Transcript Cell-Matrix Interactions

Cell-Matrix Interactions
Dr. Jeff Miner
7717 Wohl Clinic
362-8235
[email protected]
Fibronectin
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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
 51
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
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
Mao and Schwarzbauer, Matrix Biol. 2005
Fibronectin is Required for
Somitogenesis: Mesenchyme to
Epithelium Transition (MET)
Dr. Christoph Winkler, Wurzburg
Integrins
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Large family of transmembrane receptors for
extracellular matrix and cell surface proteins.
Consist of an  and a subunit, both with a single-pass
transmembrane domain.
16 different  chains and 8 different chains associate
to form 22 distinct heterodimers.
Cytoplasmic tails of both  and 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 actinmyosin 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
 7-helix leads to 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 subunit tail.
• A delicate equilibrium among
the different conformation
states exists.
Fibronectin and the Mesenchyme
to Epithelium Transition
Somitogenesis
°•Basement Membrane assembly
Larsen et al., Curr Opin Cell Biol 2006
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-proteincoupled 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
Anchors
Feet
Do they
exist in
tissues
in vivo?
Sensors
Sastry and Burridge, 2000
Biogenesis of Focal Adhesions
Legate KR et al. (2006) ILK, PINCH and parvin: the tIPP of integrin signalling
Nat. Rev. Mol. Cell Biol. 7: 1–12
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.
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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
•  31,  61,  71, and  64
• They are found on the surface of many
epithelial ( 3 and  6), endothelial ( 3, α6),
and muscle ( 7) 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
Control
(Itga3 expressed by the upper cell)
Integrin  3 Knockout
Kreidberg et al., Development 1996
The Dermal-Epidermal Junction
The Dermal-Epidermal Junction
LM-332
Laminin-332
LM-332
LM-511
LM-311
LM-511
Rousselle et al., J Cell Biol. 1997
Receptor-Laminin Interactions at the
Dermal-Epidermal Junction
• Integrin  31 binds primarily to laminin-511 in the
epidermal basement membrane (EBM) and links
the EBM to the actin cytoskeleton.
• Integrin  64 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.
Laminin-332 ( 332) 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  64 and the Hemidesmosome
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Litjens et al., TCB 2006
Hemidesmosome Assembly vs. Disassembly
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The binding of integrin  64 to
plectin plays a central role in HD
assembly. Disrupting the
association between these two
proteins, through serine/threonine
phosphorylation of the 4
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 4 and
HD disassembly.
Dystroglycan
• First identified as part of the
dystrophin glycoprotein complex
(DGC) in skeletal muscle.
• Consists of two polypeptides or
subunits ( and -dystroglycan)
– derived by proteolytic cleavage*** of
a single large protein
• Highly glycosylated
• α-DG is extracellular and binds to
Laminin
the LG4-5 domains of laminin  1
and α2 better than to  5
• -DG is transmembrane and binds
to  -DG outside the cell and to
dystrophin inside the cell.
K. Sekiguchi
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”)
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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
Glycosylation of  -Dystroglycan
Barresi, R. et al. J Cell Sci 2006;119:199-207
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.
Evans Blue Assay
Straub et al. J. Cell Biol. 1997
Mutations in Lama2 Cause
Congenital Muscular Dystrophy
• Laminin-211 ( 211) 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
A
polymorphism/mutatio
n in LTBP4 impacts
disease in a mouse
model of muscular
dystrophy.
Heydemann et al., J. Clin. Invest. 2009
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 1 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