Cell junctions and the

extracellular matrix

Know the terminology:

Cadherins, integrins, tight junction, desmosome,
adhesion belt, hemidesmosome, gap junction,
connexin/connexan,

Basal lamina, connective tissue, fibroblast,

proteoglycan, GAG (glycosaminoglycan), hyaluronin,
aggrecan, collagen, fibronectin,
 Overview
Multicellular organisms combine cells into tissues.

Extracellular matrix is a complex network of

macromolecules (proteins, glycosaminoglycans,
proteoglycans) secreted by cells

Specialized ECMs include connective tissue, basal

lamina, exoskeletons, cartilage/bones,

Tissues are formed from cell-cell connections and

cell-matrix connections.
 Outline
How are cells organized into tissues?
I. Cell junctions
-types of cell junctions
-tight junctions, adherens junctions,
desmosomes, focal adhesions, hemidesmosomes,
gap junctions

II. Extracellular matrix proteins

-types of ECM macromolecules
-synthesis and properties of hyaluronan,
aggrecan, collagen, fibronectin
 Cell types and tissues
• Cell types ---?
• Tissues
– Epithelial tissue
– Connective tissue
– Muscular tissue
– Nervous tissue
– Blood
Tissue composition
 Complexity-arrangement
• To form various organs
• Information from surrounding
• Distinct mechanical properties
• Permiability barriers
• Chemically and functionally distinct
compartments (e.g. stomach)
• Sophisticated regulation of biological
function
 Assembly of cells-tissues
• Molecular interactions at cellular
level
• Polar structure-ordered
• Specialized integral membrane
proteins – (CAMs)
• CAMs- cluster together into cell
junctions
• Cell – cell adhesion
• Cell- matrix adhesion
 Cell Polarity
• The epithelial cells remain in a perfect
state of orientation of apical and basal
domains.
• This polarity of epithelial cells
organizes its plasma membrane into a
free apical domain and a basolateral
domain contacting the neighbouring
cells and extracellular matrix.
Cell-Cell/Cell-matrix
 Cell Adhesion molecules
(CAMs)

Tight J Cell-cell

Gap J Cell-cell

Adheren Cell-cell
CAMs
J
Anchoring
Desmoso
Junctions Cell-cell
mes
Hemides
Cell-matrix
mosomes
 CAMs
• Cams are mosaics of multiple distinct
domains
• Many are found in one CAM (repeats)
• Exist in multiple times in the same
molecule
• Adhesive properties of CAMs are through
their extracellular domains
• Interactions- same cell type/ different
cell type/ between same CAMs/ different
CAMs
 Interactions
• Homophilic-same CAM on both the cells
• Heterophilic- one CAM binds to
another CAM on the other cell
• Long lasting- e.g. nerve cells
• Short lived: moving blood cells
Schematic model
 Interactions

Cell - Cell -
Matrix Cell
Intra cellular Intercellular

Lateral
Trans
Cis interactions
With the
Cell cytosol to adjacent cell
extracellular
Cell-cell
 Variables that determine
the nature of adhesion
• Thermodyanamic properties
• Kinetic properties
• Geometric properties
• Biochemical properties
• Mechanical properties
 Adapter proteins
• The cytosol facing domains of CAMs
recruit a set of multifunctional
adapter proteins
• These adapters act as linkers that
directly or indirectly connect CAMs
to elements of cytoskeleton
• They also recruit intracellular
molecules that function in signalling
pathways and gene expression
 Cell junctions
Four types of cell junctions:
1. Occluding/tight junctions: seal cells together
into sheets (forming an impermeable barrier)
2. Communicating/gap junctions: allow exchange of
chemical/electrical information between cells
3.Anchoring junctions: attach cells (and their
cytoskeleton) to other cells or extracellular matrix
(providing mechanical support)
4. Signal relaying junctions: allows signal to be
relayed from cell to cell cross their plasma
membranes
Four functional classes of cell junctions in animal tissues
Anchoring junctions
 Anchoring junctions
Integral membrane proteins connect a cell’s
cytoskeleton to another cell or extracellular matrix
 Anchoring junctions
Integral membrane proteins connect a cell’s
cytoskeleton to another cell or extracellular matrix
 Anchoring junctions
Cytoskeletal fibers (MF, intermediate filaments)

connect to a

Membrane protein receptor

which attaches to another protein in either:

-the extracellular matrix
or
-another cell membrane
 Anchoring junctions
Cadherins Integrins
Cell-matrix
Cell-Cell attachment
attachment

Link to Link to intermediate

Link to actin Link to actin filament
intermediate
filament

Desmosome
Actin
linked cell Hemidesmosome
matrix
From adheren junctions
junctions
 Cell-Cell Ajunctions
• Cadherins-link cytoskeleton of one cell to
cytoskeleton of other
• Primary function- resist the external
forces that pull the cells apart
• But must also be dyanamic- i.e may be
altered or rearranged when tissues are
repaired / remodeled etc…
 Cadherins
• Present in all multicellular animals
• Also present in sponges and
choanoflagellates
• Name- came from Ca+2 depedence
• E-cadherin
• N-cadherin
• P-cadherin
• Non-classical cadherins
– Brain ( 50) - 180
– Protocadherins/desmocollins/desmogleins
 Cadherins
• Spacing between cell membranes
• All members of cadherin family have an
extracellular portion consisting of
several copies extracellular cadherin
(EC) domain
• Homophilic binding- N terminal tips
• Knob-nearby pocket
• Rigid unit-joins the next cadherin by a
hinge
• Ca+2 ions bind near each hinge (prevent
it from flexing)
 Cadherins and developing tissues
• Cadherins are not like glue, making cell
surfaces generally sticky
• Rather, they mediate highly selective
organization enabling cells of the simillar
type stick together and to stay
segregated from other types of cells
• Sorting out
Sorting out
Development
 Qualitative and Quantitative
roles
• L cells
• Transfected with E cad
• Cell-cell adhesion
• L cells with different
Cads
• L cells with different
amounts of Cads
 Epithelial–Mesenchymal Transitions Depend
on Control of Cadherins
• Twist, slug, snail
• The assembly of cells into an epithelium is a reversible
process.
• By switching on expression of adhesion molecules, dispersed
unattached mesenchymal cells, such as fibroblasts, can come
together to form an epithelium.
• Conversely, epithelial cells can change their character,
disassemble, and migrate away from their parent epithelium
as separate cells.
• Such epithelial–mesenchymal transitions play an important
part in normal embryonic development and disease (cancer)
• Most cancers originate in epithelia, but become dangerously
prone to spread—that is, malignant—only when the cancer
cells escape from the epithelium of origin and invade other
tissues.
• Experiments with TWIST
 Catenins as adapter proteins
• Catenins link cadherins to actin
cytoskeleton
Assembly of adherens junctions
• In the mature form Ajs are enormous protein complexes
containing hundreds to thousands of cadherin molecules,
packed to dense, regular arrays that are linked on the
extracellular side by lateral connections between cadherin
domains

• On cytoplasmic side, a complex network of catenins, actin

regulators and actin bundles hold the cluster of cadherins
together and links it to actin cytoskeleton

• Assembling a structure of this complexity is not a simple

task and it involves a complex sequence of events
 Assembly of adherens junctions

Assembly begins when two unattached epithelial cell precursors explore their surroundings with
membrane protrusions, generated by local nucleation of actin networks

When cells make contact, small cadherin and catenin clusters take shape at contact sites and associate
with actin leading to activation of the small monomeric GTPase Rac.
Mechanotransduction
 Tissue remodelling
• Depend on coordination of actin mediated
cytoskeleton
• Ajs essential for modeling shapes
• By indirectly linking the actin filaments in
one cell to neighbours – they enable cells
in the tissue to use their actin
cytoskeletons in a coordinated way
• Ajs occur in various forms – small
punctate, linear
Cadherins participiate in adherens junctions

In epithelia, they
form the adhesion
belt – zonula
adherans just
beneath the apical
face

They surround the

cell, forming a belt
 Tissue remodelling
• The coordination of cell-cell adhesion and
actin – development
• Gastrulation – germ band extension – cells
converge inwards towards dorso-ventral axis
• Intercalation – actin dependent contraction
due to loss of cadherins
 Selectins mediate cell cell adhesion
and migration in blood stream
• Selectins are cell-surface carbohydrate-binding proteins that mediate a
variety of transient, cell-cell adhesion interactions in the bloodstream.
• Their main role, in vertebrates at least, is in inflammatory responses
and in governing the traffic of white blood cells.’
• White blood cells lead a nomadic life, roving between the bloodstream
and the tissues, and this necessitates special adhesive behavior. The
selectins control the binding of white blood cells to the endothelial
cells lining blood vessels, thereby enabling the blood cells to migrate
out of the bloodstream into a tissue.
• Each selectin is a transmembrane protein with a conserved lectin
domain that binds to a specific oligosaccharide on another cell (Figure
l9-l9A). There are at least three types: L-selectin on white blood cells,
P-selectin on blood platelets and on endothelial cells that have been
locally activated by an inflammatory response, and E-selectln on
activated endothelial cells
• In a lymphoid organ, such as a lymph node
or a tonsil, the endothelial cells express
oligosaccharides that are recognized by L-
selectin on lymphocytes, causing the
lymphocytes to loiter and become trapped.
• At sites of inflammation, the roles are
reversed: the endothelial cells switch on
expression of selectins that recognize the
oligosaccharides on white blood cells and
platelets, flagging the cells down to help
deal with the local emergency
• Selectins do not act alone, however; they
collaborate with integrins, which strengthen
the binding of the blood cells to the
endothelium.
• The cell-cell adhesions mediated by both
selectins and integrin s are heterophilic- that
is, the binding is to a molecule of a different
type: selectins bind to specific
oligosaccharides on glycoproteins and
glycolipids, while integrins bind to other
specific proteins.
 Adhesions at synapse
• To make a synapse, the pre and post synaptic cells have to
do more than recognize one another and adhere: they have
to assemble a complex system of signal receptors, ion
channels, synaptic vesicles, docking proteins, and other
components
• Thus, cadherins are generally present, concentrated at spots around the
periphery of the synapse and within it, as well as Ig superfamily
members and various other types of adhesion molecules.
• In fact, about 20 different classical cadherins are expressed in the
vertebrate nervous system, in different combinations in different
subsets of neurons, and it is likely that selective binding of these
molecules also plays a part in ensuring that neurons synapse with their
correct partners.
Many Types of cell adhesion molecules
act in parallel to create a synapse
Cadherin - functions
 Anchoring junctions
Cadherins Integrins
Cell-matrix
Cell-Cell attachment
attachment

Link to Link to intermediate

Link to actin Link to actin filament
intermediate
filament

Desmosome
Actin
linked cell Hemidesmosome
matrix
From adheren junctions
junctions
 Desmosomes
• Structurally simillar to AJ but –
cadherins – linked to Ifs
• Their main function is to provide
mechanical strength
• Imp in vertebrates – but not in
DROSO
• Present in high amounts in tissues
that are subject to high levels of
mechanical stress – heart , skin
 Desmosomes
• Typically appear as
button like spots of
adhesion
• Inside the cell,
bundles of ropelike
intermediate
filaments are
anchored to form a
structural framework
for great tensile
strength
• Ifs
• Keratin – most epithelial cells
• Desmin – heart muscle cells
– pemphigus
Type Protein Size (kd) Site of expression
I Acidic keratins 40–60 Epithelial cells
(~15 proteins)
II Neutral or basic keratins 50–70 Epithelial cells
(~15 proteins)
III Vimentin 54 Fibroblasts, white blood
cells, and other cell types
Desmin 53 Muscle cells
Glial fibrillary acidic protein 51 Glial cells
Peripherin 57 Peripheral neurons
IV Neurofilament proteins
NF-L 67 Neurons
NF-M 150 Neurons
NF-H 200 Neurons
α-Internexin 66 Neurons
V Nuclear lamins 60–75 Nuclear lamina of all cell
types
VI Nestin 200 Stem cells of central
nervous system
 Cells-to-ECM attachments:
Focal adhesions and hemidesmosomes
Cytoskeletal fibers attach to transmembrane
receptors (integrins) that are attached to
extracellular matrix components
•Focal adhesions
•Hemidesmosomes
 RGD seq
Mg+2
Ca+2

• Integral membrane proteins

• 2 chains
• 17 alpha
• 8 beta
• Most cells possess a variety of integrins
 Functions
• Adhesion of cells to substratum
• Transmission of signals from the
external environment to cells interior
• Signals are transmitted by change in
conformation
• Motility, growth, and survival
• Primary cells- in suspension
• Malignant cells in suspension
Four functional classes of cell junctions in animal tissues
 Tight Junctions
separate apical from lateral plasma membrane

• Continuous belt around

circumference of cell

• Anchorage for terminal web

• adhesive contact between cells

• Control of paracellular
permeability. Transepithelial
resistance.
 TEM of tight junction
Tight junction
The tight junction forms a regulated barrier to paracellular
transport of solutes and ions. The barrier contains aqueous
channels capable of discriminating charge and size.

apical
Cell 1

Cell 2

139037
Tight Junctions
Tight seals
Tight junctions

• The TJ strands visible in EM

are composed mainly of the
proteins claudins and occludin.

• Claudins polymerize within the

plasma membranes to assemble
the backbone of the fibrils seen
in freeze fracture.
Claudins-occludins-ZO1
 Occludins
• An integral membrane protein that copolymerizes with the clauding polymers.
Occludin is an 60-kDa tetraspan membrane protein that forms two extracellular
loops separated by a short cytosolic loop, and both amino- and carboxy-terminal
domains are cytosolic
• The carboxy-terminal domain is rich in serine, threonine, and tyrosine residues,
which are targets for a number of protein and tyrosine kinases.
• The two extracellular loops have an unusual amino acid composition, consisting
in
• the first loop of a high content (61%) of tyrosine and glycine residues, whereas
the second loop is rich (18%) in tyrosine residues.
• Very few charged amino acids are present in either loop, and at neutral pH they
are predicted to have no net charge. Phosphorylated occludins, TEER
 Claudins
Claudin family (1-20). Small integral membrane proteins 20-24 kDa.
Might create the selective paracellular properties
Claudins, like occludin, are tetraspan proteins with relatively short cytoplasmic
amino and carboxy termini flanking a first extracellular loop of 53 amino acids and
a second shorter loop of 24 amino acids in length
The COOH terminus of all claudins, except claudin-12, ends in
YV, a site that binds to the PDZ domains of proteins including ZO-1, -2, and -3
 JAMs
• JAM-1. JAM-1, a 43-kDa glycosylated protein that belongscto the IgG superfamily, is
characterized by two extracellular V-type Ig domains, a single transmembrane domain,
and a short intracellular COOH terminus that has a PDZ binding motif that binds to the
PDZ3 domain of ZO-1
• JAM-1 forms homophilic contacts between the first V-type Ig domain of apposing
JAM-1 molecules
• It is expressed at both endothelial and epithelial TJs where it facilitates the migration
of monocytes through the paracellular pathway.
• Three additional family members, JAM-2 (8), JAM-3 (113), and JAM-4 (65), have
been identified.
• JAM-2 is expressed on endothelial cells, whereas JAM-3 on T cells functions as a
counter receptor, thereby facilitating lymphocyte migration between endothelial cells
• JAM-4 is localized to the renal glomerulus and intestinal epithelial cells.
 Zona occludins
• ZO-1, ZO-2, and ZO-3: all three bind claudins
• Provide structural support, scafold proteins
• Concentrated beneath the plasma membrane at TJs

• ZOs link TJs to actin-based cytoskeleton

• Belong to MAGUK family
– Membrane-associated guanlyate kinase proteins
– All MAGUK protein characterized by:
PDZ domains: post synaptic density protein (PSD95),
Drosophila disc large tumor suppressor (Dlg1), and zonula
occludens-1 protein (zo-1)
SH3 domain: Src homology 3. Mediates protein
interactions
GK domain: homologous to enzyme that catalyzes GMP
to GDP.
 Functional cross-talk between AJs and TJs.

• ZO-1 is almost exclusively localized to

tight junctions in kidney epithelial cells.

• In cells that are less-well polarized or in

stratified epithelial, ZO-1 is associated with
adhesions junctions.
Four functional classes of cell junctions in animal tissues
 Gap junctions

Gap junctions allow cells to exchange electrical

and/or chemical signals

Composed of proteins that form channels that allow

small molecules to pass.

Subunits of these channels are connexins that are

assembled together to make connexons. The
connexons from 2 cells join together to make a gap
junction.
Gap junctions
 SELECTINS

• The selectins are cell surface lectins

that mediate adhesion of white blood
Leukocytes cells to endothelial cells and
platelets.

Endothelial • They recognize fucosylated,

sialylated and in some cases sulfated
ligands expressed on glycoproteins
Platelets
• Physiologically important in
inflammation, immunological
responses, and homing of bone
marrow stem cells and lymphocytes.

• Role in atherosclerosis, ischemia-

reperfusion injury, inflammatory
diseases, and metastatic spreading of
some cancers
 The selectin family of glycoprotein adhesion molecules (P-
selectin, E-selectin, and L-selectin) has been implicated in the
pathogenesis of a number of inflammatory disease states. The
selectins modulate the early adhesive interactions between
circulating neutrophils and the endothelium.
 Cell adhesion is important in
. acute inflammation and

metastasis. Pg. 277

1. Walls of small vein (venule) respond to signals from damaged tissue, causing
activation of endothelial cells that are more adhesive to circulating neutrophils

2. Adhesion is mediated by transient display of P- and E-selectins on endothelial surface

3. Neutrophils bind to selectins, transendothelial migration is slowed to a “roll”.

4. Integrins on neutrophils become activated (role for platelet activating factor), tighter binding.
Integrins bind with high affinity to ICAMS (IgSF) on endothelial cell surface

5. Bound neutrophil undergoes dynamic shape changes (THINK ABOUT CELL

MOTILITY AND THE CYTOSKELTON) to pass through endothelial layer (extravasation)
 Regulation of connectivity

When might a cell want to alter its connections to

other cells?

How do cells alter these connections?

-alter the profile of cytoskeletal connections,
receptors, and extracellular matrix
-alter the binding affinity of receptors
-many are Ca2+ dependent
-many are affected by protein kinases
Summary
Summary