MULTICELLULAR ORGANISMS

• Cell-Cell Adhesion

• Cell-Matrix Adhesion

• The Extracellular Matrix, ECM

M. Habibi-Rezaei

Cell-Cell Interactions

Cleavage

MULTICELLULAR ORGANISMS

• The appearance of multicellular organisms allows specialization of cells and formation of organs

• Vertebrates have more than 100 specialized cell types (plants have more than 15)

• A special matrix, the extracellular matrix, ECM, fills out the space between cells

Cell Signals

• Direct contact

• Paracrine signaling

• Endocrine signaling– hormones

• Synaptic signaling– neurotransmitters

Cell Signaling

Cell Surface Receptors

MULTICELLULAR ORGANISMS

• By means of cell adhesion molecules, CAMs, cells are capable of recognizing each other

• Plasma membrane receptors take care of cell-ECM interactions

CELL-CELL ADHESION MOLECULES

• Cadherins

• Ig superfamily CAMs

• Selectins

• Integrins

Functional Categories of Cell Junctions

Occluding

Tight Junctions

Anchoring Communication

Adherens Junctions

Desomosomes

Focal Adhesions

Hemi-desmosomes

Gap Junctions

Chemical Synapses

Plasmodesmata (Plants)

Cadherin

Integrin

Septate Junctions (Invertebrates)

Adhesion Molecules and Extra Junctional Adhesion:

Types of cell-cell adhesion

Cell adhesion

• Emphasis on cell migration:–Embryogenesis–Immune cell chemotaxis–Tumor cell metastasis

• Types of adhesion molecules involved in these processes

Major Families of Cell Adhesion Molecules (CAMs)

Cadherins: participate in adherens junctions (adhesion belts) & desmosomes

Immunoglobulin-like CAMs (ICAMs): only extrajunctional

Integrins: cell matrix adhesion, hemidesmosomes, focal contacts

Selectins: transient adhesion of leucocytes to blood vessels

Integral membrane proteoglycans

Cell-Cell Recognition and Adhesion

• What are the different categories of adhesion receptors?

• What is the difference between homophilic and heterophilic interactions?

CADHERINS

• A family of Ca2+-dependent CAMs

• Ca2+ causes dimerization of Cadherins

• The binding is homophilic

Cadherins – Ca++ dependent

Homophilic binding

Cadherins are Responsible for Cell-Cell Adhesion

Cadherins are homophilic, calcium dependent adhesion molecules

Primary tumor suppressor function of E- cadherin

http://www.chembio.uoguelph.ca/educmat/chm736/cancer.htm

Sequesteration of cytoplasmatic pools

of ß-catenin

Which prevents ß-catenin Of entering the nucleus

and starting transcription programm

Cadherins Mediate Cell Sorting Due to Homophilic Binding

1) The type of cadherins they express

2) The level of cadherins they express

Cells in culture sort themselves based upon:

Adherens Junctions Help Fold Epithelial Cells

Fig. 19-10

Embryogenesis & Cadherins

• Expression of specific cadherins accompanies morphogenetic movements during embryogenesis

Integrins - Binding to extracellular matrix

Protease cleaves Talin; binding/uncoupling with actin

Tripeptide binding sequence

Immunoglobulin Superfamily CAMs (ICAMs)Important during neural development

Binding is calcium-independent

SELECTINS• Selectins are involved in extravasation• Inflammatory signals activate endothelial cells

making P-Selectin undergo exocytosis• P-Selectin on the surface of endothelial cells binds

a specific carbohydrate ligand (Sialyl Lewis -x) on leukocytes

• The leukocytes attach to the endothelial wall and roll slowly on it

• PAF and integrins are then activated and the leukocytes start to extravasate

Selectins mediate adhesions of white blood cells (leukocytes) during extravasation (migration of cells out of blood vessels)

Adhesion is weak & transient

Involved in other process, including adhesion of early embryo to uterine wall

Model of Extravasation

Integrins are Heterodimers

dimerized single-transmembrane proteins

consist of alpha and beta subunits,

(18 a-subunit members and 8 b-subunit members)

combine to form at least 25 different integrin receptors

fibronectin, vitronectin, laminin, collagen etc .

focal adhesion complex.

ECM molecules

INTEGRINS serve as a velcro for cell migration

In resting cell,

most of the integrins are inactive (Not ligand-binded)

In moving cell, integrins are turned on

in “front” of cell, griping to ECM and pulling.

At back, integrins are off. Internalised and recycled

The cell moves by actin-driven "ruffling" it's membrane.

Focal Adhesions: Connect Cells to the EC Matrix

• Integrins

• Attach actin filaments from cell to matrix

•Connect to cytoplasmic anchor proteins

Adhesion Proteins

Anchor ProteinsFig. 19-12

Circ Res 89:211. 2001

Integrins and their ligands

J Biol Chem 275:21785, 2000

Integrin Clustering Mediates Intracellular Signaling

http://www.nature.com/nrc/journal/v2/n2/fig_tab/nrc727_F1.html

Extracellular Matrix

Extracellular Matrix Prominent in Connective Tissues

Figure 4.1

Extracellular Matrix is Contains of 3 Main Components:

Proteoglycans

Collagens

Multi Adhesive Matrix Proteins

PerlecanLarge ( aggrecan, versican)Small ( decorin, Biglycan, Fibromodulin, Lumicin)

At least 12 types

•Fibronectin•Laminin•Nidogen•Entactin

ECM

Basement Membrane

• thin extracellular layer made up of– basal lamina

• closest to epithelial cells

• secreted by epithelial cells

• Components

– reticular lamina• deep to basal lamina

• part of connective tissue layer

• produced by fibroblasts

Composition of The Extracellular Matrix

Bone Cornea

MAJOR ECM CONSTITUENTS

• Hyaluronan

• Proteoglycans

• Collagens

• Elastin

• Fibronectin

• Laminin

• Enactin/nidogen

Protein in green, glycosaminoglycan in red.Protein in green, glycosaminoglycan in red.

PLASMA MEMBRANE PROTEOGLYCANS

Annu Rev Biochem 68:729,’99

Glycosaminoglycan (GAG) Chains are Composed of a Repeating Disaccharide Sequence

Contains carboxyl & sulfate groups

(-) Charge

Hyaluronan is a GAG Chain Composed of a Repeating Disaccharide Sequence

Does not form proteoglycans; Contains only carboxyl groups

(-) Charge

19-38

HYALURONAN

Relative volumes

Some Common Proteoglycans

Proteoglycans = GAG Chain + Core Protein

19-39

Proteoglycans & Hyaluronan Associate to Form Large Complexes in the ECM

19-41

Basal Lamina

• Some functions:– Provide structural definition & integrity to tissues

– Acts as selective filter of small compounds

– Determines cell polarity

– Organize cell surface proteins on adjacent cell membranes

– Promotes cell survival, proliferation, differentiation

– Serves as “pathway” for cell migration

Basil Lamina: a specialized sheet of ECM

21_019.jpgScanning Electron Micrograph of an Epithelium

Composition of Basal lamina

Collagen

COLLAGENS A helix comprised of homotrimer &

heterotrimer polypeptides (alpha chains)

Major proteins of ECMs

Many different alpha chains

Multiple structures (involves cross-linking of chains) fibrils fibril-associated network forming

Fig. 19-40

Some types of collagen & their properties

Formation of Collagen FIBRILS and FIBERS

Formation of Collagen Networks

COLLAGEN ASSEMBLIES

Ann Med 33:7, 2001

LAMININ

• Heterotrimeric glycoprotein

• Basal lamina constituent

• Multiple binding domains

74

2. Laminini. a cross-shaped-protein with four binding sites for:

i. cells trans-membrane proteins including integrinsii. other laminins, iii. proteoglycans and iv. collagen. : thus forming networks of extracellular fibers, including an inter-

laced web in the basement membrane

3. entactin. another web-forming protein found in the basement membrane. Lin

Binding Domains of Laminin

• Self assembly• Type IV collagen• Heparan sulfate• Enactin/nidogen• Cell Surface

– integrin

– nonintegrin

J. Anat. 193:1, ‘98

Cell Suface Binding Sites

FIBRONECTIN (FN)• Extracellular dimeric

glycoprotein• Differential splicing • Multiple functional

domains– cell binding

• RGD sequence of FN

• other specificities

– heparin binding

– collagen binding

– fibrin binding

• Organized into a matrix

Fig. 19-51 A,C

78

5. fibronectin. Important multi-valent linker. Multiple recognition sites on each of two peptides.

• constructed of two similar (not identical) peptides joined by disulfide bridges. A family of proteins.

– Each one has modular construction, with multiple binding sites for components of the ECM: i. collagen, ii. proteoglycans,

– integrins an arg-gly-asp amino acid (RGD) sequence

– functions to make a web of proteins, proteoglycans and cells by cross linking them

– Especially important as binding sites for cells within the ECM due to integrin binding.

H2N s sss

COOH

H2N COOH

Collagen proteoglycan laminin integrin

Matrix MetalloproteasesMMPs

Genes Dev 14:2123,’00

Genes Dev 14:2123,’00

Matrix Metalloproteases

Distintegrin and Metalloproteinase (ADAM)ADAM proteins are members of the same superfamily as MMPs, namely the Metzincins, named for their zinc binding domains and their structurally important C-terminal conserved methionine residue. The name ADAM stands for “A Disintegrin And Metalloprotease” and like the name suggests, ADAM proteins are cell surface proteins that possess both an adhesion domain as well as a protease domain (Wolfsberg, TG et al. J Cell Biol 1995; 131:275–278).

There are more than 35 members of the ADAM family of proteins; the precise function of many the ADAM family members are unknown, but some, such as ADAM17 (a.k.a. tumor necrosis factor–a converting enzyme) have known biological functions. An additional class of ADAM related proteins are known as the ADAMTS proteins. ADAMTS proteins are structurally homologous to ADAM proteins, but they contain at least one C-terminal thrombospondin type 1 (TSP1) repeat and are secreted rather than membrane bound. ADAMTS1 and ADAMTS8 are inhibitors of angiogenesis, and others, such as ADAMTS5, cleave extracellular proteoglycans such as aggrecan.

ADAM A Disintegrin And Metalloprotease

ADAM A Disintegrin And Metalloprotease

Trends Genet. 16:83, ‘00

A disintegrin is a molecule that binds to an integrin.

T = transmembrane domain

Trends Genet. 16:83, ‘00

Tumor Cell Metastasis

Proteases allow cells to move through ECM, basal lamina

•White blood cells

•Tumor cells

CELL JUNCTIONS

• Adherens junctions

• Gap junctions

• Tight junctions

• Desmosomes/Hemidesmosomes

• Focal adhesions

Cell-Matrix Interactions

Lectin family Typical saccharide ligands Subcellular location Examples of functions

Calnexin Glc1Man9 ERProtein sorting in the endoplasmic reticulum.

M-type lectins Man8 ERER-associated degradation of glycoproteins.

 L-type lectins Various ER, ERGIC, GolgiProtein sorting in the endoplasmic reticulum.

P-type lectins Man 6-phosphate, others Secretory pathwayProtein sorting post-Golgi, glycoprotein trafficking, ER-associated degradation of glycoproteins, enzyme targeting.

C-type lectins VariousCell membrane,

extracellularCell adhesion (selectins), glycoprotein clearance, innate immunity (collectins).

Galectins -Galactosides Cytoplasm, extracellularGlycan crosslinking in the extracellular matrix.

I-type lectins (siglecs) Sialic acid Cell membrane Cell adhesion.

R-type lectins Various Golgi, Cell membraneEnzyme targeting, glycoprotein hormone turnover.

F-box lectins GlcNAc2 Cytoplasm Degradation of misfolded glycoproteins.

Ficolins GlcNAc, GalNAcCell membrane,

extracellularInnate immunity.

Chitinase-like lectins Chito-oligosaccharides Extracellular Collagen metabolism (YKL-40).

F-type lectins Fuc-terminating oligosaccharides Extracellular Innate immunity.

Intelectins Gal, galactofuranose, pentosesExtracellular/cell

membrane Innate immunity.  Fertilization and embryogenesis.

Summary of lectin families

 

Common name Gene Isoforms[a] LOF phenotype(s)[b] References

Collagen IV α1 emb-9 1 Emb (2-3X)Guo et al., 1991; Gupta

et al, 1997

Collagen IV α2 let-2 2 Emb (2-3X) Sibley et al., 1993, 1994

Collagen XVIII cle-1 4 Neuro, gonad morphAckley et al., 2001,

2003

Fibulin-1 fbl-1 2 Gonad morphHesselson et al., 2004;

Kubota et al., 2004

Hemicentin him-4 2Tissue adhesion,

aneuploidyVogel and Hedgecock,

2001

Laminin αA lam-3 1 Emb, Acc Huang et al., 2003

Laminin αB epi-1 2 Emb, Acc, Ste Huang et al., 2003

Laminin β lam-1 1 N.D.  

Laminin γ lam-2 1 N.D.  

Nidogen nid-1 3 NeuroKang and Kramer,

2000; Kim and Wadsworth, 2000

Osteonectin/SPARC ost-1 1 Acc (L1-L2)Fitzgerald and

Schwarzbauer, 1998

Papilin ppn-1 3 Emb (hyp enclosure) Kramerova et al., 2000

Perlecan unc-52 >5 PatRogalski et al., 1993,

1995

COLLAGENS

• The most abundant animal protein• At least 16 types exist• The structural unit is composed of three 300 nm

long coiled subunits in a triple helix• The helical structure depends on the abundant

presence of glycin, proline (and hydroxyproline) making a motif gly-pro-x, which is necessary for twisting together the three strands

COLLAGENS 2• Collagens are synthesized as precursors called

procollagens• They are glycosylated in ER and Golgi adding Gal

and Gly to hydroxy-lysine residues and long oligosaccharides to selected asparagine residues

• Proline and lysine are hydroxylated• Disulphide bonds are made between the N- and C-

terminal parts of the propeptides• After exocytosis the N- and C-terminals are

“trimmed”, only then can the fibrils be formed

COLLAGENS 3

• Lack of vitamin C prevents hydroxylation impaired fibrils

• Mutations or deletions of -chains in Collagen I can lead to the disease Osteogenesis imperfecta

LAMININ•Laminin is a key component of the basal lamina

DISEASES OF THE BASAL LAMINA

• Alport’s syndrome appears as impaired ultrafiltration in the kidney resulting in renal failure and hearing loss. Mutations in collagen IV -chains result in this syndrome.

• Antibodies against 3-chains of collagen IV lead to pulmonary hemorrhage and renal failure (Goodpasture’s syndrome)

FIBRONECTIN

• Fibronectins attach cells to collagens

• Fibronectins are dimers

• Fibronectins express the RGD sequence recognized by integrins

PROTEOGLYCANS 1

• The Polysaccharides in proteoglycans are long repeating polymers of dissacharides called Glucosaminoglycans (GAGs)

• One sugar of the dissacharides is a uronic acid and the other is an aminosugar (e.g. N-acetylglucosamine)

• One or both sugars contain one or two sulphate residues

HYALURONAN (HA)

• HA is a GAG found in ECM• HA is also a key component of complex proteoglycans• HA consists of approx. 50,000 disaccharides in a random

coil. It can be bound to the surface receptor CD44• HA gives strength, flexibility and smoothness to the

ECM and forms a viscous hydrated gel in which cells can migrate

• HA makes the ECM able to resist compression

PROTEOGLYCANS 2

• Heparin sulphate and chondroitin sulphate are added to a 3-sugar “linker” (Xyl-Gal-Gal) added to a Serine in the core protein

• Proteoglycans are found both in ECM and attached to the plasma membrane

PROTEOGLYCANS IN THE ECM

• In cartilage the key proteoglycan is aggrecan• The central component of aggrecan is a carbohydrate,

hyaluronan• At 40 nm intervals aggrecan core proteins are attached (assisted

by a linker protein) to a decasaccharide sequence in hyaluronan• Attached to the aggrecan core protein are multiple GAGs (via

the trisaccharide linker)• The GAGs in aggrecan are chondroitinsulphate and keratin

sulphate• MW of an aggrecan 2 x 108

PROTEOGLYCANS ON THE CELL SURFACE

• A typical example is syndecan• The core protein spans the membrane with a short

cytosolic domain• The GAGs are attached via the trisaccharide linker to

serine residues• The GAGs in syndecan are heparan sulphate chains• Syndecan binds extracellularly to collagens and

fibronectin and intracellularly to the cytoskeleton

DISEASES OF GAG

Rare genetic defects in enzymes required for the synthesis of

Dermatan sulfate lead to defects in bones, joints, muscles, and skin. The

individuals do not grow to normal hight and appear prematurely aged.

NEURONAL CELL ADHESION MOLECULESLEARNING AND MEMORY

• Male humans with L1-mutations develop– Mental retardation– Hydrocephalos– Adducted thumbs

• NCAM knock-out animals develop– Morphological changes in bulbus olfactorius and

hippocampus– Impaired learning– Emotional disturbances

• Modulation of NCAM and L1-function interferes with LTP and learning and memory

CELL-MATRIX ADHESION

• Integrins

• Collagens

• Laminin and Fibronectin

• Proteoglycans and Glucosaminoglycans

CELL MATRIX ADHESION

• Integrins on the cell surface mediate cell-ECM binding

• Integrins are composed of an- and a -chain• There are 3 different -chains and more than 10

types of -chains• The chain composition determines the ligand

specificity• The affinity is generally low (Kd 10-6 -10-8)

INTEGRINS

• Integrins can be activated through a signal from the interior of the cell

• Activation involves conformational changes of the integrin

• Various integrins recognize specific sequences in their ligands. E.g. 41 recognizes EILDV (in VCAM-1 and in fibronectin) and 51 recognizes RGD in many ECM proteins

INTEGRIN CONTAINING JUNCTIONS

• A junction consists of an exterior ligand, a transmem-brane protein, a linker, and a cytoskeletal component

• An adherence junction connects an ECM component with an integrin linked to an adapter (e.g. vinculin) and F-actin

• A hemidesmosome connects an ECM-component to integrin and via an adapter (e.g. plectin) to intermediate filaments (keratins)

INTEGRIN DISEASES

• Genetic defects in integrin 2 lead to leucocyte-adhesion deficiency. The patient becomes susceptible to bacterial infections

DISINTEGRINS

• Disintegrins contain the RGD sequence and interfere with integrin-ECM adhesion allowing deadhesion and cell migration

• The ADAMs (A Disintegrin And a Metalloprotease) “remodel” surface proteins; f.x. at the fusion of sperm and egg, the fusion of myoblasts during myogenesis, release of TNF from the surface