Lectures 11-13 Notes

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

CHAPTER 19 CELL JUNCTIONS, CELL ADHESION, AND THE EXTRACELLULAR MATRIX •

Major tissue types in vertebrates: o Nerve o Blood o Lymphoid o Connective ƒ ƒ ƒ

Make lots of extracellular matrix, including collagen. Cell-cell junctions are rare. Provides a lot of mechanical stress resistance.

o Epithelial (skin) Fig. 19-1 ƒ ƒ ƒ ƒ ƒ

Very little extracellular matrix made. Many strong cell-cell junctions make a strong epithelial sheet. Junctions are attached to the cytoskeleton providing resistance to mechanical stress. Epithelial sheets include the skin and the lining of your gut. Form barriers to water, other molecules, and cells.

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Epithelial sheets rest on a bed of connective tissue.

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Molecules must move through the cell.

Extracellular matrix is formed from secreted macromolecules that provide the substratum upon which cells can attach and move around on. Cells are also bound to each other via cell-cell junctions. Groups of tissue that have specific purposes are called organs, i.e. epithelial tissues are part of the organ “skin”

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MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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Cell Junctions • • •

Points of cell-cell attachment, and cell-matrix attachment. Highly abundant in epithelial cells. Three functional groups o Occluding junctions ƒ Like ‘zip-lock’ bags, nothing gets past o Anchoring junctions ƒ How cells hold on to each other and the matrix ƒ Are attached to the cytoskeleton o Communicating junctions ƒ Provides a passage for communication between cells. ƒ Small molecules pass through the junctions.

Occluding Junctions Form a Selective Permeability Barrier Across Epithelial Cell Sheets • •

Also called tight junctions in vertebrates. The ‘zip-lock’ seal prevent molecules and cells from permeating between cells from the gut of an animal. Fig. 19-2 o

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Actually cells regulate this too, to let certain molecules (like amino acids, certain ions) past.

The tight junctions also prevent transmembrane carrier proteins on basal and lateral side of the cell from migrating over to the luminal side (apical surface). Model for tight junctions Fig. 19-4a, Fig. 19-5 o Claudins and occludins are integral membrane proteins that form the tight junctions. o Both form homophilic interactions that bridge the extracellular space

Anchoring Junctions Connect the Cytoskeleton of a Cell Either to the Cytoskeleton of Its Neighbors or to the Extracellular Matrix/Basal Lamina • • • •

They attach to each other across membranes between cells. Fig. 19-7 Inside the cell they are attached to the cytoskeleton. Cell membrane alone does not provide significant structural stability to cells. Two major functional forms: o Cell cell junctions ƒ adherens junctions use classical cadherins attached to actin filaments via anchoring proteins such as catenins, vinculin, and α-actinin. (Fig 19-9) • Connect Bundles of Actin Filaments from Cell to Cell

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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desmosomes contain non-classical cadherin family proteins that attach to intermediate filaments such as keratin filaments. Fig. 19-11 • Connect Intermediate Filaments from Cell to Cell

o Cell matrix junctions: focal adhesions and hemidesmosome attachments to the extracellular matrix. ƒ Focal adhesions use integrins attached to actin filaments via anchoring proteins such as α-actinin and vinculin. Fig. 19-12 ƒ Hemidesmosomes use integrins attached to intermediate filaments. Fig 19-13 Summary • cell-cell o Adherens junctions – classical cadherins attach to actin filaments o Desmosomes – non classical cadherins family proteins that attach to intermediate filaments. • cell-matrix o Focal adhesions – integrins attach to actin filaments o Hemidesmosomes – integrins attach to intermediate filaments.

Anchoring Junctions Formed by Integrins Bind Cells to the Extracellular Matrix: Focal Adhesions and Hemidesmosomes Gap Junctions Allow Small Molecules to Pass Directly from Cell to Cell o Allow small molecules (<1000 Daltons) to pass directly between cells o gap-junction connexon hemi channel consists of 6 transmembrane connexin subunits o Two connexons in adjacent cells form a channel between two adjacent cells. Fig. 9-15 o Gap junctions are used for communication via small molecule second messengers o In some neurons and heart muscle cells GAP junctions serve as electrical synapses, allowing ionic currents to pass and spread between cells o Different connexins can form heteromeric connexons Can small proteins pass through GAP junctions? How small?

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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The Permeability of Gap Junctions Is Regulated, i.e. junctions close in response to low pH or high Ca2+ (protection from dying cells !) In Plants, Plasmodesmata Perform Many of the Same Functions as Gap Junctions Summary • See Fig. 19-19 • Multicellular animals use a variety of junctions as permeability barriers, structural support, cell-cell communication.

CELL–CELL ADHESION Animal Cells Can Assemble into Tissues Either in Place or After They Migrate Dissociated Vertebrate Cells Can Reassemble into Organized Tissues Through Selective Cell–Cell Adhesion Cadherins Mediate Ca2+-dependent Cell–Cell Adhesion • • • • • • •

Any cell adhesion molecule is generically referred to a CAM. Cadherins contain repeats that are structurally related to antibodies Fig. 19-24 But are NOT members of the Ig superfamily of cell adhesion molecules (see later) Virtually all cells in a multicellular organism express some sort of cadherin. Different family members are expressed in different cell types Cadherins are single-pass glycoproteins. They bind calcium. What do you do to lift cultured cells from the surface of a Petri dish, or to dissociate a piece of animal tissue into single cells ?

Cadherins Mediate Cell–Cell Adhesion by a Homophilic Interaction • •

Three different possibilities shown in Fig. 19-26 Cells expressing one type of cadherin tend to seek out similar cells. Fig. 19-27

Cadherins Are Linked to the Actin Cytoskeleton by Catenins Fig. 1929 Anchoring to cytoskeletal proteins is essential for the mechanical strength

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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of the connection

Selectins Mediate Transient Cell–Cell Adhesions in the Bloodstream Fig 19-30 • • • •

L-, P, E-selectins are receptors for polysaccharides Involved in recruiting white blood cells to endothelial cells that line blood vessel In response to cytokines released by inflamed underlying tissue, endothelial cells express L-selectins that are recognized by oligosaccharides on white blood cells. Conversely, at sites of inflammation, endothelial cells switch on selectins to flag down white blood cells and platelets o o

This interaction is weak, allowing the white blood cell to move around. But this induces the expression of integrins which allow stronger binding and penetration of the white blood cell into the tissue.

Members of the Immunoglobulin Superfamily of Proteins Mediate Ca2+-independent Cell–Cell Adhesion Fig 19-31 •

The most notable one is N-CAM (neural cell adhesion molecule o

Homophilic interactions

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Another one is I-CAM. (Intercellular Cell Adh Molec)

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Modification of N-CAM with sialic acid prevents cell adhesion. N-CAM directed cell-cell interaction are not as strong as that generated by cadherins. N-CAMs might be important for nerve cell-cell interactions.

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Heterophilic interactions with integrins

Multiple Types of Cell-Surface Molecules Act in Parallel to Mediate Selective Cell–Cell Adhesion Nonjunctional Contacts May Initiate Cell–Cell Adhesions That Junctional Contacts Then Orient and Stabilize • •

This allows cells to move past one another. If permanent residence is taken up then they need to set up cell junctions.

Summary see Fig. 19-32 • Calcium dependent cell-cell adhesion is mediate by cadherins, via homophilic interactions. • N-CAMs play a role in neural development.

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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THE EXTRACELLULAR MATRIX OF ANIMALS • • •

A vast network of proteins and polysaccharides upon which cells move and attach. It is made by the cells that occupy and traverse the matrix. Fig. 19-35 The matrix can be diverse in function. o o o

Calcification of the matrix gives rise to bones and teeth. It can become transparent to give rise to the eye cornea Rope-like organization gives rise to tendons.

Source of Extracellular Matrix • • •

Fibroblast cells secrete the matrix over much of the body Chondroblasts make cartilage Osteoblasts make bone.

Composition of Extracellular Matrix •

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Proteoglycans containing glycosaminoglycan (GAGs) polysaccharides linked to a core protein. GAGs … o are 2-sugar repeats o produce gel-like matrix that provides a spongy protection and milieu for cells to move around in. o are usually covalently linked to proteins and hence form so called proteglycans o occupy large amounts of space and form hydrated gels Fig. 19-37, Fig. 19-38 o make up < 10% of weight of the extracellular matrix but contribute most of the volume Fibrous proteins of extracellular matrices: o collagen - provides mechanical strength o elastin – provides stretchability/elasticity o fibronectin o laminin How does this relate to lamin?

Proteoglycans o = GAGs linked to core protein o added to membrane or secreted proteins after translocation into the ER lumen while they reside in the Golgi (before being exocytosed) o linkage involves special link tetra-saccharide o Regulate activities of secreted proteins o some signaling proteins bind to the extracellular matrix o carbohydrate content of up to 95% (compared to 1 - 60% for standard glycoproteins = standard cell surface proteins with carbohydrate modifications o Core proteins of proteoglycans are highly diverse with no common

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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structural motif o The number of different types of GAGs linked to a single type of core protein is also highly heterogeneous

Hyaluronan Is Thought to Facilitate Cell Migration During Tissue Morphogenesis and Repair Fig 19-37, 38 • • • • • • • •

Hyaluronan = simplest of the GAGs but unusual: up to 25,000 non sulfated disaccharides not typically linked to proteins contains no sulfated sugars (less negatively charged) synthesized by an enzyme integral to the membrane unlike GAGs of proteoglycans excellent lubricant of joints. holds cell-free space and makes it available to other cells to move into excess hyaluronan is degraded by hyaluronidase

What properties of GAGs make them so great as hydrating gels?

GAG Chains May Be Highly Organized in the Extracellular Matrix • • • •

Fig. 19-41 i.e. Aggrecan aggregate: Consists of about 100 aggrecan molecules linked non-covalently to a hyaluronan molecule via 2 linkage proteins Each aggrecan consists of a 3000 amino acid core protein (decorin) that is linked to >100 GAG molecules (keratane sulfate and chondroitin sulfate)

Cell-Surface Proteoglycans Act as Co-Receptors

• Some proteoglycans represent carbohydrates attached to integral membrane proteins • Can collaborate with conventional (protein only) receptors in binding cells to the extracellular matrix and regulating the response of cells to extracellular signals • Best characterized: Syndecans o Contain a membrane spanning core protein o Extracellular domain interacts with matrix o Intracellular domain interacts with actin cytoskeleton o Bind Fibroblast Growth Factor (FGF) and present it to the FGF receptor o Analogously, betaglycan binds TGF-β and is thought to presents it to TGF-β receptors

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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Collagens Are the Major Proteins of the Extracellular Matrix • • • • • • • •

Most abundant proteins in mammals (25% of protein mass) Most abundant extracellular matrix protein, Major component of skin, bone Typical triple helix Fig. 19-43 Glycine is present at every third amino acid. Why At least 25 isoforms of polypeptide chains encoded by separate genes Selectively assembled into approximately 20 different triple helices

Collagens Are Secreted with a Nonhelical Extension at Each End • • •

Pro-α chains with non helical extensions at N and C termini are synthesized and injected into lumen of rER In the ER, select Lys and Pro residues are hydroxylate A chains are assembled into triple helices

After Secretion, Fibrillar Procollagen Molecules Are Cleaved to Collagen Molecules, Which Assemble into Fibrils • • •

Pro α-chain Æ hydroxylated Pro α-chain Æ Pro triple helices Æ secretion Æ triple helix collagen Æ assembly into collagen fibrils Æ aggregation into collagen fibers Fig. 19-47 Crosslinking of collagen helps provide mechanical strength 19-46

Elastin is a protein that Gives Tissues Their Elasticity (much more than rubber). Fig. 19-52 • •

Skin, blood vessels, lungs, bladder lining, need to be both strong and elastic In elastin fibers elastin is covered with a sheet of inelastic fibrillin; together they produce a structure that is both strong and elastic .

Basal Laminae Are Composed Mainly Of Type IV Collagen, Laminin, Nidogen And Heparin Sulfate Proteoglycan Fig 19-56, 58 • • • •

Basal Lamina = flexible, thin mat of specialized extracellular matrix that underlies the epithelial cell sheets and tubes Surrounds individual muscle cells, fat cells, Schwann cells and separates these cells and epithelia from the extracellular matrix Synthesized by cells that rest on it Attached to integrins and dysroglycan, integral membrane proteins of cells that rest on it and act as receptors for laminin and type IV collagen

MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

The Controlled Degradation of Matrix Components Helps Cells Migrate • •

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Ability of cells to degrade extracellular matrix is as important as making it Required for o tissue repair o to allow cells to divide and migrate through the extracellular matrix (white blood cells migrating across the basal lamina of a blood vessel, or to allow proliferating cancer cells to invade a tissue) o for the remodeling of bone to adapt to mechanical stress (i.e. orthodontics) Marix degradation is controlled by matrix metalloproteases (i. e. collagenases) and serine proteases o These proteases are often secreted as inactive precursors that can be locally activated o i.e. plasminogen gets activated by the protease tissue plasminogen activator (tPA), which results in production of the highly active serine protease plasmin. What s the clinical application of tPA?

Integrins • • • • • • • • • •

= principle receptors for most extracellular matrix proteins including collagen, fibronectin, laminin Large family of homologous membrane proteins Can also serve focal adhesion between cells and cells and extracellular matrix (Fig 19-32) Unlike typical signaling receptors, integrins are much more abundant and have very low affinity for the matrix. Why? o allows a cell to move rapidly over the matrix. = Transmembrane Heterodimers Fig. 19-64 24 different α subunits + 9 β subunits -> DIVERSE HETERMOERS depend on divalent cations for proper folding and matrix binding. Most interact with actin filaments via α-actinin, filamin, and other adaptor proteins. Clustering of integrins as a result of matrix binding results in reorganization of actin filament, and thus altered cell movement and shape. Conversely clustering of actin filament by other signals also re-enforces the clustering of integrins allowing them to bind the matrix more efficiently.

Integrins Must Interact with the Cytoskeleton to Bind Cells to the Extracellular Matrix Fig 19-65 •

Cells can regulate the activity of their integrins

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MICRB/BMB 252 lecture 11-13 notes (prepared by B. Luscher)

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In white blood cells, integrins are normally in a conformation that does not bind to the matrix. Î allows cells to move around until a signal triggers them to stick Î signals emanating from damaged tissue bind to cell surface receptors and elicit an intracellular signal transduction cascade that results in activation of integrins. Î white blood cell then stick around the damaged tissue longer Similar mechanism induces platelets to aggregate Human leukocyte adhesion deficiency is due to loss of β2 integrin function β3 integrin deficiency leads to Glanzman disease (excessive bleeding due to defect in platelet aggregation) Many integrin deficiencies (KO mice) are lethal

Integrins Activate Intracellular Signaling Pathways •Integrins bound to the matrix may induce localized cytoplasmic changes. •This is particularly true in axon guidance in developing nerves, where local interactions of transmembrane adhesion proteins affect the placement of actin fibers and the plasma membrane Summary • The extracellular matrix is composed of polysaccharides and glycoproteins. • The polysaccharides are highly hydrated providing a gel for movement of cells and a cushion against physical pressure. • Some matrix proteins provide mechanical strength, like collagen. • Others, like elastin, provide elasticity. • Cells move through the matrix by secreting proteases. • Integrins are the principle transmembrane receptor used to bind the matrix. • They link the matrix to the cytoskeleton or basal lamina • They can direct an intracellular signal transduction cascade that directs cell movement, localized growth, and survival.