multicellular organisms cell-cell adhesion cell-matrix adhesion the extracellular matrix, ecm m....
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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