Cell biology 2014 (revised 12/2-13)
Lecture 10:
The eukaryotic kingdom
Cell Biology interactive  media  ”video” or ”animation”
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The four major tissues in the human body
Metazoan cells form organs with specialized tissues:
- Epithelial
Cells
- Muscle
- Connective
Cells +
ECM
- Nerve
2
Different types of cell adhesion
Homophilic binding
Heterophilic binding
3
Cell-cell contacts in columnar epithelia
Tight junction
4
Restricting movement of
extra-cellular fluids
Adherens junction
Desmosome
Gap junction
hemidesmosome
Cell-cell adhesion
Connection allowing local
communication
Cell-ECM adhesion
Basal lamina
I. Tight junctions: function
Tight junctions seal epithelial sheets to
block passage of fluids in between cells
Intestine
Glucose
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Active and selective transport
Blood
through the cytosol of cells by e.g.,
vessels Glucose
the Na+ driven glucose symport
II. Tight junctions: Architecture
Tight junctions are made up by occludin and claudin. These are
transmembrane proteins, which form tight connections across
the extracellular space
Linking protein attaches occludin and
claudin to the cortical actin cytoskeleton
The appearance of tight junctions
resemble stitches across the plasma
membranes of the two cells
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I. Cadherins: adherence junctions
Adherence junctions from stable cell-cell
adhesion points between adjacent cells
a-catenin
Cell #1
P.M.
b-catenin
Ca2+
P.M.
Cell #2
b-catenin
a-catenin
Many cadherins are known:
E-cadherin in Epithelia
N-cadherin in Neural cells
Cadherin (calcium-dependent adhesion)
Linkers of cadherins
to the actin cytoskeleton
Actin
filament
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video 19.1- adhesion_junctions
II. Cadherins: growth arrest at cell-cell contact
4. Sequestering of cytosolic b-catenin at the adherence junctions
formed after cell proliferation (i.e., at ”density arrest”)
Wnt
2. ..but is stabilized
Ca
by Wnt signaling
P.M.
2+
3. b-catenin enters the nucleus:
 G1 cyclin transcription
 cell proliferation
b-catenin
a-catenin
b-catenin
b-catenin
1. Cytosolic b-catenin is
by default unstable.....
(Ubiq. dep. degradation)
TCF
G1
G1 cyclin
gene
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III. Cadherins: organization of cells into organs
Cells expressing
different cadherins
Cells expressing
different amounts of
the same cadherin
+Ca2+
+Ca2+
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Cadherins are important for organ formation during development
Structure and function of the desmosome
Linkers
Desmosomes hold cells together
like rivets. Through linkage to
IFs, they distribute shear forces
evenly within the cell
Cell #1
P.M.
Cadherin
family protein
P.M.
Cell #2
Ca2+
Linkers
Intermediate
filament (IF)
animation 16.4- intermediate_filament
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Structure and regulation of gap junctions
Connexon
Connexin
Different connexins –
different pore size
Cell #1
Cell #2
= ~1.5 nm
Free passage of: Amino acids
Nucleotides
Sugars
Ions
”2nd messengers”
Regulation of pore size
PP
P P
PP
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I. Integrins: Structure and ligand specificity
i)
Hetero-dimeric proteins consisting of a- and b- chains
ii) At least 21 cell-type specific isoforms of a/b-chain pairs
iii) Integrin ligands include ECM components (collagen,
fibronectin, laminin) and structures on neighboring cells
Integrins linked to actin
(focal adhesions: fibroblasts)
by ay (ligand: fibronectin)
ECM: connective tissue
Integrins linked to IF
(hemidesmosomes: epithelia)
bx ax (ligand: laminin)
ECM: Basal lamina
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II. Integrins: Anchorage to ECM
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Integrins linked to IF (hemidesmosomes)
 Static cell-ECM interactions , e.g. epithelial sheets
Basal lamina: barrier towards connective tissue
ECM: Basal lamina
Integrins linked to actin (focal adhesions)  Dynamic cell-ECM
interactions, e.g., during migration of fibroblasts or leukocytes
Inactive
integrin
ECM: connective tissue (contains residual migratory cells)
III. Integrins: Architecture of the focal adhesion
Focal adhesions exist only in motile cells (i.e., not in epithelia)
The dynamic nature of focal adhesion is dependent on both
“Inside-out” and “Outside-in” signaling
Tyr- P
Linker
FAK
Talin
Tyr- P
FAK
Talin
FAK: Focal adhesion kinase
 integrin dependant signaling
P. M.
Active
integrin
ECM
Tyr- P  recruitment of SH2-domain signaling proteins
(Clustering of FAK trans-phosphorylation, i.e. the same principle as for
tyrosine kinase receptors, which are dimerized by ligand binding)
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IV. Integrins: Regulation of ligand-affinity
The a- and b-chains of integrins have affinity for both “each
other” and ECM ligands the concept of competing affinities
Outside-in activation of ECM-binding
1. Default state:
The a- and b-chains are
tightly associated
1.
2.
2. Activated state:
a- and b-chains are pushed
apart and clustered by talin
 High affinity/avidity ECMassociation
Inside-out activation of ECM-binding
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V. Integrins: Inside-out activation
1. Activation of talin by a
RTK ligand (e.g. EGF)
3.
3.
2. Separation of a- and b-chains
3. High affinity ECM-binding
2.
4.
2.
4. Integrin clustering
increased avidity
Albert et al. Fig 19-49
1.
Inactive talin
P
FAK
Activated talin:
i)
ii)
iii)
iv)
Pushes a- and b-chains apart
Clusters the cytosolic parts of integrin b-chains
Links b-chains with actin filaments
Recruit focal adhesions proteins (vinculin, FAK etc)
 generation of a focal adhesion point
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VI. Integrins: Outside-in activation
1. Binding by (very) high affinity ECM ligands……..
2. breaks the interaction between the a- and b-chains
3. The exposed b-chain talin-binding site……..
4. ….activates talin  Generation of a focal adhesion point
1.
2.
outside-in + inside-out
= positive feedback
inactive
talin
2.
3.
4.
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VII. Integrins: survival and cell proliferation signals
Motile cell types requires ECM for both growth and survival
Survival
P
PKB/Akt
P
Bad
3
P
FAK
P
P
MAPK
P
-Tyr- P
-Tyr- P
PI-3 K
-Tyr- P
14-3-3
P
Cell cycle entry
myc
GTP
Ras
P
Plasma membrane
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I. The extra-cellular matrix (ECM)
- Provides mechanical support to tissues
- Organizes cells into tissues
- ‘Instructs’ cells as to where they are and what they should do
- Reservoir for extra-cellular signaling molecules
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II. The extracellular matrix (ECM)
1.
Composed of polymeric networks
of several types of macromolecules.
Secreted by connective tissue cells,
such as fibroblasts & chondrocytes.
2.
3.
1. Proteoglycan molecules form highly hydrated gel-like “ground
substance” in which the fibrous proteins are embedded
2. Structural proteins, such as collagen and elastin, strengthen
and organize the matrix
3. Multi-adhesive proteins, such as fibronectin and laminin,
facilitate cell attachment to the ECM
The aqueous phase of the ECM permits diffusion of nutrients
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III. ECM: general structure of proteoglycan
Linking saccharides
Glucosaminoglycans (GAGs)
linear polymers
of repeating
disaccharides
Polysaccharide
sidechain
Protein core
Na+
Ca2+
Osmosis
H2O
O-linked sugar
-
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Negatively charged saccharides attract counter ions
and water, giving the ECM the property to resist
compression and bounce back to its original shape
IV. ECM: Proteoglycan aggregates
Proteoglycans can form huge aggregates onto hyaluronan. These
aggregates can be up to 4 mm in length
Linker protein
Hyaluronan,
up to 50 000
repeating
disaccharides
These aggregates have a very high shock absorbing capacity
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and are highly enriched in cartilage
V. ECM: Collagen architecture
Collagen is the most common protein in body, it forms strong and
flexible fibers. Many types (at least 15)
Collagen a-chain
(single helix)
Collagen molecule
(triple helix)
Assembled
in ER
Collagen fibril
Assembled
outside
the cell
Collagen fiber
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VI. ECM: Elastic elastin networks
In cases there ECM is very flexible, e.g., in skin, lungs and blood
vessel walls, some of the collagen is replaced by elastin.
Cross-linked elastin behaves like a rubber band!
Single elastin
molecule
Stretching
Crosslinking
Relaxation
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VII. ECM: different types of connective tissue
”Normal” connective tissue
Cartilage
Bone
Ca10(PO4)10(OH)2
Ca10(PO4)10(OH)2
Fibroblast
Chondrocyte
Osteoblast
Physical properties of the tissue depend on the content of the
ECM, which is determined by the residual cell type
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Summary: ECM – a sticky business!
Fibronectin - Present in all ECM and primary high-affinity
ligand for focal adhesions
Laminin - Present in basal lamina of epithelia and the
ligand for hemidesmosomes
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Differential means to achieve mechanical strength
Epithelial cells
Basal lamina
(dense ECM)
Connective tissue
(ECM + cells)
Cells resistant
to mechanical
stress
ECM (but not
cells) resistent
to mechanical
stress
Fig. 19-1:
Epithelial tissue: The intermediate filaments of the cells themselves (linked from
cell to cell by desmosomes) provides mechanical strength. Hemidesmosomes
(integrin binding to laminin) are only found in the epithelial cells that connect to the
basal lamina. These epithelial cells are normally essentially non-motile.
Connective tissue: ECM provides the mechanical strength, the sole role of the
residual cells (fibroblasts) is to produce the ECM components. These residual cells
move around and may migrate to e.g. a site of tissue damage.
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“Recommended reading”
Chapter 19
1131-1145
1150-1162
1164-1194
Alberts et al
5th edition
Focus on the general principles
and topics highlighted in
the lecture synopsis
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