Bio2000 Lecture 2004-2

Cell-Cell Adhesion

Cell-ECM Adhesion

Part II: Cell-ECM adhesion and Integrin Signaling Joy Yang Department of Cell Biology Johns Hopkins University School of Medicine [email protected]

To be learned in this lecture: • Extracellular matrix (ECM) - ECM in epithelium and connective tissue - Biological functions of ECM macromolecules - A case study: the role of ECM in epithelial branching

• Signaling pathways activated by interactions between integrins and ECM

Extracellular Matrix (ECM) in connective tissue

Extracellular matrix - A meshwork of proteins and sugars secreted, assembled and organized by cells - Two types of ECM: - ECM of connective tissues - Basal lamina (basement membrane)

Collagen fibrils shaped by fibroblasts Sawhney and Howard (2002) J. Cell Biol. 157:1083

Fibroblasts

- Predominant constituent of connective tissue

traction

- Contains embedded cells (fibroblasts) that remodel ECM

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Basal Lamina (basement membrane)

ECM Macromolecules

Epithelium

- Structural: collagens, glycosaminoglycans, proteoglycans and elastic fibers - Instructive: adhesive glycoprotein (fibronectin and laminin) Basal Lamina

Connective tissue

Glycosaminoglycans and Proteoglycans

Collegens Fibrillar collagen (Type I, II)

Sheet-forming collagen (Type IV)

Glycosaminoglycan (GAG)

- Water: compressive strength: - Extended: space for diffusion of molecules and migration of cells - Reservoir for growth factors

Proteoglycans

in connective tissues

GAG Core protein

- Co-receptors for growth factor receptors and integrins

in basal lamina Hyaluronan

• Major structural component of ECM • Tightly packed triple-helical domains: tensile strength to tissues

ECM of connective tissue Collagen glycosominoglycans

Proteoglycan

Proteoglycan-GAG complex in cartilage

Adhesive glycoproteins are mediators between ECM and cells • Fibronectin

Fibronectin

• Laminin • Vitronectin • Thrombospondin Inside of cell • Osteopondin

Structural : collagens, glycosaminoglycans and proteoglycans Instructive: adhesive glycoproteins (fibronectin)

- Play instructive roles to regulate cell behaviors

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Fibronectin reverts malignant tumor phenotype without FN

with FN

Mautner and Hynes (1977) J. Cell Biol. 75:743

Cell-ECM attachment and detachment are coupled to mitosis

Anchorage-dependent cell proliferation and survival

- Adhesion glycoproteins are essential for cell cycle progression

Differentiation of Mammary Epithelium

without ECM

Fibronectin instructs neural crest cell to migration

with laminin

Role of Laminin-1 in axon path finding

Coated with laminin

Coated with polylysine Rovasio et al. (1983) J. Cell Biol. 96:462

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Adhesive glycoprotein: fibronectin

Fibronectin fibrils co-align with actin filaments

• At least 20 isoforms derived from a single gene by alternative splicing • RGD motif • Mediator between cells and ECM: binding to collagens, GAGs and integrins

- Insoluble as fibrils at the cell surface - Soluble in blood serum

Adhesive glycoprotein: laminin

Basal lamina (basement membrane)

• At least 15 isoforms derived from different combinations of several a, b and g laminin genes • Major components of basal lamina • Mediator between cells and basal lamina: binding to Collagen IV, GAGs and integrins

- Type IV collagen (sheet forming) - Perlecan (proteoglycan) - Laminin (adhesive glycoprotein )

Laminin-1 is essential for basal lamina assembly

Laminin-1 is required for basal lamina assembly during development

- Laminin-1 knockout in mouse ES cells - Embryoid body as a model system: Basal lamina

ES cells

Embryoid body

Li et al. (2002) J. Cell Biol. 157:1279

Laminin-1 Collagen IV

Li et al. (2002) J. Cell Biol. 157:1279

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Summary: major functions of ECM • Provides structural and mechanical support to tissues (collagens, GAGs and proteoglycans) •

Provides a reservoir for growth factors and space for molecular diffusion and cell migration (GAGs and proteoglycans)

• Plays instructional roles in a variety of cellular activities via cell surface receptors (adhesive glycoproteins)

How do ECM molecules function in a complex biological process?

Example: epithelial branching morphogenesis

- Cell

proliferation, differentiation, migration and survival

Epithelial Branching occurs during the development of

Salivary gland branching as a model system to study epithelial branching Bud

Clefts and lobules

Branching

-

Lung - Kidney - Mammary gland - Salivary gland - Growth factors but not apoptosis are required - How about ECM ?

Glycosaminoglycans in salivary gland branching

The role of collagens in branching

- Pulse-chase experiment shows faster turnover of GAGs at the lobular tips. - Depends on mesenchymal cells Bernfield and Banerjee (1982) Dev. Biol. 90:291

Nakanishi et al. (1986) Dev. Biol. 113:210

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w/o anti-laminin-1

w/ anti-laminin-1

Fibronectin in branching morphogenesis of salivary gland

Sakai et al. (2003) Nature 423:876

Kadoya et al. (1995) J. Cell Biol. 129:521

Fibronectin in branching morphogenesis of salivary gland

Fibronectin in branching morphogenesis of salivary gland

Sakai et al. (2003) Nature 423:876 Sakai et al. (2003) Nature 423:876

- Cleft formation: fibronectin down-regulates E-cadherin

Temporal and spatial regulation of ECM assembly and degradation Promoting cell migration

Promoting proliferation

Breaking cell-cell adhesion Basal lamina formation: cell-ECM adhesion

Coordination of ECM assembly/degradation, cell-cell adhesion and cell-ECM adhesion

(4)

Inhibiting cell proliferation

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Remaining questions 1. How is FN biosynthesis and matrix assembly regulated? 2. How does FN down-regulates E-cadherin in epithelial cells? 3. How does FN promote the migration of mesenchymal cell? FN Matrix deposition by epithelial cells Loss of cell-cell adhesion Promote migration of mesenchymal cells

Remaining questions - How are laminin expression and assembly into basal lamina coordinated with down-regulation of E-cadherin?

FN Matrix deposition by epithelial cells Loss of cell-cell adhesion Promote migration of mesenchymal cells

Laminin: basal lamina Gain of cell-ECM adhesion

Collagen deposition by mesenchymal cells Mechanical support

- How are adhesion junctions between epithelial cells and basal lamina assembled?

Remaining questions 1. How is collagen biosynthesis and deposition by mesenchymal cells regulated? 2. Are there additional functions of collagen in branching besides providing mechanical support?

FN Matrix deposition by epithelial cells Loss of cell-cell adhesion Promote migration of mesenchymal cells

Laminin: basal lamina Gain of cell-ECM adhesion

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The role of collagens in branching

Collagen deposition by mesenchymal cells Mechanical support

High GAG turnover Cell proliferation

FN Matrix deposition by epithelial cells Loss of cell-cell adhesion Promote migration of mesenchymal cells

Laminin: basal lamina Gain of cell-ECM adhesion

Nakanishi et al. (1986) Dev. Biol. 113:210

Summary

Remaining questions -

What regulates the turnover rate of GAGs during branching?

-

Is degradation of other ECM components required (metalloproteases)?

-

How is cell proliferation at the branching tip regulated?

Tissue morphogenesis and remodeling requires - Temporal and spatial regulation of ECM assembly and degradation - Integrin signaling - Coordination between cell-cell and cell-ECM adhesion •

Cultured cells plated on fibronectin or other ECM proteins

How do cells receive signals from ECM? Integrin-mediated signaling

Differential interference microscopy

IF of Actin filaments

(C) Transmission EM

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Focal adhesions - adhesion junctions

Inside of cell

formed by cultured cells plated on ECM

Focal adhesion functions as a signaling centers

• • • •

Transmembrane receptor: integrin Cytoplasmic adaptor proteins: talin, vinculin Linked to actin cytoskeleton Signaling proteins: FAK etc.

Two-way signaling through integrins

Inside-out signaling

Inside-out signaling

Outside-in signaling

Outside-in signaling

Discovery of integrin-mediated FAK pathway

(polylysine)

(anti-phosphotyrosine)

Guan et al. (1991) Gene Regul. 2:951

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Integrin transduces signals from ECM into cells via a FAK-mediated pathway

Integrin-ECM binding induces FAK autophosphorylation

Guan and Shalloway (1992) Nature 358:690 Schlaepfer et al. (1994) Nature 372:786

ECM Integrin

FAK: focal adhesion kinase Ta

lin

FAK Scr

P

Grb2

P

• Autophosphorylation of FAK at tyrosine residues • The phospho-tyrosine residues bind to SH2 domains of downstream signaling molecules Conclusion: FAK was tyrosine-phosphorylated and bound to Src when cells were plated on fibronectin

FAK activation requires integrin clustering and ligand engagement

Clustering of integrins is achieved by focal adhesion assembly

Hato et al. (1998) J. Cell Biol. 141:1685

Focal adhesion assembly and signaling

Integrin signaling is targeted to cell cycle progression ECM Integrin Ta

lin

FAK P Grb2 Scr

• Focal adhesion assembly requires multivalent ligand-binding and actin-cytoskeletal association • Focal adhesions recruit a large number of signaling proteins and act as signaling center

Ras GTP SOS

P

MAPK

JNK

Cyclin D

Cell cycle progression

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Shc-mediated pathway in epithelial cells Laminin Actin filaments

Intermediate filaments

Basal lamina

Intermediate filaments Basal lamina

Integrin

Maintain the integrity of Epithelial tissues

Dans et al. (2001) J. Biol. Chem. 276:2494

Skin tissue renewal

Disassembly of hemidesmosome during cell proliferation and migration

dead cells

EPIDERMIS (epithelium)

DERMIS (connective tissue) dividing cells

basal lamina

dividing cells

basal lamina

How does basal lamina regulate proliferation of epithelial cells in the skin?

Shc pathway is targeted to cell cycle progression

Phosphorylation of b4 integrin cytoplasmic tail leads to disassembly of HD

Shc-mediated pathway in epithelial cells Laminin

Dans et al. (2001) J. Biol. Chem. 276:2494

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Collaborative signaling ECM

Growth Factors

Integrin

Anchorage-dependent cell proliferation and survival

Growth Factor Receptor Shc

FAK

RAS

JNK

MEK

ERK JUN/Fos Cyclin D

Crosstalk between integrins and growth factor receptors

Cell Cycle Progression

EGF-indep and integrin-dep activation of EGFR avb3 :

Ag1478: inhibitor for EGFR kinase activity

- Src activation is required for avb3-dependent activation of EGFR Moro et al. (2002) J. Biol. Chem. 277:9405

EGFR and integrin avb3 form a complex by Src activation

avb3

EGFR Src Src

Src

P P Cas

P

Moro et al. (2002) J. Biol. Chem. 277:9405

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Mechanisms for integrin-growth factor receptor crosstalk

• Multiple pathways converge on the same target - cross talk • One pathway has multiple targets - Cell proliferation

- Cell survival - Cell migration - Cell differentiation - Collaborative signaling - Integrin-dependent activation of growth factor receptors - Growth factor-dependent activation of integrins

The targets of FAK-mediated pathways

The targets of Shc-mediated pathways Laminin

ECM Integrin Rac GTP

Paxillin

P

Ta

lin

FAK P Grb2 Scr

Actin cytoskeleton

Cell migration

Ras GTP SOS

P

MAPK

JNK

Cyclin D

Cell cycle progression

Mobilized a6b4 integrin promotes cell migration

Cell proliferation

Actin

Cell migration

Cell Migration requires coordination among four events

Focal adhesions

Actin polymerization Membrane extension

Adhesion at the front

Contraction

Migration of epithelial cells occurs in wound healing and tumor metastasis

De-adhesion at the rear

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Migration of CHO cells expressing a4b1 integrin on fibronectin

Rho family of small GTPases are central regulators for membrane protrusions

Research topics on cell migration •

Cell polarity: set up front and back

•

Membrane protrusions: - regulation of actin dynamics - activation and targeting of Rac

•

Focal adhesion assembly and turnover

•

Regulation of contraction

•

Rear end de-adhesion and retraction

Spatial regulation of Rac and Rho activities lamellipodia GTP-Rho

Stress fibers GTP-Rac Focal adhesions Actin polymerization

lamellipodia Rho: stress fiber formation Rac: membrane ruffling and lamellipodia protrusion Cdc42: filopodia protrusion and polarity

FRET: GTP-Rac/effector binding A

B

Bead coated with FN

A. GTP-Rac/effector coupling is locally enhanced in lamellipodia. B. GTP-Rac/effector coupling is locally induced by fibronectin/integrin binding

How does integrin signaling lead to targeting of Rac-GTP to plasma membrane?

Del Pozo et al. (2002) Nat. Cell Biol. 4:232

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Integrins regulate targeting of GTP-Rac to plasma membrane via lipid rafts

The role of integrin signaling in Rac function

Lipid raft

Adaptor protein Rac GEF

?

- Translocation of activated Rac to the plasma membrane

Del Pozo et al. (2004), Science 303:839

- Activation of Rac via FAK and downstream Rac GEFs

Assembly and turnover of focal adhesions

The role of integrin signaling in cell migration

Focal adhesions

Targets GTP-Rac to plasma membrane at the front

Assembly of nascent focal adhesions

GFP-paxillin

- Mature focal adhesions provide anchors for contraction - Turnover of focal adhesions

FAK-mediated signaling pathway is required for turnover of focal adhesions -

FAK is required for FA turnover

The role of integrin signaling in cell migration

Focal adhesions

Targets GTP-Rac to plasma membrane at the front

Assembly of nascent focal adhesions - Mature focal adhesions provide anchors for contraction - Turnover of focal adhesions De-adhesion Webb et al. (2004) Nat. Cell Biol. 6:154

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Remaining questions - How is assembly of nascent focal adhesions regulated spatially and temporally?

Epithelial-mesenchymal transition (EMT) Epithelium

- Disassembly of AJ and desmosome - Disassembly of HD - disruption of basal lamina - Attachment to connective tissue ECM (FA assembly)

- How does FAK pathway regulate focal adhesion turnover? - How is activation of integrins coordinated with protrusion, adhesion and de-adhesion?

Mesenchymal cells

How are these events coordinated?

The targets of integrin/FAK pathway ECM Integrin Rac GTP

Paxillin

P

Ta

lin

FAK P Grb2 Scr

Actin cytoskeleton

Cell migration

Ras GTP SOS

P

Problem: a pathway has multiple targets

How do cells interpret signals to execute specific functions?

MAPK

JNK

Cyclin D

Cell cycle progression

Integrin-mediated signaling network

Summary 1. Integrins transduce bi-directional signals by an allosteric mechanism 2. Outside-in signaling from ECM requires: - Engagement of ECM ligands with integrins - Clustering of integrins - Recruitment of signaling molecules to the integrin complex (focal adhesions) 3. Integrin signaling is targeted to: - Cell cycle progression: cell proliferation - Cytoskeleton via Rac and Rho: cell migration - etc.

Cell proliferation

Cell survival

Cell migration

4. Signaling network: cross-talk

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