Topic

26

Cell interactions II-cell-matrix interactions

Connective tissue
Connective tissue contains a variety of cells that contribute to the production of the extracellular matrix.
The predominant cell type that produces extracellular matrix in connective tissue are fibroblasts.

In specialized tissues, like cartilage and bone,

cells from the fibroblast family make special
matrix molecules: chondroblasts make
cartilage and osteoblasts make bone

Extracellular matrix
The extracellular matrix in connective tissue is
made from the two classes of macromolecules:
1.Glycosaminoglycan polysaccharide chains,
linked to proteins-called proteoglycans
2.Fibrous proteins such as collagen

Fibroblasts in connective tissue

Extracellular matrix of collagen fibrils can be seen

Extracellular matrix molecules

Molecules of extracellular matrix:
1.Glycoproteins-laminin, fibronectin and nidogen
2.Proteoglycans-perlecan, hyaluronan,aggrecan
3.Fibrous Proteins-collagen

Green-protein
Red-glycosaminoglycan

Glycosaminoglycans (GAGs)
1. Glycosaminoglycans are unbranched polysaccharide chains composed of repeating disaccharide units
2. One of the two sugars is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), these are frequently
sulfated making the molecule highly negatively charged
3. The second sugar is usually a uronic acid (glucuronic or iduronic)

There are four main GAGs:

A.Hyaluronan
B.Chondroitin sulfate and
dermatan sulfate
C.Heparan sulfate
D.Keratan sulfate

GAGs
Polysaccharide chains are too stiff to fold and are strongly hydrophillic
Thus, GAGs tend to form highly extended conformations that occupy a huge volume
relative to their mass and form gels at even low concentrations
Their negative charge attracts cations like Na+ which tends to cause large amounts of
water to be sucked into the tissue
This attraction of water causes swelling or tugor that enables the matrix to withstand
compressive forces, an example is the cartilage in the knee joint

In plants, cellulose is packed into ribbon arrays that form the cell walls
In insects, crustaceans and other arthropods, chitin forms the main
component of the exoskeleton

Hyaluronan
Hyaluronan is the simplest of the GAGs:
1.It consists of a repeating sequence of up to 25,000 disaccharide units
2.Unlike many GAGs it contains no sulfated sugars, has identical repeating sugars and is not linked covalently to any proteins
3.Also, unlike other GAGs which are synthesized in cells and released by exocytosis, hyaluronan is synthesized by a complex
embedded in the membrane
4.Hyaluronan is degraded by the enzyme hyaluronidase
5.It is produced in wound healing and is important in joint fluid where it acts as a lubricant

Proteoglycans
Most GAGs are linked to proteins, called proteoglycans
1.The proteins are made by membrane bound ribosomes and then assembled onto polysaccharide chains in the Golgi
2.This begins initially by attaching a linkage tetrasaccharide to a serine side chain followed by the addition of sugars one at a
time by glycosyl transferase
3.Proteoglycans can contain 95% carbohydrate by weight and have about 80 sugars, in contrast glycoproteins are usually less
than 50% carbohydrate by weight and have short branched sugar trees

Proteoglycans
Compared to proteins, proteoglycans are huge:
Aggrecan has a mass of 3 x 106 daltons and has over 100 GAG chains

Proteoglycans
Molecules of aggrecan assemble with hyaluronan in cartilage to form aggregates as big as bacterium!

Proteoglycans
Proteoglycans play a role in chemical signaling between cells:

They bind secreted signal molecules, such as growth factors, and control
their diffusion through the matrix. For example: the heparan sulfate chains on
proteoglycans bind fibroblast growth factors (FGFs) which stimulates a variety
of cells to proliferate. The binding induces oligomerization which enables them
to bind and activate transmembrane tyrosine kinases
In inflammatory responses, heparan sulfate proteoglycans immobilize
chemotactic attractants, called cytokines, on the endothelial cell surface of a
blood vessel
TGF and VEGF also bind to proteins in the matrix

Proteoglycans

Collagen
Collagens are fibrous proteins that are components of the
extracellular matrix
They are secreted by connective tissue cells and are a major
component of skin and bone
They are the most abundant protein in mammals where they
constitute 25% of the total protein mass

Collagen is a long, stiff, triple stranded helix of three collagen polypeptide

chains (called a-chains)
The protein is rich in proline and glycine; proline stabilizes the helix and
glycine, spaced at every third residue, has the smallest side chain which
allows tight packing of the helices

Collagens in the Extracellular Matrix

A fibroblast surrounded
by collagen fibrils in the
connective tissue of
embryonic chick skin.
The fibrils are organized
into bundles that run
approximately at right
angles to one another.
The collagen fibrils are
produced by fibroblasts.

Collagen
The human genome encodes 42 different collagen genes resulting in the formation
of many different collagens:
1.The most common is type I-found in skin and bone, belongs to the class of
fibrillar collagens or fibril forming collagens that are long ropelike structures
with few or no interruptions. They assemble into higher order polymers called
collagen fibrils that often aggregate even further to form cable like bundles
called collagen fibers
2.Types IX and XII are fibril associated collagens because they cover collagen
fibrils
3.Type IV is a network-forming collagen, a major component of basal laminae
4.Type VII form dimers that form anchoring fibrils that help attach basal lamina
to connective tissue

Collagen

Collagens undergo post-translational modifications

Collagens are injected into the ER lumen as precursors called pro- chains, they have additional amino acids at both their N
and C- termini called propeptides that are clipped off.
1. In addition, some of the lysines and prolines are hydroxylated to form hydroxylysine and hydroxyproline which are thought
to stabilize the triple helix
2. These are found infrequently in other proteins. Conditions that prevent proline hydroxylation, like deficiency in vitamin C,
causes scurvy which is characterized by the formation of defective chains that are rapidly degraded. Within a few months,
loss of preexisting collagen causes fragile blood vessels, loose teeth and lack of wound healing

Collagen crosslinking
In addition to hydroxylation, collagen fibers are cross linked to each other by covalent cross links between lysines
on collagen molecules.
These cross links create higher order structures called fibrils and greatly strengthen the fibrils, the extent of cross
linking varies from tissue to tissue.

Steps of collagen fibril formation

Secreted Fibril-Associated Collagens

Collagen fibrils in the tadpole skin.

1.This electron micrograph shows the
arrangement of the fibrils.
2.This organization is also found in
mature bone and in the cornea.

Collagen fibrils
The formation of collagen fibrils is a complex process, cells secrete other molecules that influence the organization of these
structures. These include the fibrous protein, fibronectin, which helps guide the organization and fibril associated collagens.
Fibril associated collagens, include Type IX and Type XII, differ from fibrillar collagens in several ways:
1.Their triple stranded structure is interrupted by short non-helical domains that make them more flexible
2.They are not cleaved after secretion and therefore retain their propeptides
3.The do not aggregate to form fibrils, instead they bind fibrils formed by fibrillar collagens.
They mediate interactions of collagen fibrils with one another and with other matrix molecules to help organize the fibrils
in the matrix

Type IX collagen
bound to type II
fibrils

Cells Help Organize the Collagen Fibrils They

Secrete by Exerting Tension on the Matrix
The shaping of the extracellular
matrix by cells.
This micrograph shows a region
between two pieces of embryonic
chick heart (rich in fibroblasts as
well as heart muscle cells) that
were cultured on a collagen gel for
4 days.
A dense tract of aligned collagen
fibers has formed between the
explants, presumably as a result of
the fibroblasts in the explants
tugging on the collagen.

Elastin
Many tissues need to be strong and elastic in order to function. This is accomplished by the formation of elastic fibers.
The main component of elastic fibers is elastin, a hydrophobic protein that is rich in proline and glycine
Unlike collagens, elastin is not glycosylated and does not contain hydroxylysine
It is synthesized as tropoelastin that is secreted and assembled into elastic fibers close to the plasma membrane
The individual molecules become highly cross linked to generate an extensive network of fibers and sheets.

An aorta segment
showing elastic fibers

Elastin stretching
Elastin protein is composed of short segments that alternate along the polypeptide chain-hydrophobic segments which are
responsible for the elastic properties of the molecule, and alanine-lysine rich -helical segments which form cross links between
molecules

It is uncertain how the conformation

of elastin in fibers leads to elasticity
Models suggest that parts of the
polypeptide chain are in a random
coil configuration that allows the
entire grid to stretch and recoil

Fibronectin and Other Multidomain

Glycoproteins Help Organize the Matrix
Complex glycoproteins of the extracellular matrix.
1.Many matrix glycoproteins are large scaffold
proteins containing multiple copies of specific
protein-interaction domains.
2.Each domain is folded into a discrete globular
structure, and many such domains are arrayed
along the protein like beads on a string.
Each protein contains multiple repeat domains:
Fibronectin, for example, contains numerous
copies of three different fibronectin repeats (types
IIII, labeled here as FN1, FN2, and FN3).
The scaffolding and regulatory functions of many
matrix proteins are further expanded by assembly
into multimeric forms, as shown at the right.

Fibronectin
The matrix contains a number of noncollagen proteins that have multiple domains involved in binding other matrix
molecules, cell surface receptors and themselves
These molecules help organize the matrix, help cell attach to it and help guide cells to certain locations
One such protein is fibronectin, which is a large glycoprotein that exists as a dimer joined by disulfide bonds on one end
Humans have only one gene but it contains 50 exons and thus forms many different fibronectin isoforms

Each isoform is made from bringing in different exons

that encode different binding regions
Within those regions are subdomains that have
specific binding capabilities (shown in colors)

Fibronectin
Fibronectin can exist in two forms: a soluble form that can circulate in the blood stream and as insoluble fibronectin fibrils,
in which dimers are crosslinked to each other to form part of the matrix
Fibronectin does not spontaneous form fibrils, it only assembles into fibrils on the cell surface and only where fibronectin
binding proteins, like integrins, are
When this binding occurs, the attachment to the cytoskeleton via integrins provides tension on the fibronectin revealing
binding sites that allow fibril formation:
Attachment points to integrins
and the cytoskeleton

Revealed fibril forming domain

Fibronectin
The region used by fibronectin to bind to integrins, and thus cells, has be
identified as a specific tripeptide (Arg-Gly-Asp, or RGD)
Called the RGD sequence, it can compete with fibronectin for binding to cells
and inhibit the attachment of cells to a fibronectin matrix
Cells will also stick to a surface that has this peptide attached to it

Fibronectin
Front end of a migrating fibroblast
Green-fibronectin
Red-actin

Fibronectin initially appears as small dots near the leading

edge associated with focal adhesions
It becomes associated with actin filaments as tension
causes the exposure of the fibril forming domains

Organization of basal lamina

The basal lamina or basement membrane, is extracellular matrix that consists of a thin, tough, flexible sheet of matrix
molecules and is an essential foundation of all epithelia
It is found in all multicellular animals, is typically 40-120 nm thick and plays many roles including providing structural
support, filtering, determining cell polarity, organizing proteins in adjacent plasma membrane, promoting cell survival,
proliferation and differentiation

Organization of basal lamina

The basal lamina is synthesized by the cells
lying on either side of it:
Epithelial cells
Underlying connective tissue (stroma)
Consists of two main classes of extracellular
molecules-
1. Fibrous proteins (glycoproteins)
2. Polysaccharide chains
(glycosaminoglycans or GAGs), linked
to proteins. Called proteoglycans.

Structure of laminin
1. Laminin is thought to be the primary organizer of the sheet structure; Laminin-1 consists of three long
polypeptide chains (, and ) held together by disulfide bonds.
2. The molecules twist around each other for part of the length and the N-terminal heads remain separate.
3. These molecules can self assemble via interactions between the head groups.
4. There are several isoforms of each subunit so they can associate in a variety of different combinations

Type IV collagen
1.The second major component of the basal
lamina is type iv collagen
2.Type IV collagen consists of three protein chains
that twist together to form a ropelike helix:

1. The collagen molecules interact via their terminal domains to assemble

extracellularly into a flexible, highly cross linked, structure.
2. This structure provides tensile strength to the basal lamina
Both end and lateral
binding occur

Molecular structure of the basal lamina

1. Laminin molecules generate the initial sheet structure by joining to each other while bound to receptors on the cells that
produced them
2. These receptors are often from the integrin family or another called dystroglycan
3. Then the laminin heads interact to form a two-dimensional network
4. The perlecan and nidogen molecules then
serve as linkers to connect the two meshworks
together: the laminin and collagen type IV

Cell interactions with the matrix

Integrins
Matrix receptors link information from inside the cell to the
extracellular matrix
These consist of several different types of molecules but the
principle receptors on animal cells are the integrins
Integrins can transmit signals in both directions across the
plasma membrane:
1. Binding to matrix components on one side of the
membrane can relay a signal to the interior of the cell
2. Interior conditions can signal to regulate the binding of
the integrin to the matrix and thus the cell to the matrix

Integrins
There are at least 24 different integrins in humans but they all
have conserved features:
Two non-covalently associated glycoprotein subunits called
and
Both subunits span the cell membrane with short
intracellular C-terminal tails and large N-terminal extracellular
domains
The extracellular domains bind specific sequences in matrix
proteins like laminin or fibronectin or ligands on other cells
The intracellular domain binds complexes that link to the
cytoskeleton-usually this is a linkage from talin to the actin
cytoskeleton

These structures can either be transient or large and durable. The latter type is called a focal adhesion

Integrins at hemidesmosomes
In epithelia the most prominent cell-matrix attachments are hemidesmosomes
They incorporate a special integrin, 64, that is attached to keratin filaments inside the cell instead of actin:

Integrin activation
Integrins can switch between an active and an inactive state, necessary in cells that are moving
on a solid surface
The attachment points must be broken and reformed and be coupled to the assembly and
disassembly of the cytoskeleton
The molecular basis for this switch is allosteric regulation: binding or detaching from ligand on
one side of the molecule causes structural changes that are transmitted across the membrane to
the other end of the molecule

Regulation of integrin ligand binding

The allosteric changes can be seen using high resolution electron microscopy:
In the absence of binding extracellular matrix, the dimer is compact and v-shaped
After addition of a peptide containing the recognition sequence for the integrin, the dimer opens up
Further study has shown that this opening causes an increase in binding to the matrix protein as well as to the intercellular
protein, talin

This is called outside-in activation

Integrin activation by signaling

The chain of cause can also happen in reverse from inside to outside-talin binding to an inactive dimer causes the two
subunits to spring apart and drives the extracellular portion into the active state
This is called inside-out activation and is triggered by intracellular regulatory molecules that are produced in response to
receptor signaling like G-protein coupled receptors and receptor tyrosine kinases

Talin is a tension sensor at cellmatrix junctions

Tension across cellmatrix junctions stimulates the local recruitment of vinculin and other actin-regulatory
proteins, thereby strengthening the junctions attachment to the cytoskeleton.
(a)The C-terminal region of talin is divided into a series of folded
domains, some of which contain vinculin-binding sites (dark green
lines) that are thought to be hidden and therefore inaccessible.
(b)This experiment tested the hypothesis that tension stretches the 12helix domain, thereby exposing vinculin-binding sites:
1.A fragment of talin containing this domain was attached to an
apparatus in which the domain could be stretched, as shown here.
2.The fragment was labeled at its N-terminus with a tag that sticks to
the surface of a glass slide on a microscope stage. the C-terminal
end of the fragment was bound to a tiny magnetic bead, so the talin
fragment could be stretched using a small magnetic electrode.
3.The solution around the protein contained fluorescently tagged
vinculin proteins so that after the talin protein was stretched, and
excess vinculin solution was washed away, a microscope was used
to determine if any fluorescent vinculin proteins were bound to the
talin protein.
4.In the absence of stretching (top), most talin molecules did not bind
vinculin. When the protein was stretched (bottom), two or three
vinculin molecules were bound (only one is shown here for clarity).

Integrins

Integrins
Integrins bind with low affinity and depend on the formation of integrin clusters to provide
effective adhesion
Like other cell adhesion molecules this strategy makes the formation and destruction of the
adhesion plaques easier
Stable adhesions, focal adhesions, are maintained by the activation of Rho GTPases that
promote recruitment of actin filaments and integrins

Matrix attachments regulate cell survival

Integrins activate cellular pathways-cells in culture will not grow unless the are attached to extra-cellular matrix
For some cells, cell survival also requires attachments-when they lose attachment they undergo apoptosis. This dependence
is called anchorage dependence

Cells also need to spread on the

matrix for survival-cells that have
multiple adhesions at widely
separate sites survive better than
cells that have few such
attachment sites

Focal adhesions
One of the best studied models for integrin signaling involves a cytoplasmic protein tyrosine kinase called focal adhesion
kinase or FAK.
Focal adhesions are prominent sites of tyrosine phosphorylation and FAK is localized to these sites.
FAK is recruited by talin or paxillin (binds -integrin) to sites of integrin mediated adhesion
Cross phosphorylation results in the formation of phosphotyrosine binding sites which recruits Src family kinases that bind
those sites
These kinases further phosphorylate FAK which, in turn creates more binding sites that effectively recruit other intracellular
signaling proteins

Actin-green
Phosphotyrosine-Red

Focal adhesions
Loss of FAK in mice results in an increase in the number of focal adhesions
In fact the cells make too many focal adhesions and thus cell spreading and migration are extremely slow
This suggests that FAK is involved in the disassembly of focal adhesions
Cancer cells often have elevated levels of FAK which may explain why they are frequently more motile that their normal
counterparts

Staining for vinculin to reveal

the location of focal adhesions

Adhesion families

Matrix proteases
Cells also have mechanisms to destroy the matrix. This is essential in tissue repair, bone remodeling and other
processes
For individual cells, destroying the matrix is important in cell division as well as cell migration
Matrix degradation is mediated by proteases that are locally produced to degrade the matrix
These proteases are either matrix metalloproteases, which depend on Ca2+ or Zn2+ for activity, or serine proteases
Many of these proteases are substrate specific; collagenases, for example, only digest collagen. Others are not as
specific but are tethered to the region where they are needed
As a group, these activities must be regulated and there are three basic mechanisms in place:
Local activation-precursor are secreted locally and only activated when needed
Confinement by cell-surface receptors-receptors bind and localize proteases to the desired region
Secretion of inhibitors-protease specific inhibitors are secreted except where matrix degradation is needed

Summary
Connective tissue
Cells in connective tissue are imbedded in a matrix that binds cells together and influences survival,
development, shape, polarity and migration
The matrix consist of various protein fibers interwoven in a network of glycosaminoglycan chains
(GAGs)
GAGs are negatively charged polysaccharide chains that are frequently covalently linked to proteins
to form proteoglycans
The negative charges attract counterions that draw water into the matrix causing it to swell
Fiber forming proteins give the matrix strength and resiliance
They form structures to anchor cells via large glycoproteins like laminin and fibronectin that bind
integrins on the cell surface
Elasticity is provided by elastin which forms a network that can stretch and recoil
Matrix components are degraded by extracellular proteases whose activities are regulated by
various mechanisms

Summary
Basal lamina
1.The basal lamina is a tough, thin sheet of extracellular matrix that lies near epithelia
2.It is organized on a framework of laminin molecules linked together and to cells by binding to integrins and
other receptors
3.Type IV collagen molecules are recruited to this structure to assemble into a sheetlike mesh
4.Basal laminae provide mechanical support for epithelia and form an interface between epithelia and
connective tissue
Integrins
1.Integrins are receptors used by animal cells to bind to the extracellular matrix
2.They are transmembrane linkers between the extracellular space and the cytoskeleton
3.They are heterodimers and dramatically change conformation upon binding ligand
4.This binding creates an allosteric change in the receptor that conveys signals both inside to out and from
outside to in
5.Many processes are regulated by assembly of complexes on integrins including: proliferation, survival,
anchorage dependence, cell polarity and migration guidance

Questions?

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Cellular Pathologies-Cancer