Physiology Seminar11/2/2013PowerPoint® Seminar Slide Presentation prepared byDr. Anwar Hasan Siddiqui, Senior Resident, Dept of Physiology, JNMCIntercellular ConnectionsandMolecular Motors
Learning Objectives Cell Adhesion Molecules Intercellular Connections. Brief description of each type and theirfunction. Molecular motors. What are they and whatthey do?
Cell Adhesion Molecules (CAMs) Important cell surface proteins moleculespromoting cell–cell and cell–matrixinteractions. Important for many normal biological processes-embryonic cell migration, immune systemfunctions, wound healing. Involved in intracellular signaling pathways(primarily for cell death/survival, secretion etc.)
Cell Adhesion Molecules (CAMs) Express 3 major domains:• The extracellular domain allows one CAM to bind toanother on an adjacent cell.• The transmembrane domain links the CAM to theplasma membrane through hydrophobic forces.• The cytoplasmic domain is directly connected to thecytoskeleton by linker proteins.
Cell Adhesion Molecules (CAMs) Interactions between CAMs can be mediated by:Binding of anadhesion molecule onone cell to the sameadhesion molecule ona second cellCadherin - cadherinAn adhesion moleculeon one cell type bindsto a different type ofcell adhesion moleculeon a second cellSelectins – mucinsThe linker molecule inmost cases isLaminin, a family oflarge cross shapedmolecules withmultiple receptordomains.
Principal classes of cell-adhesion molecules Identified by using specific monoclonalantibodies (mAbs). genes encoding these molecules has shown thatthey are structurally different from each other. These cell adhesion molecules can be dividedinto 4 major families• The cadherin superfamily• The selectins• The immunoglobulin superfamily and• The integrins
The Cadherin superfamily Cadherins are the most prevalent CAMs invertebrates. 125 kD transmembrane glycoproteins - mediateintercellular adhesion in epithelial andendothelial cells by Ca2+ dependent homophilicadhesion. Primarily link epithelial and muscle cells totheir neighbors• Form desmosomes and adherens junctions Play critical role during development (cellsorting). Do not interact with extracellular matrix.
The Cadherin superfamily Contain a short transmembranedomain and a relatively longextracellular domain containing fourcadherin repeats (EC1-EC4), each ofwhich contains calcium bindingsequences Cadherins interact with specificcytoplasmic proteins, e.g., catenins(α, β and γ), as a means of beinglinked to the actin cytoskeleton. The binding of cadherins to thecatenins is crucial for cadherinfunction.
The Cadherin superfamily E-cadherin is thought to be important duringembryonic development, and is also involved ingenerating and maintaining epithelial layers inadult tissues. The loss of E cadherin expression has beenlinked to the invasive behavior of tumour cells
The Cadherin superfamilyCa binds in the hinge regions between cadherindomains, and prevent the flexing. Without Ca themolecule is floppy and adhesion fails
The Selectins Involved in heterophilic cell-cell interactions. Family of Ca+2 dependent carbohydratebinding proteins, mediate the initial attachmentof leukocytes to the endothelium on the bloodvessel wall during the rolling step of leukocyteextravasation in inflammation. Selectins recognize fucosylated carbohydrateligands, especially structures containing Sialyl-Lewis x (sLex) and Sialyl-Lewis a (sLea), whichare heavily expressed on neutrophils andmonocytes
The Selectins Structural features of selectinsinclude:• NH2-terminal C-type Ca2+dependent lectin like bindingdomain, which determines theability of each selectin to bind tospecific carbohydrate lingands.• an epidermal growth factor-likeregion.• a number of repeat sequences.• a membrane-spanning region and• a short cytoplasmic region
The Selectins Selectin family• Leukocyte-expressed L-selectin(CD62L)• Endothelial-expressed E-selectin(CD62E)• P-selectin(CD62P) which is expressed by both platelets andendothelial cells
The Selectins• Recently elevated levels of L-selectin have been observed in theserum of patients with AIDS and leukemia (1)• E selectin has been found to regulate adhesion of human coloncancer cells to the endothelium by binding to sLea and sLexcarbohydrate ligands (2)
Immunoglobulin Superfamily Molecules Have a series of globular Ig-likedomains, formed by disulfide bonds. Mediate Ca-independent cell adhesion. Primarily homophilic cell-cell adhesion but alsosome heterophilic. Activate intracellular signaling pathways. Play critical role during morphogenesis anddifferentiation of muscle, glial and nerve cells In neurons promote the formation of myelin In vascular endothelial cells leukocyte adhesionand extravasation.
Immunoglobulin Superfamily Molecules Consists of more than 25 molecules. Important ones being:• Intracellular adhesion molecule 1(ICAM1; CD54)• Intercellular adhesion molecule 2 (ICAM2),• Vascular cell adhesion molecule1 (VCAM1; CD106),• Platelet endothelial cell adhesion molecule 1 (PECAM1; CD31) and• the mucosal addressin cell adhesion molecule 1(MAdCAM1).
The integrins Cell adhesion receptors responsible for the cellextracellular matrix adhesion Important signal transduction receptors forregulation of cell growth Present in membranes of all cells excepterythrocytes. Composed of heterodimers consisting of twonon-covalently associated subunits,α and β, bothof which are necessary for adhesive binding.
The integrins Fifteen different α and eightdifferent β subunits give rise to overtwently different heterodimericcombinations at cell surfaces. Bind epithelial and muscle cells tolaminin in the basal lamina Allow platelets to stick to exposedcollagen in a damaged blood vessel Allow fibroblasts and white bloodcells to adhere to fibronectin andcollagen as they move
Tight Junctions Also known as Zona Occludens. Surround the apical margins of the cells inepithelia such as the intestinal mucosa, the walls ofthe renal tubules, and the choroid plexus. Made up of ridges—half from one cell and halffrom the other—which adhere so strongly at celljunctions that they almost obliterate the spacebetween the cells. Permit the passage of some ions and solute inbetween adjacent cells (paracellular pathway) andthe degree of this ―leakiness‖ varies, depending inpart on the protein makeup of the tight junction.
Tight Junctions Basic architectural principle - transmembraneproteins are linked to a cytoplasmic plaque that isformed by a network of scaffolding and adaptorproteins, signalling components and actin-binding cytoskeleton
Tight Junctions TRANSMEMBRANE TIGHT JUNCTION PROTEINS: Tight Junctions contain two principal types ofTransmembrane protein components – tetraspanand single-span transmembrane proteins. The tetraspan proteins are:• occludin and the claudins• have both the N- and C-termini in the cytosol.• form the paracellular permeability barrier and determinethe capacity and the selectivity of the paracellulardiffusion pathway. The single-span transmembrane proteins are thejunctional adhesion molecules (JAMs),
Functions of Tight Junctions Paracellular permeability:• allow the passive selective diffusion of ions and smallhydrophilic molecules through the paracellularpathway across epithelia and endothelia.• the claudin composition of TJs is a major determinantof the permeability properties of a tissue.• Occludin regulates the paracellular diffusion of smallhydrophilic molecules, and regulates thetransepithelial migration of neutrophils.• The passage of solute depends upon its size andcharge.
Functions of Tight Junctions Cell proliferation, polarity and differentiation:• Several studies have linked TJs to the regulation ofcell proliferation and cell polarity.• Occludin suppresses oncogenic Raf-1 signalling(Wang et al., 2005) and interacts withZONAB, thereby regulating gene expression, cellproliferation and epithelial morphogenesis (Matterand Balda, 2007; Sourisseau et al.,2006)• Occludin has also been linked to the regulation ofvarious subcellular signalling pathways, such as MAP-kinase-dependent pathways.
Gap Junction Gap junctions are clusters of intercellularchannels that allow direct diffusion of ions andsmall molecules between adjacent cells. At gap junctions, the intercellular space narrowsfrom 25 nm to 3 nm. gap junctions were first discovered inmyocardium and nerve because of theirproperties of electrical transmission betweenadjacent cells (Weidmann 1952; Furshpan andPotter 1957).
Gap Junction The intercellular channels are formed by head-to-headdocking of hexameric assemblies (connexons) oftetraspan integral membrane proteins, the connexins(Cx) (Goodenough et al. 1996).
Gap Junction Electron microscopy of gap junctions joining adjacent hepatocytesin the mouse. The gap junction (GJ) is seen as an area of closeplasma membrane apposition
Function of Gap Junction The diameter of the connexon channel is normally about2 nm, which permits the passage of ions, sugars, aminoacids, and other solutes with molecular weights up toabout 1000 Dalton. Function as suppressors of somatic cell mutations -lossof a critical metabolic enzyme or ion channel in one cellcompensated by its neighbours. Are particularly important in cardiac muscle: the signalto contract is passed efficiently through gapjunctions, allowing the heart muscle cells to contract intandem. A gap junction located in neurons referred to as anelectrical synapse are important in neurotransmitterrelease
Disease associated with Gap Junctions 20 different genes code for connexins in humans, andmutations in these genes can lead to diseases that arehighly selective in terms of the tissues involved. In humans, mutations in Cx32 underlie X-linkedCharcot-Marie-Tooth syndrome, a common peripheraldemyelination neuropathy. mutations in Cx47 result in a central demyelinatingcondition. disorders of the skin and the auditory systemaccompany mutations in Cx31 andCx30. Familial cataracts are commonly associated withmutations in either Cx46 or Cx50.
Desmosomes Also known as macula adherens is a cell structurespecialized for cell-to-cell adhesion. Are molecular complexes of cell adhesion proteins andlinking proteins that attach the cell surface adhesionproteins to intracellular keratin cytoskeletal filaments. The cell adhesion proteins of the desmosome, desmogleinand desmocollin, are members of the cadherin family. On the cytoplasmic side of the plasma membrane, thereare two dense structures called the Outer Dense Plaque(ODP) and the Inner Dense Plaque (IDP).• The Outer Dense Plaque is where the cytoplasmic domains of thecadherins attach to desmoplakin via plakoglobin and plakophillin.• The Inner Dense Plaque is where desmoplakin attaches to theintermediate filaments of the cell.
Hemidesmosomes Hemidesmosomes look like half-desmosomesthat attach cells to the underlying basal lamina. Rather than usingdesmogleins, hemidesmosomes usedesmopenetrin cell adhesion proteins,which aremembers of Integrin family. The integrin molecule attach to one of manymulti-adhesive proteins such aslaminin, resident within the extracellularmatrix, thereby forming one of many potentialadhesions between cell and matrix.
Molecular Motors Molecular motors composed of motor proteins. These proteins bind to a polarized cytoskeletalfilament and use the energy derived fromrepeated cycles of ATP hydrolysis to movesteadily along it Power movements of subcellular components Create local forces leading to cell shape changes• Muscle contraction Power cell movements
Molecular Motors Dozens of different motor proteins coexist inevery eucaryotic cell. They differ in the type of filament they bind to(either actin or microtubules), the direction inwhich they move along the filament, and the―cargo‖ they carry. There are three super families of molecularmotors:• kinesin,• dynein, and• myosin.
Molecular Motors, Kinesin The conventional form of kinesin is a doubleheadedmolecule that tends to move its cargo toward the ―+‖ends of microtubules. Dimer of two heavy chains Each heavy chain complexes with a light chain Three domains• Two globular head domains• Long central coiled-coil stalk• Two small globular tail domains(contain light chains
Molecular Motors, Kinesin Kinesin accomplishes transport by "walking" along amicrotubule. Two mechanisms have been proposed toaccount for this movement.• In the "hand-over-hand" mechanism, the kinesin heads step pastone another, alternating the lead position.• One head binds to the microtubule and then bends its neck whilethe other head swings forward and binds, producing almostcontinuous movement• In the "inchworm" mechanism, one kinesin head alwaysleads, moving forward a step before the trailing head catches up.
Molecular Motors, Dyenin Dynein transports various cellular cargo by "walking"along cytoskeletal microtubules towards the minus-endof the microtubule. Composed of two or three heavy chains (that include themotor domain) and a large and variable number ofassociated light chains. Dyneins can be divided intotwo groups:• cytoplasmic dyneins and• axonemal dyneins, which arealso called ciliary or flagellar dyneins.
Molecular Motors, Dyenin Cytoplasmic dyneins are found in all eucaryotic cells -important for vesicle trafficking, and for localization ofthe Golgi apparatus near the center of the cell. Axonemal dyneins, are highly specialized for the rapidand efficient sliding movements of microtubules thatdrive the beating of cilia and flagella. Dyneins are the largest of the known molecularmotors, and they are also among the fastest: axonemaldyneins can move microtubules in a test tube at theremarkable rate of 14 μm/sec
Molecular Motors, Myosin Myosins comprise a family of ATP-dependent motorproteins and are best known for their role in musclecontraction and their involvement in a wide range ofother eukaryotic motility processes. Most myosin molecules are composed of ahead, neck, and tail domain.• The head domain binds the filamentous actin, and uses ATPhydrolysis to generate force and to "walk" along the filamenttowards the barbed (+) end (with the exception of myosinVI, which moves towards the pointed (-) end).• the neck domain acts as a lever arm for transducing forcegenerated by the catalytic motor domain.• The tail domain generally mediates interaction with cargomolecules and/or other myosin subunits. In some cases, the taildomain may play a role in regulating motor activity
Molecular Motors, Myosin 18 different families (identified by geneticanalysis) Have different functions• Myosin II powers muscle contraction and cytokinesis• Myosins I transport of endocytic vesicles• Myosin V phagocytosis and transport of cellularelements• Myosins VI and VII – transport endocytic vesiclesinto the cell. Found in the inner ear and mutations inthe gene coding for myosin VII cause deafness in miceand humans.
Molecular Motors, Myosin A myosin II molecule is composed of two heavy chains(each about 2000 amino acids long (green) and fourlight chains (blue). the long coiled-coil tail bundles itself with the tails ofother myosin molecules forming bipolar ―thickfilaments‖ that have several hundred myosin heads,oriented in opposite directions at the two ends.
Molecular Motors, Myosin How does the myosin move? Cyclic attachment and detachment of myosin head fromactin filament, each coupled to hydrolysis of one ATP ATP binding to myosin opens the cleft and disruptsactin binding Release of actin from myosin head ATP hydrolysis - bending of the head to the newposition (generation of movement) After ATP hydrolysis the cleft closes on the next actinmolecule