Transcript 08 cell adhesion

Molecular control of gastrulation and morphogenesis
Does each cell in the blastula have detailed
instructions in the DNA that tell it exactly
where to go during gastrulation?
If an embryo is disaggregated into individual
cells, each should know exactly where to go
to reform a new embryo.
Molecular control of gastrulation and morphogenesis
Does each cell in the blastula have detailed
instructions in the DNA that tell it exactly
where to go during gastrulation?
If an embryo is disaggregated into individual
cells, each should know exactly where to go
to reform a new embryo.
When this experiment is performed, a degree
of reorganization occurs, but it is not
complete. Embryoids: are slightly similar to
embryos but they lack the real organization.
Conclusions:
1. Genes impart only partial instructions for
assembly of the embryo
2. Like cells all stick together, revealing
distinct adhesive properties.
3. The relative positions of aggregates reflect
the relative positions in the embryo (skin
outside, heart inside).
Cell adhesion is the driving force in gastrulation
When cells from an embryo are disaggregated and recombined, they can be
readily ranked according to their ability to form the central portion.
(chondrocytes > heart cells > liver cells is the hierarchical order)
Why?
Cell adhesion is the driving force in gastrulation
When cells from an embryo are disaggregated and recombined, they can be
readily ranked according to their ability to form the central portion.
(chondrocytes > heart cells > liver cells is the hierarchical order)
Why?
Differential adhesion hypothesis: the cell type with
maximal adhesiveness (chondrocytes) will form a
core that is surrounded by concentric spheres of
cells with progressively lower adhesiveness.
Cell adhesion can be measured by the ‘pancake
test’. When aggregates of different cell types are
subjected to a flattening force (centrifugation to
induce a centrifugal force), the cells that adhere
most tightly form a ball, while those that adhere
more loosely form a flatter, pancake structure.
Cell adhesion is a major factor that regulates
aggregation of like cells and controls position
during morphogenesis.
What regulates how tightly or loosely cells attach?
Cells adhere by cell junctions, cell adhesion molecules,
or substrate adhesion molecules
Cell junctions: large, complex structures that form slowly but generate very
strong and durable connections (tight junctions, desmosomes, and gap
junctions).
Cell adhesion molecules (CAMs): single molecules that traverse cell
membranes and allow cells to adhere to one another. Adhesions form
quickly, they are selective, but they are relatively weak in comparison to cell
junctions.
Substrate adhesion molecules (SAMs): a group that consists of
extracellular matrix molecules and matched receptors that are expressed
on the cell surface.
3 types of specialized attachments hold embryo cells together
Tight junctions are regions where membranes of adjacent cells actually fuse.
They encircle the whole cell and provide a barrier for leakage between cells
(common in the GI tract to prevent leakage of food out of the gut). The also
prevent membrane proteins from moving freely from apical to basal regions.
Desmosomes are spot rivets that
weld cells together. A cytoplasmic
plaque is connected by inter
cellular protein filaments, and the
plaques are connected to the
intermediate filaments within the
cytoplasm (common in cells that
are stretched a lot such as skin).
Gap junctions are small channels
between cells that allow for
intercellular communication. They
are found in smooth muscle and
allow signals (small ions) to
spread between cells so that all
muscle cells contract as one.
CAMs firmly anchor adjacent cells to the cytoskeleton
Cell adhesion molecules (CAMs) are glycoproteins with 3 major domains:
The extracellular domain allows one CAM to bind to another on an adjacent
cell. The binding can be to the same type of cell (homotypic) or to a
different cell type (heterotypic).
The transmembrane domain links the CAM to the plasma membrane
through hydrophobic forces.
The cytoplasmic domain is directly connected to the cytoskeleton by linker
proteins. This anchoring is important to prevent lateral diffusion of
adhesion molecules in the membrane.
Three major
types of CAMs
are immuno
globulin-like
CAMs, cadherins,
and lectins.
Neural cell adhesion molecule is typical of
immunoglobulin (IgG)-like CAMs
N-CAM was one of the first to be discovered.
The extracellular domain has IgG like repeats that are thought to allow
binding to other N-CAMs by interdigitation between loops. Insects have IgG
CAMs but no IgG. Thus, IgGs may have evolved from IgG-like CAMs.
Polysialic acid region (PSA): 3 long
carbohydrate chains with negative
charge are attached to the 5th loop. The
overall charge varies on different NCAMs. Large PSA regions induce a large
negative charge which repels cells
(embryonic cells during gastrulation).
Small PSA regions allow attachment due
to low charges, and these are common
on adult cells.
N-CAMs have isoforms: one N-CAM
gene can generate over 100 different
molecules by post transcriptional and
post translational modification. The
most common are 120, 140, and 180 kD.
Cadherins mediate calcium-dependent cell adhesion
Cadherins are the most prevalent CAMs in vertebrates. They are rapidly
degraded by proteases in the absence of Ca++. There are 4 major types:
E cadherins in epithelial cells
P cadherins in placenta
N cadherins in neural tissue
L cadherins in liver
Each associates with its own type.
125 kD transmembrane glycoproteins that
bind homotypically using the first 113 AA
The differences in cadherin expression are
responsible for the differential adhesiveness
seen in disaggregated tissue. Cells that
express more cadherin = tissues that form a
ball in the center of cell aggregates.
Catenins are proteins that link cadherins to
the cytoskeleton. If this linkage is disturbed,
cadherins do not work, and embryonic
development is disrupted (disrupt catenins in
neural tissue = brain forms improperly).
Lectins bind heterotypically to sugars on the cell surface
Lectins are the third group of CAMs.
They bind weakly and heterotypically
to oligosaccharides of many types
through the large extracellular
domain.
Selectins are lectins that are
expressed in endothelial cells.
Glycosyltransferases are lectinrelated CAMs. They are enzymes that
transfer monosaccharides to an oligo
saccharide chain on an adjacent cell.
In the absence of monosaccharides,
the enzyme links one cell to another
by binding to the oligosaccharide
chain (can’t let go). In the presence
of mono saccharides, the binding is
lost. A simple way to regulate cell
adhesion by mono saccharides.
Substrate adhesion molecules (SAMs) and the
extracellular matrix (ECM)
Spaces between cells are filled
with ECM that consists of:
1. Amorphous ground substance:
a gel-like material that absorbs
water.
2. Meshwork of fibers that
reinforce the ground substance.
The ECM influences cell migration,
cell shape, cell gene expression,
and cell differentiation.
Mesenchymal cells are surrounded
by a diffuse ECM. Epithelial cells
rest on a dense sheet of ECM
called the basement membrane.
The ECM is actively secreted by the cells living there. What molecules
compose the ECM?
Glycosaminoglycans and proteoglycans
form the amorphous ground substance
Glycosaminoglycan: long unbranched polysaccharide chains composed of
repeating units of disaccharides. One sugar is an amino sugar (n-acetyl
glucosamine) and the other is a uronic acid (glucuronic acid). The most
abundant glycosaminoglycans are hyaluronic acid, chondroitin sulfate,
heparin, and heparin sulfate.
Proteoglycans: glycosaminoglycans are covalently linked to core proteins
The core proteins have have many side chains of glycosaminoglycans.
They attract Na+ and water and expand to form gels that occupy space
between cells. They also bind and selectively release growth factors.
Glycosaminoglycan (hyaluronic acid)
Proteoglycan
Fibrous glycoproteins make up the meshwork of the ECM
Proteoglycans are carbohydrate with some protein (>50% carbohydrate).
Glycoproteins are proteins with some carbohydrate attached (> 50% protein).
ECM consists of 3 major fibrous glycoproteins:
Collagen: the most abundant protein in mammals (>25% total protein). There
are numerous genes that encode different collagen molecules. Collagen
forms very strong fibers that are abundant in bones, skin, and connective
tissue.
Fibronectin: a fibrous protein that has binding sites for cells and other ECM
proteins. It links cells to the ECM. The RGD sequence of fibronectin
(argenine, glycine, aspartate) binds to cells avidly. Fibronectin is important
for motility.
Laminins: abundant in basement membranes where they promote adhesion
to many types of cells.
How do cells stick to fibrous proteins of the ECM?
Integrins mediate adhesion to ECM
Integrins are a family of
transmembrane glycoproteins that
are composed of 2 chains, a and b.
There are 40 different types of a
chains and 8 types of b chains that
can combine to form a large number
of different integrin molecules.
The a chain has binding sites for
Ca++ and Mg++ which are needed for
integrins to adhere. The 2 subunits
form the site that binds to the RGD
domain on ECM.
The cytoplasmic tail of integrins is
connected to a linker protein that
connects to the cytoskeleton. A
bridge from ECM to cytoskeleton.
Cell surface proteoglycans also link cells to ECM
These molecules often have an
extracellular and intracellular domain.
Proteoglycans consist of
glycosaminoglycans linked to a core
protein in the extracellular domain.
These interact with collagen,
fibronectin, and other ECM
molecules.
Proteoglycans can be released from
the cell by cleavage at protease
sensitive sites.
Syndecan is a common proteoglycan
Proteoglycans and integrins have
another important function:
They are receptors that transduce
signals from the extracellular matrix
to the nucleus.
If CAMs and SAMs were important for gastrulation, their
expression might reflect that fact.
1.
They might be expressed selectively on specific cells of the blastula
that are destined to migrate to selected areas to form organs.
2.
Expression might change as cells left the surface and ingressed or
migrated into the blastocoel.
3.
Blocking expression of CAMs or SAMs might interfere with the normal
process of gastrulation.
CAM expression during gastrulation is correlated with cell fate
Fate map: it is possible to predict which parts of the blastula will develop into
specific structures after gastrulation.
Expression map of CAMs: it is possible to localize expression of CAMs using
in situ hybridization and immunostaining of the blastula.
Cells with different fates express different CAMs. Cells destined to become
neural tissue express high levels of N-CAM. Cells destined for epidermis
express E-cadherin.
The respective cell adhesion
molecules are expressed
before the cells actively start
to form the adult tissue. This
suggests that CAM expression
is important in fate
determination.
During gastrulation, cells go
where their CAMs lead them
If CAMs and SAMs were important for gastrulation, their
expression might reflect that fact.
1.
They might be expressed selectively on specific cells of the blastula
that are destined to migrate to selected areas to form organs.
2.
Expression might change as cells left the surface and ingressed or
migrated into the blastocoel (changes in CAM drive movement).
3.
Blocking expression of CAMs or SAMs might interfere with the normal
process of gastrulation.
Changes in cell adhesion are important for gastrulation
Gastrulation in the sea urchin is initiated by specific changes in cell
adhesion. One of the first steps is ingression of mesenchymal cells from
the vegetal plate into the blastocoel to form the skeleton of spicules.
The mesenchymal cells lose their adhesion to hyaline and the adjacent
nonmesenchymal blastomeres. They start to increase adhesion to the
basement membrane and material within the blastocoel. These changes can
be measured by isolating specific cells and testing adhesion in culture.
E-cadherin is lost from the ingressing cells due to endocytosis of specific
areas where it was expressed. Levels of b-catenin are also reduced on
these cells.
CAMs promote formation of cell junctions
CAMs allow cells to attach quickly but not tightly. They allow reversible
changes in adhesion that aid in migration and intercalation.
Cell junctions (tight junctions, desmosomes, gap junctions) are long term,
tight, cell-cell attachments. They take longer to form. CAMs facilitate
formation of cell junctions by ‘holding cells in place while the glue sets’.
Compaction: cells of the blastula
become polarized and form tight
junctions which compacts the surface.
Just before compaction, cells express
high levels of E-cadherin where
blastomeres touch (future tight
junction).
If blastulas are placed in medium with
antibodies to E-cadherin to disrupt
function, no compaction occurs.
Mature tight junctions are associated
with an area of abundant E-cadherin
expression under the cell surface = the
zonula adherens (think of a zipper).
If CAMs and SAMs were important for gastrulation, their
expression might reflect that fact.
1.
They might be expressed selectively on specific cells of the blastula
that are destined to migrate to selected areas to form organs.
2.
Expression might change as cells left the surface and ingressed or
migrated into the blastocoel.
3.
Blocking expression of CAMs or SAMs might interfere with the normal
process of gastrulation.
Fibrous ECM components provide contact guidance
to migrating cells during gastrulation
The movement of cells during gastrulation may also depend upon
expression of ECM. ECM allows migrating cells to attach transiently while
moving over the surface.
During gastrulation in amphibians, cells move into the blastocoel and
migrate over the inside of the roof.
If a portion of the roof is cut out and inverted, no movement of gastrulating
cells occurs here. This suggests that some CAMs or SAMs may be missing.
What molecules would this be?
Fibronectin on the inner roof of the blastocoel
is critical for gastrulation
Immunostaining of the blastocoel shows that fibronectin was expressed in
abundance on the inner roof. Fibronectin binds to integrins on the membrane.
Neutralizing antibody to fibronectin was injected into the blastocoel to test the
role of fibronectin. This aborted gastrulation. Since no epidermal cells could
migrate into the blastopore, many cells accumulated on the surface, forming
deep folds. If an unrelated antibody was injected, there was no inhibition.
Fibronectin binds integrins through an RGD sequence. Similar results were
obtained by injecting the tripeptide RGD. Furthermore, blocking the integrin
receptor with injected antibodies also inhibited gastrulation. Fibronectin is
important for contact guidance of migrating cells during gastrulation.
CAMs and SAMs are major regulators of cell movement
during gastrulation / morphogenesis.
Do these molecules also directly influence gene
expression or cell differentiation during morphogenesis?
During gastrulation cells migrate and are rearranged in the developing
embryo. Many new cell-cell contacts are established when cells reach their
new positions.
1.
Do specific CAMs on one cell influence how the adjacent cell expresses
genes or undergoes differentiation?
2.
Would cells have different gene expression or differentiation depending
upon the different types of ECM that they rested upon?
Neural differentiation is triggered by N-CAMs or N-cadherins
N-cadherins and N-CAMs are expressed on presumptive neural tissue during
and after gastrulation. What would happen if N-cadherin or N-CAM expression
were experimentally abolished?
Neural differentiation is triggered by N-CAMs or N-cadherins
N-cadherins and N-CAMs are expressed on presumptive neural tissue during
and after gastrulation. What would happen if N-cadherin or N-CAM expression
were experimentally abolished?
Dominant negative mutants of N-cadherin: a mutant that blocks normal
function dominantly (an N-cadherin molecule with the binding site for other
cadherins cut off).
This mutant was injected into a
blastomere of presumptive neural
tissue before gastrulation. The
portion of the brain formed by
daughter cells of this blastomere
failed to develop. The other half of
the brain that developed from
uninjected blastomeres was normal.
N-cadherin is critical for early brain
development.
Does N-cadherin directly influence
neural differentiation?
PC12 cells resemble chromaffin cells which can differentiate into neurons.
When they convert to the neural phenotype they express N-CAM and Ncadherin on their cell surface.
When PC12 cells are grown on cells that do not express N-CAM or N-cadherin
(3T3 cells) they retain the undifferentiated chromaffin phenotype.
If the PC12 cells are grown
on the same cells that have
been transfected with NCAM or N-cadherin genes,
they convert to the
neuronal phenotype.
They form long dendrites
and express neuronal
genes.
Differentiation is
accompanied by opening
of calcium channels
CAMs are necessary for neuronal differentiation
Conversion of PC12 cells to the
neuronal phenotype can be
inhibited by adding antibodies
that neutralize either N-CAM or Ncadherin on the 3T3 cells.
Thus, adhesion through CAMs is
necessary for neuronal
differentiation in these cells.
Neuronal differentiation is also
inhibited by drugs that block
calcium channels. This suggests
that intracellular signaling by
calcium is an important
requirement for neuronal
differentiation.
CAMs and SAMs are major regulators of cell movement
during gastrulation / morphogenesis.
Do these molecules also directly influence gene
expression or cell differentiation during morphogenesis?
During gastrulation cells migrate and are rearranged in the developing
embryo. Many new cell-cell contacts are established when cells reach their
new positions.
1.
Do specific CAMs on one cell influence how the adjacent cell expresses
genes or undergoes differentiation?
2.
Would cells have different gene expression or differentiation depending
upon the different types of ECM that they rested upon?
Collagen directly activates epithelial cells to form stroma
During development, the cornea (outer eye) and lens (inner eye) interact.
The cornea consists of outer epithelial cells and inner stromal cells.
Formation of the inner layer depends upon interaction with the underlying
lens which is covered with collagen.
This interaction has been studied in culture. When corneal cells are grown on
top of the lens (i.e., in contact with collagen), they differentiate to form a
lower layer of stroma.
When corneal cells are cultured on artificial substrate, no stroma forms.
When the cells are cultured on a collagen substrate, a normal stroma
develops.
Interaction with collagen is critical for corneal differentiation.