Extracellular Matrix,

Cell Junctions and Cell

Adhesions

Fundamentals for cells to

form tissue
Outline
‡ Extracellular Matrix
„ Heterophilic polysaccharides
‡ GAG chains in hyaluronon
‡ Proteoglycans
„ Fibrous proteins
‡ Collagen
‡ Elastin
‡ Fibronectin
‡ ECM receptor - Integrin
‡ Cell Junctions
„ Occluding junctions
„ Anchoring junctions
„ Communicating junctions
‡ Cell Adhesions
The Importance of ECM
‡ Secreted in many cases by cells as precursor molecules
‡ Significantly modified before assembly with other
components into functional polymers
‡ Formation is unidirectional, irreversible
‡ Polymers reconstituted in lab with components
extracted from ECM do not have all properties as when
assembled by cells
‡ ECM is also modified by cells as they proliferate,
differentiate, and migrate
‡ Cells continually interact with matrix
‡ Communication pathway
‡ ECM influences cell shape, fate and metabolism
The Extracellular Matrix
‡ Giant molecules: GAGs (glycosaminoglycans)
‡ Protein-carbohydrate complexes (proteoglycans)
‡ Structural proteins (collagens, etc.)
‡ Signal and functional molecules (fibronectin, elastin,
etc.)
Glycosaminoglycans (GAGs)
‡ Long, unbranched polysaccharide chains
composed of repeating sugar units
‡ 70-200 sugar residues long
‡ Highly negatively charged due to sulfate and
carboxyl groups
‡ One of two sugar residues in repeating
disaccharide is always an amino sugar
„ N-acetylglucosamine
„ N-acetylgalactosamine
GAGs

GAGs attract a great deal of water. Hydroxyl groups

form hydrogen bonds, and the many negative charges
attract clouds of cations (Na+) that induce an osmotic
movement of water. These hydrated gels resist
compression (useful for joints).
GAGs
‡ Four main groups of GAGs, distinguished by
sugar residues, type of linkage between
residues and number and location of
sulfate groups
„ Hyaluronic acid (HA)
„ Chondroitin sulfate and dermatan sulfate
„ Heparan sulfate and heparin
„ Keratan sulfate
Proteoglycans

‡ Except for HA, all GAG’s found linked to protein

‡ Usually easily distinguishable from glycoproteins by
nature and arrangement of sugar side chains
‡ Glycoproteins 1-60% carbohydrate by weight, 300, 000
Da or less
‡ Proteoglycans – up to 95% carbohydrate by weight – 3,
000, 000 Da or more
‡ Potential for limitless heterogeneity
‡ Can differ markedly in protein content, molecular size,
number and type of GAGs
‡ Very difficult to characterize and classify
Function of Proteoglycans
‡ Bind various secreted signaling molecules in vitro
‡ Form gels of varying pore size and charge density,
functioning as sieves to regulate traffic of molecules
and cells
‡ Difficult to determine arrangement in vivo since
highly water soluble and readily washed away
Hyaluronan
Very simple GAG, consisting of 10,000+ repeats of
glucuronic acid and N-acetylglucosamine

Spun directly from cell membranes by a surface enzyme

complex

Attracts water and fills spaces between cells with non-

compressible gel (found around joints)

Some cells secrete it to isolate themselves from other

cells (e.g. myoblasts). These cells can secrete
hyaluronidase to break it down, allowing contact
Hyaluronan

Very long
macromolecule
that hydrates and
fills enormous
volumes
Aggrecan
One of the largest macromolecules, consisting of a
core protein with GAGs attached to form a feather-
like appearance.
Aggrecan
An aggrecan core protein is very large but also binds
many (different) GAGs (shown in red)
Aggrecan aggregates
Each aggrecan can
be attached to a
hyaluronan backbone
to form an aggrecan
aggregate
Collagens
‡ Major scaffold proteins of ECM
‡ Family of proteins
‡ Most abundant protein in mammals, up to 30% of all
proteins
‡ Responsible for functional integrity of tissues such as
cartilage, skin, tendon
‡ 13 collagen types present in human tissues
‡ Synthesized as pro-collagen monomers (pro-α
collagen);,Peptide modified by hydroxylation and
glycosylation; Prior to secretion they self- assemble into
trimers; Upon secretion the trimers are processed by
proteolytic enzymes then assemble into fibrils
‡ High tensile strength, equivalent to steel when
compared on cross-sectional area, factor of three
greater on a per weight basis
Collagen alpha chains
are highly repetitive; Assembly of the
they almost always molecules is governed
have the sequence by the need to
Gly-X-Y, where X is prevent spontaneous
often Pro and Y is fibril assembly until
often HydroxyPro AFTER the molecules
leave the cell.
BUT this structure is Imagine collagen
only uniform for the fibers assembling
fibril-forming internally…it would
collagens; sheet- destroy the cell
forming collagens are
a bit different
Collagen
Collagen
Collagen fibers
Collagen proteins (trimers) are then cross-linked to
form collagen fibers (stiff, not very elastic)
Medical notes
‡ Defects in assembly of the molecules of the
extracellular matrix can be devastating
‡ One particularly nasty example is Osteogenesis
Imperfecta, a suite of syndromes in which bones are
poorly formed and break easily---this is a mutation in
a fiber-forming collagen (usually) in which the
molecules are kinked and do not easily form fibrils
‡ A less-severe form exists in which an
underproduction of collagen leads to rickets-like bone
bending
‡ The lethal form is due to loss of collagen production
or mutation in decorin protein
Medical notes
Shown here is type III
OI. Note the rickets-
like bending in the long
bones and the metal
rods inserted to brace
the bones.

OI is not limited to the

legs. The long bones of the
arm, and indeed ALL the
bones are susceptible to
numerous breaks. Again,
the bones are badly bent

Sadly, it is not uncommon for parents of OI children to be accused of chronic child

abuse and for social programs to attempt removal of the child. Doctors are usually
able to diagnose OI accurately…if they are allowed to examine the child.
The Basal Lamina
‡ The basal lamina is primarily made of a framework of
collagen IV
‡ Due to Collagen IV’s unique structure, it assembles
into sheets with connective extensions both above
and below the plane of the sheet (see figure next
slide)
‡ It can therefore form a multilayered structure that
is very strong
The Basal Lamina
The first level of
assembly involves
association of the
globular domains to form
dimers;
The triple helices are
then able to associate
(much like fibril-forming
collagens do) to form a
sheet;
Because of the hairpin
bends in the molecules,
extensions lie both above
and below the plane, and
can form a multilayered
network if necessary
The Basal Lamina
Note that “basal” laminae
Basal laminae have a
are not always basal; in
number of important
muscle, for example, the
functions, including:
cells are completely
surrounded by the stuff Barrier to movement of
molecules and/or cells
Tissue regeneration;
In epithelia we usually
basal lamina injury
find what we would
promotes cell migration
typically think of as a
into wounded region
basal lamina
Controlling localization of
molecules
But that’s not always the
Guidance of embryonic
case; in the kidney,
cells when migrating
glomerular epithelia are
found on either side of Sequestering signalling
what we might think of as molecules
a “central” lamina
Elastin
‡ Necessary for providing tissue with elasticity so that
they can recoil after transient stretch
‡ Extensibility that is five times that of elastic band with
same cross-sectional area
‡ Highly insoluble
‡ Elasticity driven by hydrophobic interactions, tendency
of hydrophobic segments to adopt a random coil
configuration following stretch
Elastin
‡ Abundant is tissues subjected to repetitive
deformation
„ Blood vessel wall
„ Alveolar septal interstices
„ Deep dermal layers
„ Elastic cartilage
‡ Amount varies depending on physical demands on
tissue – 30-75% of dry weight of tissue
Elastin
‡ Organized into three distinct morphological
forms
„ Elastic ligaments skin and lungs – fibers are small
and rope-like
„ In blood vessels – concentric sheets or lamellae
interconnected by fine elastic fibers
„ Cartilage – organize as trabecular network
Elastin

Proteins can provide

elastic properties in
many tissues (e.g.
elastin)
Fibronectin
Animals have a single fibronectin gene that can be
alternatively spiced into 40+ forms.

Different exons are able to bind different

proteins/GAGs (e.g. integrins, collagen, etc)

Fibronectin dimerises using 2 similar (not identical)

monomers
Fibronectin
ECM proteins, GAGs, and proteoglycans

Green-protein
Red-GAG
Control of ECM
Cells control the synthesis of ECM by altering gene
expression and co-translational import and secretion

They also control the degradation of the ECM by

secreting and activating/inactivating extracellular
enzymes.

Most important are a group of serine proteases called

matrix metalloproteinases
Receptors of the
Extracellular Matrix
‡ Integrins serve as membrane-associated receptors of
the cell matrix, allowing cells to detect and interact
with the matrix
‡ They also allow for the influence of the cytoskeleton
on the extracellular matrix and vice-versa
‡ They allow for control of cells and matrix based on
local conditions
Integrins
‡ Cell-cell and cell-ECM binding
‡ Dimeric proteins consisting of α and β
subunit, assembled non-covalently into active
dimer
‡ 15 α subunits, 8 β subunits identified,
assemble into 21 αβ combinations
Integrins
‡ The β1, β2 and β3 subclasses are most
commonly expressed integrins
‡ The β2 integrins are involved primarily in
cell-cell recognition
‡ The β1 and β3 integrins bind to numerous
proteins present in ECM
„ Collagen
„ Fibronectin
„ Vitronectin
„ vonWillebrand factor
„ Laminin
Integrins
‡ Morphological hallmarks of integrin binding
include
„ Spreading of cells
„ Extension of cellular membrane processes
„ Clustering of integrin receptors at sites of focal
contacts
„ Assembly of intracellular proteins at site of
clustered receptors
„ Interactions of transmembrane receptors with
intracellular cytoskeleton
Integrins
Integrins are dimeric
glycoproteins with a wide
variety of subunit structure
depending on cell type, etc.
The alpha chain binds divalent
cations; this implies that the
cell can regulate integrin
function by controlling local
ion concentration
Both chains bind to
extracellular matrix
components
Some integrins bind more than
one substrate; and the same
integrin can have different
binding properties in
different cells, implying
regulation via other cell
components
Their primary function is to align
the cell matrix according to the
alignment of the cytoskeleton of
the cells within the matrix
They also play an important role
in transient connections to the
matrix
Only one confirmed integrin
defect is known in humans, and
its major effect is that it
prevents neutrophils from
moving into tissues---thus
recurrent bacterial infections
are the main phenotype, as well
as an extreme neutrophil count.
Integrins
At a cellular level, this is how alignment occurs---the cells
orient as the matrix is laid down, providing a scaffold
whose structure is translated to the outside by integrin
binding

As shown in the next figure, integrins are regulated by cells. They can be
regulated by ion concentrations, signal molecules, and/or phosphorylation
events
ECM and Cells
‡ Complex chemically and physically crosslinked network
of proteins and glycosaminoglycans
‡ Organizes cells in space
‡ Provide with environmental signals to direct site-
specific cellular regulation
‡ Separate one tissue space from another
‡ Bidirectional interactions
‡ Cells accept information on environment from ECM
‡ Cells frequently remodel ECM
‡ Interactions based on binding of cell-surface proteins
and proteoglycans with matrix immobilized proteins
Cell junctions
Three types of cell junctions:
1. Occluding junctions: seal cells together into sheets
(forming an impermeable barrier)

2. Anchoring junctions: attach cells (and their

cytoskeleton) to other cells or extracellular matrix
(providing mechanical support)

3. Communicating junctions: allow exchange of

chemical/electrical information between cells
Occluding junctions

Example: Tight junctions

of intestinal epithelium
Tight junction

Each cell possesses integral membrane proteins that bind to similar

proteins in the adjacent, forming a continuous “weld”
What proteins are used?
‡ 2 proteins, occludin and claudin, are used extensively.
They are both 4-span transmembrane proteins and
form connections with each other across the
membrane
‡ The c-terminal cytosolic faces are bound to three
proteins (ZO-1, -2, -3) that mediate connections to
the cytoskeleton
Anchoring junctions
‡ Integral membrane proteins connect a cell’s
cytoskeleton to another cell or extracellular matrix
‡ Anchoring junctions are classified according to which
type of cytoskeletal element is being used
‡ In your book, 2 are shown: the adherens junction,
with actin association, and the
desmosome/hemidesmosome, with IF association
Anchoring junctions
Integral membrane proteins connect a cell’s cytoskeleton to
another cell or extracellular matrix

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
Cadherins
Cadherins in adherens junctions

Under the cell

membrane,
contractile fibers
of microfilaments
connect to cell
membrane proteins
called cadherins

They surround the

cell, forming a belt
Cadherins and Adherens Junctions

Cell to cell connections

are mediated by
cadherins.

These receptors extend

out from the cell, binding
to other cadherens
Adherens Junctions
These belts are, as you might guess, contractile, providing a critical
developmental function in folding of cell sheets (gastrulation, for
example)
Invertebrates have adherens junctions, but they also have
these septate junctions---like adhesion zones, they help
to hold cells together, are arranged in belts, and serve as
sites for actin attachment
Desmosome junctions
‡ Desmosomes are like rivets holding cells together
‡ However, another essential function is that they
evenly distribute and resist shear forces that would
otherwise tear a cell apart
‡ They come in 2 flavors: desmosomes that are cell-cell
connectors, and hemidesmosomes that connect cell-
lamina
Desmosomes
Cadherins can also form localized spot connections

Cadherins attach
to intermediate
filaments via
anchoring
proteins: a
desmosome
 Desmosome junctions
1. Cadherin proteins 3. A cytoplasmic plaque is
mediate the extracellular used as the spot weld; it
attachment. Numerous mediates the connection
types of cadherin between the extracellular
proteins are known, and cadherin and the cytosolic
we’ll look at their IFs (intermediate
structure later filaments)
2. IF attachments are 4. Other IF types may be
used to mediate the used; cardio-myocytes, for
cytoskeletal involvement; example, use desmin
these keratin filaments rather than keratin
may be imbedded in the
protein disc

Medical note: pemphigus

vulgaris, an autoimmune
diorder, results from
autoimmune attack of the
cadherin desmoglein in the
plaque, so epithelia fall
apart
Cells-to-ECM attachments:
Focal adhesions and hemidesmosomes

Cytoskeletal fibers attach to

transmembrane receptors
(integrins) that are attached
to extracellular matrix
components
‡ Focal adhesions use MF
‡ Hemidesmosomes use IF
Hemidesmosomes

Hemidesmosomes facilitate anchoring to a basal lamina. Consequently,

they look like desmosomes but have some important differences:
The extracellular anchors are integrins, not cadherins
The disks are only half-disks
The keratin fibers are unquestionably imbedded in the disc, making for a
better force resistor; but they can still break (blisters, anyone?)
Communication junctions
‡ Communication junctions allow molecules of
specific sizes across the lipid bilayer in
adjacent cells
‡ The basic unit us called a connexon
‡ The size of molecule being passed across can
apparently be regulated; but there is an upper
size limit
‡ Gap Junction
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
Since the pore of a connexon is aqueous, charged
molecules are able to pass across. Noting the size cutoff,
it is clear that proteins are largely excluded, leaving ions
and small signal molecules as the main travelers across the
pore
Gap junctions
Gap junctions
Each connexon contains six (identical?) subunits called connexins that
form a tube or channel bridging the two cell membranes by connecting
two connexons together
Medical note 1:
numerous human genetic
syndromes have been
linked to mutations in
one or another connexin;
Gap junction properties for example,
differ in different erythrokeratodermia is
tissues---this is thought caused by mutation in
to be due to the use of connexin GJB3 and
different connexin results in sharply
defined but “migrating”
proteins expressed in
patches of red,
different tissues hyperkeratinized skin
Medical note 2: another connexin used in the inner ear results in deafness when
defective; the dominant form of the disease causes a nonfunctional connexin to
bind with normal copies, thus producing a nonfunctional connexon
Current views on control of permeability focus on Ca 2+ and pH in
the cell; locally high Ca2+ or low pH may close the connexon, while
the opposite conditions open it up

Since pH can be tightly modulated, this could explain how a connexin

might “partly open” and allow smaller molecules through than it
otherwise would.
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
Cell Adhesion
‡ Two major types of interactions are considered here
‡ Calcium-dependent mechanisms mediated through
cadherins, catenins, actin
‡ Calcium independent mechanisms mediated by CAM
proteins and fasciculins
‡ Cell adhesion is a very dynamic process; adhesion may
change according to the developmental regime and
final developmental identity and location of the cells
involved
Cell Adhesion
Here’s an example of that kind of
change.
Neural crest cells loosely
aggregate at the top the neural
tube, having dropped down from
the ectoderm
They then break connection,
migrate below the neural tube, and
reaggregate, sticking to each other
Only then does differentiation
occur
Cell Adhesion
Dissociated retinal cells from a vertebrate embryo (mouse, probably, but at
this stage the terrestrial verts all look alike) will aggregate with each other
but not with liver cells

…and liver cells will aggregate

with each other but not with
retinal cells.
Not only are these cells
functionally distinct, they are
genetically so, in the sense
that they express different
cell adhesion molecules that do
not interact with each other
Cell Adhesion

These are the three basic mechanisms that could be

used by molecules facilitating cell adhesion processes.
The different molecules I’ll cover in the next several
slides will use one or another of these mechanisms

These 2 are common mechanisms in verts This is rare in verts

Cell Adhesion Receptors
‡ Four major classes of adhesion receptors
„ Three (cadherins, selectins, CAMs) involved
primarily in cell-cell adhesion
„ One (integrins) cell-cell adhesion + cell ECM
adhesion
Cadherins
As seen here, the Medical note 1: Presenilin, a component
protein contains of the cadherin/catenin complex in the
several Ca2+ binding brain, when mutant results in early-
onset familial Alzheimer’s disease---
sites; without Ca2+ ,
neurons lose touch with each other and
the protein is targeted become more susceptible to
for degradation, so the programmed cell death pathways when
Ca2+ is pretty critical excess amyloid protein present
Cadherins associate via
catenins and/or
Medical note 2: loss of function of
desmosomal proteins, cadherins is an important step in
depending on the producing metastatic cancers in many
functional fate of the tissues, including adipocytes (in flies
molecule and the and likely humans), gastric epithelia,
junction it is involved in colon epithelia, etc. Inherited
mutations have many effects
The “X factor” is an depending on tissue the protein is
adaptor protein linking used in (Usher syndrome type ID,
the cadherins to the resulting in deafness and early retinal
actin cytoskeleton cancer, is a cadherin defect)
Cell Adhesion

This figure clearly shows why cytoskeletal attachment in cell adhesion is so

important; without attachment, the cells pull away, but with attachment, the
cells adhere