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ANAT 212 - Teacher 2.docx

15 Pages

Anatomy & Cell Biology
Course Code
ANAT 212
Thomas Duchaine

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Multicelluar organisms are held together by 1) cell junctions/2) adhesions 1) Cell junctions Occluding Junctionsoccludes/prevents shit from diffusing throughTight Junctions Anchoring JunctionsSubdivided between 4 different junctions i) Adherens Junctionscell-cell junction Junctions that connect intracellularly to actin ii) Focal adhesionscell-matrix junctionExtracellular matrx(ECM) junctions connectng to actin iii) Desmosomes cell-cell junctions Connect intracellularly to intermediate filaments iv) Hemidesmosomescell-matrix junctionthru extracellular matrx to intermediate filaments Recall: 3 cytoskeleton filamentsActin, Intermediate filaments, &&& Microtubules Communicating Junctionsused for communicating chemically or metabolicallyGap junctions Occluding junctions: Is importantin gut for glucose uptake: -only located right under microvilli(near lumen) where cell touches(near apical surface only, not at the base surface) There are channels located in the microvilli called Na+ driven Glucose Symport, which faces the lumen of the GUT, and brings Na+ from lumen into the cell due to the Na+ gradient that is greater in the lumen this Na+ driven glucose symport provides energy for glucose uptake since the glucose is greater in the cell than the lumen[unlike Na+ conc’] Glucose co -transports thru the Na+ driven Glucose symport The Glucose that enters through the Lumen of the cell will move across the cell to its base, and diffuse out through passive glucose channels which are located at the base of the cell, facing the blood stream Importance of Occluding junctions(tight junctions): 1) keep the Na+ driven glucose symport separated from the Passive glucose channels so that the glucose DOES NOT diffuse OUT into the lumen due to gradient 2) Also to keep the glucose concentrations controlled and not freely diffusing into the cell 300-700nm is the size of the wavelengths in the visible light rangecan NOT see much of the things in the nm ranges using Light microscopy -However, using electron Microscopy, you can see much smaller objects since the electrons can be accelerated and see smaller wavelengths -Disadvantage is that electrons cannot easily pass through air medium. collisions with air molecules So not all the electrons would reach the screen, so an ultra VACUUM is used so suck out the air in the column -Disadvantage is that the specimens must be very very thin for the electrons to be able to move through and reach the camera -Since organic material(Carbon, etc) is not electron dense, there will not be as much scattered electrons for the image to be good, so a chemical called Osmium tetroxide has high electron density and is used to stain the cells for the electrons to be scattered betterOsmium tetroxide binds to lipid bilayer and shit… Intermediate filaments: keratin, vimentin, desmin Occluding junctions (Tight junctions)- seal cells together in an epithelium no leaking -each bilayer of adjacent cells are attached to eachother by membrane proteins(claudin & Occludin) Claudin: are 20-27kDA hs 24 members that are related, expressed differently in different epithelial cells Occludins: 65kDA much larger than Claudin -Two isoforms of occludin that result from alternative splicing… however have similar distribution -Localization of occludins are regulated by phosphorylation Common feature of claudin and Occludin: BOTH N- and C- terminals are facing the inside of the cell!!!! Claudin are more important in terms of Function • : claudin in kidney epithelia (claudin-16 is required for Mg2+ to be reabsorbed from the urine into the blood. A mutation in the gene encoding this claudin results in excessive loss of Mg2+ in the urine) These proteins make up the Zonula Occludens, which anchor the junction to the cytoskeleton(actin) Anchoring Junctions Hold cells together and stably in place -Desmosomes(cell-cell contact) -Adherens Junctions(cell-cell contact) -Hemidesmosomes(cell- matrix contact) -Focal Adhesion(cell- matrix contact) Desmosomes(cell-cell contact) - Attach to intermediate filaments within the cell -Has anchoring complex within the cell that attach to Intermediate filament -Has Transmembrane protein(Cadherin) that connect two adjacent cells Adherens Junctions(cell-cell contact) – Attach to Actin -Has anchoring complex within the cell that attach to Actin -ALSO has Transmembrane protein(Cadherin) Hemidesmosomes(cell- matrix contact) – Attach to intermediate filaments -Has transmembrane proteins called Integrins Focal adhesions(cell- matrix contact) - Attach to Actin -Also has Integrin transmembrane proteins These Four anchoring junctions are explained below! Intermediate filaments: keratin, vimentin, desmin i) Adherens junction -Found right under Zonula occludens -Attached to Actin belt that goes across the cell important for contraction due to actin and myosin present Contraction of the actin belt causes invagination of epithelial sheet which allows it to be pinched offimportant for neural tube development -transmembrane Cadherins will form a dimer with another Cadherin(Homodimerize) through homophilic interactions that is Calcium-dependant Anchored within the cell by proteins: Catenins, Vinculin, and Alpha-actinin Not anchored by Actin… only attached to actin ii) Desmosomes - transmembrane Cadherin family Desmoglein & Desmocollin -Desmoglein and Desmocollin attach to a plaque that is made of Desmoplakin and Plakoglobin -Desmoplakin and Plakoglobin then attach to Intermediate filaments within the cell Keratin in Epithelial cells, and Desmin in the heart cardiac cells The plaques attached to Intermediate filaments in parallel, which means that the Intermediate filaments do not end into the plaque, but curve parallel and continue bak away from the plak iii) Focal Adhesion -Attach to the ECM through Integrins, -The Integrin are also attached within the cell (cell-matrix) to alpha-actinin, Vinculin, and Actin -Integrins HETEROdimerize, with an Alpha version and Beta version which make it fully functional Both subunits are required! iv) Hemidesmosomes -Hemidesmosomes attach to Keratin intermediate filaments within the cell -Attached to Matrix (Basement membrane for Hemidesmosomes) -BM is made up of Laminin and Collagen IV -Transmembrane protein Alpha-6-Beta-4-Integrin -TM protein attach to Plaque made of Plectin Epidermolysis Bullosa:caused by mutation in Keratin, Plectin, alpha6beta4-integrin, Laminin, Collagen IV Hemidesmosomal Epidermolysis: caused by mutation in Plectin Intermediate filaments: keratin, vimentin, desmin Communication Junctions (Gap junctions) -Molecules up to 1000Daltons in size can diffuse through to neighboring cells through Gap junctions E.g. H2O, Glucose, AND, ions&Protons&electrons [Not only calcium??] thus metabolic & electrical coupling -Gap junctions are made up of Connexins( multipass of 4-pass of transmembrane proteins) -Both C-terminal and N-terminal in the 4-pass TM protein is sticking into the cytosol side There are 14 different types of Connexin 6 connexins make up a Connexon If more than 1 type of connexins make a connexon, then its called a Heteromeric connexon If all the connexins are the same type of connexins, then its called Homomeric connexon 2 connexONS make up a gap junction, one connexon form each cell membrane If two Homomeric connexons connect, then it is a Homotypic Gap junction If two Heteromeric connexons connect ,OR, 1 homomeric and 1 heteromeric connect then it will be called Heterotypic Gap junction Gap junctions are dynamic structures, they are NOT ALWAYS OPEN It OPENS when Ca++ concentration WITHIN the cell is LOW [high pH] It CLOSES when Ca++ concentration WITHIN the cell is HIGH [low pH] Inside the Cell, the Ca++ levels are in the Micromolar level Outside cell, Ca++ is 1000x much greater, in Millimolar level(2mM)extracelular level!even in blood! Dynamic regulation of gap junction(closing&opening) is very important because if too much calcium flushes into the cell, the cell will die Intermediate filaments: keratin, vimentin, desmin 1) Cell Adhesions -Unlike Cell junctions which are nearly permanent… Cell adhesions are more transient anchors -Like Cell junctions, there are cadherins and Integrins here too! -Cell adhesion form before cell junctions are established! • -Neural tube that is developing involves Nerual crest cells that aggregate together throughout the process by cell adhesions… The differentiating cells change patterns of Cell Adhesion molecules(CAM) cadherins, Integrin, etc… -Different cells are able to recognize eachother because of the Cadherinsthe cells that aggregate with eachother have the same type of cadherin! -Embryonic tissue is not highly structured and can be easily dissociated using 1. Trypsin protease used to dissociate cell adhesion molecules, 2. Ethylenediaminetetraacetic Acid(EDTA) used to bind metal ions like Ca++ and it has such high affinity for it that it will rip them away from the cells -Cells of two different embryonic organs(liver&spleen) can be dissociated, and mixed… -two types of fibroblasts which contain either E-cadherin or N-cadherin will separately aggregate where N-cadherin cells will aggregate separately from cells with E-cadherins two cells types where they have EITHER high E-cadherin levels, OR, low E-cadherin levels WILL ALSO SEPARATELY aggregate! THREE MECHANISMS OF Cell-Cell adhesions 1) Homophilic binding where the cells are connected through same proteins sticking out of each cell 2) Heterophilic binding where the cells have different proteins sticking out 3) Linker-dependent binding occurs through a third protein in between the proteins sticking out  Linker-dependent binding occurs the LEAST Heterophilic binding is prefered Homophilic binding is preferred AND occurs the MOST Intermediate filaments: keratin, vimentin, desmin Cadherin Superfamily • Classical Cadherins (at least 17 in Homo sapiens) • E-cadherin (Epithelial cells) • N-cadherin (Neurons and nerve related cells like muscle cells) • P-cadherin (Placenta, epidermis) • Non-classical Cadherins: • Protocadherins; expressed in the brain • desmosomal cadherins(Desmoglein & Desmocollin) There are Calcium-dependent and –independent CAMs Cadherins are Ca++Dependant CAM Cadherin superfamily structures Classical Cadherins: Single pass TM protein with 5 extracellular domains Flamingo cadherins: Multipass TM proteins Desmosomal cadherins: Single pass TM protein Protocadherins: Single pass TM protein MOST CADHERINS ARE SINGLE PASS TRANSMEMBRANE PROTEINS T-cadherins are NOT TM proteins…their anchored proteins glycosylphosphatidylinositol (GPI) anchor Classical Cadherin Structure Single pass TM protein with 5 extracellular domains leads to the N-terminus that is outside the cell in the Cytosol side there is the C-terminus which dimerize to form a single cadherin Each of th 5 domains are called cadherin repeats, cadherin domains -In between each cadherin repeat, there are Ca++ binding sites indicated in red below At the extracellular Ca++ concentration(2mM), all the Ca++ binding sites are saturated -when saturated with Ca++, the Cadherins repeats will be locked and become stiff and can interact with neighboring cells - when exposed to EDTA, all the Ca++ will be pulled off of the Cadherins, and the cadherins will become floppy and be susceptible to by proteolytic enzymes and be degraded and loses Cell-cell adhesion EDTA Degradation Like shown in the representation above, the cadherins do not overlap!! ONLY The N-terminal tip of each homodimer cadherin will fit into eachother and bind! Intermediate filaments: keratin, vimentin, desmin -Binding strength of the homodimer cadherin is weak! BUT since the homodimerss all cluster beside one another, they will bind to the next homodimer and the one after and so on…THIS makes the overall binding strength/affinity very strong! Cadherin diversity Protocadherins diversity is possible because they have 15 variable exons which can go through alternative splicing and since each variable exon has its own promotor 1 of them is chosen to be the N-terminal [different to the antibody diversification by site-specific recombination] and 3 constant regions that are always the samethus the C-terminal is always the same for them! The one Variable exon that is chosen will make up the entire extracellular component! Constant regions make up the Intracellular component (NOT intercellular which is between cells) Classical Cadherin E-cadherin -Has exterior cadherin dimer ready to bind another cadherin -Has its interior component attached to adaptor proteins within the cell(p120 & alpha/beta-Catenins ) -These proteins are attached to Actin filament within the cell cytosol Catenin regulate/signal how actin filaments are to assemble -When the Cadherins from each cell are not connected to eachother, Rac1 GTPase is bound to GDP and is inactive and is bound to GDP dissociation inhibitor (GDI) which prevents it from rebinding GTP -When the cadherins bind to eachother, GDI dissociates from Rac1…Rac1 moves to bind the membrane on the cytosolic side -When Rac1 binds the membrane, the E-cadherin signals a Phosphate Kinase enzyme to activate GEF(guanine exchange factor ) which puts GTP on Rac1 -This Active Rac1 will signal the cell to polymerize Actin Globules into Actin filaments and bind the cadherins Actin filaments will pull the two cells together allowoing more homodimer cadherins to bind eachother like a Feedback mech… a single cadherin couple positive feedback on more cadherins to form! Cold Spring Harb Perspect Biol 2009;1:a003020 END OF CADHERINS Intermediate filaments: keratin, vimentin, desmin Selectin adhesion proteins -Single TM protein with Large extracellular portion too -intracellular portion binds to anchor proteins -Anchor proteins beind to Actin filament -at Tip has a Lectin domain which interacts with Oligosaccharides -Calcium dependantbecome reactive when bound to calcium L-selectin – found on lymphocytes(WBC) E-selectin – found endothelial cells that line blood vessel P-selectin – platelets and endothelial cells When you have a wound, there are WBC that crawl out of the blood and move to the area -Cells in blood move very fast and need to be slowed down for them to squeeze through endothelial cells -due to wound, there are cytokines that signal the Endothelial cells to express more Selectin -WBC have oligosaccharides on their membrane, which interact with Selectin of the endothelial cells and the WBC consequently start slowing down while the rest of the RBC are flowing fast -Since the Selectin-WBC interaction is weak, the cells keep letting go and catch back to following selectin as they are being dragged by the force of the blood flow -this weak adhesion causes the WBC to roll across the blood vessels endothelial wall Due to strong interactions with Integrins of the Endothelial cells, the WBC will end up stopping allowing themselves to squeeze between the endothelial cells and diffuse to the wound these WBC lymphocytes DO NOT move out of arteries.. ONLY veins.. because the blood flow is much stronger in the Arteries than the veins..
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