is called the EC domain that is involved in the leading specificity. The rest enhances the interaction between the cells. CSB331 Page 7 Closer look: EC domain have ig like folds. They both are E cadherins, and similar. They have homophilic interactions. The distance here, when they a re sticking like this in the end, is like 40nm apart. But this collapses down to like 20nm when you have full maximum interactions. What's so special is, This shows what Ig like folds look like. You have a loop domain with 2 antiparallel beta plated sheets looking like this . the protruding loops is what gives you specificity. Small AA acid changes in the loop domain have a major impact on what molecular interactions they can form. In some instances they have beta sheets, you can have beta stranded strings that augments the interactions. But that’s not …? No need to memorize the molecular design of the Ig. Just to show that you can stabilize them with disulfide bridges. There is an antigen bindings domain and the loop domain. CSB331 Page 8 How do they get together? Light integrin's binds to their ligands. They are weak forces. They are not like the typical growth factor binding to its cognate receptor. What cognate means is two partners. What it looks like form molecular studies is that it starts with monomers. This is two opposing cells, and youa re looking at the PM. And there is a the cadherin and this forms a cis dimer. These homodimers forms a trans interaction and zippers down to 20nm. So from 40 --> 20nm. So they lock in nicely at the end. Models for the intercellular adhesion activities of cadherin and nectin. (A) Cadherin. (B) Nectin. This is a strategy for each other. There is like a bell curve Like arrangement. They don’t have lot of affinity to each other, but when it starts to combining in aggregates it ends up been __ in 100s of moelcules tat are visible at the ___ where it is becomes really tight The models for this forms, where the zipper like binding or forming the parallel like binding assembly is not 100% resolved, but msot literature would point to the zipper like arrangement. But to all purposes its not 100% certified. Models for the trans-interactions of nectins and cadherins and for those among nectins, nectin-like molecules (Necls), and other immunoglobulin (Ig)-like molecules. (a) Two nectin molecules of the same plasma membrane first form cis-dimers and then form a trans- interaction by the cis-dimers on apposing cells through each first Ig- like loop or in a zipper-like fashion with all three Ig-like loops (upper panel). Similar to nectins, two cadherin molecules of the same plasma membrane first formcis-dimers, followed by a trans-interaction by the cis-dimers on apposing cells through each EC1 ectodomain or in a zipper-like fashion with all five ectodomains (middle panel). A recent study proposed that this trans-interaction may be formed by a parallel-like multiply bonded system with four ectodomains (lower panel) From CSB331 Page 9 EC1 domain: What we see in the knob like way, what you can see highlighted ebtween 2 EC1 domians, te same type of cadherins, is there is a Ca ions gone into the defelxible hinge regions to promote the interaction. There is a knob here which is actually a tryptophan residue which promotes the exchange. So you will see within the knob structure the tryptophan going into the knob opposing going into the region here exchanging the tryptophan interacting with the gap/loop. So that’s the specificity. But what promtoes it? As opposed to the pockers, this is important for the stabilization . What actually happens: Once you add Cal to the hinge region you create an unstable change. How you resolve this is, (stabilize it in terms of energy is) go to undergo conformational change now you can exchange the tryptophan residues. In this you can see that in the hydrophobic pocket, containing in the same cadherin you add Ca and you get an exchange, you put the trytophan in the opposing cadherin EC1 domain and vice versa. That stabilizes the interaction. Theya re cis tryptophan residues. Cadherins are cell adhesion proteins that are important for tissue formation and integrity. Cell–cell adhesion occurs through the formation of the strand-crossover dimer between identical cadherins on the surface of neighboring cells. The strand-crossover dimer forms exclusively between their EC1 domains via swapping of the βA sheet by undocking the conserved tryptophan 2, W2, from its own hydrophobic pocket and docking it into the hydrophobic pocket of its adhesive partner. An interesting aspect of the system is the fact that critical noncovalent interactions in the monomer re-form in the dimer. Thus, as these noncovalent interactions are conserved, what drives the formation of dimer? Moreover, why is dimer formation calcium-dependent? Thus, to probe the structural and energetic effects of calcium on the noncovalent interactions that are necessary for dimer formation, we performed spectroscopic, stability, and assembly studies of wild-type and two mutants, W2A and E89A, of neural (N-) cadherin. We find that while the ionic interaction involving E89 has a minimal effect on the general stability of the closed conformation of the βA sheet, the hydrophobic interaction involving W2 is the source of the calcium requirement for adhesive dimer formation. The binding of calcium creates strain in the W2–hydrophbic pocket interaction through direct connection of E11 at the C-terminus of the βA sheet to calcium. To overcome this unfavorable condition in the monomer, N-cadherin forms a dimer. Taken together, our data provide a thermodynamic basis for the calcium dependence of strand-crossover dimer formation in N-cadherin. From CSB331 Page 10 How can we make a newly made embryo fall apart? The idea is that in the EC space there is 1mM of Ca. so if we can use a chealor, You can use EDTA, but there is a better chealtor called EGTA. . What B)Thee xtracellulapra rto f each is the differencve between something that binds polypeptidec onsistso f a and a chelator? serieso f compactd omainsc alledc adherin Chealator means high affnity. repeatsjo, inedb y flexibleh If you remove, go down to the [] of Ca allmost at inger egionsC. a2+b indsi n the the same level of wahts inside the cell, what neighborhood actually happens to Ca is that it comes away from of eachh inge,p reventingi t the hinge regions --> the cadherence falls apart, from flexing.I n the absenceo f and loses the association. And in actual fact this Ca2+t,h e cadherence is now very close to be broken down moleculeb ecomesfl oppya nd by proteases. There is a special type of proteases adhesionfa ils that are embedded in the membrane that can shed the molecules off the surface and that’s a major mechanism of turning things over. In the rod like arrangement there is a strong degradation by proteases. In the floppy state without Ca it is much more prone for it. Taking a look at this, cell 1 and cell 2. and the clustering. This is self explantory in terms of the itneractions we talked about. Now we are talking about the intracellular proteins associated with it. C)A t a typicaljunction, manyc adherinm oleculeas rea rrayedin parallelf,u nctioningli keV elcroto holdc ellst ogetherC. adherinosn the samec ella ret houghtt o be coupled by side-to-sidien teractionbse tweent heirN - terminahl eadr egionsa, ndv ia the attachmentosf theiri ntracellulatar ilst o a mat of otherp roteins( not shown here CSB331 Page 11 E cadherin interactions: are alpha and beta catenin and so on. Todays focus: p 120 catenin and beta catenin. Beta catenin binds to the end of the tail of cadherin. - This is a complex mixture of proteins. (p120) This is a very dynamic state of regulation. Whats missing from this is, there is another group of molecules called polartity complex molecules. - There is a bunch of adaptor molecules that help anchor the actin filaments here at this moments, and beta catenin is the moelcule with interacts with alpha catenin which directly interacts with actin filament. CSB331 Page 12 You can see that it gets a little bit more complicated. We have p120, alpha and beta catenin. An there is a whole bunch of different molecules depenidng on what epithlia you are looking at and what cell stage, and if it undergoes EMT. Don’t memorize them yet. Figure 1. Factors required to polarize an epithelium. (A) E-Cadherin can dimerize and form trans-homophilic interactions to form cadherin clusters. Ca 2+ ions are required to stiffen the extracellular domain and are essential to form homophilic interactions. The E-cadherin intracellular domain contains binding sites for the catenins p120 and β-catenin, thereby forming the cadherin–catenin complex. p120 catenin links cadherin to microtubules and is also important to prevent cadherin endocytosis and degradation. β-Catenin binds α-catenin, which in turn binds actin and several actin-associated proteins, including α-actinin, vinculin, and formin-1. The cadherin– catenin complex also binds many other proteins, including signaling proteins, and cell surface receptors and forms a hub for protein–protein interactions From Whats so special about beta catenin> - It is important for promoting and stabilizng adherence structures. It is also capable of doing - Helps communication of actin dynamics. - Importantly, it Beta cadherin: it is an improtant thing to promoting and stabilizing the structure. CSB331 Page 13 Beta catenin: you can see that adherence moelcules was also seen in the nucleus. The problem with nucleur staiing it can be highly unspecific, so it was ignored for a while thinking it was an antibody. Because antibodies are pathological liars. Lol. They found it and part of it was in the Wnt signaling pathway (don’t memorize yet). The idea here is that a subset was it coming from a membrane or from a separate pool of catenin? But the idea is that if you don’t have Wnt signaling, this complex which involves the _____-synthases (check book), kinase will phsophorylate beta catening and have destruction. One in Wnt signal you end up taking a subset of beta catenin and goes to the nucleus and acts in combination of other transcription factors and you regulate the gene expression. That’s why we need beta catenin in the context of adherence juctions. Specifically with the calssical Wnt signaling pathway. This is also called canonical signaling poathway ebcayse this was the first one found with Wnt signaling. Don’t memorize pathway. Key points: berta catenin has a dual function in regulating the communication of adherene to the the actin cytoskelton and also as a TF. How is it kept on the membrane? One way to do that is: If you don’t want to destroy molecules, and keep them there is a few strategies.. We have the classical E cadherin. It is promoted one way that use a lot to maintain the junctions, not only for the cadherin, because remember if you brigning in the cadherin complex for endodomain to the other molecule, you are also going to internalize the adaptor molecules. Re think about that region that between all the adaptor molecules are concentrated like sibling hots? So if you are bringing in E cadherin, I am talking about cell cell adhesion as focus, your own sibling effect that is going on inside cells. So cadherin do promote differntiation. But threre are 2 choices CSB331 Page 14 Theres 2 choices. Taking in by endosomes can go to late endosomes and be degraded. One way of silencing Or you can bring it to the recycling endosomes and bring it back to the cell surface. Phosphorylation events are part of that. There's also this vision of neighbor bonds, small monomeric GTPases of RAS super family. Keeping the dynamic state is all promoted by the endosomes recycling, or if you want to silence the event you can degrade it by proteasomes. But if you want to go in the proteasome direction you will want to silence the expression of the gene. so you have the expression of repressors that will block the expression of E cadherin. What will be another way of doing this? Ectoshedding One level of regulation: 1. Recycling 2. Or bring it back to the target… If i asked will a cell alter its Ca level in the extracellular space? That doesn’t occur because the Ca [] In the extracellular space is so large so either you are going to turn over? And one of the primary actions is by recycle the endocytic pathway either for degradation or recycling. CSB331 Page 15 This p120 catenin actually serves to stabilize the interactions here by preventing the ubiquitination of beta catenin. This covers the sensitive region P120 catenin primarily serves to stabilize the interactions by preventing the ubiquitination of the beta catenin. What it actually does is that, it covers the sensitive region which you will have the ubiquitinin monomer and prevents the targeting of degradation. If you remove p120 it tends to target beta
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