Test bank

Chapter 7

Interactions Between Cells And Their Environment

Objectives

§ Define the general structure and function of the glycocalyx and extracellular matrix.

§ Describe the function and structure of basement membranes (basal lamina).

§ Describe the structures of the components of the extracellular matrix (collagen, GAGs, proteoglycans, glycoproteins) and differentiate between them.

§ Clarify the steps involved in the adhesion of cells to a noncellular surface.

§ Describe the membrane proteins involved in the adhesion of cells to noncellular surfaces.

§ Compare and contrast the structures and functions of the different cell junctions in plants and animals.

§ Describe the membrane proteins involved in cell-cell adhesion.

§ Contrast the structures of plant and bacterial cell walls despite the similarities in their function.

Lecture Outline

Introduction

I. Materials present outside the plasma membrane play an important role in the cell's life

A. Most cells in multicellular plants & animals are organized into clearly defined tissues in which component cells maintain a defined relationship with one another

1. They also maintain a relationship with the extracellular materials that lie between the cells

2. Even cells that are not found in solid tissue (white blood cells) must interact in highly specific ways with other cells & extracellular materials with which they come into contact

B. These interactions regulate a number of diverse activities: cell migration, cell growth, cell differentiation

1. They also determine the 3D organization of tissues & organs that emerges during embryonic development

II. Example of cell interactions in a tissue – human skin

A. The skin's outer layer (epidermis) is a type of epithelial tissue (epithelia line spaces within the body)

1. Epidermis consists largely of closely packed cells attached to one another & to an underlying noncellular layer by specialized contacts

2. These contacts provide a mechanism for cells to adhere to & communicate with one another

B. The deeper layer of the skin (dermis) is a type of connective tissue

1. Like other connective tissues (tendons, cartilage), dermis consists largely of extracellular material, including a variety of distinct fibers that interact with each other in specific ways

2. Cells (fibroblasts) are scattered throughout the dermis; the outer surface of their membranes contains receptors that mediate interactions between the cell & components of its environment

3. The cell surface receptors interact not only with external surroundings, but are connected at their internal ends to various cytoplasmic proteins

4. Such receptors exhibiting dual attachment are well suited to transmit messages between a cell & its environment

The Extracellular Space: Background

I. Glycocalyx (cell coat) - mediates cell-cell & cell-substratum interactions; mechanical protection for cells; barrier to particles moving toward plasma membrane

A. Made of short sugar chains (oligosaccharides); project outward from virtually all integral proteins & some lipids in plasma membrane; closely applied to outer surface of plasma membrane

B. Also contains additional extracellular materials secreted by cell into external space, where they stay closely associated with cell surface

C. Very prominent in some types of cells like epithelial cells lining mammalian digestive tract

II. Extracellular matrix (ECM) - organized network of extracellular materials found beyond the immediate vicinity of membrane

A. More than inert packing material or nonspecific glue that holds cells together; it plays a key regulatory role in determining cell shape & activities

1. Experiment: digest ECM surrounding cultured cartilage or mammary gland epithelial cells with enzymes —> get decrease in secretory & synthetic activities of cells

2. Add back ECM materials into culture —> restores differentiated state & cells produce usual products

B. May consist of ill-defined, amorphous associations of proteins & polysaccharides (like loose connective tissue) or may be in the form of a distinct structure

III. ECM takes diverse forms in different tissues & organisms, but is composed of similar proteins

A. Most proteins in cells are compact & globular; those of extracellular space are extended & fibrous

B. Among their diverse functions, ECM proteins serve as scaffolds, girders, mortar & wire

C. Alterations in amino acid sequence of extracellular proteins can lead to serious disorders

IV. ECM very prominent in connective tissues (cartilage, bones, tendons, corneal stroma)

A. In connective tissue, cells occupy a small fraction of tissue volume

B. ECM, not cells, gives tissues their identifiable properties: bone matrix hardness, cartilage matrix toughness & flexibility, tendon matrix tensile strength, corneal stroma matrix transparency

V. Components of the ECM - members of a small number of molecular families

A. Collagens – one of most important & ubiquitous ECM molecules; fibrous glycoprotein family

1. Functions only as part of ECM & only found there

B. Proteoglycans - protein-polysaccharide complex

C. Fibronectin, Laminin, ECM Proteins

The Extracellular Space: Basement Membrane as an Example of ECM & Functions of ECM Proteins

I. Basement membrane (basal lamina); a continuous ~50 - 200 nm thick sheet; one of best defined examples; found in the following places:

A. It surrounds muscle & fat cells

B. It underlies basal surface of epithelial tissues (skin epidermis, digestive & respiratory tract linings)

C. It underlies the inner endothelial lining of blood vessels

II. Functions of basement membrane

A. Provides mechanical support for the attached cells

B. Generates signals that maintain cell survival

C. Maintains epithelial cell polarity

D. Serves as a substratum for cell migration & determines cell migration path

E. Separates adjacent tissues within an organ (compartmentalization)

F. Acts as barrier to passage of macromolecules & errant cancer cells – capillary basement membranes prevent passage of proteins out of blood & into tissues (kidney – good example)

1. Kidney glomerulus - blood filtered under high pressure through double-layered basal lamina separating glomerular capillaries from kidney tubule wall

2. Basal lamina around glomeruli may thicken abnormally in long-term diabetics —> kidney failure

III. ECM may take diverse forms in different tissues & organisms, but it tends to be composed of similar macromolecules

A. Unlike most proteins found inside cells, which are compact, globular molecules, proteins of extracllular space are typically extended, fibrous species

B. These proteins can self-assemble in extracellular space into an interconnected 3D network

C. ECM proteins have diverse functions; they serve as:

1. Roadmaps

2. Scaffolds

3. Girders

4. Wire

5. Glue

D. Alterations in amino acid sequence of extracellular proteins can lead to serious disorders

Components of the Extracellular Matrix: Collagens

I. Comprise a fibrous glycoprotein family; present only in ECMs; found throughout animal kingdom; one of most important, and ubiquitous, ECM molecules

A. Noted for high tensile strength (resistance to pulling forces); it is estimated that a 1 mm dia collagen fiber can suspend a 10 kg [22 lb] weight without breaking

B. It is the single most abundant protein in human body (constitutes >25% of all protein); reflects widespread occurrence of extracellular materials

C. Collagen molecules provide the insoluble framework that determines many ECM mechanical properties

II. Made mostly by fibroblasts (found in various connective tissue types), smooth muscle & epithelial cells

A. To date, 27 distinct human types identified; each restricted to particular sites in body; ≥2 can be present together in same ECM; get functional complexity by mixing several types in same fiber (heterotypic)

1. Heterotypic fibers are biological equivalent of metal alloys

2. Different structural & mechanical properties result from different mixtures of collagens in fibers

B. Many differences among collagen family members, but all share 2 important structural features:

1. All collagen molecules are trimers consisting of 3 polypeptide [] chains - may be identical or 2 or 3 different chains

2. Along at least part of length, the 3 chains wind around each other; form unique, rodlike triple helix

C.  chains of collagen molecules contain large amounts of proline; many of the proline (& lysine) residues are hydroxylated after synthesis of the collagen polypeptide

1. The hydroxylated amino acids are important in maintaining the stability of the triple helix by forming H bonds between component chains

2. Failure to hydroxylate collagen chains has serious consequences for connective tissue structure & function; evident from scurvy symptoms (results from vitamin C [ascorbic acid] deficiency

3. Scurvy is characterized by inflamed gums & tooth loss, poor wound healing, brittle bones & weakening of blood vessel linings, which causes internal bleeding

4. Ascorbic acid is required as a coenzyme by the enzymes that add hydroxyl groups to the lysine & proline amino acids of collagen

D. Some are fibrillar collagens (I, II, III) - assemble into rigid, cable-like fibrils, which in turn assemble into thicker fibers that are typically large enough to be seen in light microscope

E. Fibrils are strengthened by covalent cross-links between lysine & hydroxylysine residues on adjacent collagen molecules - if disrupted weakened

1. Cross-linking process continues through life

2. May contribute to decreased skin elasticity & increased brittleness of bones among elderly

III. Collagen provides insoluble framework that determines many of the ECM mechanical properties & 3D organization of collagen molecules often correlates with properties of tissue in which it is found

A. Tendons (connect muscles to bones) must resist large pulling forces during muscle contraction - collagen fibrils aligned parallel to long axis of tendon & parallel to direction of pulling force

B. Cornea – serves as durable, transparent (so light can pass through), protective layer at eyeball surface

1. Stroma (thick middle layer) – fibrils relatively short, in orthogonal layers (fibers in layer parallel to each other, but nearly perpendicular to those in adjoining layers) like plywood (gives strength)

2. Fiber size uniformity & ordered packing promote tissue transparency (minimizes light scattering of incoming light rays)

C. Basement membrane (very thin, mechanically supportive sheets); type IV collagen (only seen here)

1. Type IV collagen is nonfibrillar collagen & is organized in flattened network; it provides mechanical support & serves as a lattice for deposition of other ECM material

2. Type IV collagen trimer has some interspersed nonhelical segments, not a long, uninterrupted triple helix like Type I (gives flexibility)

3. Also have globular heads on each end that serve as interaction sites between molecules & give the complex its lattice-like character

IV. Given their abundance & widespread distribution, serious disorders can be caused by abnormalities in fibrillar collagen formation

A. Burns or traumatic injuries to internal organs can cause scar tissue buildup (largely fibrillar collagen)

B. Type I collagen mutations - osteogenesis imperfecta, potentially lethal condition characterized by extremely fragile bones, thin skin & weak tendons

C. Type II collagen mutations - alter cartilage properties; causes dwarfism & skeletal deformities

D. A number of collagen gene mutations - cause various distinct but related collagen matrix structure defects (Ehler-Danlos syndromes) – one causes hyperextendable joints & highly extensible skin

E. Type IV collagen gene mutations - Alport syndrome, an inherited kidney disease in which the glomerular basement membrane is disrupted

Components of the Extracellular Matrix: Proteoglycans

I. Basement membranes & other ECMs contain large amounts of distinctive type of protein-polysaccharide complex called a proteoglycan

A. Consist of core protein to which glycosaminoglycan (GAG) chains are covalently attached

B. GAGs - repeating disaccharides (2 different sugars; -A-B-A-B-); very acidic due to both carboxyl & sulfate groups on their component sugar rings

C. ECM proteoglycans may assemble into gigantic complexes by linking core proteins to hyaluronic acid (a nonsulfated GAG); can occupy very large volumes (equivalent to that of bacterial cell)

II. Due to sulfated GAG negative charges, proteoglycans bind large numbers of cations, which, in turn, attract lots of H₂O

A. They form porous hydrated gel; that fills extracellular space & acts like packing material to resist crushing (compression) forces

B. This complements adjacent collagens, which resist pulling forces & provide scaffold for proteoglycans

C. Together they give cartilage & other ECMs strength & resistance to deformation (wiggle your ears)

D. The ECM of bone is also made of collagen & proteoglycans, but it is hardened by impregnation with calcium phosphate salts

III. In addition to serving as major ECM component, one group of proteoglycans, the heperan sulfate proteoglycans (HSPGs), function directly at the cell surface

A. Members of this group of proteoglycans include the syndecans, whose core protein spans the plasma membrane

1. The GAGs, which are attached to the protein's extracellular domain, interact with a variety of proteins, including important growth factors

2. It is speculated that the GAGs might protect the growth factor from degradation & influence the interaction of the growth factor with its own receptor at the cell surface

B. The GAGs of these proteoglycans are not constructed of identical disaccharides, but instead exhibit structural diversity

1. One region may contain sulfated sugars, whereas another region may lack sulfation

2. These differences in composition within a GAG chain are thought to influence the binding properties of various regions of these molecules

Components of the Extracellular Matrix: Fibronectin, Laminin & Other ECM Proteins

I. Matrix implies a structure made up of a network of interacting components; this is apt for ECM

A. It contains a number of proteins, in addition to collagen & proteoglycans that interact with one another in highly specific ways

B. Many ECM proteins occur in families (more than 1 form; each formed by alternate mRNA splicing)

1. Different family members made in different tissues & at different times during development

2. Different protein forms may have different properties (characteristics may not apply to all forms)

II. Fibronectin (fibrous) - one of best studied ECM proteins; has features common to most other matrix components & ECM proteins; consists of linear array of distinct building blocks (a modular construction)

A. Each fibronectin polypeptide is constructed from a sequence of ~30 independently folding Fn modules of 3 distinct types (FnI, FnII & FnIII)

1. Fn modules were first found in fibronectin, but they are found as part of many other proteins

2. Found in proteins from blood clotting factors to membrane receptors

3. Presence of shared segments among diverse proteins suggests that many present-day genes have arisen during evolution by fusion of parts of separate ancestral genes

4. In fibronectin, the 30 or so structural modules combine to form 5 or 6 larger functional domains

B. Each of the two polypeptide chains making up fibronectin contains:

1. Binding sites for other ECM components (collagen, proteoglycans, etc.); link these molecules into stable, interconnected networks

2. Binding sites for cell surface receptors (form stable ECM-cell attachments); endothelial cell will adopt shape unlike it does in body when it spreads over a square surface coated with fibronectin

C. Importance of fibronectin & other ECM proteins is particularly evident during embryonic development

1. Development involves waves of cell migration over pathways containing ECM proteins; different cells follow different routes from one part of embryo to another

2. Migrating cells guided by proteins like fibronectin contained in landscape over which they pass

D. Example: neural crest cells follow fibronectin pathways out of developing nervous system into virtually all parts of embryo; they traverse pathways rich in fibrils composed of fibronectin

1. Inject fibronectin-binding antibodies into embryos —> neural crest cells can no longer interact with fibronectin molecules in surrounding matrix & their cellular movement is inhibited

2. Antibodies to fibronectin bind/block recognition sites on fibronectin —> this inhibits cell movements

E. Example: anti-fibronectin antibodies disrupt other developmental processes

1. A number of body organs (salivary gland, kidney, lung) are formed by process of branching in which epithelial layer becomes divided by series of clefts; fibronectin important in forming them

2. Cleft formation & branching is entirely abolished as result of anti-fibronectin antibody-influenced fibronectin molecule inactivation after incubation of salivary gland with anti-fibronectin antibodies

F. Example: Also mice lacking functional fibronectin gene —> abnormal; do not survive past early development

III. Laminin also has specific domains – family of extracellular glycoproteins; consist of 3 different polypeptide chains linked by disulfide bonds; organized into a cross with 3 short arms & 1 long arm

A. Extracellularly, it greatly influences cell's potential for migration, growth & differentiation

1. Guides embryonic axon tips as grow outward from central nervous system to distant targets

2. Critical role in primordial germ cell (PGC) migration – PGCs follow laminin paths from yolk sac (outside embryo) through blood & embryonic tissues to developing gonad, become eggs or sperm

a. During migration, PGCs traverse surfaces particularly rich in laminin

b. PGCs possess cell surface protein that adheres strongly to one of laminin's subunits

B. Certain cells migrate over laminin-containing matrix that they secrete (keratinocytes – skin cells)

1. Isolate keratinocytes from mice genetically engineered to lack genes for this type of laminin

2. Their migratory ability is vastly diminished

C. Also binds tightly to other laminins, proteoglycans, basal lamina components, cell surface receptors

1. Basal lamina type IV collagens & laminin are thought to form separate, but interconnected, networks

2. These interwoven networks give basal lamina both strength & flexibility

D. In fact, basement membranes fail to form in mouse embryos that are unable to synthesize laminin, causing the death of the embryo around the time of implantation

E. ECM can also exhibit dynamic properties, both in space & over time

1. ECM fibrils stretch several times their normal length as they are pulled on by cells; they contract when tension is relieved

2. Temporally, ECM components are subject to continual degradation & reconstruction; renews the matrix & allows it to be remodeled during embryonic development or after tissue injury

3. Even the calcified matrix of bone is subject to continual restoration

F. ECM degradation, along with that of cell surface proteins, is accomplished largely by a zinc-containing enzyme family (matrix metalloproteinases [MMPs])

1. MMPs are either secreted into extracellular space or anchored to plasma membrane

2. As a group, MMPs can digest nearly all of the diverse ECM components, but individual family members are limited as to the types of extracellular proteins they can attack

3. Physiological roles of MMPs are not well understood, but they are thought to be involved in tissue remodeling, embryonic cell migration, wound healing & blood vessel formation

3. Excessive or inappropriate activity of MMPs is likely to cause disease; implicated in a number of pathological conditions (arthritis, hepatitis, atherosclerosis, tooth/gum disease, tumor progression)

IV. Other ECM molecules

A. Tenascin - large, oligomeric glycoprotein; on many cell types & a variety of cancer cell surfaces depending on cell type, can promote or discourage cell adhesion

B. Entactin - component of basement membranes; thought to play role in adhesion & penetration of early mammalian embryo into uterine lining

C. Thrombospondin - secreted into ECM by many cells; prominent in matrix surrounding lining of mature blood vessels; inhibits formation of new blood vessels (angiogenesis)

V. Interactions among ECM materials can be very complex

A. Some may contain binding sites with opposite activities (one may promote & another inhibit cell adhesion); thus effects on cell behavior may change from time to time

B. Spatial arrangement of various components may ultimately determine their effect on cell behavior

C. ECM components like fibronectin, laminin, proteoglycans & collagen are capable of binding to receptors situated on the cell surface

Interactions of Cells with Noncellular Substrates: Integrins

I. Integrins - integral membrane protein family; composed of 2 membrane spanning chains ( & ; linked noncovalently); thought to be on surface of virtually all vertebrate cell types (found only in animals)

A. Most important family of receptors that attach cells to their extracellular microenvironment; they bind to specific substances (ligands) in the extracellular environment

B. 18 different  & 8 different  subunits identified on cell surfaces; thus many heterodimers (only ~24 integrins identified on cell surfaces), each with specific distribution within body

1. >100 possible  &  pairings are possible theoretically

2. Most cells have variety of different integrins; most integrins present on a variety of different cell types

C. EM pictures (late 1980s) suggest that the 2 subunits are oriented to form a globular extracellular head connected to the membrane by a pair of elongated "legs"

1. The legs of each subunit extend through lipid bilayer as a single transmembrane helix & end in a small cytoplasmic domain of ~20 – 70 amino acids

2. An exception to this is ₄ chain, which has extra 1000 or so amino acids as part of its cytoplasmic domain; this huge addition makes ₄ integrins able to extend much more deeply into cytoplasm

D. Each integrin can bind a number of divalent cations like Ca²⁺, Mg²⁺ & Mn²⁺, although the effect of each ion on the structure & ligand-binding capabilities of the protein remains unclear

E. 2001 – the first X-ray crystallographic structure of an integrin extracellular portion displayed a highly unexpected feature

1. Rather than "standing upright", the integrin _v₃ was bent dramatically at the "knees" so that the head faces the plasma membrane outer surface

II. Many integrins can exist on surface of cell in an inactive conformation - they can be activated rapidly by events within the cell that alter the conformation of cytoplasmic domains of the integrin's subunits

A. These changes are propagated through the molecule, increasing the integrin's affinity for an extracellular ligand

B. Example – platelet aggregation during blood clotting occurs only after cytoplasmic activation of _IIb₃ integrins, which increases their affinity for fibrinogen

1. This type of alteration in integrin affinity is triggered by changes occurring inside the cell & is called "inside-out" signaling

2. Without the inside-out signal, the integrin remains inactive, protecting the body against the formation of an inappropriate blood clot

C. Evidence suggests strongly that the bent conformation of an integrin corresponds to the inactive state that cannot bind a ligand

1. An _v₃ integrin containing a bound ligand when analyzed by X-ray diffraction, no longer exhibits the bent structure, but is present instead in the upright conformation

2. The ligand is bound to the integrin's head in a region where the  &  subunits come together

3. If these conformations respectively represent the active & inactive forms, what triggers the transformation?

D. Studies suggest that ligand-binding ability of integrin head projecting from plasma membrane outer surface depends upon spatial arrangement of  &  cytoplasmic tails present on membrane inner side

1. Cytoplasmic domains of integrins bind a wide array of proteins; one, called talin, causes separation of the  &  subunits

2. Mutations in talin that block its interaction with -integrin subunits also prevent activation of integrins & adhesion to the ECM

3. Separation of the cytoplasmic ends of integrins by talin is thought to send a change in conformation through the integrin legs

4. This causes the molecule to assume an upright position in which the head of the protein is capable of interacting specifically with its appropriate ligand

a. Several structural studies indicate that a modified conformation of a bent integrin can bind certain types of ligands

b. This modified form of the bent conformation might represent an intermediate stage of integrin activation

5. The increased affinity of individual intergins is often followed by clustering of activated integrins; this greatly enhances overall strength of interaction between cell surface & its extracellular ligands

E. Integrins are implicated in 2 major types of activities: adhesion of cells to their substratum (or to other cells) & transmission of signals from external environment to cell interior (outside-in signaling)

1. Binding of integrin extracellular domain to a ligand (like fibronectin or collagen) can induce a conformational change at the opposite, cytoplasmic end of the integrin

2. Changes at the cytoplasmic end can, in turn, alter the way the integrin interacts with nearby cytoplasmic proteins, modifying their activity

3. Thus, as integrins bind to an extracellular ligand, they can trigger the activation of cytoplasmic protein kinases, like FAK (focal adhesion kinase) & Src

4. These kinases can then phosphorylate other proteins initiating a chain reaction; in some cases, the chain reaction leads al the way to the nucleus, where a specific group of genes may be activated

III. Outside-in signals transmitted by integrins & other cell-surface molecules can influence many aspects of cell behavior, including differentiation, motility, growth & even cell survival

A. Influence of integrins on cell survival is best illustrated by comparing normal & malignant cells

1. Most malignant cells can grow while suspended in liquid culture medium; normal cells, in contrast, only grow & divide if cultured on a solid substratum; they die in suspension cultures

2. Normal cells may die in suspension culture since their integrins cannot interact with extracellular substrates &, as a result, cannot transmit life-saving signals to the cell interior

3. On the other hand, malignant cell survival no longer depends on integrin binding

B. Linkage between integrins & their ligands mediates adhesions between cells & their environment

1. Individual cells may express a variety of different integrins on their cell surface; thus, such cells can bind to a variety of different extracellular components

2. Despite the apparent overlap, most integrins do appear to have unique functions – knockout mice that lack different integrin subunits exhibit distinct phenotypes

3. For example, ₈ knockouts show kidney defects; ₄ knockouts exhibit heart defects; ₅ knockouts display vascular defects

C. Most extracellular proteins that bind integrins have tripeptide sequence arginine-glycine-aspartic acid (RGD)

1. RGD is found in cell-binding sites of proteoglycans, fibronectin, collagen, laminin, other ECM proteins

2. The cell-binding domain of fibronectin has an extended RGD-containing loop

IV. Applications & examples of integrin involvements – discovery of RGD sequences' importance has opened the door to new treatments of medical conditions involving receptor-ligand interactions

A. When the wall of a blood vessel is injured, the damaged region is sealed by the controlled aggregation of blood platelets, which are non-nucleated cells that circulate in the blood

1. When it occurs at an inappropriate time or place, the aggregation of platelets can form a potentially dangerous blood clot (thrombus) that can block the flow of blood to major organs

2. This is one of the leading causes of heart attack & stroke

3. Platelet aggregation requires interaction of platelet-specific integrin (_Iib₃) with soluble RGD-containing blood proteins (fibrinogen, von Willebrand factor), which link platelets together

4. RGD-containing peptides can inhibit blood clot formation (shown in animal experiments); stop platelet aggregation by preventing platelet integrin from binding to blood proteins

5. Led to design of new class of antithrombotic agents (like Aggrastat & Integrelin) that resemble RGD structure but bind selectively to platelet integrin

6. A specific antibody (ReoPro) directed against the RGD binding site of _IIB₃ integrins can also prevent blood clots in certain patients undergoing high-risk vascular surgeries

7. Other compounds that target integrins involved in other diseases are in clinical trials

B. Integrin cytoplasmic domains contain binding sites for a variety of cytoplasmic proteins, including several that act as adaptors to link the integrin to actin filaments of the cytoskeleton

1. The role of integrins in connecting the ECM & the cytoskeleton is best seen in 2 specialized structures: focal adhesions & hemidesmosomes

Interactions of Cells with Noncellular Substrates: Focal Adhesions & Hemidesmosomes

I. Focal contacts anchor cells to their substratum – it is much easier to study cell adhesion to a surface in vitro (in a culture dish) than with an extracellular matrix inside an animal

A. Much of our knowledge of cell-matrix interactions is derived from studies of cells adhering to various substrates in vitro

B. Steps in cell adhesion to culture dish

1. Cell initially has rounded morphology like most animal cells suspended in aqueous medium

2. As cell contacts substratum, it sends out projections that make increasingly stable attachments

3. Over time, cell flattens & spreads itself out on substratum with rearrangement of cytoskeleton (may be mediated by transmembrane signaling)

4. Bottom surface of fibroblasts or epithelial cells is not pressed uniformly against substratum, but is anchored to surface (as close as 10 nm) at scattered, discrete sites (focal contacts or focal adhesions)

C. Focal adhesions – dynamic structures; can be rapidly disassembled if the adherent cell is stimulated to move or enter mitosis

1. The plasma membrane in region of focal adhesion contains large clusters of integrins (often _V₃)

2. Integrin cytoplasmic domains are connected by various adaptors to actin filaments of cytoskeleton

3. Binding of an extracellular ligand (fibronectin, laminin) can activate protein kinases (like FAK or Src) that can transmit signals throughout the cell, including cell nucleus

D. Focal adhesions have been implicated in cell adhesion and/or cell locomotion although the precise roles of these structures have been debated

1. Focal adhesions are capable of creating or responding to mechanical forces, which might be expected from a structure that contains actin & myosin, two of the cell's major contractile proteins

E. Attach cultured cell to gelated surface that can be deformed by local forces (original surface has uniform grid pattern)

1. Grid can be distorted by traction (gripping/pulling) forces generated by focal adhesions at cell undersurface

F. Conversely, mechanical forces applied to cell surfaces can be converted by focal adhesions into cytoplasmic signals

1. For example, cells were allowed to bind to beads that had been covered with a fibronectin coating

2. When membrane-bound beads were pulled by an optical tweezer, the mechanical stimulus was transmitted into the cell interior where it generated a wave of Src kinase activation

3, Within the body, activation of protein kinases can dramatically alter cell behavior, including transformation of cells to a cancerous state

III. Focal adhesions are most commonly seen in cells grown in vitro, but similar types of adhesive contacts are found in certain tissues, like muscle & tendon

A. In body, the tightest attachment between a cell & ECM is at epithelial cell basal surface where they are anchored to underlying basement membrane by specialized adhesive structure (hemidesmosome)

B. They contain dense plaque on membrane inner surface with filaments coursing outward into cytoplasm

1. Filaments are thicker than actin of focal adhesions & made of keratin (intermediate filaments)

2. Primarily supportive rather than contractile; keratin-containing filaments of hemidesmosome are linked to ECM by membrane-spanning integrins (₆₄)

3. These integrins, like focal adhesion integrins, also transmit signals from the ECM that affect the shape & activities of attached epithelial cells

C. Bullous pemphigoid – rare autoimmune disease (people make antibodies against hemidesmosome plaque proteins, bullous pemphigoid antigens); demonstrates importance of hemidesmosomes

1. Autoimmune disorders are caused by production of antibodies (autoantibodies) directed against one's own tissues; responsible for a wide variety of conditions

2. Presence of autoantibodies causes lower epidermal layer to lose attachment to underlying basement membrane & thus to underlying connective tissue layer of dermis

3. Causes severe blistering of skin when fluid leaks into space under epidermis

D. Epidermolysis bullosa, a similar inherited blistering disease - seen in patients with genetic alterations in any one of a number of hemidesmosomal proteins (₆ or ₄ integrin subunit, collagen VII, laminin)

Interactions of Cells with Other Cells – Experimental Approaches

I. Little is known about the mechanisms that generate complex 3-D cell arrangements in developing organs – it is presumed to depend heavily on selective interactions between cells of same & different types

A. Evidence indicates that cells can recognize the surfaces of other cells, interacting with some & ignoring others

II. Experiments demonstrating cell adhesion – difficult to study cell interactions occurring in small organs of a developing embryo

A. Early experiments involved removal of developing organ from chick or amphibian embryo, dissociating its tissues to single cells & observing their ability to reaggregate in culture

B. If one disaggregates cells from 2 different developing organs & mixes them —> they form mixed clump then sort themselves out; each cell adheres long term only to cells of the same type

1. Once separated into a homogeneous cluster, these cells often differentiate into many of the structures they would have formed within an intact embryo

2. Add labeled cells to dishes with monolayers of varied cell types —> cells stick to own type better

C. Little was known about nature of cell-cell adhesion molecules until techniques developed for purifying integral membrane proteins & recently for the isolation/cloning of genes encoding these proteins

1. Dozens of proteins involved in cell adhesion have now been identified

2. Different arrays of these proteins on different cells are responsible for specific interactions between cells within complex tissues

III. Four distinct families of integral membrane proteins play a major role in mediating cell-cell adhesion

A. Selectins

B. Certain members of immunoglobulin superfamily (IgSF)

C. Certain members of the integrin family

D. Cadherins

Interactions of Cells with Other Cells – Families of Integral Membrane Proteins

I. Selectins – comprise an integral membrane glycoprotein family; binds to specific sugar arrangement in oligosaccharides that project from other cells' surfaces

A. The name of this class of cell-surface receptors comes from lectin, a term for a compound that binds to specific carbohydrate groups

B. During 1960s - remove lymphocytes from peripheral lymph nodes, label them radioactively & reinject —> go back to site of origin from which they were derived (lymphocyte homing)

1. Homing also studied in vitro by allowing lymphocytes to adhere to frozen tissue sections of lymphoid organ —> selectively adhere to venule endothelial lining of peripheral lymph nodes

2. Binding could be blocked by antibodies against specific glycoprotein on lymphocyte surface (glycoprotein was called LEU-CAM1 & later L-selectin)

C. Selectins possess a small cytoplasmic domain, a single membrane-spanning domain, & a large extracellular segment consisting of a number of separate modules (outermost domain acts as the lectin)

D. 3 known types - E-selectin (endothelial cells); P-selectin (platelets, endothelial cells); L-selectin (present on all types of leukocytes or white blood cells)

1. All 3 recognize a particular grouping of sugars found on ends of carbohydrate chains of certain complex glycoproteins; binding of selectins to their carbohydrate ligands requires calcium

2. As a group, selectins mediate transient interactions between circulating leukocytes & vessel walls at inflammation & clotting sites

II. Immunoglobulins (Igs) & Integrins - blood-borne antibody (immunoglobulin) structure was elucidated in the 1960s; this was a milestone in the understanding of the immune response

A. Igs are a large family of proteins; they consist of polypeptide chains composed of a number of similar domains; most are present on lymphocyte surfaces as integral proteins

1. Each Ig domain is composed of 70 – 110 amino acids organized into tightly folded structure

2. Ig-type domains were seen in wide variety of proteins, which together constitute the immunoglobulin superfamily (IgSF)

B. Most IgSF members are involved in various aspects of immune function, but some of them mediate calcium-independent cell-cell adhesion

1. Ig-like domains were discovered in cell-adhesion receptors in invertebrates, animals that lack a classic immune system

2. This suggests that Ig-like proteins originally evolved as cell-adhesion mediators & only secondarily took on their functions as effectors of the vertebrate immune system

C. Most IgSFs mediate specific interactions of lymphocytes with cells needed for immune response (other lymphocytes, macrophages, target cells) but some mediate adhesion between nonimmune cells

1. VCAM (vascular cell-adhesion molecules)

2. NCAMs (neural cell-adhesion molecules)

3. L1 – NCAMs & L1 play roles in nerve outgrowth, synapse formation & other events during nervous system development

D. Like fibronectin & many other cell-adhesion proteins, IgSF cell-adhesion molecules exhibit modular construction; composed of individual domains similar in structure to domains in other proteins

E. L1 importance in neural development has been revealed in several ways - human L1 gene mutations can have devastating consequences

1. In extreme cases, babies are born with a fatal condition of hydrocephalus (water on the brain)

2. Children with less severe forms of the mutation typically exhibit mental retardation & difficulty in controlling limb movements (spasticity)

3. Autopsies of those who died of L1-deficiency disease are often missing 2 large nerve tracts: one that runs between the 2 halves of brain & the other running between the brain & spinal cord

4. Nerve tract absence suggests L1 is involved in axon growth within embryonic nervous system

F. Various types of proteins serve as ligands for IgSF cell-surface molecules - may bind to same or different IgSFs on other cells or a few integrins

1. Most integrins facilitate adhesion of cells to their substratum, but a few integrins mediate cell-cell interactions by binding to proteins on other cells

2. Leukocyte surface integrin (₄₁) binds VCAM (an IgSF protein on the endothelial lining of certain blood vessels)

III. Cadherins – large family of glycoproteins mediating Ca²⁺-dependent cell-cell adhesion; also transmit signals from ECM to cytoplasm; found in many different cell types with specific body distribution

A. Typically join cells of similar type to one another mostly by binding to the same cadherin present on neighboring cell surface; this was demonstrated by the following experiment:

1. Genetically engineer nonadhesive cells to express one of a variety of cadherins —> then mix them in various combinations & their interactions were monitored

2. Cells expressing one species of cadherin preferentially adhered to other cells expressing the same cadherin (may mold cohesive tissues in embryo & hold them together in adult)

B. Like selectins & IgSF molecules, cadherins have a modular construction

1. The best-studied cadherins are E-cadherin (epithelial), N-cadherin (neural), P-cadherin (placental)

2. Cadherins appear to be important in holding cells together in tightly cohesive tissues

C. These "classical" cadherins have a relatively large extracellular segment consisting of 5 tandem domains of similar size & structure, 1 transmembrane segment & a small cytoplasmic domain

1. Cytoplasmic domain is often associated with members of catenin family of cytosolic proteins

2. Catenins (like integrins) have 2 roles: tie cadherins to cytoskeleton & transmit signals to cytoplasm

D. X-ray crystallography has been carried out on the extracellular portions of cadherins

1. Cadherins from the same cell surface associate laterally to form parallel dimers; studies also shed light on role of calcium, which for decades has been known to be essential for cell-cell adhesion

2. Ca²⁺ ions form bridges between successive domains of a given molecule, not between molecules from different cells, as had long been presumed

3. Ca²⁺ ions apparently maintain rigid conformation of extracellular portion of each cadherin needed for cell adhesion

E. Cell-cell adhesion results from interaction between extracellular domains of cadherins from opposing cells to form a "cell adhesion zipper", which, if extensive, would hold cells together with great strength

1. Controversy has arisen over degree to which cadherins from opposing cells overlap with one another

2. Different cell types bearing different cadherins likely engage in different types of interactions

3. Thus, more than one (or all)configuration may occur within an organism

4. Cadherin interdigitation can be compared to a zipper; cadherin clusters can be compared to Velcro.

5. The greater the number of interacting cadherins in a cluster, the greater the strength of adhesion between apposing cells

F. Cadherin-mediated adhesion may be responsible for ability of like cells to sort out of mixed aggregates

1. Cadherins may be the single most important factor in molding cells into cohesive embryonic tissues & holding them together in the adult

2. The loss of cadherin function may play a key role in the spread of malignant tumors

G. Embryonic development is characterized by changes in gene expression, cell shape, cell motility, cell adhesion, etc.

1. Cadherins thought to mediate many of the dynamic changes in adhesive contacts that are required to construct the tissues & organs of an embryo, which is known as the process of morphogenesis

2. Many embryo development morphogenetic events involve group of cells changing from epithelium (tightly adherent, organized cell layer) to mesenchyme (loose, mostly nonadhesive cells) or vice versa

H. Mesodermal cell movement in gastrulation exemplify mesenchymal-epithelial transition

1. Usually, cells leave cohesive epithelial layer at early gastrula surface & wander into interior regions as mesenchymal cells

2. Later, some of them get adhesive again & form epithelium (somites along embryo dorsal midline)

3. Even later, some somite cells lose adhesiveness & wander again as mesenchyme into developing limb (become cartilage or muscle) or beneath developing epidermis (become dermal tissues)

I. Cadherins & other cell-adhesion molecules play a key role in such activities by changing cell adhesive properties

1. Aggregation of cells into an epithelium (as in somite) correlates with N-cadherin appearance on cell surfaces; this event would be expected to promote cell-cell adhesion

2. In contrast, cell dispersion from epithelium is correlated with disappearance of N-cadherin from cell surface

3. Cadherins are typically distributed diffusely over cell surfaces of 2 adherent cells but they also participate in formation of specialized intercellular junctions

Anchoring Cells to Other Cells: Adherens Junctions and Desmosomes

I. Cells of certain tissues (epithelia, cardiac muscle) are notoriously difficult to separate from one another, because they are held together tightly by specialized Ca²⁺-dependent adhesive junctions

A. Two main types of adhesive junctions: adherens junctions & desmosomes

B. Other types of epithelial cell junctions (gap junctions & tight junctions) are also located along lateral cell surfaces near apical lumen

C. When these junctions are in specific array, the assortment of surface specializations called junctional complex

II. Adherens junctions – found in a variety of body sites; particularly common in epithelia (like the lining of the intestine)

A. Occur as belt encircling each cell at apical end; binds cell to its surrounding neighbors; may transmit signals between them, too; called zonulae adherens

1. Cells of adherens junction are tightly held together by Ca²⁺-dependent linkages formed between extracellular cadherin domains bridging 30 nm gap between neighboring cells

2. Cytoplasmic cadherin domains in junctions are linked by - & -catenins to a variety of cytoplasmic proteins, like actin filaments of cytoskeleton

B. Thus, junction cadherins, like focal adhesion integrins, connect the external environment to actin cytoskeleton & provide a pathway for signal transmission from cell exterior to the cytoplasm

1. Adherens junctions between endothelial cells lining blood vessel walls transmit signals that ensure cell survival

2. Mice lacking an endothelial cell cadherin are unable to transmit these survival signals —> these animals die during embryonic development as result of the death of cells lining vessel walls

III. Desmosomes (maculae adherens) - disk-shaped adhesive junctions (~1 µm in diameter) found in a variety of tissues (most notably in epithelia where they are basal to the zonulae adherens)

A. Particularly numerous in tissues subjected to mechanical stress (skin epithelial layers, cardiac muscle, gingiva [gums], uterine cervix epithelial layers)

B. Like adherens junctions, they contain cadherins that link the 2 cells across a narrow (30 nm) extracellular gap

1. They have a different domain structure from that of adherens junction classical cadherins; they are called desmogleins & desmocollins

C. Dense cytoplasmic plaques on inner membrane surfaces serve as sites of anchorage for looping intermediate filaments (like those in hemidesmosomes)

1. These filaments extend into cytoplasm & connect with other desmosomes

2. The 3D network of ropelike intermediate filaments gives structural continuity & tensile strength to the entire cell sheet

3. Intermediate filaments also link to desmosomal cadherin cytoplasmic domains via additional proteins

D. Region between cells (desmoglea) filled with lightly staining material, which may act as glue

E. Autoimmune disease pemphigus vulgaris illustrates importance of cadherins in maintaining epithelium structural integrity

1. Antibodies made against a desmoglein causing loss of epidermal cell-cell adhesion & severe blistering of skin

The Role of Cell Adhesion Receptors in Transmembrane Signaling

I. All 4 types of cell-adhesion molecules have the potential to transfer information across plasma membrane

A. Integrins & cadherins can transmit signals from extracellular environment to cytoplasm via links with cytoskeleton & with cytosolic regulatory molecules, like protein kinases & G proteins

1. This is an example of transmembrane signaling

2. Protein kinases activate (or inhibit) their target proteins through phosphorylation, whereas G proteins activate (or inhibit) their protein targets through physical interaction

B. Engagement of an integrin with its ligand can induce a variety of cell responses via transmembrane signaling

1. Changes in cytoplasmic pH

2. Changes in Ca²⁺ ion concentration

3. Changes in protein phosphorylation

4. Changes in gene expression

C. Such changes can alter subsequent cell behavior - growth potential, migratory activity, differentiation state, survival; an example is mammary gland epithelial cells

II. Remove cells from mammary gland & grow them on bare culture dish, in absence of extracellular glycoproteins (laminin & fibronectin)

A. They appear as flattened, undifferentiated cells & lose their ability to produce milk proteins

B. Add laminin or other extracellular molecules to culture —> it binds cell surface integrins —> cells regain their differentiated appearance

1. They also become organized into milk-producing, glandlike structures (milk protein production rises 50 X)

C. Laminin may work by binding cell surface integrins & activating kinases at inner membrane surface

Tight Junctions: Sealing the Extracellular Space

I. A simple epithelium (lining of intestine or lungs) is comprised of a layer of cells that adhere tightly to one another to form a thin cellular sheet

A. Certain types of epithelia (frog skin, urinary bladder wall) can be mounted between 2 compartments containing different solute concentrations

B. When this is done, the membranes allow very little diffusion of ions or solutes across the wall of the epithelium from one compartment to the other

C. Plasma membranes are impermeable so solutes cannot diffuse freely through cells of the epithelial layer but why can't solutes pass between cells by a paracellular pathway?

II. Tight junctions (TJs; zonula occludens) - discovered in 1960s; found between adjacent epithelial cells at the very apical end of the junctional complex between those cells (frog skin, urinary bladder wall)

A. In tight junctions, the membranes make contact at intermittent points; not fused over large surface area

B. The points of cell-cell contact are sites where integral proteins of the 2 adjacent membranes meet within the extracellular space

1. Freeze fracture (allows observation of internal membrane faces) shows that plasma membranes of a TJ contain interconnected strands (or grooves in opposite face of fractured membrane)

2. The interconnected strands run mostly parallel to one another & to the epithelium's apical surface; they wrap around the cell like a belt around waist

3. These strands or grooves in the opposite face of the fractured membrane correspond to paired rows of aligned integral membrane proteins

4. The integral TJ proteins form continuous fibrils that completely encircle the cell like a gasket & contact neighboring cells on all sides

C. Thus, TJs serve as a barrier (a seal) to free diffusion of water & solutes from extracellular compartment on one side of epithelial sheet to that on the other side

1. Place piece of tissue in lanthanum (electron-dense heavy metal) & take EM pictures —> lanthanum penetrates between cells as far as upper & lower edge of tight junctions

D. TJs also serve as fences that help maintain the polarized character of epithelial cells; they block diffusion of integral proteins between the apical domain of membrane & its lateral & basal domains

1. Like other cell adhesion sites, TJs are also involved in signaling pathways that regulate numerous cellular processes

III. TJs are occluding junctions & form a continuous permeability barrier, but not all of them exhibit the same permeability properties

A. TJs with several parallel strands form better seals than those with one or a couple of strands

1. Leakiest junctions (proximal renal tubule) - a single strand & little resistance

2. Tightest junctions (bladder wall, brain capillaries) - a number of parallel, interconnected strands; high resistance

B. Some TJs are permeable to specific ions or solutes to which other TJs are impermeable

1. All cells of human kidney tubule are connected to neighbors by TJs

2. Only one small region of tubule has TJs permeable to Mg²⁺ ions (thick ascending limb; TAL)

3. TAL is site along tubule where Mg²⁺ ions are reabsorbed from tubular fluid back into blood

C. Until 1998, TJ strands were thought to be composed of single protein, occludin

1. But it was found that cultured cells that lacked occludin gene (& could not make the protein), but were still able to form TJ strands of normal structure & function

2. S. Tsukita et al., Univ. of Kyoto – discovered family of proteins (claudins) that form major structural TJ strand component; occludin & claudin are found together in linear TJ fibers

3. At least 24 different claudins have been identified; differences in their distribution may explain selective differences in TJ permeability

D. Example of permeability differences is seen in one small region of a human kidney tubule, the thick ascending limb (TAL); the TAL has TJs that are permeable to magnesium (Mg²⁺) ions

1. It is thought claudin-containing TAL strands have pores that are selectively permeable to Mg²⁺ ions

2. Support for this idea comes from the finding that one specific member of the claudin family (claudin-16) is expressed primarily in TAL

3. 1999 - patients were found to have rare disease characterized by abnormally low Mg²⁺ levels in their blood; they had mutations in both copies of their claudin-16 gene

4. Their blood levels of Mg²⁺ were low since TJs with the abnormal claudin were impermeable to Mg²⁺, thus Mg²⁺ fails to be reabsorbed from the tubule & is simply excreted in the urine

IV. Another important TJ function came to light in 2002

A. For decades, mammalian skin impermeability to water was solely thought to be a property of the outer, cornified layer of the skin, which contains tightly packed protein filaments & associated lipids

B. Tsukita et al. (2002) – it was discovered, however, that mice lacking a gene for claudin-1 died shortly after birth as a result of dehydration

1. Further study revealed that cells in one of the outer layers of normal epidermis are connected to one another by TJs

2. Animals lacking the gene for claudin-1 were unable to assemble watertight epidermal TJs & thus suffered from uncontrolled water loss

V. TJs also present between endothelial cells lining capillary walls; they are particularly evident in brain

A. They help form blood-brain barrier (prevents substances from passing from bloodstream into brain)

B. Although small ions & even water molecules may not be able to penetrate the blood-brain barrier, immune system cells are able to pass across endothelium through these junctions

1. These cells are thought to send a signal that opens the junction, allowing the cells to pass

C. While protecting the brain from unwanted solutes, the blood-brain barrier also prevents access of many drugs to the central nervous system (CNS)

1. Major pharmaceutical industry goal is to develop drugs that open brain TJs allowing passage of therapeutic compounds

Mediating Intercellular Communication: Gap Junctions

I. Gap junctions - plasma membranes of adjacent cells come very close together (~3 nm) but they make no direct contact; they are sites between animal cells that are specialized for intercellular communication

A. Very fine strands span gap between cells, form molecular pipeline connecting adjacent cell cytoplasms

B. Link cells of most mammalian tissues, except skeletal muscle & most nerve tissue

II. Gap junctions have simple molecular composition made entirely of an integral membrane protein connexin (multigene family; ~12 reported)

A. Connexins clustered in membrane to form multisubunit complex (connexon); totally spans membrane

1. Each connexon is composed of 6 connexin subunits arranged around a central opening (annulus; ~1.5 nm in diameter at its extracellular surface)

2. Connexons assembled between connexin production in RER & arrival at membrane

3. Some cells make >1 connexin; don't know if single connexon contains >1 connexin

B. During gap junction formation, connexons in membranes of apposing cells become tightly linked to one another through extensive interactions of connexin subunit extracellular domains

1. Once aligned, connexons in apposing membranes form complete intercellular channels connecting neighboring cell cytoplasms

2. The channels become clustered in specific membrane regions; form gap-junction plaques that can be visualized when the membrane is split down the middle by freeze-fracture

III. Gap junctions mediate gap junction intercellular communication (GJIC) – communication sites between adjacent cell cytoplasms

A. GJIC is revealed through passage of ionic currents or low MW dyes (fluorescein) from one cell to its neighbors

1. Mammalian gap junctions allow diffusion of molecules with molecular mass < ~1000 daltons like ions & dyes like fluorescein

2. Unlike highly selective ion channels connecting a cell to the external medium, gap junction channels are relatively nonselective

B. Gap junctions are also thought to be gated; channel closure is probably triggered primarily by phosphorylation of connexin subunits by protein kinase

1. Closure can also be triggered by abnormally high Ca²⁺ concentrations

2. Several treatments can cause closure of gap junctions, including abnormally elevated intracellular [Ca²⁺]; inject Ca²⁺ ions —> gap junctions close

IV. While skeletal muscle cells are stimulated by chemicals released from the tips of nearby nerve cells, cardiac or smooth muscle stimulation occurs by a very different process, one involving gap junctions

A. Contraction of mammalian heart is stimulated by an electrical impulse generated in small region of specialized heart muscle (sinoatrial node); acts as the heart's pacemaker

1. Impulse spreads rapidly as ion current flows from 1 cardiac cell to its neighbors via gap junctions, causing the cells to contract in synchrony

2. Intestinal/esophageal wall smooth muscle coordinated peristaltic waves that move down the length of wall are due to current (ion) flow through gap junctions interconnecting smooth muscle cells

B. Gap junctions also occur between the presynaptic & postsynaptic membranes of adjacent nerve cells in certain parts of brain

1. They allow nerve impulses to be transmitted directly from one neuron to another without requiring the release of chemical neurotransmitters

V. Gap junctions can put many cells of tissue into intimate cytoplasmic contact; has important physiological consequences; with respect to molecules small enough to pass, connected cells form 1 giant compartment

A. Highly active regulatory molecules (cAMP, inositol phosphates) pass through gap-junction channels

1. Thus, individual connected cells' activities can be integrated so that they act as a functional unit

B. Connected cells respond together even if only a small portion of the cells are stimulated by hormone

1. If only a few cells near a particular blood vessel happen to be stimulated by a hormone, the stimulus can be rapidly transmitted to all cells of the tissue

C. Gap junctions also allow cells to cooperate metabolically by sharing key metabolites (ATP, sugar phosphates, amino acids, many coenzymes), small enough to pass through these intercellular channels

1. This is particularly important in tissues, like the lens, which are avascular, (i.e., lack blood vessels)

VI. Connexins (Cx), the proteins of which gap junctions are made, are members of a multigene family

A. ~20 different connexins with distinct tissue-specific distributions have been identified

1. Connexons made of different connexins —> marked variations in conductance, permeability, regulation

2. Sometimes, connexons in neighboring cells made of different connexins can dock & form functional channels, other times they cannot

B. These compatibility differences may play important roles in either promoting or preventing communication between different types of cells in an organ

1. Connexons joining cardiac muscle cells are made of connexin Cx43; those joining cells of heart’s electrical conduction system made of Cx40 - they are incompatible, can’t form working channels

2. These cells are electrically insulated from each other, even though they are in physical contact

C. Some inherited disorders associated with mutations in connexin-encoding genes; consequences of the disorders include deafness, blindness, cardiac arrhythmias, skin abnormalities or nerve degeneration

D. Recently, a new type of communication system was discovered consisting of thin, highly elongated tubules capable of conducting cell-surface proteins & cytoplasmic vesicles from one cell to another

1. To date, these tunneling nanotubes have only been observed between cells growing in culture

2. It remains to be seen whether they have a significant physiological role within the body

Mediating Intercellular Communication: Plasmodesmata

I. What are plasmodesmata? - a little like gap junctions of animal cells; cylindrical cytoplasmic channels (30 - 60 nm diameter) that pass through the cell walls of adjacent cells; plasmodesma is singular

A. Unlike animals, whose cells are in intimate contact with each other, plant cells are separated by cell wall

B. Plants lack the specialized junctions of animal tissues; but most plant cells connected by plasmodesmata; thus, plants lack the cell adhesion molecules typical of animals

II. They are lined by plasma membrane & usually contain a dense central structure (desmotubule)

A. Serve as sites of intercellular communication, allowing plant tissue to function as a metabolic unit

B. Desmotubule is derived from smooth endoplasmic reticulum of the two cells - passage usually limited to space between desmotubule & inner membrane surface

1. Like gap junctions between animal cells, plasmodesmata serve as sites of cell – to – cell communication, as substances pass through the annulus surrounding the desmotubule

C. Until recently, it was thought that they were impermeable to molecules > ~1 kDa

1. Based on studies in which different-sized fluorescent dyes were injected into cells

2. More recent studies suggest that some plasmodesmata allow much larger molecules (up to 50 kDa) to pass between cells

D. Unlike gap junctions, which have fixed openings, the plasmodesmatal pore is capable of dilation so larger molecules (RNA, protein) can pass

1. Insight into this dynamic property was gained from studies on plant viruses in the 1980s

2. These viruses spread from one cell to another through plasmodesmata

3. Viral infections increase plasmodesmata permeability so virus particles/nucleic acids can pass

4. Virus encodes a movement protein that interacts with plasmodesma wall, increasing pore diameter

E. It was shown later that plant cells make their own movement proteins that regulate the flow of proteins & RNAs from cell to cell

1. Some of these macromolecules find their way into the plant vascular system, where they integrate plant-wide activities (growth of new leaves & flowers or defense against pathogens

2. Example: one protein moves from one type of plant tissue (the stele), where it was synthesized, to an adjoining tissue (the endodermis)

a. The protein is seen concentrated in the spherical nuclei of the endodermal cells where it acts to stimulate gene expression

Cell Walls

I. Naked cells covered by a <10 nm lipid-protein membrane are extremely fragile structures (offers minimal protection for cells; cells of nearly all organisms but animals are enclosed in a protective outer envelope

A. Protozoa have thickened outer coat

B. Bacteria, fungi & plants have distinct cell walls (plant cell walls were first cell structures observed with light microscope)

II. Vital functions of plant cell walls:

A. Plant cells develop osmotic turgor pressure that pushes against their surrounding wall —> the wall gives the enclosed cell its characteristic polyhedral shape

B. In addition to providing support for individual cells, cell walls serve collectively as a type of "skeleton" for the entire plant

1. A tree without cell walls might resemble, in many respects, a human without bones

C. Cell walls also protect the cell against damage from mechanical abrasion & pathogens

D. They also mediate cell-cell interactions

E. Like the ECM at the animal cell surface, a plant cell wall can also be a source of signals that alter the activities of the cells that it contacts

F. Primary barrier to large molecular substance penetration, while ions/small molecules pass freely

III. Cell wall structure – often likened to fabricated materials like reinforced concrete or fiberglass; contain fibrous element embedded in nonfibrous, gel-like matrix

A. Cellulose is fibrous component of cell wall; organized into microfibrils that confer rigidity on cell wall & provide resistance to tensile or pulling forces

1. Microfibrils - ~5 nm in diameter; typically composed of bundles of 36 cellulose molecules oriented parallel to one another & held together by H bonds

2. Walls of many plant cells are made of layers in which the microfibrils of one layer are oriented at ~90° to those of adjacent layers (similar to collagen fiber layers in corneal stroma)

B. Proteins & pectin provide the matrix

IV. Cellulose molecules are polymerized at cell surface - glucose subunits are added to end of growing cellulose molecule by multisubunit enzyme complex embedded in membrane (cellulose synthase)

A. Subunits of enzyme are organized into 6-membered ring (rosette), which is embedded in membrane

1. Cortical microtubules just under membrane set microfibril orientation at outer membrane surface

B. In contrast, materials of the matrix are synthesized within the cytoplasm & carried to the cell surface in secretory vesicles

1. The matrix is highly complex, requiring hundreds of enzymes for its synthesis & degradation

V. Cell wall matrix is made of 3 types of macromolecules - hemicelluloses, pectins, structural proteins

A. Hemicelluloses - branched polysaccharides whose backbone consists of 1 sugar like glucose & side chains of other sugars like xylose

1. Hemicellulose molecules bind to cellulose microfibril surfaces, cross-linking them into a complex & resilient structural network

B. Pectins - heterogeneous class of negatively charged polysaccharides, containing galacturonic acid

1. Form extensive, hydrated gel filling space between fibrous elements (attract H₂O like animal GAGs)

2. When plant is attacked by pathogens, pectin fragments released from wall trigger defensive plant cell response

3. When purified, pectin is used commercially to provide gel-like consistency of jams & jellies

C. Proteins – functions not well understood, but they mediate dynamic activities

1. One class, the expansins, facilitate cell growth; they cause localized relaxation of cell wall, which allows the cell to elongate at that site in response to turgor pressure generated within cell

2. Cell wall-associated protein kinases span the plasma membrane & are thought to transmit signals from the cell wall to the cytoplasm

VI. Percentages of these various materials in cell walls are highly variable

A. Depends on type of plant, type of cell & stage of wall

B. Cell walls are dynamic structures modified in response to changing environmental conditions, like animal connective tissue ECMs

VII. Cell walls arise as a thin cell plate formed between membranes of newly formed daughter cells after cell division

A. Cell wall matures by incorporating additional materials assembled inside cell & then secreted into the extracellular space

B. Walls of young, undifferentiated cells must be able to grow along with the enormous growth of cell it surrounds along with providing mechanical support & protection from foreign agents

1. Such walls of growing cells are called primary walls & they possess extensibility that is lacking in the thicker secondary walls present around many mature plant cells

2. As cell enlarges, primary cell wall grows by insertion of materials into existing wall structure

C. Transformation from primary to secondary wall occurs as wall increases in cellulose content &, in most cases, incorporates lignin (a phenol-containing polymer) that provides structural support

1. Lignin is also the major component of wood & thus the most abundant organic molecule on Earth

2. Lignin in water-conducting xylem cell walls gives support needed to move H₂O through the plant

The Human Perspective: The Role of Cell Adhesion in Inflammation and Metastasis

I. Inflammation is one of the primary responses to infection – although it is a protective response, it also produces negative side effects (fever, swelling due to fluid accumulation, redness, pain)

A. If a part of the body were to become contaminated by bacteria (as with a puncture wound in skin), the site of injury would become a magnet for a variety of white blood cells

B. White blood cells (leukocytes) that would normally stay in bloodstream instead are stimulated to traverse the endothelial layer lining the smallest veins (venules) in the region & enter the tissue

C. Once in tissue, the leukocytes move in response to chemical signals toward the invading microorganisms, which they ingest

II. Inflammation can also be triggered inappropriately

A. Damage to tissues of the heart or brain can occur when blood flow to these organs is blocked during a heart attack or stroke

1. When blood flow to organ is restored, circulating leukocytes may attack the damaged tissue, causing a condition known as reperfusion damage

B. An overzealous inflammatory response can also lead to asthma, toxic shock syndrome & respiratory distress syndrome

III. Research has focused on questions related to the above conditions – answers to such questions have focused on 3 types of cell-adhesion molecules: selectins, integrins & IgSF proteins

A. How are leukocytes recruited to sites of inflammation?

B. Why do they stop flowing through the bloodstream and adhere to vessel walls?

C. How do they penetrate the walls of the vessels?

D. How can some of the negative side effects of inflammation be blocked without interfering with the beneficial aspects of the response?

IV. Chain of events proposed to occur during acute inflammation – this cascade of events involves several different types of cell-adhesion molecules

A. Walls of venules become activated in response to chemical "signals" from nearby damaged tissue

1. Endothelial cells lining these venules become more adhesive to circulating neutrophils, a type of phagocytic leukocyte that carries out a rapid, nonspecific attack on invading pathogens

2. This change in adhesion is mediated by a temporary display of P- & E-selectins on the surfaces of the activated endothelial cells in the damaged area

B. When neutrophils encounter the selectins, they form transient adhesions that dramatically slow their movement through the vessel

1. Neutrophils can be seen to roll slowly along the wall of the vessel

2. Companies are trying to develop anti-inflammatory drugs that act by interfering with binding of ligands to selectins, especially P-selectin

3. Anti-selectin antibodies block neutrophil rolling on selectin-coated surfaces in vitro & suppress inflammation & reperfusion damage in animals

4. A similar type of blocking effect has been attained using synthetic carbohydrates that bind to P-selectin, thereby competing with carbohydrate ligands on the surfaces of the neutrophil

C. As neutrophils interact with the inflamed venule endothelium, integrins present on the neutrophil surface become activated, causing a marked increase in their binding activity

D. The activated integrins bind with high affinity to IgSF molecules (ICAMs) on the surface of the endothelial cells, causing the neutrophils to stop their rolling & adhere firmly to the vessel wall

E. The bound neutrophils then change their shape & squeeze between adjacent endothelial cells into the damaged tissue

1. Invading neutrophils appear capable of disassembling the adherens junctions that form the major barrier between cells of the vessel wall

F. The above events ensure attachment of blood cells to blood vessel walls & their subsequent penetration occurs only at sites where leukocyte invasion is required

V. The importance of integrins in the inflammatory response is demonstrated by a rare disease called leukocyte adhesion deficiency (LAD)

A. People with this disease cannot produce the ₂ subunit as part of a number of leukocyte integrins

B. The leukocytes of these individuals lack the ability to adhere to the endothelial layer of venules, a step required for their exit from the bloodstream

C. Patients suffer from repeated, life-threatening bacterial infections; the disease is best treated by bone marrow transplantation, which provides patient with stem cells that can form normal leukocytes

D. Administration of antibodies against the b₂ subunits can mimic the effects of LAD, blocking the movement of neutrophils & other leukocytes out of blood vessels

1. Such antibodies might prove useful in preventing inflammatory responses associated with diseases like asthma & rheumatoid arthritis or with reperfusion

VI. Cancer is a disease in which cells escape from the body's normal growth control mechanisms & proliferate in an unregulated manner

A. If malignant cells remained in a single mass (as often occurs in some types of skin cancer or thyroid cancer), most cancers would be readily cured by surgical removal of the diseased tissue

B. Most malignant tumors, however, spawn cells that are capable of leaving the primary tumor mass & entering the bloodstream or lymphatic channels

1. This thereby initiates the growth of secondary tumors in other parts of the body

2. The spread of a tumor within the body (metastasis) is the reason why cancer is so devastating

C. Metastatic cells (cancer cells that can initiate the secondary tumors) are thought to have special cell-surface properties that are not shared by most other cells in the tumor

1. Metastatic cells must be less adhesive than other cells to break free of the tumor mass

2. They must be able to penetrate numerous barriers, (ECMs of surrounding connective tissues, basement membranes that line the blood vessels that carry them to distant sites

3. They must be able to invade normal tissues if they are to form secondary colonies

D. The penetration of ECMs is accomplished largely by ECM-digesting enzymes, most notably the matrix metalloproteinases (MMPs)

1. In some cases, cancer cells secrete their own MMPs, but usually growing tumors induce the synthesis & secretion of these enzymes by the surrounding "host" cells

2. Either way, these enzymes degrade the proteins & proteoglycans that stand in the way of the cancer cell migration

3. In addition, cleavage of certain ECM proteins by MMPs produces active protein fragments that act back on the cancer cells to stimulate their growth & invasive character

E. Because of their prominent role in malignant tumor development, MMPs became a major target of the pharmaceutical industry

1. Synthetic MMP inhibitors were found that could reduce metastasis in mice; clinical trials ensued on patients with a variety of advanced, inoperable cancers were done

2. Unfortunately, these inhibitors have shown little promise in stopping late-stage tumor progression & have sometimes led to joint damage

3. Thus far, the only FDA-approved MMP inhibitor (Periostat) is us3ed to treat periodontal disease

4. The failure of these studies have been evaluated in light of potential flaws in the ways that anti-cancer drugs are selected for testing

F. Changes in the numbers & types of various cell-adhesion molecules (& thus the ability of cells to adhere to other cells or to ECMs) have also been implicated in the promotion of metastasis

1. Major studies have focused on E-cadherin, the predominant epithelial cell-cell adhesion molecule of the adherens junctions that hold epithelial cells in a cohesive sheet

2. E-cadherin loss from normal epithelial cells, as in embryonic development, is associated with cell conversion to a more mobile, mesenchymal phenotype very similar to that of most cancer cells

3. Surveys of a variety of epithelial cell tumors (e.g., breast, prostate & colon cancers) confirm that these malignant cells have greatly reduced levels of E-cadherin

4. The lower the level of E-cadherin expression, the greater the cell's metastatic potential

5. Conversely, when malignant cells are forced to express extra copies of the E-cadherin gene, the cells become much less capable of causing tumors when injected into host animals

6. Thus, the presence of E-cadherin is thought to favor the adhesion of cells to one another & suppress the dispersal of tumor cells to distant sites

7. E-cadherin may also inhibit the signaling pathways within the cell that lead to tissue invasion & metastasis

8. E-cadherin importance - study of native New Zealander family (lost 25 members to stomach cancer over 30-years); DNA analysis shows that susceptible individuals have E-cadherin gene mutations