• Cells of a given type often aggregate into tissue to cooperatively perform a common function: muscle contracts; nervous tissues conduct electrical impulses; xylem tissue in plants transports water

• Different tissues can be organized into an organ, again to perform one or more specific functions

• The adult form of the roundworm Caenorhabditis Elegans contains a mere 959 cells, but these cells fall into 12 different general cell types and many distinct subtypes

• The complex and diverse morphologies of plants and animals are examples of the whole being greater than the sum of the individual parts

  • More technically described as the emergent properties of a complex system

• Cells in tissues can adhere directly to one another (cell-cell adhesion) through specialized integral membrane proteins called cell-adhesion molecules (CAMs) that often cluster into specialized cell junctions

6.1 - Cell-Cell and Cell-Matrix Adhesion: An Overview

Cell-Adhesion Molecules Bind to One Another and to Intracellular Proteins

• A large number of CAMs fall into four major families: the cadherins, immunoglobulin (Ig) superfamily, integrins, and selectins

• CAMs mediate, through their extracellular domains, adhesive interactions between cells of the same type (homotypic adhesion) or between cells of different types (heterotypic adhesion)

• A CAM on one cell can directly bind to the same kind of CAM on an adjacent cell (homophilic binding) or to a different class of CAM (heterophilic binding)

• Cell-cell adhesions can be tight and long-lasting or relatively weak and transient

• The associations between nerve cells in the spinal cord or the metabolic cells in the liver exhibit tight adhesion

• The cytosol-facing domains of CAMs recruit sets of multifunctional adapter proteins

• These adapters act as linkers that directly or indirectly connect CAMs to elements of the cytoskeleton

  • They can also recruit intracellular molecules that function in signaling pathways to control protein activity and gene expression

• The formation of many cell-cell adhesions entails two types of molecular interactions

  • First, CAMs on one cell associate laterally through their extracellular domains or cytosolic domains or both into homodimers or higher-order oligomers in the plane of the cell’s plasma membrane

    • These interactions are called intracellular, lateral, or cis interactions

  • Second, CAM oligomers on one cell bind to the same or different CAMs on an adjacent cell; these interactions are called intercellular or trans interactions

The Extracellular Matrix Participates in Adhesion and Other Functions

• Certain cell-surface receptors, including some integrins, can bind components of the extracellular matrix (ECM)

  • Indirectly adhering cells to each other through their interactions with the matrix

• Although the extracellular matrix generally provides mechanical support to tissues, it serves several other functions as well

• Different combinations of ECM components tailor the extracellular matrix for specific purposes: strength in a tendon, tooth, or bone; cushioning in cartilage, and adhesion in most tissues

• Changes in ECM components, which are constantly being remodeled, degraded, and resynthesized locally, can modulate the interactions of a cell with its environment

Diversity of Animal Tissues Depends on Evolution of Adhesion Molecules with Various Properties

• Cell-cell adhesions and cell-matrix adhesions are responsible for the formation, composition, architecture, and function of animal tissues

• Adhesion molecules of animals are evolutionarily ancient and are some of the most highly conserved proteins among multicellular (metazoan) organisms

• The diversity of adhesive molecules arises in large part from two phenomena that can generate numerous closely related proteins,

  • Called isoforms, that constitute a protein family

• Sometimes the different members of a protein family are encoded by multiple genes that arose from a common ancestor by gene duplication and divergent evolution

6.2 - Sheetlike Epithelial Tissues Junctions and Adhesion Molecules

• Generally, external and internal surfaces of organs are covered by a sheetlike layer of epithelial tissue called an epithelium

• Cells that form epithelial tissues are said to be polarized because their plasma membranes are organized into at least two discrete regions

• The basal surface usually contacts an underlying extracellular matrix called the basal lamina, whose composition and function

• Epithelia in different body locations have characteristic morphologies and functions

• Stratified (multilayered) epithelia commonly serve as barriers and protective surfaces (e.g., the skin)

  • Whereas simple (single-layer) epithelia often selectively move ions and small molecules from one side of the layer to the other

• The simple columnar epithelium lining the small intestine has numerous fingerlike projections (100 nm in diameter) called microvilli (singular, microvillus) that extend from the luminal (apical) surface

Specialized Junctions Help Define the Structure and Function of Epithelial Cells

• All epithelial cells in a sheet are connected to one another and the extracellular matrix by specialized cell junctions consisting of dense clusters of CAMs

• But, hundreds of individual CAM-mediated interactions are sufficient to cause cells to adhere, junctions play special roles in imparting strength and rigidity to a tissue, transmitting information between the extracellular and the intracellular space

  • Controlling the passage of ions and molecules across cell layers, and serving as conduits for the movement of ions and molecules from the cytoplasm of one cell to that of its immediate neighbor

• Three major classes of animal cell junctions are prominent features of the intestinal epithelium

• Anchoring junctions and tight junctions perform the key task of holding cells together into tissues

• These junctions are organized into three parts

• Gap junctions permit the rapid diffusion of small, water-soluble molecules between the cytoplasm of adjacent cells

• However, present in epithelia, gap junctions are also abundant in nonepithelial tissues and structurally are very different from anchoring junctions and tight junctions; they also bear some resemblance to an important cell-cell junction in plants

• Epithelial and some other types of cells, such as smooth muscle, are also bound tightly together by desmosomes

  • Button-like points of contact sometimes called spot desmosomes

• Hemidesmosomes found mainly on the basal surface of epithelial cells, anchor an epithelium to components of the underlying extracellular matrix

  • Much like nails holding down a carpet

• Desmosomes and hemidesmosomes also transmit shear forces from one region of a cell layer to the epithelium as a whole

  • Providing strength and rigidity to the entire epithelial cell layer

• These junctions are especially important in maintaining the integrity of skin epithelia

Ca2+ -Dependent Homophilic Cell-Cell Adhesion in Adherens Junctions and Desmosomes Is Mediated by Cadherins

• Primary CAMs in adherens junctions and desmosomes belong to the cadherin family

• In vertebrates and invertebrates, this protein family of more than 100 members can be grouped into at least six subfamilies

• Cadherins are key molecules in cell–cell adhesion and cell signaling, and they play a critical role during tissue differentiation

• The “classical” E-, P-, and N-cadherins are the most widely expressed, particularly during early differentiation

• Classical Cadherins: The results of experiments with L cells, a line of cultured mouse fibroblasts grown in the laboratory, demonstrated that E-cadherin and P-cadherin preferentially mediate homophilic interactions

• L cells express no cadherins and adhere poorly to themselves or to other types of cultured cells

• The adhesiveness of cadherins depends on the presence of extracellular Ca2, the property that gave rise to their name (calcium adhering)

• Each classical cadherin contains a single transmembrane domain, a relatively short C-terminal cytosolic domain, and five extracellular “cadherin” domains

• The results of domain swap experiments, in which an extracellular domain of one kind of cadherin is replaced with the corresponding domain of a different cadherin

  • Have indicated that the specificity of binding resides, at least in part, in the most distal extracellular domain, the N-terminal domain

• The C-terminal cytosolic domain of classical cadherins is linked to the actin cytoskeleton by a number of cytosolic adapter proteins

• These linkages are essential for strong adhesion, apparently owing primarily to their contributing to increased lateral associations

• Although E-cadherins exhibit primarily homophilic binding, some cadherins mediate heterophilic interactions

• Each classical cadherin has a characteristic tissue distribution

• In the course of differentiation, the amount or nature of the cell-surface cadherins changes, affecting many aspects of cell-cell adhesion and cell migration

• Desmosomal Cadherins: Desmosomes contain two specialized cadherin proteins, desmoglein, and desmocollin, whose cytosolic domains are distinct from those in the classical cadherins

Tight Junctions Seal Off Body Cavities and Restrict Diffusion of Membrane Components

• For polarized epithelial cells to carry out their functions as barriers and mediators of selective transport

  • Extracellular fluids surrounding their apical and basolateral membranes must be kept separate

• Tight junctions prevent the diffusion of macromolecules and to varying degrees impede the diffusion of small water-soluble molecules and ions across an epithelial sheet in the spaces between cells

• They are composed of thin bands of plasma-membrane proteins that completely encircle a polarized cell and are in contact with similar thin bands on adjacent cells

• Very high magnification reveals that rows of protein particles 3–4 nm in diameter form the ridges seen in freeze-fracture micrographs of tight junctions

• The two principal integral-membrane proteins found in tight junctions are occludin and claudin

• Initially, investigators thought that occludin was the only essential protein component of tight junctions

• But, when investigators engineered mice with mutations inactivating the occludin gene, the mice still had morphologically distinct tight junctions

  • This technique is called gene knockout

• The claudin multigene family encodes numerous homologous proteins (isoforms) that exhibit distinct tissue-specific patterns of expression

• Recently, a group of junction adhesion molecules (JAMs) have been found to contribute to homophilic adhesion and other functions of tight junctions

• These molecules, which contain a single transmembrane helix, belong to the Ig superfamily of CAMs

• Plasma-membrane proteins cannot diffuse in the plane of the membrane past tight junctions

• These junctions also restrict the lateral movement of lipids in the exoplasmic leaflet of the plasma membrane in the apical and basolateral regions of epithelial cells

Differences in Permeability of Tight Junctions Can Control Passage of Small Molecules Across Epithelia

• The barrier to the diffusion provided by tight junctions is not absolute

• Owing at least in part to the varying properties of the different isoforms of claudin located in different tight junctions

  • Their permeability to ions, small molecules, and water varies enormously among different epithelial tissues

• The leakiness of tight junctions can be altered by intracellular signaling pathways, especially G protein-coupled pathways entailing cyclic AMP and protein kinase C

Many Cell-Matrix and Some Cell-Cell Interactions Are Mediated by Integrins

• The integrin family comprises heterodimeric integral membrane proteins that function as adhesion receptors, mediating many cell-matrix interactions

• Invertebrates, at least 24 integrin heterodimers, composed of 18 types of ox subunits and 8 types of B subunits in various combinations

• In epithelial cells, integrin ox6B4 is concentrated in hemidesmosomes and plays a major role in adhering cells to the matrix in the underlying basal lamina

• Integrins typically exhibit low affinities for their ligands with dissociation constants KD between 10-6 and 10-8 mol/L

• Parts of both the ox and the B subunits of an integrin molecule contribute to the primary extracellular ligand-binding site

• Integrins can mediate outside-in and inside-out transfer of information

6.3 - The Extracellular Matrix of Epithelial Sheets

• In animals, the extracellular matrix helps organize cells into tissues and coordinates their cellular functions by activating intracellular signaling pathways that control cell growth, proliferation, and gene expression

• Three types of molecules are abundant in the extracellular matrix of all tissues.

1. Highly viscous proteoglycans, a group of glycoproteins that cushion cells and bind a wide variety of extracellular molecules

2. Collagen fibers, which provide mechanical strength and resilience

3. Soluble multi adhesive matrix proteins, which bind to and cross-link cell-surface adhesion receptors and other ECM components

The Basal Lamina Provides a Foundation for Epithelial Sheets

• In animals, epithelia and most organized groups of cells are underlain or surrounded by the basal lamina, a sheetlike meshwork of ECM components usually no more than 60–120 nm thick

• The basal lamina is structured differently in different tissues

• In columnar and other epithelia (e.g. intestinal lining, skin), it is a foundation on which only one surface of the cells rests.

• Most of the ECM components in the basal lamina are synthesized by the cells that rest on it

  • Four ubiquitous protein components are found in basal laminae

1. Type IV collagen, trimeric molecules with both rodlike and globular domains that form a two-dimensional network

2. Laminins, a family of multi-adhesive proteins that form a fibrous two-dimensional network with type IV collagen and that also bind to integrins

3. Entactin (also called nidogen), a rod-like molecule that cross-links type IV collagen and laminin and helps incorporate other components into the ECM

4. Perlecan, a large multidomain proteoglycan that binds to and cross-links many ECM components and cell-surface molecules

Sheet-Forming Type IV Collagen Is a Major Structural Component in Basal Laminae

• Type IV collagen, the principal component of all basal lamina

  • Is one of more than 20 types of collagen that participate in the formation of the extracellular matrix in various tissues

• The collagen triple helix can form because of an unusual abundance of three amino acids: glycine, proline, and a modified form of proline called hydroxyproline

• Glycine is essential because its small side chain, a hydrogen atom, is the only one that can fit into the crowded center of the three-stranded helix

Laminin, a Multiadhesive Matrix Protein, Helps Cross-link Components of the Basal Lamina

• Multiadhesive matrix proteins are long, flexible molecules that contain multiple domains responsible for binding various types of collagen, other matrix proteins, polysaccharides, cell-surface adhesion receptors, and extracellular signaling molecules

• Laminin, the principal multi adhesive matrix protein in basal laminae, is a heterotrimeric, cross-shaped protein with a total molecular weight of 820,000

• LG domains are found in a wide variety of proteins and can mediate binding to steroids and proteins as well as carbohydrates

Secreted and Cell-Surface Proteoglycans Are Expressed by Many Cell Types

• Proteoglycans are a subset of glycoproteins containing covalently linked specialized polysaccharide chains called glycosaminoglycans (GAGs), which are long linear polymers of specific repeating disaccharides

• One or both of the sugars contain at least one anionic group (carboxylate or sulfate)

• GAGs are classified into several major types based on the nature of the repeating disaccharide unit: heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan

• Biosynthesis of Proteoglycans: With the exception of hyaluronan, which is discussed in the next section, all the major GAGs occur naturally as components of proteoglycans

  • Like other secreted and transmembrane glycoproteins, proteoglycan core proteins are synthesized on the endoplasmic reticulum

  • The GAG chains are assembled on these cores in the Golgi complex

• Diversity of Proteoglycans: The proteoglycans constitute a remarkably diverse group of molecules that are abundant in the extracellular matrix of all animal tissues and are also expressed on the cell surface

  • The latter include integral membrane proteins (syndecans) and GPI-anchored proteins (glypicans); the GAG chains in both types of cell-surface proteoglycans extend into the extracellular space

  • The sequences and lengths of proteoglycan core proteins vary considerably, and the number of attached GAG chains ranges from just a few to more than 100

• Perlecan, the major secreted proteoglycan in basal laminae, consists of a large multidomain core protein (≈400 kDa) with three or four specialized GAG chains

• Syndecans are expressed by epithelial cells and many other cell types

• These cell-surface proteoglycans bind to collagens and multi adhesive matrix proteins such as the fibronectins

Modifications in Glycosaminoglycan (GAG) Chains Can Determine Proteoglycan Functions

• The case with the sequence of amino acids in proteins

• The arrangement of the sugar residues in GAG chains and the modification of specific sugars (e.g., the addition of sulfate) in the chains can determine their function and that of the proteoglycans containing them

• For years, the chemical and structural complexity of proteoglycans posed a daunting barrier to an analysis of their structures and an understanding of their many diverse functions

6.4 - The Extracellular Matrix of Non-Epithelial Tissues

• These are similar to CAMs and adhesion receptors

Fibrillar Collagens Are the Major Fibrous Proteins in the Extracellular Matrix of Connective Tissues

• Connective tissue, such as tendon and cartilage, differs from other solid tissues in that most of its volume is made up of extracellular matrix rather than cells

• This matrix is packed with insoluble protein fibers and contains proteoglycans, various multi-adhesive proteins, and hyaluronan, a very large, non-sulfated GAG

• The most abundant fibrous protein in connective tissue is collagen

• Rubberlike elastin fibers, which can be stretched and relaxed, also are present in deformable sites (e.g., skin, tendons, heart)

Formation of Collagen Fibrils in the Endoplasmic Reticulum and Is Completed Outside the Cell

• Collagen biosynthesis and secretion follow the normal pathway for a secreted protein

• The collagen ox chains are synthesized as longer precursors, called pro-ox chains, by ribosomes attached to the endoplasmic reticulum (ER)

• The pro-ox chains undergo a series of covalent modifications and fold into triple-helical procollagen molecules before their release from cells

• Short non-triple-helical segments at either end of the collagen ox chains are of particular importance in the formation of collagen fibrils

• Lysine and hydroxylysine side chains in these segments are covalently modified by extracellular lysyl oxidases to form aldehydes in place of the amine group at the end of the side chain

Type I and II Collagens Form Diverse Structures and Associate with Different Non Fibrillar Collagens

• Collagens differ in their ability to form fibers and to organize the fibers into networks

• Type I collagen fibrils are also used as the reinforcing rods in the construction of bone

• Bones and teeth are hard and strong because they contain large amounts of dahllite, crystalline calcium- and phosphate-containing mineral

• In many connective tissues, type VI collagen and proteoglycans are noncovalently bound to the sides of type I fibrils and may bind the fibrils together to form thicker collagen fibers

• Type VI collagen is unusual in that the molecule consists of a relatively short triple helix with globular domains at both ends

• The fibrils of type II collagen, the major collagen in cartilage, are smaller in diameter than type I fibrils and are oriented randomly in a viscous proteoglycan matrix

• The rigid collagen fibrils impart strength and compressibility to the matrix and allow it to resist large deformations in shape

Hyaluronan Resists Compression and Facilitates Cell Migration

• Hyaluronan, also called hyaluronic acid (HA) or hyaluronate, is a non-sulfated GAG formed as a disaccharide repeat composed of glucuronic acid and N-acetylglucosamine by a plasma-membrane-bound enzyme (HA synthase) and is directly secreted into the extracellular space

• Hyaluronan molecules range in length from a few disaccharide repeats to ≈25,000

• The typical hyaluronan in joints such as the elbow has 10,000 repeats for a total mass of 4 x 106 Da and length of 10 μm (about the diameter of a small cell)

• With a large number of anionic residues on its surface, the typical hyaluronan molecule binds a large amount of water and behaves as if it were a large hydrated sphere with a diameter of ≈500 nm

• As the concentration of hyaluronan increases, the long chains begin to entangle, forming a viscous gel

• Hyaluronan is bound to the surface of many migrating cells by a number of adhesion receptors (e.g., one called CD44) containing HA-binding domains, each with a similar three-dimensional conformation

Association of Hyaluronan and Proteoglycans Form Large, Complex Aggregates

• The predominant proteoglycan in cartilage, called aggrecan, assembles with hyaluronan into very large aggregates

• The backbone of the cartilage proteoglycan aggregate is a long molecule of hyaluronan to which multiple aggrecan molecules are bound tightly but noncovalently

• A single aggrecan aggregate, one of the largest macromolecular complexes known, can be more than 4 mm long and have a volume larger than that of a bacterial cell

• The aggrecan core protein (≈250,000 MW) has one N-terminal globular domain that binds with high affinity to a specific decasaccharide sequence within hyaluronan

Fibronectins Connect Many Cells to Fibrous Collagens and Other Matrix Components

• Many different cell types synthesize fibronectin, an abundant multi adhesive matrix protein found in all vertebrates

• The discovery that fibronectin functions as an adhesive molecule stemmed from observations that it is present on the surfaces of normal fibroblastic cells, which adhere tightly to Petri dishes in laboratory experiments

  • But is absent from the surfaces of tumorigenic cells, which adhere weakly

• Fibronectins help attach cells to the extracellular matrix by binding to other ECM components

  • Particularly fibrous collagens and heparan sulfate proteoglycans, and to cell-surface adhesion receptors such as integrins

• Fibronectins are dimers of two similar polypeptides linked at their C-termini by two disulfide bonds; each chain is about 60–70 nm long and 2–3 nm thick

• Partial digestion of fibronectin with low amounts of proteases and analysis of the fragments showed that each chain comprises six functional regions with different ligand-binding specificities

• One of the type III repeats in the cell-binding region of fibronectin mediates binding to certain integrins

• A three-dimensional model of fibronectin-binding to integrin based on structures of parts of both fibronectin and integrin has been assembled

• Microscopy and other experimental approaches (e.g., biochemical binding experiments) have demonstrated the role of integrins in cross-linking fibronectin and other ECM components to the cytoskeleton

• The force needed to unfold and expose functional self-association sites in fibronectin is much less than that needed to disrupt fibronectin–integrin-binding

• Thus fibronectin molecules remain bound to integrin while cell-generated mechanical forces induce fibril formation

6.5 - Adhesive Interactions and Non-Epithelial Cells

• After adhesive interactions in epithelia form during differentiation

• They often are very stable and can last throughout the lifespan of epithelial cells or until the cells undergo differentiation into loosely associated nonpolarized mesenchymal cells, the epithelial-mesenchymal transition

Integrin-Containing Adhesive Structures Physically and Functionally Connect the ECM and Cytoskeleton in Non-Epithelial Cells

• As already discussed in regard to epithelia, integrin-containing hemidesmosomes connect epithelial cells to the basal lamina and, through adapter proteins, to intermediate filaments of the cytoskeleton

• Integrin containing adhesive structures have been studied most frequently in fibroblasts grown in cell culture on flat glass or plastic surfaces (substrata)

  • These conditions only poorly approximate the three-dimensional ECM environment that normally surrounds such cells in vivo

• When fibroblasts are cultured in three-dimensional ECM matrices derived from cells or tissues, they form adhesions to the three-dimensional ECM substratum, called 3D adhesions

• These structures differ somewhat in composition, shape, distribution, and activity from the focal or fibrillar adhesions seen in cells growing on the flat substratum typically used in cell-culture experiments

• Observations indicate that the topological, compositional, and mechanical (e.g., flexibility) properties of the extracellular matrix all play a role in controlling the shape and activity of a cell

• Tissue-specific differences in these matrix characteristics probably contribute to the tissue-specific properties of cells

Diversity of Ligand-Integrin Interactions Contributes to Numerous Biological Processes

• Most cells express several distinct integrins that bind the same ligand or different ligands, many integrins are expressed predominantly in certain types of cells

• All integrins appear to have evolved from two ancient general subgroups: those that bind RGD-containing molecules (e.g., fibronectin) and those that bind laminin

• The I-domain in some integrins mediates binding to various collagens

• Other integrins containing subunits with I-domains are expressed exclusively on leukocytes and hematopoietic cells

  • These integrins recognize cell-adhesion molecules on other cells, including members of the Ig superfamily (e.g., ICAMs, VCAMs), and thus participate in cell–cell adhesion

• The diversity of integrins and their ECM ligands enables integrins to participate in a wide array of key biological processes

  • Including the migration of cells to their correct locations in the formation of the body plan of an embryo (morphogenesis) and in the inflammatory response

Cell-Matrix Adhesion Is Modulated By Changes in the Binding Activity and Numbers of Integrins

• Cells can exquisitely control the strength of integrin-mediated cell–matrix interactions by regulating the ligand-binding activity of integrins or their expression or both

• Many, if not all, integrins can exist in two conformations: a low-affinity (inactive) form and a high-affinity (active) form

• The results of structural studies and experiments investigating the binding of ligands by integrins have provided a model of the changes that take place when integrins are activated

• These structural models also provide an attractive explanation for the ability of integrins to mediate outside-in and inside-out signaling

• The binding of certain ECM molecules or CAMs on other cells to the bent, low-affinity structure would force the molecule to straighten and consequently separate the cytoplasmic tails

• The binding of platelet to collagen or thrombin in a forming clot induces from the cytoplasm an activating conformational change in oxIIbB3 integrin that permits it to tightly bind clotting proteins and participate in clot formation

• Persons with genetic defects in the 3 integrin subunit are prone to excessive bleeding, attesting to the role of this integrin in the formation of blood clots

• The attachment of cells to ECM components can also be modulated by altering the number of integrin molecules exposed on the cell surface

• For these hematopoietic cells to proliferate and differentiate, they must be attached to fibronectin synthesized by supportive (“stromal”) cells in the bone marrow

Molecular Connections Between the ECM and Cytoskeleton Are Defective in Muscular Dystrophy

• Dystroglycan is synthesized as a large glycoprotein precursor that is proteolytically cleaved into two subunits

• The subunit is a peripheral membrane protein, and the B subunit is a transmembrane protein whose extracellular domain associates with the ox subunit

• Multiple O-linked oligosaccharides are attached covalently to side-chain hydroxyl groups of serine and threonine residues in the subunit

• The transmembrane segment of the dystroglycan B subunit associates with a complex of integral membrane proteins; its cytosolic domain binds dystrophin and other adapter proteins, as well as various intracellular signaling proteins

• The resulting large, heterogeneous assemblage, the dystrophin-glycoprotein complex (DGC), links the extracellular matrix to the cytoskeleton and signaling pathways within muscle cells

• Mutations in dystrophin, other DGC components, laminin, or enzymes that add the O-linked sugars to dystroglycan disrupt the DGC-mediated link between the exterior and the interior of muscle cells and cause muscular dystrophies

Ca2+-Independent Cell-Cell Adhesion in Neuronal and Other Tissues Is Mediated by CAMs in the Immunoglobulin Superfamily

• Numerous transmembrane proteins characterized by the presence of multiple immunoglobulin domains (repeats) in their extracellular regions constitute the Ig superfamily of CAMs, or IgCAMs

• The Ig domain is a common protein motif, containing 70–110 residues, that was first identified in antibodies, the antigen-binding immunoglobulins

• One type, the NCAMs, primarily mediate homophilic interactions. First expressed during morphogenesis, NCAMs play an important role in the differentiation of muscle, glial, and nerve cells

• Their role in cell adhesion has been directly demonstrated by the inhibition of adhesion with anti-NCAM antibodies

• An NCAM comprises an extracellular region with five Ig repeats and two fibronectin type III repeats, a single membrane-spanning segment, and a cytosolic segment that interacts with the cytoskeleton

• The covalent attachment of multiple chains of sialic acid, a negatively charged sugar derivative, to NCAMs alters their adhesive properties. In embryonic tissues such as the brain, polysialic acid constitutes as much as 25 percent of the mass of NCAMs

Movement of Leukocytes into Tissues Depends on a Precise Sequence of Combinatorially Diverse Sets of Adhesive Interactions

• In adult organisms, several types of white blood cells (leukocytes) participate in the defense against infection caused by foreign invaders (e.g., bacteria and viruses) and tissue damage due to trauma or inflammation

• To fight infection and clear away damaged tissue, these cells must move rapidly from the blood, where they circulate as unattached, relatively quiescent cells, into the underlying tissue at sites of infection, inflammation, or damage

• Extravasation requires the successive formation and breakage of cell-cell contacts between leukocytes in the blood and endothelial cells lining the vessels

• Some of these contacts are mediated by selectins, a family of CAMs that mediate leukocyte–vascular cell interactions

• For tight adhesion to occur between activated endothelial cells and leukocytes, B2-containing integrins on the surfaces of leukocytes also must be activated by chemokines or other local activation signals such as platelet-activating factor

• The selective adhesion of leukocytes to the endothelium near sites of infection or inflammation thus depends on the sequential appearance and activation of several different CAMs on the surfaces of the interacting cells

• Different types of leukocytes express specific integrins containing the B2 subunit

• Many of the CAMs used to direct leukocyte adhesion are shared among different types of leukocytes and target tissues

• Often only a particular type of leukocyte is directed to a particular tissue

Gap Junctions Composed of Connexins Allow Small Molecules to Pass Between Adjacent Cells

• Early electron micrographs of virtually all animal cells that were in contact revealed sites of cell-cell contact with a characteristic intercellular gap

• In many tissues (e.g., the liver), large numbers of individual cylindrical particles cluster together in patches

• This property has enabled researchers to separate gap junctions from other components of the plasma membrane

• When the plasma membrane is purified and then sheared into small fragments, some pieces mainly containing patches of gap junctions are generated

• The effective pore size of gap junctions can be measured by injecting a cell with a fluorescent dye covalently linked to molecules of various sizes and observing with a fluorescence microscope whether the dye passes into neighboring cells

• Gap junctions between mammalian cells permit the passage of molecules as large as 1.2 nm in diameter

• In nervous tissue, some neurons are connected by gap junctions through which ions pass rapidly, thereby allowing very rapid transmission of electrical signals

• Impulse transmission through these connections, called electrical synapses, is almost a thousandfold as rapid as at chemical synapses

• Some extracellular hormonal signals induce the production or release of small intracellular signaling molecules called second messengers that regulate cellular metabolism

• There are probably more than 20 different connexin genes in vertebrates, and different sets of connexins are expressed in different cell types. Some cells express a single connexin; consequently, their gap-junction channels are homotypic, consisting of identical connexons

• The generation of mutant mice with inactivating mutations in connexin genes has highlighted the importance of connexins in a wide variety of cellular systems

6.6 - Plant Tissues

• Plant tissues are organized into just four main organ systems

  • Stems have support and transport functions; roots provide anchorage and absorb and store nutrients; leaves are the sites of photosynthesis, and flowers enclose the reproductive structures

• At the cell, tissue, and organ levels, plants are generally less complex than most animals

The Plant Cell wall Is a Laminate of Cellulose Fibrils in a Matrix of Glycoproteins

• The plant cell wall is ≈0.2 μm thick and completely coats the outside of the plant cell’s plasma membrane

• This structure serves some of the same functions as those of the extracellular matrix produced by animal cells

  • Even though the two structures are composed of entirely different macromolecules and have a different organization

• Because a major function of a plant cell wall is to withstand the osmotic turgor pressure of the cell, the cell wall is built for lateral strength. It is arranged into layers of cellulose microfibrils—bundles of long, linear, extensively hydrogen-bonded polymers of glucose in B glycosidic linkages

• The layers of microfibrils prevent the cell wall from stretching laterally

• Cellulose microfibrils are synthesized on the exoplasmic face of the plasma membrane from UDP-glucose and ADP-glucose formed in the cytosol

• The polymerizing enzyme, called cellulose synthase, moves within the plane of the plasma membrane as cellulose is formed, in directions determined by the underlying microtubule cytoskeleton

• Unlike cellulose, pectin and hemicellulose are synthesized in the Golgi apparatus and transported to the cell surface where they form an interlinked network that helps bind the walls of adjacent cells to one another and cushions them

• The cell wall is a selective filter whose permeability is controlled largely by pectins in the wall matrix

• Whereas water and ions diffuse freely across cell walls, the diffusion of large molecules, including proteins larger than 20 kDa, is limited

Loosening of the Cell Wall Permits Elongation of Plants Cells

• The cell wall surrounding a plant cell prevents the cell from expanding, its structure must be loosened when the cell grows

• The amount, type, and direction of plant cell growth are regulated by small-molecule hormones (e.g., indoleacetic acid) called auxins

• The auxin-induced weakening of the cell wall permits the expansion of the intracellular vacuole by uptake of water, leading to elongation of the cell

• The cell wall undergoes its greatest changes at the meristem of a root or shoot tip

• These sites are where cells divide and expand

• Young meristematic cells are connected by thin primary cell walls, which can be loosened and stretched to allow subsequent cell elongation

Plasmodesmata Directly Connect the Cytosols of Adjacent Cells in Higher Plants

• Plant cells can communicate directly through specialized cell-cell junctions called plasmodesmata, which extend through the cell wall

• Like gap junctions, plasmodesmata are open channels that connect the cytosol of a cell with that of an adjacent cell

• The diameter of the cytosol-filled channel is about 30–60 nm, and plasmodesmata can traverse cells walls as much as 90 nm thick

• Molecules smaller than about 1000 Da, including a variety of metabolic and signaling compounds, generally can diffuse through plasmodesmata

• Although plasmodesmata and gap junctions resemble each other functionally, their structures differ in two significant ways

• The plasma membranes of the adjacent plant cells merge to form a continuous channel, the annulus, at each plasmodesmata, whereas the membranes of cells at a gap junction are not continuous with each other

Only a Few Adhesive Molecules Have Been Identified in Plants

• Systematic analysis of the Arabidopsis genome and biochemical analysis of other plant species provide no evidence for the existence of plant homologs of most animal CAMs, adhesion receptors, and ECM components

• Arabidopsis proteins have a single transmembrane domain and an intracellular cytosolic tyrosine kinase domain

• The results of in vitro binding assays combined with in vivo studies and analyses of plant mutants have identified several macromolecules in the ECM that are important for adhesion

• Disruption of the gene encoding glucuronyltransferase 1, a key enzyme in pectin biosynthesis, has provided a striking illustration of the importance of pectins in intercellular adhesion in plant meristems

6.7 - Growth and Use of Cultured Cells

• Many technical constraints hamper studies on specific cells or subsets of cells in intact animals and plants

• One alternative is the use of intact organs that are removed from animals and perfused with an appropriately buffered solution to maintain their physiologic integrity and function

• Cultured cells have several advantages over intact organisms for cell biology research

• A major disadvantage of cultured cells is that they are not in their normal environment and hence their activities are not regulated by the other cells and tissues as they are in an intact organism

Culture of Animal Cells Requires Nutrient-Rich Media and Special Solid Surfaces

• The most bacterial cells, which can be cultured quite easily, animal cells require many specialized nutrients and often specially coated dishes for successful culturing

• To permit the survival and normal function of cultured tissues or cells, the temperature (37 °C for mammalian cells), pH, ionic strength, and access to essential nutrients must simulate as closely as possible the conditions within an intact organism

• Media for culturing animal cells must supply histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine; no cells in adult vertebrate animals can synthesize these nine essential amino acids

• The other necessary components of a medium for culturing animal cells are vitamins, various salts, fatty acids, glucose, and serum—the fluid remaining after the noncellular part of the blood (plasma) has been allowed to clot

• Unlike bacterial and yeast cells, which can be grown in suspension, most animal cells will grow only on a solid surface

• This highlights the importance of cell adhesion molecules

Primary Cell Cultures and Cell Strains Have a Finite Life Span

• Normal animal tissues (e.g., skin, kidney, liver) or whole embryos are commonly used to establish primary cell cultures

• To prepare tissue cells for primary culture, the cell–cell and cell–matrix interactions must be broken

• Often connective tissue fibroblasts divide in culture more rapidly than other cells in a tissue, eventually becoming the predominant type of cells in the primary culture, unless special precautions are taken to remove them when isolating other types of cells

• When cells removed from an embryo or an adult animal are cultured, most of the adherent ones will divide a finite number of times and then cease growing

• Cell strains can be frozen in a state of suspended animation and stored for extended periods at liquid nitrogen temperature

  • Provided that a preservative that prevents the formation of damaging ice crystals is used

Transformed Cells Can Grow Indefinitely in Culture

• To be able to clone individual cells, modify cell behavior, or select mutants, biologists often want to maintain cell cultures for many more than 100 doublings

• Such prolonged growth is exhibited by cells derived from some tumors

• A culture of cells with an indefinite lifespan is considered immortal and is called a cell line

• Most cell lines have lost some or many of the functions characteristic of the differentiated cells from which they were derived

• Such relatively undifferentiated cells are poor models for investigating the normal functions of specific cell types

Hybrid Cells Called Hybridomas Produce Abundant Monoclonal Antibodies

• Each normal B lymphocyte in a mammal is capable of producing a single type of antibody directed against (can bind to) a specific chemical structure (called a determinant or epitope) on an antigen molecule

• If an animal is injected with an antigen, B lymphocytes that make antibodies recognizing the antigen are stimulated to grow and secrete the antibodies

• The resulting mixture of antibodies that recognize different epitopes on the same antigen is said to be polyclonal

• Such polyclonal antibodies circulate in the blood and can be isolated as a group and used for a variety of experiments

• During cell fusion, the plasma membranes of two cells fuse together, allowing their cytosols and organelles to intermingle

• Treatment with certain viral glycoproteins or the chemical polyethylene glycol promotes cell fusion

• Some of the fused cells can undergo division and their nuclei eventually coalesce, producing viable hybrid cells with a single nucleus that contains chromosomes from both “parents”

HAT Medium Is Commonly Used to Isolate Hybrid Cells

• The principles underlying HAT selection are important not only for understanding how hybridoma cells are isolated but also for understanding several other frequently used selection methods

  • Including a selection of the ES cells used in generating knockout mice

• Most animal cells can synthesize the purine and pyrimidine nucleotides from simpler carbon and nitrogen compounds

• Cells with a TK mutation that prevents the production of the functional TK enzyme can be isolated because such cells are resistant to the otherwise toxic thymidine analog 5-bromodeoxyuridine

• Cells containing TK convert this compound into 5-bromodeoxyuridine monophosphate, which is then converted into a nucleoside triphosphate by other enzymes

• The triphosphate analog is incorporated by DNA polymerase into DNA, where it exerts its toxic effects