cell-matrix interactions dr. jeff miner 7717 wohl clinic 362-8235 [email protected]
TRANSCRIPT
Fibronectin
• A glycoprotein associated with many extracellular matrices and present in blood plasma
• Alternative splicing generates many isoforms that heterodimerize covalently via S-S bonding
• Fibroblasts make it, assemble it, stick to it, and respond to it
• FN harbors the famous “RGD” motif (III-10) which serves as a ligand for various integrins, especially α5β1
• Fn-/- mouse embryos die at E8.5 due to defects in the vasculature and in heart development
Mao and Schwarzbauer, Matrix Biol. 2005
Molecular Interactions of Fibronectin
Fibronectin and Branching Morphogenesis
Sakai et al., Nature 2003
Fibronectin and Branching Morphogenesis
Inhibiting FN expression with siRNA reduces branching
Adding FN promotes branching
Sakai et al., Nature 2003
Integrins Direct FN Fibril Formation
Mao and Schwarzbauer, Matrix Biol. 2005
Compact soluble FN binds integrin
FN binding induces reorganization of actin and signaling
Cell contractility leads to changes in FN conformation, exposing FN interaction domains and allowing fibril formation
Fibronectin is Required for Somitogenesis: Mesenchyme to
Epithelium Transition (MET)
Dr. Christoph Winkler, Wurzburg
Integrins
• Large family of transmembrane receptors for extracellular matrix and cell surface proteins.
• Consist of an α and a β subunit, both with a single-pass transmembrane domain.
• 16 different α chains and 8 different β chains associate to form 22 distinct heterodimers.
• Cytoplasmic tails of both α and β chains mediate cell signaling events in response to ligand binding.
• Some integrins bind to a specific site on matrix proteins, such as Arg-Gly-Asp (RGD), which is found in fibronectin, vitronectin, tenascin, et al.
• Ligand binding absolutely requires divalent cation• As mechanotransducers, integrins link the
extracellular matrix to the force generating actin-myosin cytoskeleton. This allows the cell to influence the nature of the extracellular matrix, and allows the ECM to influence cellular architecture and behavior.
Integrin Family Members and Their Ligands
Hynes (2002) Cell 110:673
Integrins Need to be Activated
• Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling.
• Ligand binding transduces signals from the cellular environment to the interior of the cell through outside-in signaling.
• Protein structure analyses have provided insights into the mechanisms whereby integrins become activated to bind ligand and how ligand binding translates to changes in intracellular signaling.
Adair and Yeager, Meth. Enzymol. 2007
Model for Integrin Activation
• Involves a switchblade-like motion when the headpiece extends
• Downward movement of the α7-helix leads to β subunit hybrid domain swing out, separation of the knees, and opening of the headpiece for high affinity ligand binding
• Activation can occur by PKC stimulation, GPCR activation, or binding of proteins such as talin to the β subunit tail.
• A delicate equilibrium among the different conformation states exists.
Fibronectin and the Mesenchyme to Epithelium Transition
Larsen et al., Curr Opin Cell Biol 2006
Somitogenesis
°•Basement Membrane assembly
Integrin Signaling Pathways
Signal transduction proteins associated with, or activated by, integrins. Signaling molecules, such as FAK, bind to and recruit additional signaling molecules, creating a complex signaling network that is intimately connected to the cytoskeletal network.
Integrins and growth factor receptors cooperate in cell cycle regulation. Both growth factors and cell adhesion are required for transmitting signals to the Ras/Raf/Mek/Erk signaling pathway.
Miranti and Bruge (2002) Nature Cell Biol. 4:83
Integrin Signaling
The major integrin signal transduction pathways and many of the key players, leading to the effects on cell behavior, often acting in concert with G-protein-coupled or kinase receptors.From: Hynes (2002) Cell 110:673
Different pathways by which integrins can link to the actin cytoskeleton.
From: Brakebusch & Fässler (2003) EMBO J. 22:2323
Anoikis
• Apoptosis induced by inadequate or inappropriate cell/matrix interactions.
• Resistance to anoikis can lead to metastasis of epithelium-derived cancer cells.
Focal Adhesions are Organized by Integrins and Form Along Actin Stress Fibers
Sastry and Burridge, 2000
Do they exist in tissues in vivo?
Anchors
Feet
Sensors
Legate KR et al. (2006) ILK, PINCH and parvin: the tIPP of integrin signallingNat. Rev. Mol. Cell Biol. 7: 1–12
Biogenesis of Focal Adhesions
Receptors for the Basement Membrane
• Cells are thought to recognize the basement membrane through receptors that interact with specific basement membrane components, primarily with laminin.– Integrins– Dystroglycan– Ig-superfamily transmembrane receptor (Lutheran/B-CAM)– Syndecans--transmembrane receptors with HSPG side
chains in their ectodomains.
• Binding of receptors to the basement membrane can result in signal transduction and alterations in cell behavior
Laminin-Binding Integrins
• α3β1, α6β1, α7β1, and α6β4• They are found on the surface of many
epithelial (α3 and α6), endothelial (α3, α6), and muscle (α7) cells.
• They bind primarily to laminin α chains and demonstrate some specificity.
• Their activities are modulated by members of the tetraspanin family of 4-pass transmembrane proteins– CD9, CD81, CD151
Tetraspanin
Integrins Regulate Basement Membrane Architecture
Kreidberg et al., Development 1996
Control Integrin α3 Knockout
(Itga3 expressed by the upper cell)
The Dermal-Epidermal Junction
The Dermal-Epidermal Junction
Rousselle et al., J Cell Biol. 1997
LM-511
Laminin-332
LM-511
LM-311
LM-332
LM-332
• Integrin α3β1 binds primarily to laminin-511 in the epidermal basement membrane (EBM) and links the EBM to the actin cytoskeleton.
• Integrin α6β4 binds primarily to laminin-332 in the EBM and initiates hemidesmosome formation
• Laminin-332 is covalently linked to laminin-311, which incorporates into the EBM via nidogen/HSPG interactions
• Laminin-332 binds to collagen VII, a large collagen that binds to other matrix proteins in the dermal stroma.
Receptor-Laminin Interactions at the Dermal-Epidermal Junction
Laminin-332 (α3β3γ2) is a Required Component of the EBM
• Mutations in LAMA3, LAMB3, or LAMC2 cause junctional epidermolysis bullosa (JEB), a skin blistering disease.
• Two forms of JEB: severe (Herlitz, lethal), and non-Herlitz, non-lethal
• Type of mutation determines disease severity.– Truncating vs. missense or
reduced expression.
• Corresponding mutant mice also show blistering phenotype
Ryan et al., J. Cell Biol. 1999
Integrin α6β4 and the Hemidesmosome
Litjens et al., TCB 2006
*
Hemidesmosome Assembly vs. Disassembly
• The binding of integrin α6β4 to plectin plays a central role in HD assembly. Disrupting the association between these two proteins, through serine/threonine phosphorylation of the β4 cytoplasmic domain (perhaps by PKC and PKA), is a critical event in the disassembly of HDs.
• De-phosphorylation of residues distal to the plectin binding domain leads to unfolding of the tail, exposing the binding site for plectin.
• EGF signaling can lead to phosphorylation of integrin β4 and HD disassembly.
Dystroglycan• First identified as part of the
dystrophin glycoprotein complex (DGC) in skeletal muscle.
• Consists of two polypeptides or subunits (α and β-dystroglycan) – derived by proteolytic cleavage*** of
a single large protein
• Highly glycosylated • α-DG is extracellular and binds to
the LG4-5 domains of laminin α1 and α2 better than to α5
• β-DG is transmembrane and binds to αDG outside the cell and to dystrophin inside the cell. K. Sekiguchi
Laminin
Dystroglycan is Part of the Dystrophin Glycoprotein Complex (DGC)
Dystroglycan
• Some evidence suggests that dystroglycan is involved in and perhaps necessary for laminin polymerization at the surface of cells, which initiates basement membrane formation.
• Dystroglycan KO embryonic stem cells cannot assemble soluble laminin at the cell surface.
Dystroglycan
• Some evidence suggests that dystroglycan is involved in and perhaps necessary for laminin polymerization at the surface of cells, which initiates basement membrane formation.
• Dystroglycan KO embryonic stem cells cannot assemble soluble laminin at their cell surfaces.
• Dystroglycan KO has no Reichert’s basement membrane, dies at ~E5 to E6 (just after implantation into the uterus).
Dystroglycan Function Requires Extensive Glycosylation
• DG isolated from certain muscular dystrophy patients or mice does not bind a DG antibody with an epitope dependent on glycosylation– Also shows reduced binding to laminin
• Six glycosylation enzymes are mutated in human muscular dystrophies (called “dystroglycanopathies”)– LARGE1: MDC1D– POMT1, POMT2: Walker-Warburg syndrome– POMGnT1: Muscle-eye-brain disease– Fukutin: Fukuyama congital muscular dystrophy– FKRP: diverse group of MDs, from severe to
mild
• The protein core of DG has little receptor function on its own; glycosylation is paramount
Martin, P. T. Glycobiology 2003 13:55R-66R
Barresi, R. et al. J Cell Sci 2006;119:199-207
Glycosylation of α-Dystroglycan
Muscular Dystrophy
• Muscular dystrophy refers to a group of genetic muscle diseases that cause progressive muscle weakness due to defects in muscle proteins, which lead to death of muscle cells and tissue (necrosis).
• There are many forms of muscular dystrophy that vary in onset and severity based on the protein affected and the nature of the mutation.
• It has been proposed that the trigger of muscle cell necrosis is the focal breakdown of the plasma membrane due to contraction-induced damage.
• MD is a disease characterized by defective muscle cell/matrix interactions. Straub et al. J. Cell Biol. 1997
Evans Blue Assay
Mutations in Lama2 Cause Congenital Muscular Dystrophy
• Laminin-211 (α2β1γ1) is the major laminin in skeletal muscle fiber basement membranes and in peripheral nerve basement membranes.
• Mutations, some null, affect humans and mice.
• Skeletal muscle BM breaks down with the severe mutations, disrupting linkage of the actin cytoskeleton to the ECM.
Agrin
Engineering a Cure for CMD with “mini-Agrin”
Meinen and Ruegg, Gene Therapy 2006
Dystroglycan is Part of the Dystrophin Glycoprotein Complex (DGC)
Indirect Promoters of Muscle Pathology in Muscular Dystrophy
Heydemann et al., J. Clin. Invest. 2009
A polymorphism/mutation in LTBP4 impacts disease in a mouse model of muscular dystrophy.
Basement Membrane Proteins Regulate Mammary Cell Gene Expression:
Got ECM?
Streuli et al,J. Cell Biol. 1991
What is the Mechanism? (What Receptors?)
• Dystroglycan and integrins containing β1 cooperate to organize laminin, transduce the information from the ECM, induce cell polarization, and activate expresson of milk proteins.
Weir et al., J. Cell Sci. 2006