Bio2000 Lecture 2004-1

Cell adhesion and Integrin signaling Joy Yang Department of Cell Biology Johns Hopkins University School of Medicine [email protected]

Lecture I. Cell adhesion principles

Lecture I: Cell adhesion principles • Cell adhesion receptors • Adhesion junctions • Principles of cell adhesion • Regulation of cell adhesion - epithelial-mesenchymal transition

Lecture II. Extracellular matrix and Integrin signaling

Cell Adhesion - a fundamental way that the cells communicate by attachment

- leukocyte extravasation

Cell adhesion is an essential process that allows cells to form tissues

Homophilic cell-cell adhesion - Maintains integrity and architecture of tissues

Heterophilic cell-cell adhesion

- Signal transduction

Cell-extracellular matrix adhesion - Homophilic: between cells of the same type - Heterophilic: between cells of different types

Cell adhesion regulates a variety of cell behaviors

• • • • •

Cell adhesion receptors (Cell adhesion molecules)

Proliferation Survival Migration Differentiation Specific cell-cell interactions ~25 members

~25 members

24 members 3 members

~100 related

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Principle of cell adhesion #1

Cells in different tissues have different adhesive activities Epithelium

Specificity • Specificity of cell adhesion is

achieved when each cell type expresses a unique set of cell adhesion receptors

Connective tissue • Epithelium: direct cell-cell contact • Connective tissue: no direct cell-cell contact, cells attach to extracellular matrix

Distinct adhesive activities of cells are critical for tissue function Epithelium: barrier and polarity between inside and outside

Lecture I

Cadherins - homophilic cell-cell adheison

Lecture II Epithelium Integrins - cell-ECM adhesion

Connective tissue: mechanical strength, constantly remodeled by cells in the tissue Connective tissue

White blood cells adhere to endothelial cells lining blood vessels in response to infection Integrins, IgCAMs and selectins: - heterophilic cell-cell adhesion

Discovery of E-cadherin Question: How do multicellular organisms form epithelial sheets? Hypothesis: Cells are connected to one another by transmembrane molecules.

Infected Tissue

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Aggregation assay - by Takeichi and colleagues

Aggregation assay - identification of two cell adhesion systems without Ca2+

CIDS

Low trypsin (Molecules #1 and #2 remained) without Ca2+

High trypsin (Molecule #1 remained)

- Are cell surface molecules responsible for the aggregation? Urushihara et al. (1976) J. Cell Sci. 22: 685

with Ca2+

CADS

High trypsin (Molecule #1 remained)

Urushihara et al. (1976) J. Cell Sci. 22: 685

Evidence for a role of E-cadherin in calcium-dependent cell-cell adhesion • Identified a monoclonal antibody that blocked CADS

Epithelium

Untransfected L cells

L cells expressing E-cadherin

• Recognized a protein of 120kDa: Ecadherin • Expression cloning of E-cadherin cDNA

Connective tissue

Fibroblasts (L cells) Nagafuchi et al. (1987) Nature 329:341

Cadherin superfamily • Classical (type I) cadherins (~ 5 members) E-cadherin N-cadherin P-cadherin Calcium-independent

• Type II cadherins (~ 12 members)

Calcium-dependent

• Desmosomal cadherins desmogleins (3 members) desmocollins (3 members) CADS

CIDS

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Cadherin Superfamily

Cell-type specific expression of different cadherins

- Differential adhesion may provide a mechanism for tissue morphogenesis and remodeling

The role of cadherins in cell sorting

Classical cadherins • Single-pass transmembrane homodimer

ectoderm + mesoderm

• Homotypic binding

Townes&Holtfreter (1955) J. Exp. Zool. 128:53

Friedlander et al. (1989) PNAS 86:7043

Identification of cadherin-catenin complex

• Ca2+ binding - rigidity

- Based on the crystal structures of EC1-2 fragments in classical cadherins: a zipper model

E-cadherin-catenin complex forms adherens junctions

L-cells expressing E-cadherin

Metabolically label the cells with [35S]Met

Lyse the cells with mild detergent

IP with an anti-E-cadherin antibody

SDS-PAGE gel and autoradiograph

Ozawa and Kemler (1992) J. Cell Biol. 116:989

• Transmembrane receptor: E-cadherin • Cytoplasmic adaptor proteins: b-catenin, a-catenin, etc. • Linking to actin cytoskeleton

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Cell junctions in epithelium

Cell junctions in epithelium

• Tight junction: barrier, sets polarity Tight junction

Tight junction

Adherens junction

• Adherens junction and desmosome: mediate adhesion between adjacent epithelial cells

Actin filaments

Intermediate filaments

• Hemidesmosomes: mediate adhesion between cells and basal lamina

Basal lamina

Desmosome

Hemidesmosome

Formation of epithelial sheets during organ formation in embryonic development Mesenchymal cells

- Assembly of cell junctions - Assembly of basal lamina Intermediate filaments

mesenchyme

Intermediate filaments Basal lamina

Integrin

Epithelium

(Mesenchyme: embryonic connective tissue)

Ca2+-dependent assembly of adherens junctions by epidermal keratinocytes

How is adherens junction assembled?

No Ca2+

Add Ca2+

Green: E-cadherin Red: Phalloidin staining of actin cytoskeleton Vasioukhin et al. (2000) Cell 100:209

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Vasioukhin et al. (2000) Cell 100:209

The actin cytoskeleton is required for E-cadherinmediated adherens junction formation

Green: vinculin Red: actin filaments

•

Formation of adherens junctions involves - clustering of E-cadherins - binding of cytoplasmic adaptor proteins - actin cytoskeleton association - zippering of opposing cadherin clusters

Vasioukhin et al. (2000) Cell 100:209

E-cadherin-catenin complex forms adherens junctions

Cell B

Cell A Actin filament or Intermediate filament

(weak affinity)

(strong avidity)

Cytoplasmic adapter proteins

Anchoring junctions mediate strong adhesion by - Clustering of many receptors of the same type • Transmembrane receptor: E-cadherin • Cytoplasmic adaptor proteins: b-catenin, a-catenin, etc. • Linking to actin cytoskeleton

- Association with the cytoskeleton

Embry

Principle #2 Affinity vs. Avidity • Cell adhesion receptors have weak ligand-binding affinity – Transient adhesion mediated by non-junctional receptors

Principle #3 Cytoskeletal association is essential for the formation and functions of anchoring junctions

• Clustering of the receptors results in high avidity and strong adhesion – Mediated by adherens junction and other anchoring junctions

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Epithelial-mesenchymal transition (EMT)

Epithelial-mesenchymal transition during embryonic development

Epithelium

Gastrulation

Neural crest migration

Mesenchyme Mesenchymal cells

Epithelial-mesenchymal transition

Epithelial-mesenchymal transition in progression of epithelial cancers

Epithelium • Loss of cell-cell contacts - down-regulation of E-cadherin - disassembly of adhesion junctions • Cells become migratory

Mesenchymal cells

Down-regulation of E-cadherin in cancer cells

- E-cadherin is down-regulated in a majority of epithelial cancers (carcinoma)

Transcriptional repression of E-cadherin in EMT during development and tumor metastasis by Snail

• Mutations/deletions of E-cadherin • Transcriptional regulation of E-cadherin expression

- Transcriptional repressor Snail

• Endocytosis and turnover of E-cadherin complex • Adhesion function of E-cadherin (disassembly of AJ)

Cano et al. (2000) Nat. Cell Biol. 3:76

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EGF-induced endocytosis of E-cadherin (A mechanism for EGF-promoted tumor metastasis) EGF:

-

Regulation of E-cadherin turnover by p120-catenin

+

Endocytosis

• No EGF: E-cadherin is localized at cell-cell junctions (yellow) • With EGF: E-cadherin is internalized by endocytosis (green)

Decision: recycled or degrated? Lu et al. (2003) Cancer Cell 4:499

Regulation of E-cadherin turnover by p120-catenin

Pulse-chase experiment Epithelial cells with or without expressing p120-ctn siRNA

Grow cells in culture medium with [35S]Met. (pulse)

Replace medium with non-radioactive medium and grow cells for various time intervals

Lyse the cells and IP with an anti-E-cadherin antibody

Davis et al. (2003) J. Cell Biol. 163:525

Regulation of E-cadherin turnover by p120-catenin

SDS-PAGE gel and autoradiograph

Regulation of E-cadherin

Endocytosis

Lactacystin: proteosome inhibitor

- p120-catenin inhibits proteosome-mediated degradation of E-cadherin Davis et al. (2003) J. Cell Biol. 163:525

- Biosynthesis: expression level - Turnover: stay or endocytose, recycled or degraded - AJ assembly or disassembly

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The roles of E-cadherin in tumor progression

Down-regulation of E-cadherin leads to: • cells breaking away from primary tumor • loss of control of cell proliferation

Loss of contact inhibition by malignant tumor cells

b-catenin has dual functions

• in AJs: linking cadherins to actin cytoskeleton • in cytoplasm/nucleus: regulates gene expression

Model for contact inhibition

Cancer cells

Normal cells

• Sequestration of b-catenin in AJ

Summary • Cadherins mediate Ca2+-dependent homophilic cellcell adhesion

Current research interests • How are the assembly of adherens junction regulated?

• E-cadherin forms a complex with a- and b-catenins in adherens junctions of epithelium

• How is the turnover of cadherins regulated?

• Adherens junction provides strong adhesion in epithelium: clustering (affinity vs. avidity) and cytoskeletal association

• Is there crosstalk between cadherin and growth factor receptors?

• Regulation of E-cadherin • Roles of cadherins in cell sorting, EMT and cancer

• Coordination among different cadherins • Cross talk between AJ and tight junctions for setting up polarity of epithelium

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Adhesion junctions in epithelium

Junctional vs. non-junctional adhesion

Actin filaments

Intermediate filaments

Junctional: stable adhesion

- Maintain tissue integrity Basal lamina

- mediate relatively stable adhesion - maintain integrity of tissues

Migration of leukocytes from blood circulation to a wound

Nonjunctional: transient adhesion Embryonic development - Wound healing - Leukocyte trafficking

-

Leukocyte extravasation in response to infection • Weak adhesion - Rolling • Stronger adhesion (but weaker than junctional adhesion) • Invasion

- Extravasation

leukocyte

leukocyte

Rolling

- P-selectin is presented on cell surface by exocytosis when endothelial cells are activated - Weak adhesion and rolling

Endothelial cell of blood vessel

Endothelial cell of blood vessel

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leukocyte

Leukocyte activation

Leukocyte adhesion

Leukocyte invasion

- aLb2 integrin is activated upon neutrophil activation - Relatively strong adhesion

Endothelial cell of blood vessel

Leukocyte adhesion deficiency-I

Principle #4 Cell adhesion receptors can be regulated at different levels • Selective surface expression - Transcriptional (all) - Post-translational • Released from storage granules by exocytosis (selectins) • Endocytosis (cadherins)

• Modulation - the state of clustering or dispersal: avidity - Results from null mutation of b2 integrin

(integrins and cadherins - assembly and disassembly of cell adhesion junctions)

- the state of activation: affinity (integrins)

Integrins - Heterodimer with a and b subunits

How is the ligand-binding affinity of integrins regulated?

- Cell-ECM adhesion - Heterophilic cell-cell adhesion - A large family with at least 24 members

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The Structure of Integrin

Integrin Superfamily

I /A

(Fibronectin)

Integrin I/A domain

I /A

MIDAS site

Closed (yellow) and open (blue) conformations

- Conformational change in the head is propagated to the legs and vice versa

Conformations of avb3 integrin visualized by electron microscopy

Inactive (closed) and active (open) conformations of avb3 integrin

cDNAs encoding the extracellular domains of av and b3 Co-transfect into cultured cells

Purify the recombinant avb3 ecto-domain heterodimer

Ca2+: inactive bent, legs close together Mn2+: partially activated extended, legs close together RGD and Mn2+: fully activated fully extended, legs separated

Treat with Ca2+, Mn2+ or Mn2+/RGD (ligand)

Electron microscopy Takagi et al. (2002) Cell 110:599

When locked in bent conformation, ligand failed to bind. Takagi et al. (2002) Cell 110:599

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Integrins in closed and open conformations From outside to inside: 1. Mn2+ opens the head and straightens the legs 2. Ligand binds 3. Legs separated

Activated aLb2

From inside to outside: 1. Legs straightened and separated 2. Opens the head 3. Ligand binds

ICAM-1

PAF

Talin as a cytoplasmic integrin activator

How is integrin activation regulated? - inside-out signaling pathway

Separation of integrin legs detected by Fluorescence resonance energy transfer

Kim et al. (2003) Science 301:1720

Fluorescence resonance energy transfer (FRET)

CFP: donor YFP: acceptor

No FRET

FRET Kim et al. (2003) Science 301:1720

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Talin is an integrin activator Adhesion

Activation of integrin by an inside-out signaling pathway

Separation of legs

Intracellular signaling cascade

Talin head - enhanced the adhesive activity of integrin - decreased FRET between a and b tails: separation of the tails Kim et al. (2003) Science 301:1720

Summary • Non-junctional adhesion receptors mediate transient cell-cell and cell-ECM interactions

Principle #5 Cell adhesion receptors transduce signals across the plasma membrane

• Cell adhesion is regulated at different levels • Integrins have open and closed conformations • Inside-out signaling by integrins

Principles of cell adhesion • Specificity • Affinity vs. avidity • Cytoskeletal association (cadherins and integrins) • Regulation • Signaling

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