Bio2000 Notes-2004-2

Bio2000, 2004 Joy Yang

CELL ADHESION AND INTEGRIN SIGNALING Joy Yang Department of Cell Biology Johns Hopkins University, School of Medicine 725 N. Wolfe Street Baltimore, MD 21205 U.S.A Tel: 410-614-5938 FAX: 410-955-4129 [email protected]

Lecture I. CELL ADHESION PRINCIPLES To be covered: a. Cell adhesion receptors a. The principles of cell adhesion a. Adhesion junctions a. Regulation of cell adhesion • Epithelial-mesenchymal transition • Leukocyte extravasation 1. CELL ADHESION RECEPTORS • Cell adhesion play a wide variety of roles in multicellular organisms - Cell proliferation and survival - Cell differentiation - Cell migration - Tissue architecture - Specific cell-cell adhesion events such as egg-sperm fusion, etc. • The cell surface receptors that mediate cell adhesion are called cell adhesion receptors (also named cell adhesion molecules or CAMs). There are four major classes of cell adhesion receptors: - Cadherins - Integrins - Immunoglobulin superfamily of cell adhesion molecules (IgCAMs) - Selectins • All four families mediate cell-cell adhesion - Homophilic cell-cell adhesion: adhesion between cells of the same type cadherins and IgCAMs - Heterophilic cell-cell adhesion: adhesion between cells of different types IgCAMs, selectins and integrins Integrin is the only family that mediates cell-extracellular matrix adhesion. Cell adhesion principle #1- Specificity In multicellular organisms, each cell type has its unique adhesive properties that allow the cells to interact with their neighbors in a specific way. This specificity is achieved when each cell type expresses a unique set of cell adhesion receptors. 1

Bio2000, 2004 Joy Yang 2. CADHERINS Identification Classical experiments a. Two types of cell adhesion mechanism, calcium-dependent system (CADS) and calciumindependent system (CIDS), co-existed on the same embryonic cells (Urushihara et al., 1976), which could be distinguished under the following experimental conditions: Trypsin Ca2+ Aggregation Ca2+-dependent concentration (adhesion) adhesion high + + + CADS low + CIDS high b. Takeichi and colleagues identified a monoclocal antibody that blocked the calcium-dependent form of adhesion in epithelial cells. The antibody recognized a protein of 120 kDa. Several groups of researchers independently identified this protein using similar approaches. The protein was named E-cadherin, the first cadherin in this superfamily to be identified. Later, the adhesion receptor mediating calcium-independent form of adhesion was identified as NCAM, a member of the IgCAM family. c. Takeichi and colleagues (Nagafuchi et al., 1987) took the following approaches to study the role of E-cadherin in calcium-dependent cell adhesion. •

Expression-cloning of E-cadherin cDNA using the anti-E-cadherin antibody.

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Transfect E-cadherin cDNA into L cells (fibroblasts isolated from connective tissue) lacking E-cadherin and showed that the L cells acquired strong Ca2+-dependent aggregating activity by expressing E-cadherin. This study provided direct evidence that E-cadherin mediates Ca2+-dependent cell-cell adhesion.

Cadherin superfamily • Classical cadherins (E-cadherin, Pcadherin, etc.) • Desmosomal cadherins (desmogleins and desmocollins) • Type II cadherins • Other subgroups of related proteins Cell sorting during embryonic development As discussed above, each tissue or cell type has its distinct adhesive properties that contribute to a specific tissue function or Figure 1. Cell sorting by cadherin-mediated cell adhesion. cellular event. A classical example is the role of cadherins in cell sorting during embryonic development.

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Bio2000, 2004 Joy Yang Classical experiments: a. Cell-cell recognition in embryonic tissues Classical experement: When cells dissociated from two different embryonic tissues (e.g. ectoderm and mesoderm or liver and kidney) were mixed together, they initially formed a mixed clump, but remarkably, with time, moved around in the aggregate and sort themselves out by tissue type (Figure 1A, (Townes and Holtfreter, 1955). b. Homophilic adhesion by cadherins as a cell-cell recognition mechanism A similar segregation of cells occurred when L cells expressing different amount of the same cadherin (Figure 1B, (Friedlander et al., 1989), or different types of cadherins, were mixed. The structure of classical cadherins Single-pass transmembrane homodimers with conserved extracellular repeats (EC repeats) • Homotypic binding achieved by a zipper-like interaction, based on the crystal structures of the EC1-2 domains of classical cadherins. • This binding requires Ca2+, which contributes to the rigidity of the molecule. The rigidity allows the cadherin monomers to form dimers and extend from the surface to interact with a cadherin dimer of the same type on an adjacent cell. •

Figure 3. Mechanism of calcium-dependent adhesion (Kock et al. 1999).

Figure 2. Structure of cadherin. (Cell Biology, Pollard and Earnshaw, Mol. Cell Biol., 5th ed.)

E-cadherin forms adherens junctions in epithelial tissues A number of cytoplasmic proteins coimmunoprecipitate with cadherins, including b-catenin and a-catenin (Ozawa and Kemler, 1992), which are cytoplasmic adaptor proteins. E-cadherin and the associated cytoplasmic proteins form adherens junctions in epithelial tissues. Adherens junction is one type of Figure 4. Adherens junction. (Mol. Cell Biol., 5th ed.) adhesion junctions. In epithelial cells, there are three types of adhesion junctions: adherens junction, desmosomes and

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Bio2000, 2004 Joy Yang hemidesmosomes. In an adherens junction, many E-cadherin molecules are clustered. The cytoplasmic tail of E-cadherin binds to b-catenin, which in turn binds to a-catenin and other proteins, forming a large complex. a-catenin also binds to actin filaments. The actin cytoskeletal association via cytoplasmic adaptor proteins is required for the formation of adherens junctions. Other adhesion junctions have similar melocular compositions. Assembly of adherens junction Experiment (Vasioukhin et al., 2000): E-cadherin-mediated cell-cell adhesion was studied in a tissue culture model system. Primary keratinocytes (skin epithelial cells) did not adhere to one another at low calcium. Under this condition, the cell-cell boundaries lacked E-cadherin expression (Figure 5A). When the cells were plated in high calcium, anti-E-cadherin labeled two rows of puncta at sites of intercellular contact (Figure 5B). By immunofluorescence staining, all of the Figure 5. Adherens junction assembly (upper panel) in cultured well-known cytoplasmic components keratenocytes was examined by immunofluorescence staining of actin filaments (red) and E-cadherin (yellow). The cells were of adherens junction, including a- and photographed at various time-points after addition of Ca2+. b-catenin, were localized to the puncta, Adherens junctions failed to form when actin cytoskeleton was and each puncta associated with an disrupted by cytochalasin D (lower panel). (Vasioukhin et al., actin bundle. Subsequently, the two2000) row ‘zipper’ was closed to form a continuous line (Figure 5C). This ‘zipper’ formation was dependent on actin cytoskeleton since it failed to form in the presence of cyochalasin D, an inhibitor of actin filament formation (Figure 5 D-F). Therefore, the adherens junction and other anchoring junctions achieve strong adhesion by • clustering of many adhesion receptor molecules of the same type • association with the actin cytoskeleton Adhesion principle #2. Affinity vs. avidity • Cell adhesion receptors have weak ligand-binding affinity. The weak ligand-binding affinity allows non-junctional adhesion receptors to mediate transient adhesion. • In junctional complexes, many receptors of the same type work together to achieve strong and relatively stable adhesion (high avidity). Adhesion principle #3. Cytoskeletal association is essential for the formation and functions of anchoring junctions.

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Bio2000, 2004 Joy Yang Epithelial-mesenchymal transition - Epithelial cells: direct cell-cell contact, organized into a sheet - Mesenchymal cells (in embryos) and cells in connective tissues (in adults): no direct cell-cell contact, migratory The term ‘epithelial-mesenchymal transition’ is used to describe changes that occur when epithelial cells lose cell-cell contracts and become migratory, which occurs during embryonic development, tissue remodeling and tumor metastasis. During epithelial-mesenchymal transition, E-cadherin is down-regulated and adherens junctions are disassembled. E-cadherin is down-regulated during tumor progression Tumor progression in cancer involves down-regulation of E-cadherin by different mechanisms: • Mutations/deletions of E-cadherin gene • Activation of a transcriptional repressor of E-cadherin (Slug/Snail). • Removal of cadherin complex from the cell surface by endocytosis in response to growth factor signaling (Lu et al., 2003) • Disassembly of adherens junctions. Down-regulation of E-cadherin has two consequences: • Loss of control of cell proliferation • Loss of cell-cell attachment: metastasis Dual functions of b-catenin – cell adhesion and gene regulation b-catenin not only serves as an adaptor protein for cytoskeletal association, but also enters the nucleus to regulate gene transcription that regulates cell proliferation. The level of free cytoplasmic b-catenin is regulated by a b-catenin-specific proteosome-mediated degradation machinery. Disruption of this degradation machinery leads to cancer. It is proposed that E-cadherin in adherens junctions may inhibit cell proliferation by sequestering free cytoplasmic b-catenin.

Figure 6. Dual functions of b-catenin. APC: Adenomatous polyposis coli. (Cell biology, Pollard and Earnshaw)

5. INTEGRINS, IgCAMS AND SELECTINS IN LEUKOCYTE EXTRAVASATION Junctional vs. non-junctional cell adhesion • In anchoring junctions, cadherins (adherens junctions and desmosomes) and integrins (hemidesmosomes) mediate stable cell adhesion, which plays a key role in maintaining tissue architecture and integrity.

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Bio2000, 2004 Joy Yang •

Adhesion receptors can also be non-junctional. Cell adhesion mediated by non-junctional adhesion receptors is transient, which play important roles in cell migration and other dynamic cellular events.

Leukocyte extravasation Under normal conditions, leukocytes travel in circulating blood and they are not adhesive to the endothelial wall of blood vessels. Upon wounding and infections, these cells are stimulated to emigrate from the blood stream into the wounded tissues through a multistep process (Figure 8) involving two pairs of cell adhesion receptors: P-selectin and its counterreceptor (with sugar residues that bind to selectins), aLb2 integrin and ICAM-1 (an IgCAM family member). 1. At resting state, P-selectin is absent from the surface of endothelial cells, and aLb2 integrin is present but inactive on the surface leukocytes. 2. Upon infection, the walls of the blood vessel respond to “signals” from infected tissue. The endothelial cells that line the blood vessel are activated. This activation leads to a temporary display of P-selectin on the surfaces of endothelial cells, increasing the adhesiveness of these endothelial cells to leukocytes. When leukocytes encounter P-selectin, they form transient adhesions that slow down their movement (rolling). 3. As the leukocytes interact with the endothelial cells, PAF (a chemokine) is secreted by the endothelial cells. PAF activates an intracellular signaling pathway in the leukocytes via its receptor. This signaling cascade activates b2 integrins on the surfaces of the leukocytes. 4. The activated b2 integrins bind to ICAM-1 on the surface of the endothelial cells. The leukocytes stop rolling and adhere firmly to the wall of the vessels. 5. The bound leukocytes change their shape and squeeze through the endothelial layer into infected tissue.

Figure 7. Leukocyte extravasation (Mol. Cell Biol. 5th ed.)

Cell adhesion principle #4. Cell adhesion receptors are regulated at different levels.

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Bio2000, 2004 Joy Yang Cells regulate adhesion by: •

Selective expression of cell adhesion receptors at the cell surface: - Transcriptional: b2 integrin genes are expressed in leukocytes and the ICAM gene is expressed in vascular endothelial cells, allowing specific adhesion between these two cell types. All adhesion receptors are subjected to transcriptional regulation. -

•

Post-translational: Selectins are stored in storage granules and released transiently onto the cell surface by exocytosis in response to infection. Cadherins can be removed from the cell surface by endocytosis.

Modulation - Avidity modulation: integrins can also modulate cell adhesion by clustering or dispersion of the integrin molecules in the plane of the plasma membrane as adhesion junctions assemble or disassemble. -

Affinity modulation: b2 integrins are always present on the surface of leukocytes, but they have very low affinity to their ligands when the cells are circulating in the blood stream. In response to infection, these integrins are activated into a high affinity state by changing their conformation.

6. INTEGRINS • • •

Single-pass transmembrance heterodimer with an a and a b subunit Mediate heterophilic cell-cell adhesion and cell-ECM adhesion A superfamily with at least 24 members.

The structure of integrins Analyses of the crystal structures of the integrin extracellular domains show that an integrin heterodimer is compose of a globular ‘head’ and two ‘legs’. The a and Figure 8. The integrin superfamily. Each integrin has an a b subunits are non-covalently associated at and a b subunit. (Hynes. Cell 110:673. 2002). the head domain. The head domain forms a ligand-binding site that is composed of an I/A domain with a metal ion-dependent adhesion site (referred to as the MIDAS motif). This I/A domain resides in the a subunit of some integrins (collagenbind and leukocyte-specific integrins) and in the b subunit of others (RGD-binding integrins). In either case, the I/A domain has either an ‘open’ or a ‘closed’ conformation.

7 Figure 9. The model for the closed and open conformation of integrin. Hynes, Cell 110:673

Bio2000, 2004 Joy Yang Conformational changes in the MIDAS motif propagate to the integrin legs. At inactive state, integrin legs have a bent shape with the legs close together. As the head ‘opens’, the legs adopt an extended shape with the legs separated. To be fully activated and able to bind to its ligand, the integrin must be ‘open’. Integrin with a ‘closed’ conformation is inactive. Experiment (Takagi et al., 2002): A soluble extracellular fragment of aVb3 integrin was examined by electron microscopy after incubating with Ca2+ (prevents activation), Mn2+ (activation reagent) or an RGD peptide (ligand). The integrin molecule had three distinct shapes: (1) a compact V shape in the presence of Ca2+, (2) an extended shape with the legs close together in the presence of Mn2+, and (3) a fully extended shape with the legs separated in the presence of the RGD peptide. The EM images were identical to the projected views calculated from the crystal structures of the molecule under the same conditions (Figure 10). By introducing mutational disulfide bonds between the a headpiece and the b tailpiece, the integrin extracellular fragment was locked in a closed conformation, which failed to bind to its ligand. These studies showed directly that an integrin with a bent conformation (closed conformation) is in an inactive state, and the integrin is activated when it undergoes conformational change that straightens and separates the legs of the molecule (open conformation).

Figure 10. A-E, EM images. B-F, projected view calculated from the crystal structures of the entire extracellular fragment (B) or the headpiece (D and F). A, B and H, close conformation in the presence of Ca2+; C-F and I, extended conformation with a closed (C and D) or an open (E and F) headpiece in the presence of Mn2+ (C and D) or RGD peptide (E and F), respectively. (Takagi et al., 2002)

Integrin activation by an inside-out signaling pathway During leukocyte extravasation, aLb2 integrin is activated when leukocytes roll along the endothelial wall of blood vessels. As leukocytes roll on endothelial cells, PAF (a chemokine) is secreted by endothelial cells, which binds to a chemokine receptor on the surface of leukocytes. This binding activates an intracellular signaling cascade, leading to activation of talin, a cytoplasmic adaptor protein. The activated talin in turn binds to the cytoplasmic domain of b2 integrin, leading to a

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Figure 11. Inside-out signaling pathway.

Bio2000, 2004 Joy Yang separation of the a and b legs. This conformational change propagates to the head domains, leading to a conformational change at the ligand-binding motif and an increase in the ligandbinding affinity of the integrin. This pathway is named inside-out signaling pathway. As we will discuss in Lecture II, integrins also tranduce signals from the ECM into cells in an outsidein signaling pathway. Experiment (Kim et al., 2003): Fluorescence activated energy transfer (FRET) is a quantum mechanical phenomenon that occurs between a fluorescence donor and a fluorescence acceptor in close proximity (usually <100 Å of separation) if the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor. Under optimal FRET conditions, illumination at the excitation wavelength of the donor results in transfer of energy to the acceptor. This transfer of energy reduces illumination from the donor (also increases illumination from the acceptor).

Figure 12. FRET analysis of aLb2 integrin in living cells. (A) The donor is a monomeric cyan fluorescent protein (mCFP) fused to aL, and the acceptor is a monomeric yellow fluorescent protein (mYFP) fused to b2, both are transfected into cultured cells. (B) Left panels showed a control, in which aLb2 integrin subunits were fused with fluorescent proteins with long linkers. The long linkers separated the donor and the acceptor such that no FRET was generated. The right panels showed FRET from aLb2 subunits that were fused with mCFP and mYFP with very short linkers. FRET was detected as a decrease in illumination from mCFP, shown in the right panel by the fluorescent micrograph of a cell in the middle row and the pixel histogram in the lower row. In the pixol histogram, the left peak was before photo-bleaching, and the right peak was after photo-bleaching of YFP. Post-bleaching provided a control for illumination from mCFP in the absence of mYFP. The upper row showed that photo-bleaching of YFP indeed eliminated the ability of the acceptor to absorb energy. (C) Adhesion assay on ICAM. Grey and white bars represent cells treated with Mn2+ or CBR LFA-1/2, which are activation reagents. Black bar (con) represents cells without treatment with activation reagents. Parent: cells without transfection. TH: talin head, which is a constitutively active form of talin. TH-null: cells transfected with a plasmid lacking the talin head; TH middle and TH high: cells transfected with middle and high levels of talin head, respectively. Note that when treated with activation reagents, cells (with or without expression of talin head) had an increase in adhesion to ICAM-1 (C) and decrease in FRET (D), whereas in the absence of activation reagents (con), adhesion enhancement and FRET reduction occurred only when talin head is expressed (TH middle and TH hight).

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Bio2000, 2004 Joy Yang FRET analysis was conducted to investigate cytoplasmic conformational changes in aLb2 integrin in living cells (Figure 12). This study shows directly that the activation of aLb2 integrin involves separation of the a and b cytoplasmic domains, and talin induces this separation and activation. Principle #5. Cell adhesion receptors play important roles in signal transduction.

7. SUMMARY OF CELL ADHESION PRINCIPLES • specificity • affinity vs. avidity • cytoskeletal association (integrins and cadherins) • regulation • signaling

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Bio2000, 2004 Joy Yang

Lecture II. EXTRACELLULAR MATRIX AND INTEGRIN-MEDIATED SIGNALING To be covered: 1. Extracellular matrix (ECM) a. ECM in tissues a. Biological functions of ECM macromolecules a. A case study: the role of ECM in epithelial branching 1. Signaling pathways activated by interactions between integrins and ECM 1. EXTRACELLULAR MATRIX ECM is a meshwork of proteins and sugars that are secreted, assembled and organized by cells. a. ECM in Tissues There are two structurally and functionally distinct types of ECM in tissues: ECM of connective tissues • Predominant constituent connective tissues. • Contains embedded cells that continuously remodel the ECM. Basal lamina (also referred to as basement membrane) • A thin dense sheet composed of ECM macromolecules • Underlying the epithelial cell sheet in epithelial tissues • Surrounding muscle cells and other cell types b. Biological functions of ECM macromolecules The structural components of ECM: • Collagens: - Fibrillar collagens (Type I and Type II collagens) in connective tissue Sheet-forming collagen (Type IV collagen) in basal lamina - Function: provide strong tensile strength to tissues •

Glycosaminoglycans and proteoglycans - Glycosaminoglycans (GAGs) are linear polysaccharides with repeating disaccharide units. - Proteoglycans: GAGs covalently attached to core proteins - Peoteoglycan-hyaluronan complex: proteoglycans are non-covalently attached to a very long GAG (hyaluronan) to form a complex, found in cartilage. - Functions: ß compressive strength ß space for diffusion of molecules and migration of cells ß modulators for growth factor activities ß co-receptors for growth factor receptors and integrins

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Bio2000, 2004 Joy Yang The instructive components of ECM: • Adhesive glycoproteins Adhesive glycoproteins act as mediators between cells and the ECM. They all have functionally distinct domains with (1) binding sites for the structural components of ECM, such as collagens, and (2) binding sites for cell surface receptors (integrins). Examples of adhesive glycoproteins include fibronectin (FN) and laminin. Fibronectin is abundant in connective tissue ECM and laminin is a major component of basal lamina. Adhesive glycoproteins play instructive roles that regulate a variety of cellular activities via integrins: • Cell proliferation and survival Experiment(classical): Hynes and colleague discovered that when malignant tumor cells were plated on FN-coated surface, the cells resumed normal growth and cytoskeletal organization, suggesting that FN may play an important role in controlling cell proliferation. Anchorage dependent cell growth: A majority of normal cells proliferate only when they are attached to an ECM-coated surface and they die by apoptosis when detached from ECM. This is referred to as anchorage dependent cell growth. Malignant tumor cells lose anchorage dependence and they can proliferate and survive without attaching to ECM. The key players in anchorage dependent cell growth are the adhesive glycoproteins including FN and laminin, which play an essential role in cell cycle progression. • Cell migration Experiment: The role of FN in cell migration is demonstrated by emigration of neural crest cells from chicken embryonic explants along strips of fibronectin-coated surface. This migration was mediated by integrins since an anti-integrin antibody that specifically inhibited binding between the integrin and FN blocked the migration.

Figure 13. Fibronectin in neural crest cell migration. (Rovasio et al., 1983)

Experiment: Laminins are mediators in axon path finding. • Cell differentiation Experiment: An instructional role of ECM was demonstrated by a study on differentiation of mammary gland epithelial cells. The cells were cultured in the absence of ECM. Cells were flat and did not synthesize milk Figure 14. The role of ECM in cell differentiation. (Celll and Mol. Biol. 2nd ed. Karp) proteins (Figure 14A). When ECM was added back cells regained differentiated appearance and synthesize milk proteins (Figure 14B).

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Bio2000, 2004 Joy Yang Fibronectin • There are at least 20 FN isoforms, all of which are generated by alternative splicing of the RNA transcript of a single FN gene. • FN contains an RGD motif that binds to integrins. This motif can be found in many adhesive glycoproteins. • FN is a mediator between cells and ECM. It has a number of functional domains for binding to collagen, GAGs (heparin) or integrin.

Figure 15. Functional map of fibronectin. (Cell and Mol. Biol. 2nd ed. Karp)

• FN molecules are assembled into fibrils at the cell surface (outside), which co-align with the actin cytoskeleton (inside). FN is also present in blood serum as a soluble protein. Laminin • At least 15 laminins have been identified, which are derived from different combinations of several a, b and g laminin genes. • Laminins are major components of basal lamina. • Laminins function as mediators between cells and basal lamina by providing binding sites for Type IV collagen, GAGs and integrins. • Laminins are essential for assembly of basal lamina.

Figure 16. Functional map of laminin-1. (Modified from Mol. Cell Biol., 5th ed. )

Summary of the functions of ECM macromolecules • Provides structural and mechanical support to tissues (collagens, GAGs and proteoglycans) • Modulate activites of growth factors and other soluble factors (GAGs and proteoglycans) • Provides substrata for migrating cells (adhesive glycoproteins) • Plays instructional roles in a variety of cellular activities via cell surface receptors (adhesive glycoproteins and collagens) - Cell growth and survival - Cell differentiation - Cell motility and invasion - Morphogenesis and tissue remodeling

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Bio2000, 2004 Joy Yang c. The role of ECM in branching morphogenesis- a case study Morphogenesis and tissue remodeling are complex events involving combined effects on growth factor stimulation, ECM assembly and turnover, cell-ECM and cell-cell adhesion, cell migration and cell proliferation. A classical model for studying morphogenesis and tissue remodeling is epithelial branching during salivary gland development. Branching of epithelium occurs during the formation of salivary gland, lung and kidney when an unbranched bud is divided by clefts, yielding lobules on either side of the clefts. These lobules grow to create branches. Epithelial branching can be observed when a salivary glad bud from a mouse embryo is placed in serum-free medium. The effects of growth factors and ECM components, including GAGs, collagens and fibronectin, on this organ culture system were examined. These studies demonstrated that salivary branching depends upon regulation of the local synthesis, deposition, and turnover of ECM components, as well as cell-cell and cell-ECM adhesion. These events control the cell migration and proliferation. As illustrated in Figure 33, the following steps are involved:

Figure 17. The roles of ECM components in salivary gland branching.

(1) Fibronectin (FN) plays an essential role in cleft formation during the initiation of branching. Experimental evidence (Sakai et al., 2003): - Immunofluorescence: FN fibrils appeared in the cleft-forming region of an epithelial bud (note: FN is normally absent from epithelial cell-cell junctions). - Perturbation of FN using small interfering RNA and inhibitory anti-FN antibody reduced branching; - Adding exogenous FN to the organ culture accelerated branching. To be answered: How are the expression and fibril assembly of FN regulated? (2) FN initiates cleft formation by down-regulating E-cadherin, laminin-1 participates in the formation of basal lamina. Experimental evidence (Kadoya et al., 1995; Sakai et al., 2003): - Immunofluorescence: E-cadherin was down-regulated in areas adjacent to the FN fibrils at the cleft-forming region.

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Bio2000, 2004 Joy Yang -

Aggregates of fibronectin fibrils made in vitro were placed on top of cultured salivary gland epithelial cells. E-cadherin localization was suppressed at plasma membrane sites adjacent to the FN fibril aggregates. - At the same time, laminin-1 was transiently expressed in the same region and its inhibition by an anti-laminin-1 antibody prevented the formation of basal lamina and inhibited branching. Thus, replacement of direct cell-cell adhesion in the cleft-forming region with cell-basal lamina adhesion is required for branching. To be answered: How does FN down-regulate the expression of E-cadherin? How is the expression of laminin-1 regulated? How does laminin-1 participate in the formation of basal lamina? (3) Once cell-cell junctions at the cleft break apart, mesenchymal cells (fibroblast-like embryonic cells) migrate into the clefts. This migration is promoted by FN deposited in the clefts (based on the role of FN in cell migration). (4) Collagens play an important role in salivary gland branching. Experimental evidence (Nakanishi et al., 1986): - Scanning EM with or without collagenase inhibitor: collagen fibrils were deposited by the mesenchymal cells in the clefts but not at the lobular tips. - Addition of collagenase to the organ culture inhibited branching while addition of a collagenase inhibitor accelerated branching. Hypotheses to be tested: - Collagen fibrils may provide mechanical support of the clefts, preventing cells from invading the clefts. (5) Turnover of basal lamina glycosaminoglycans (GAGs) plays an important role in salivary gland branching. Experimental evidence: (Bernfield and Banerjee, 1982) - Pulse-chase labeling of the salivary bud in the presence or absence of mesenchymal cells: the turnover rate of basal lamina GAGs was faster at the tip of lobules than in the interlobular clefts only in the presence of mesenchymal cells, suggesting that the turnover of GAG depends upon degradation by degredation enzymes secreted by mesenchymal cells, and the GAGs at the tip are more susceptible to this degradation than those in the clefts. Hypotheses to be tested: - GAG turnover in the basal lamina at the tip may modulate growth factor availability and promote cell proliferation. Summary of branching morphogenesis • • •

Morphogenesis and tissue remodeling require temporal and spatial regulation of ECM assembly and degradation. ECM regulates cellular activities, including cell migration and cell proferation, when cells adhere to the ECM, which send signals into cells via integrin signaling. Cell-ECM adhesion and cell-cell adhesion are coordinated.

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Bio2000, 2004 Joy Yang 2. INTEGRIN-MEDIATED SIGNALING a. Focal adhesions ECM proteins regulate cells by sending signals into cells via integrins. When cultured cells attach to ECM protein-coated surface, they form focal adhesions (FAs, also referred to as focal contacts), a type of anchoring junctions (discussed in Lecture I). FAs were first identified by differential interference microscopy and transmission electron microscopy as dense plaques of close cell-ECM contacts. FAs can also be visualized by immunofluorescence staining of FA components as punctuates localized at ends of actin stress fibers.

Figure 18. Focal adhesion.

Immunofluorescence studies showed that integrins are the transmembrane components of FAs, where integrins bind to a large cytoplasmic protein complex and the actin cytoskeleton via talin and other adaptor proteins, providing a physical link between ECM and the actin cytoskeleton and strong cell-ECM adhesion. FAs also recruit a large number of signaling molecules and function as a signaling center (see below). Integrins transmit signals bidirectionally via FAs As discussed in Lecture I, integrins transduce signals across the plasma membrane bidirectionally through insideout and outside-in signaling pathways.

Figure 19. Inside-out signaling of integrin. (MBDC 4th ed.)

Inside-out signaling: Intracellular signals activated by growth factors or cytokines can activate integrins by interacting with the integrin cytoplasmic tail, leading to conformational change and activation of the integrins. •

Outside-in signaling: Multivalent ECM ligand binding allows integrin molecules to cluster into focal adhesions. As integrins bind with their ECM ligand, conformational changes occur at the integrin cytoplasmic tails. This conformational change allows the integrin tails to bind •

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Figure 20. Outside-in signaling of integrins.

Bio2000, 2004 Joy Yang intracellular signaling proteins and cytoskeletal adaptor proteins, which in turn promote more integrin clustering and more recruitment of signaling proteins into FAs, thus activating an intracellular signaling cascade. Allosteric mechanism for bidirectional signaling through integrins: The open and closed conformations of integrins revealed by structural studies are consistent with an allosteric mechanism for integrin birectional signaling. In this model, conformational changes can influence signaling events in two ways. Outside-in signaling would occur when binding of an ECM ligand leads to conformational changes in the head that in turn straightens and separates the legs, thus separating the cytoplasmic tails of the integrin and allowing interactions with cytoskeletal and signaling molecules. Inside-out signaling would occur when an intracellular activator separates the cytoplasmic domain of the integrin. This separation would in turn opens/activates the ligand-binding site in the head domain. •

• An example of integrin outside-in signaling Experiment: The first outside-in signaling pathway mediated by integrins is the focal adhesion kinase (FAK) pathway. When 3T3 fibroblasts were plated on fibronectin, FAK was tyrosine phosphorylated and bound to Src, as shown by immunoprecipitation and Western blot analyses.

Figure 21. Integrin signaling by an allosteric mechanism Hynes, (Cell 110:673) ECM Integrin Ta lin

Autophosphorylation FAK Scr

P

Grb2

Ras GTP SOS

P

MAPK

Figure 22. Tyrosine phosphorylation and Src binding of FAK in response to FN stimulation (Guan and Shalloway, 1992; Schlaepfer et al., 1994).

Figure 23. FAK pathway.

Subsequent studies show that, when integrins bind to their ECM ligands and cluster, FAK is recruited into FAs, possibly via talin, and gets autophosphorylated on a number of tyrosine residues, creating binding sites for the Src homology 2 (SH2) domain of Src and other proteins. Src binds to FAK and in turn phosphorylates a number of FA components, which then binds to

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Bio2000, 2004 Joy Yang other SH2 domain-containing molecules and activates the pathway further downstream. Activated FAK also binds to Grb2, a signaling adapter protein in the Ras-MAPK pathway. •

Integrin signaling via FAK require: - Ligand occupancy - Integrin clustering

Experiment (Hato et al., 1998): ECM ligands, such as FN and other RGD containing proteins, are filamentous polymers that have multivalent binding Figure 24. ECM-stimulated FAK activation requires both ligand sites for integrins. Cells adhesion to these binding and integrin clustering (Hato et al., 1998). multivalent ligands has two consequences: (1) the ligand binding sites of integrins become occupied; (2) integrin molecules are clustered due to multivalent binding. To examine the effects of integrin clustering on outside-in signaling, the researchers engineered the integrin molecule such that the integrins clustered in the presence of a clustering reagent. To examine the effect of ligand occupancy in the absent of clustering, a monovalent ligand was used. As shown in Figure 24, tyrosine phosphorylation became prominent only in the presence of both clustering reagent and the monovalent ligand, or in the presence of multivalent ligand. Therefore, FAK activation requires both ligand occupancy and integrin clustering. Focal adhesion functions as a signaling center Integrin binding to ECM ligands results in integrin clustering and association with the actin cytoskeleton via talin and other cytoplasmic adaptor proteins, leading to FA assembly. This is a multistep process in which the simpler and smaller focal complexes mature into the larger and more organized focal adhesions by successively recruiting more proteins into the complex. FAK and other signaling proteins are recruited into FAs and get activated. The signaling network activated in FAs is summarized in Figure 29 on the next page. •

Figure 25. Focal adhesion assembly (Giancotti and Tarone, 2003)

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Bio2000, 2004 Joy Yang Crosstalk between integrin and growth factor receptor pathways Thus, integrin-ECM interactions activate the Ras-MAPK pathway. As you have learned ECM Growth Factors earlier, the Ras-MAPK pathway is also Integrin Growth Factor Receptor activated by growth factors. For most cell types, the signals from either growth factors or FAK RAS integrins alone are not sufficient to drive the cells into the cell cycle. In malignant tumor cells, the requirement for the ECM signaling is JNK MEK bypassed due to hyper-activation of downstream signaling components in the ERK pathway. Thus, the crosstalk between integrins JUN/Fos and growth factor receptors provides a Cyclin D mechanism for anchorage dependence’ cell proliferation. Cell Cycle Progression

The role of integrins and FAs in cell migration Cell migration involves four major events as illustrated in Figure 27.

Figure 26. Convergence of integrin and growth factor receptor-mediated signaling pathways.

Integrin-mediated signaling pathways regulates Cell migration by at least two mechanisms. 1. Regulate Rho family of GTPases • Spatial regulation of Rac activities by inhibiting lipid raft internalization (del Pozo et al., 2004). • Activate Rac via the FAK pathway 2. Regulate focal adhesion assembly and turnover. FAs are assembled at the leading edge and disassembled at the trailing edge of the migrating cells, which is regulated by FAK-mediated signaling pathway. Cells deficient in FAK have reduced turnover of FAs, leading to reduced cell motility.

Figure 27. Cell migration (Mol.Cell Biol.)

Figure 28. Regulation of Rac activity by integrin signaling

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Figure 29. A signaling network mediated by focal adhesions

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