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Transcript
Chem*3560 Lecture 26: Cell adhesion and membrane fusion A distinguishing feature of complex organisms is the ability of cells to recognize each other and to adhere together as multicellular tissues (Lehninger p.404). Several sets of integral membrane proteins help mediate these interactions: Integrins Integrins are made up of one α-subunit and one β-subunit, anchored in the plasma membrane of multicellular eukaryotes by one transmembrane helix each. The extracellular region forms a globular domain which can bind and recognize the amino acid sequence -Arg-Gly-Asp- in the presence of Ca2+. This characteristic sequence is found in a number of proteins that make up the extracellular matrix (Lehninger p.313 Fig. 9-24). Extracellular matrix is the macromolecular network that holds cells together in a tissue, and different tissues have different components of the extracellular matrix. Extracellular matrix components Proteoglycan - a combination of complex heteropolysaccharides such as chondroitin sulfate and link proteins (Lehninger p.308-313). Also linked to cell membranes by the protein syndecan (Lehninger p. 312). Collagen - a triple helical protein that forms the fibrous framework of bone and cartilege. Fibronectin - fibrous proteins characteristic of the extracellular network of connective Laminin tissue (Lehninger p.313; the reference to laminin as a nuclear membrane protein on p. 473 is incorrect. The nuclear proteins are Lamins). There are 18 different α-integrins and 8 different β-integrins which are found paired up in various specific combinations in particular cell and tissue types. These give each cell type its characteristic affinities in different tissues. When integrins bind to an appropriate component of extracellular matrix, they communicate this to the interior of the cell. This occurs in part because the αand β- integrin subunits realign with each other when they bind their target, and partly because multiple integrins cluster together at a site where binding can occur, and this clustering of the intracellular domains is able to bring together key regulatory protein tyrosine kinases, and to serve as a point of anchorage for actin filaments of the internal cytoskeleton (Lehninger p. 313, Fig. 9-24). Cadherins are members of a family of Ca2+ binding proteins found on the plasma membrane surface. The extracellular structure consists of five consecutive β-sheet domains with Asp-rich junctions that bind Ca2+. Ca2+ ions can serve as bridges between two negative molecules, but β-sheets are also designed to pair up so that a cadherin only binds an identical cadherin on a neighbouring cell. Cadherins bundle together to give cells the ability to adhere to like cells and make up a homogeneous tissue . Ig-CAMs - Immunoglobulin-like cell adhesion molecules are calcium independent binding proteins. CAM molecules have a transmembrane helix that is connected to multiple immunoglobulin-like domains on the extracellular surface. Immunoglobulin domains are antiparallel β-barrel structures that perform protein-protein binding, more familiar as antibodies. N-CAM is neural cell specific, V-CAM vascular cell adhesion molecule, etc. Selectins - are extracellular adhesion molecules designed to bind to extracellular structural polysaccharides, also with a Ca2+ dependent binding site, particlarly important in blood system. Membrane fusion Membrane fusion is an extension of adhesion processes, in which two membranes approach sufficiently close to allow the bilayers to merge and the contents to combine. Membrane fusion is involved in a variety of cellular processes that transfer contents from one cellular compartment to another (Lehninger p.405): Exocytosis: vesicles bud off from the Golgi apparatus and fuse with the plasma membrane to allow secretion of proteins. Endocytosis: vesicles bud off from the plasma membrane to fuse with endosome or lysosome compartments. Endosome-Lysosome fusion: merges endosomes with lysosome for final processing Synaptic transmission: Synaptic vesicles (filled with neurotransmitter) fuse with synaptic junction membrane to release neurotransmitter into synaptic cleft. Viral infection: viral capsid fuses with plasma membrane Sperm-egg fusion Fusion of small vacuoles Fusion occurs at specific target sites in the cell Fusion specific proteins only occur at specific target locations, and the example here is the synaptic junction. Fusion is mediated by membrane proteins that distort the approaching bilayers, which otherwise repel each other due to negative charge. The protein that opened up the discovery of the mechanism was recognized because of its sensitivity to N-ethylmaleimide (NEM) which blocks enzymes, in particular ATPases, that depend on nucleophilic acion of cysteine -SH. NEM-Sensitive Fusion protein (NSF) is a cytoplasmic ATP-hydrolysing protein that is inactivated by NEM, which then blocks the fusion process. Soluble NSF attachment protein (SNAP) is a second cytoplasmic protein required for fusion. SNAP receptors or SNAREs are helical transmembrane proteins that start the fusion process. Their helical domains extend a long distance into the cytoplasm. SNAREs come in two general types: v-SNAREs (vesicle-associated), are found in the vesicle membrane. t-SNAREs (target associated) are found in the target region, e.g. inside the plasma membrane, that will be fused to. Altogether, there are 30 types of SNAREs in mammalian cells, and these would distinguish the many different vesicle/target systems in a cell (Lehninger p. 406). When the vesicle approaches the target (it may be brought there by components of the cytoskeleton) v-SNAREs and t-SNAREs associate by arranging the cytoplasmic helices into a four-helix bundle, which "docks" the vesicle into the target membrane. (Only shown as two helices in the schematic to keep figure simple; see the detail structure below) As the SNAREs wind up, they draw the vesicle closer to the surface of the cell membrane. Distortion of the bilayer by the SNARE complex then causes bilayers to merge, ultimately leading to fusion. One v-SNARE is anchored in the vesicle membrane by the transmembrane helix at its C-terminal end. This binds to two t-SNAREs that actually form three helices. The single helix t-SNARE is anchored to the cell membrane by its C-terminal transmembrane helix, and the other folds into two helices, and is anchored by two fatty acid chains. SNAP and NSF are required after fusion is over to disassemble the SNARE complex, so that the SNAREs can be re-used in another round of vesicle fusion. Synaptic vesicle fusion is blocked by Tetanus and Botulinus toxins (the most powerful known toxins, lethal at 0.1 ng per kg body mass), which act by proteolytic cleavage of SNAREs. Tetatus toxin targets inhibitory neurons, leading to spastic paralysis) whereas Botulinus toxin targets motor neurons, leading to flaccid paralysis).