1. MULTICELLULAR ORGANISMS
Cell-Cell Adhesion
Cell-Matrix Adhesion
The Extracellular Matrix, ECM
M. Habibi-Rezaei

3. Cell-Cell Interactions

4. Cleavage

5. 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

6. Cell Signals Direct contact Paracrine signaling Endocrine signaling
hormones
Synaptic signaling
neurotransmitters

7. Cell Signaling

8. Cell Surface Receptors

9. 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

12. CELL-CELL ADHESION MOLECULES
Cadherins
Ig superfamily CAMs
Selectins
Integrins

13. Septate Junctions (Invertebrates) Plasmodesmata (Plants)
Functional Categories of Cell Junctions
Occluding
Anchoring
Communication
Adherens Junctions
Tight Junctions
Gap Junctions
Cadherin
Desomosomes
Septate Junctions (Invertebrates)
Chemical Synapses
Focal Adhesions
Plasmodesmata (Plants)
Integrin
Hemi-desmosomes

14. Adhesion Molecules and Extra Junctional Adhesion:
Types of cell-cell adhesion

15. Cell adhesion
Emphasis on cell migration:
Embryogenesis
Immune cell chemotaxis
Tumor cell metastasis
Types of adhesion molecules involved in these processes

16. 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

17. Cell-Cell Recognition and Adhesion
What are the different categories of adhesion receptors?
What is the difference between homophilic and heterophilic interactions?
immunoglobulin superfamily proteins (e.g. N-CAM), cadherins, selectins, integrins
homophilic – adhesion occurs when 2 identical molecules bind, one from each cell
heterophilic – cell adhesion receptor on one cell interacts with different molecule on other cell

20. CADHERINS A family of Ca2+-dependent CAMs
Ca2+ causes dimerization of Cadherins
The binding is homophilic

21. Cadherins – Ca++ dependent
Homophilic binding
Cadherins – Ca++ dependent

22. Cadherins are Responsible for Cell-Cell Adhesion

23. Cadherins are homophilic, calcium dependent adhesion molecules

24. Primary tumor suppressor function of E- cadherin
Sequesteration
of cytoplasmatic pools
of ß-catenin
Which prevents ß-catenin
Of entering the nucleus
and starting
transcription programm

25. Cadherins Mediate Cell Sorting Due to Homophilic Binding
Cells in culture sort themselves based upon:
1) The type of cadherins they express
2) The level of cadherins they express

26. Adherens Junctions Help Fold Epithelial Cells
Fig

27. Embryogenesis & Cadherins
Expression of specific cadherins accompanies morphogenetic movements during embryogenesis

30. Integrins - Binding to extracellular matrix
Tripeptide binding sequence
Protease
cleaves
Talin;
binding/
uncoupling
with actin
Integrins - Binding to
extracellular matrix

32. Immunoglobulin Superfamily CAMs (ICAMs) Important during neural development Binding is calcium-independent

34. 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

35. 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

36. Model of Extravasation

38. Integrins are Heterodimers

39. fibronectin, vitronectin, laminin, collagen etc (18 a-subunit members
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 .
ECM
molecules
focal adhesion
complex.

40. INTEGRINS serve as a velcro for cell migration
The cell moves by actin-driven
"ruffling" it's membrane.
In moving cell,
integrins are turned on
in “front” of cell, griping to ECM and pulling.
At back, integrins are off.
Internalised and recycled
In resting cell,
most of the integrins are inactive
(Not ligand-binded)

41. Focal Adhesions: Connect Cells to the EC Matrix
Adhesion Proteins
Integrins
Attach actin filaments from cell to matrix
Connect to cytoplasmic anchor proteins
Anchor Proteins
Fig

42. Circ Res 89:

43. Integrins and their ligands
J Biol Chem 275:21785, 2000

44. Integrin Clustering Mediates Intracellular Signaling

46. Forms diverse structures
Extracellular Matrix
Forms diverse structures
Bone
Ligament
Tendon
Vessel
Connective tissue
Skin
Biological processes
Adhesion
Mechanical support
Migration
Proliferation
Signalling
Diseases
Arthritis
Atherosclerosis
Cancer
Asthma

47. Extracellular Matrix Prominent in Connective Tissues

48. Figure 4.1

49. Extracellular Matrix is Contains of 3 Main Components:
Perlecan
Large ( aggrecan, versican)
Small ( decorin, Biglycan, Fibromodulin, Lumicin)
Proteoglycans
Collagens
Multi Adhesive Matrix Proteins
At least 12 types
Fibronectin
Laminin
Nidogen
Entactin

50. 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

51. Composition of The Extracellular Matrix
Bone
Cornea

52. MAJOR ECM CONSTITUENTS
Hyaluronan
Proteoglycans
Collagens
Elastin
Fibronectin
Laminin
Enactin/nidogen
Protein in green, glycosaminoglycan in red.

53. PLASMA MEMBRANE PROTEOGLYCANS
Annu Rev Biochem 68:729,’99

54. Glycosaminoglycan (GAG) Chains are Composed of a Repeating Disaccharide Sequence
Contains carboxyl & sulfate groups
(-) Charge

55. Hyaluronan is a GAG Chain Composed of a Repeating Disaccharide Sequence
Does not form proteoglycans; Contains only carboxyl groups
(-) Charge
19-38

56. HYALURONAN
Relative volumes

57. Some Common Proteoglycans

58. Proteoglycans = GAG Chain + Core Protein
19-39

59. Proteoglycans & Hyaluronan Associate to Form Large Complexes in the ECM
19-41

61. 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

62. Basil Lamina: a specialized sheet of ECM

63. Scanning Electron Micrograph of an Epithelium
21_019.jpg

64. Composition of Basal lamina

66. Collagen

67. 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

68. Some types of collagen & their properties

69. Formation of Collagen FIBRILS and FIBERS

70. Formation of Collagen Networks

71. COLLAGEN ASSEMBLIES
Ann Med 33:7, 2001

73. Heterotrimeric glycoprotein Basal lamina constituent
LAMININ
Heterotrimeric glycoprotein
Basal lamina constituent
Multiple binding domains

74. 2. Laminin
i. a cross-shaped-protein with four binding sites for:
i. cells trans-membrane proteins including integrins
ii. 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

75. Binding Domains of Laminin
Self assembly
Type IV collagen
Heparan sulfate
Enactin/nidogen
Cell Surface
integrin
nonintegrin
Cell Suface Binding Sites
J. Anat. 193:1, ‘98

77. 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 A,C

78. 5. fibronectin. Important multi-valent linker
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.
Collagen proteoglycan laminin integrin
H2N
COOH s s s s
COOH
H2N

80. Matrix Metalloproteases MMPs

81. Genes Dev 14:2123,’00

82. Matrix Metalloproteases
Genes Dev 14:2123,’00

83. ADAM A Disintegrin And Metalloprotease
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.

84. ADAM A Disintegrin And Metalloprotease
transmembrane
domain
A disintegrin is a molecule that binds to an integrin.
Trends Genet. 16:83, ‘00

85. Trends Genet. 16:83, ‘00

87. Tumor Cell Metastasis

88. Proteases allow cells to move through ECM, basal lamina
White blood cells
Tumor cells

89. CELL JUNCTIONS Adherens junctions Gap junctions Tight junctions
Desmosomes/Hemidesmosomes
Focal adhesions

90. Cell-Matrix Interactions

97. Summary of lectin families Lectin family Typical saccharide ligands
Lectin family
Typical saccharide ligands
Subcellular location
Examples of functions
Calnexin
Glc1Man9 ER
Protein sorting in the endoplasmic reticulum.
M-type lectins
Man8
ER-associated degradation of glycoproteins.
 L-type lectins
Various
ER, ERGIC, Golgi
P-type lectins
Man 6-phosphate, others
Secretory pathway
Protein sorting post-Golgi, glycoprotein trafficking, ER-associated degradation of glycoproteins, enzyme targeting.
C-type lectins
Cell membrane, extracellular
Cell adhesion (selectins), glycoprotein clearance, innate immunity (collectins).
Galectins
-Galactosides
Cytoplasm, extracellular
Glycan crosslinking in the extracellular matrix.
I-type lectins (siglecs)
Sialic acid
Cell membrane
Cell adhesion.
R-type lectins
Golgi, Cell membrane
Enzyme targeting, glycoprotein hormone turnover.
F-box lectins
GlcNAc2
Cytoplasm
Degradation of misfolded glycoproteins.
Ficolins
GlcNAc, GalNAc
Innate immunity.
Chitinase-like lectins
Chito-oligosaccharides
Extracellular
Collagen metabolism (YKL-40).
F-type lectins
Fuc-terminating oligosaccharides
Intelectins
Gal, galactofuranose, pentoses
Extracellular/cell membrane
Innate immunity.  Fertilization and embryogenesis.

98. Hesselson et al., 2004; Kubota et al., 2004
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
Sibley et al., 1993, 1994
Collagen XVIII
cle-1 4
Neuro, gonad morph
Ackley et al., 2001, 2003
Fibulin-1
fbl-1
Gonad morph
Hesselson et al., 2004; Kubota et al., 2004
Hemicentin
him-4
Tissue adhesion, aneuploidy
Vogel and Hedgecock, 2001
Laminin αA
lam-3
Emb, Acc
Huang et al., 2003
Laminin αB
epi-1
Emb, Acc, Ste
Laminin β
lam-1
N.D.
Laminin γ
lam-2
Nidogen
nid-1 3
Neuro
Kang and Kramer, 2000; Kim and Wadsworth, 2000
Osteonectin/SPARC
ost-1
Acc (L1-L2)
Fitzgerald and Schwarzbauer, 1998
Papilin
ppn-1
Emb (hyp enclosure)
Kramerova et al., 2000
Perlecan
unc-52
>5
Pat
Rogalski et al., 1993, 1995

100. 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

102. 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

103. 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

106. LAMININ
Laminin is a key component of the basal lamina

113. 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)

114. FIBRONECTIN Fibronectins attach cells to collagens
Fibronectins are dimers
Fibronectins express the RGD sequence recognized by integrins

116. 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

120. 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

121. 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

124. 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

127. 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

131. 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.

134. NEURONAL CELL ADHESION MOLECULES LEARNING 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

135. CELL-MATRIX ADHESION Integrins Collagens Laminin and Fibronectin
Proteoglycans and Glucosaminoglycans

136. 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 )

137. 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. 41 recognizes EILDV (in VCAM-1 and in fibronectin) and 51 recognizes RGD in many ECM proteins

138. 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)

139. INTEGRIN DISEASES
Genetic defects in integrin 2 lead to leucocyte-adhesion deficiency. The patient becomes susceptible to bacterial infections

140. 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