BIOL 286 Chapter 20: Cellular Communities C.

Mouzakitis

Immunofluorescence
A cell is fixed in a solution that
cross-links all amino acids,
effectively freezing and killing
the cell. A primary antibody is
commercial produced in a
rabbit by injecting the rabbit
with foreign tubulin protein.
The antibody is extracted and
binds to the immobilized
antigen on the structure of
interest (e.g., a microtubule). A fluorescent secondary antibody binds to the primary antibody. Antibodies can
detect specific molecules, and scientists can label several structures with different colors. A cell in mitosis can
have microtubules labeled green, centromeres labeled magenta, and DNA stained (with dye, not antibody) blue.
If the primary antibody fluoresces, the brightness of the structure will not be as great as four secondary
fluorescent antibodies.

Cell Organization in Tissue

Each tissue is an organized assembly of cells held together by cell-cell adhesions, extracellular matrix (ECM),
or both. Tissues are in cooperative assemblies, meaning they cooperate to complete a task. For instance, the
epithelial tissue (e.g., surface of the skin) serves to protect the skin and keep blood inside. Epithelial tissue is
characterized by a high volume of cells, and very little extracellular matrix (few gaps). Underneath the
epithelial tissue is a basement membrane, which separates the epithelial tissue from connective tissue.
Connective tissue has fewer cells and more extracellular matrix.

Fibroblast
• Connective tissue cells that secret nonrigid ECM (e.g., collagen)
• Proliferate when tissue is injured
• May differentiate into other connective tissue cells: cartilage (chondrocytes), fat (adipocyte), bone
(osteocyte), and smooth muscle cells.

The Extracellular Space (all the cables secreted

by the fibroblasts

The cell surface is coated with sugar residues.

Proteins on the cell surface have o-linked and n-
linked glycosylation on them.
Glycocalyx, or cell coat, functions as
mechanical protection (against proteases), or in
cell interaction. Cell recognition is often based
on sugar residues. All cells have some sugar
residues, but some cells are heavily covered in a
sugary coat.

Chondrocytes are cells that release extracellular matrix that eventually becomes cartilage.
The extracellular matrix of cartilage cells is not easily viewed with a microscope, but when
the cartilage cell comes into contact with red blood cells, a halo of space is observed. This
space is the chondrocyte’s extracellular matrix secretion, which serves as a shield.

24:39 in Lecture 24
The Extracellular Matrix
• Secreted locally by fibroblasts
• Variations in amounts & components determines tissue’s physical properties (e.g., bone, cornea, tendon)
• Organizes cells into tissues and coordinates their functions
• Key regulatory role in cell shape, gene expression, cell growth and migration (along highways of ECM)
Gene expression in mammary gland epithelial cells
• When mammary gland epithelial cells are placed in a dish with incorrect ECM or without any ECM,
they assume a flat, incorrect morphology.
• When the epithelial cells are placed in the correct extracellular matrix, the cell becomes rounded and full
of fat globules. Morphology changes and the cells are able to produce milk. Thus, ECM must control
gene expression

The Extracellular Matrix contains

collagen, fibronectin, laminins, and
proteoglycans, all of which interact
with each other.

Fibrous Proteins
a. Collagen
b. Fibronectin
c. Laminin

Glycosaminoglycans: (GAGs)
Modified dipolysaccharides linked
to proteins to form proteoglycans
A. Collagens
• Family (at least 27 members) of fibrous glycoproteins
• High tensile strength (resist pulling force)
• Pound-per-pound stronger than steel
• 1 millimeter of collagen can hold 22 pounds
• Most abundant protein in body (25%)
• Produced by fibroblasts, smooth muscle, epithelial cells
• Two main types of collagen
o Fibrillar I, II, and III: structure is 300-nm fibrils (cables); tissues are skin, tendon, ligaments
o Sheet-forming IV: structure is 2-D network (sheets); tissues are basal lamina (basement membrane)

Fibrillar I, II, III vary in terms of amino acid sequence. Two

common structural features:
1) trimers of 3 polypeptide chains (called α chains)
2) 3 chains wound around each other to form rod triple
helix
Shown right, a polypeptide chain with an amino terminus
and carboxy terminus is synthesized. In the ER, the strand
becomes a triple helix via hydrogen bonding.
Hydroxyproline and hydroxylysine are responsible for this.
Outside the cell, the triple helixes laterally assemble to form
collagen fibrils via covalent lateral bonding.
In some cases, the fibrils can assemble to make thicker
fibers. Tugging on fibrils would require breaking hydrogen
and covalent bonds.
Procollagen precursors are cleaved to form mature collagen outside cell.
Shown right, the triple helix is formed in the ER. While it is in the cell, the
procollagen has two green regions attached to it, preventing the triple helix
from forming large fibrils. After release to the outside of the cell, proteinase
cleaves the terminal extensions. This cleavage allows the triple helix to self
assemble into fibrils.

Hyperextensible skin is due to incorrect collagen assembly. Vitamin C is

crucial to adding lysine and proline to the triple helix, facilitating the
hydrogen bonding. Sailors with scurvy lack this vitamin C and lose their
teeth, tendons, etc.

Sheet-forming Type IV collagen forms a distinct fence-like network that

makes up the basal membrane. It is a unique ECM that is closely associated
with laminin.

B. Fibronectin
• Has binding sites for other ECM components
• Has binding sites for cell surface receptors
• Essential for cell shape, migration, and differentiation

Fibronectin is a dimer whose monomers are held together by

disulfide bonds (bridges). Each monomer has distinct
domains that bind to different things. Example: collage-
binding domain; fibrin-binding domain; cell-binding domain
(to which integrin binds). The cell-binding domain has an
RGD loop (arginine-glycine-aspartic acid) that the integrins
on the cell surface bind to.
A fibroblast’s fibronectin is arranged by the integrins of
nearby cells. The integrins’ cytoplasmic tails are associated
with intracellular actin. Immunofluorescence shows that
intracellular actin and extracellular fibronectin arrange in
similar networks because they are indirectly linked by
integrin.
When fibronectin is placed on a glass slide, and a cell comes into contact with it, the cell will contort itself so it
stays associated with the fibronectin only and remains off
the bare glass. Inside the body, cells also follow
fibronectin pathways. Isolation from the extracellular
matrix will force a cell into apoptosis.
Neural crest cells “born” in one area of the embryo must
move to proper destination to assume their role. In vitro
neural crest cells choose fibronectin highways over other
substrates.
C. Laminin (cross-like shape shown right)
• 3 polypeptide chains
• Integral component of the basal lamina (membrane)
• Regions bind to collage, integrin, heparan sulfate
• Influences cell growth, migration

NOT I.F. LAMIN FOUND IN NUCLEUS

Primordial germ cells in the yolk sac shown right migrate into the
embryo and become gonadal cells. Laminin-lined pathways direct
primordial germ cell migration.
55:32 in Lecture 24

Glycosaminoglycans (GAGs): modified dipolysaccharide

(repeating units of ABABABAB) linked to proteins. The
combination of multiple glycosaminoglycans covalently-linked to
a core protein is called a proteoglycan.

The glycosaminoglycans are very negatively charged, so they

readily bind with extracellular Na+. Bound Na+ will then attach
water to the complex. Thus, proteoglycans are very hydrated and
useful for cushioning.
Shown left, in cartilage, the protein half of the proteoglycan is
called aggrecan (green). Aggrecan has repeating
glycosaminoglycans chondroitin sulfate and keratin sulfate.
These proteoglycans are further assembled onto a backbone of
hyaluronic acid (purple).
Many (-) charges aggregate swells by H2O attraction
GAGs form porous, hydrated gels that provide cushioning
support; e.g., cartilage
Allows diffusion of molecules in ECM
The ECM has collagen to resist stretching, as well as GAG to resist compression. Achilles tendon is purely
collagen in order to maintain rigidity, while the inner eye is all GAG. Ratios vary in different tissues.
One hyaluronan with its proteoglycans takes up a great deal of space (especially with water associated). This
space is approximately the volume of a bacterium.
Remodeling the ECM
• Dynamic in space and time
• Stretched and organized by fibroblasts and surrounding cells
• In vitro two heart explants with fibroblasts are seen tugging collagen to rearrange the network
• Continual degradation and reconstruction
• Matrix metalloproteinases (MMPs): secreted by cells to digest ECM. Metastatic cancer cells secrete
MMPs enabling them to migrate inappropriately through the ECM.

A sheet of epithelial cells has an apical and a basal surface

• Apical surface is free
• Basal surface is bound to basal lamina, a type of ECM separating
the epithelial cells from underneath connective tissue
Basal Lamina (basement membrane)
• One of the best-defined ECMs
• 50-200-nm thick continuous sheet surrounding muscle and fat
cells; underlies epithelial cells
• Mechanical support for attached cells; substrate for cell migration;
barrier for macromolecule passage (in kidney glomerulus serves as filter)
• Linked to cells via integrins and to fibrous collages
• Although called basement membrane, it is NOT a lipid bilayer
 Structure of basal lamina is different in different tissues (e.g., kidney, muscle, epithelial). This is controlled
by Type IV collagens and laminin. The height of a laminin is essentially the thickness of the basal lamina.

I. Interactions of cells with the extracellular matrix

Cell-substrate anchoring junctions: The

transmembrane proteins on the surface of
cells that interact with the extracellular
matrix are part of the integrin family.
They serve as adapter (linker) proteins,
connecting the ECM with different
internal cytoskeleton.

Two adhesions that use integrins:

a) Hemidesmosomes
b) Focal Adhesion (not epithelial)

Integrin Structure
• Integral membrane proteins
• When the extracellular portion binds to the ECM, the cytoplasmic
tails changes, and vice versa.
• Heterodimers of α and β subunits
• Ca2+ and Mg2+ required
• RGD: arginine-glycine-aspartic acid; amino acids serve as binding
site of many substrates to which integrin binds.
• Signal transduction receptors that send survival signals to nucleus

Components of heterodimer will determine ligand (substrate) specificity

Ex: α3β1 is used to bind to fibronectin

Individual cells have many types of integrins, thus are able to bind many
ECM types. Humans have at least 24 combinations of these heterodimer
components.

Shown right, the heterodimer integrin binds to the RGD

region of fibronectin, which can bind to collage fibrils.
Within the cytosol, adaptor proteins link either actin
filaments or intermediate filaments to the cytoplasmic tails of
the integrin.

Integrins are usually inactive, but switch to an active

conformation when the bind to molecules on either side of
the plasma membrane. If the inside portion binds to the
cytoskeleton, the outside portion is activated. If the outside
portion binds to the ECM, the inside portion is activated.

16:25 in Lecture 25
“Inside-out” signaling can activate integrins. An extracellular signal binds to a surface receptor. The
activated surface receptor’s tail will activate the cytoplasmic tail of integrin. This causes a
conformation change in the extracellular portion of integrin, thereby increasing its affinity for the
ECM. Alterations in affinity are triggered by inside changes.
Ex: damage in the area of the endothelial cells will activate their integrin proteins. Active integrin
proteins will allow white blood cells to bind tightly. The white blood cells will be able to squeeze
through the endothelial wall to the site of damage.

Blood clots form when platelets aggregate through fibronectin bridges

using activated integrins.

Shown left, platelets have integrin (green) on them that can recognize
fibrinogen (gray). Fibrinogen is uniquely activated ECM at sites of
damage. As the platelets flow through the blood, once they come into
contact with fibrinogen, they aggregate and form a blot to close the
site of injury.

In the second panel of the image, surgeons use RGD-containing

peptides to inhibit blood clotting, because they keep the platelets from
aggregating near fibrinogen.

Commercially, Aggrastat is used as a chemical RGD analog,

meaning it is specific for the integrin proteins on platelets and can
reduce clotting.

In order to study the anchoring of cells to the substratum (ECM) via

integrins, it is easier to employ culture dishes rather than observe the
inside of animals. In vitro mimics what occurs in animal cells.
Within 3-4 hours, cells on a dish bind to their underlying ECM and
stretch out drastically (see chapter 20, slide 44).

Two types of connections that integrins make to the cytoskeleton: focal adhesions and hemidesmosomes.

Focal adhesions (focal contacts)

• Occurs in migratory and non-epithelial cells (e.g., fibroblasts)
• Integrins link ECM on the outside with actin filaments on the inside
• Sites of tightest contact
• Linker proteins include talin, vinculin, and α-actinin

Shown left, fibronectin (bottom) binds to integrins. The cytoplasmic tails of

the integrin bind to talin, which binds to vinculin, which binds to α-actinin,
which connects to the actin filaments.

They are called focal adhesions because the entire cell is not linked to the
ECM, only specific hot spots where integrin proteins bind to the ECM.
These hot spots are the sites of tightest contact. When actin and myosin
contract, stress is put on the ECM via these focal adhesions.

35:04 in Lecture 25
 Focal adhesions are sites with many phosphotyrosines, so
immunofluorescence seeking to label these adhesions would
use antibodies for phosphotyrosine.

Shown left, integrins are signal transduction receptors.

Signals influence cell growth, motility, and survival.

Normal cells can grow only when attached to ECM;

Malignant cells can grow in suspension without any ECM.

Once bound to ECM, linkage proteins arrange the actin and

myosin with the ECM. Focal adhesion kinases also begin
phosphorylating tyrosines, which eventually tells the nucleus
to grow and survive.

Integrins binding to ECM will actually activate a whole series of kinases and other proteins. Slide 48 shows a
network of different proteins that are activated, the details of which are negligible.

Hemidesmosomes
• Epithelial cells binding to laminin of basal lamina (Type IV collage & laminin)
• Integrins (α6β4) link ECM on outside with intermediate filaments (usually keratin) on inside
• Linkage proteins include plectin

Bullous pemphigoid
• Rare disease in which body makes antibodies against hemidesmosome proteins
• Lower layer of skin (epidermis) released from basal lamina
• Fluid leakage and blistering

II. Interactions of cells with other cells (tissue)

This interaction was first recognized in an experiment called “sorting out”

1. Cells from 2 regions of early amphibian embryo, ectoderm and mesoderm, are shuffled
2. Dissociated (shuffled) cells recombine and sort themselves out properly
3. Mesoderm move to interior (proper for embryo); ectoderm move outer
4. Differentiate into proper structures
Something about the surface of these cells causes them to self-sort to find similar cells from their region
(homophilic binding). Heterophilic binding involves one cell recognizing
something different on another cell.

A) Non-junctional cell-cell adhesions (little cytoskeleton involved; break easily)

Four distinct families of integral membrane proteins: selectins,
immunoglobulin superfamily (IgSF), integrins (bind to IgSF), and cadherins
(homophilic). See the right image, which shows the proteins involved in
homophilic and heterophilic interactions. The selectin-lectin in the image is
really one protein on one cell that binds to a sugar on the partner cell.

54:20 in Lecture 25
Selectins
• “lymphocyte homing”: lymphocytes removed from a goat were radioactivately labeled and replaced in
the goat. The labeled lymphocytes were found exactly where they were extracted. When these cells
were treated with proteases to cleave surface proteins, the cells did not find their way to origin.
• When lymphoid tissue was extracted from the goat, and lymphocytes were poured over the tissue, the
lymphocytes would only stick to the lymphoid tissue and avoid muscle, etc.
• Heterophilic interaction
• White blood cells bind to the surface of endothelial cells and roll along the wall of the endothelial cell
looking to repair damage
• Lectins are compounds that bind to specific sugars (glycoproteins) on the partner cell
o L-selectin: leukocytes (WBCs) use this to stick to endothelial cells and roll
o E-selectin: endothelial cells use this when damage occurs to hold WBCs in certain spots
o P-selectin: platelets

Shown right, WBCs have L-selectins on their surface, and bind to

sugars on the endothelial cell surface. This binding allows them to
roll across the endothelium. Damaged endothelial cells will express
E-selectin, which binds to sugars on the WBC, effectively stopping
it in its tracks. The WBC will be directed through the endothelial
cell barrier to the site of damage.

B) Junctional cell-cell complexes (more permanent and involve

significant cytoskeleton anchorage). Example: intestinal
epithelial cells
1. Anchoring junctions
2. Occluding (tight junctions) – more of a sealant for cell-cell spaces
3. Communicating junctions – gap junctions to allow molecule sharing

Anchoring junctions and tight junctions use cadherins as their transmembrane receptor. Partner cells must
express the same type of cadherin to form a junction (homophilic binding). Adherens junctions form after
cadherins bind, which link the actin filaments of the partnering cells as well as the intermediate filaments of the
partnering cells.

Cadherins mediate Ca++ dependent cell-cell homophilic adhesion. Cadherins are a large family of molecules
forming homophilic interaction (joining cells of similar type). This adhesion requires calcium. If you wish to
separate cells growing on a dish from each other, a solution called a chelating agent is added to the cells, which
removes calcium and magnesium from the junctions. Trypsin (protease to chew extracellular proteins) is also
added to the dish to digest cadherins and integrins. This allows the cells to let go of each other.

In the sorting out experiment, cells expressing E-cadherin stay together and cells expressing N-cadherin stay
together. Furthermore, cells expressing a high level of E-cadherin stay together, and cells expressing a low
level of E-cadherin stay together. Adhesions are based on the cadherin type and the level of expression.

Once the first junction is formed between two E-cadherins, all the other E-cadherins on the plasma membrane
concentrate at that junction to strength the connection.

The junctions between two cells are always in the same order: 1) tight junctions, 2) adhesion junction, 3)
desmosome, 4) gap junction
Adherins Junctions use cadherins to link actin filaments
of partnering cells.

The cytoplasmic tails of the linked cadherins (green) are

associated with actin. This association is made possible
by β- and α−catenin linker proteins.

β-catenin is referred to as a moonlighting protein

because it has two jobs: linking cadherin’s cytoplasmic
tail to actin, and serving as a transcription factor.

Like focal contacts in that some molecules connecting to

the actin can act as signaling molecules (transcription
factors)

Adherens junctions are all around the cell, essentially

forming an adhesion belt. Adherens junctions are
common in epithelia. Constriction of actin and myosin
can put tension on the adherens junctions, almost like a
“purse string” (the string on cheap book bags used to
close and open the bag).

Distribution of E- and N-cadherin in the

developing nervous system:

Shown left, during development there is a layer of

epithelial cells expressing E-cadherin. Cells in
this layer that begin expressing N-cadherin will
use the adhesion belt to induce an out pocketing of
cells needed to form the neural tube.

Recall, when the cells being expressing N-

cadherin, the homophilic linkage is broken
because the N- and E-cadherins no longer
recognize each other.

30:20 in Lecture 26

Desmosomes link intermediate filaments (high tensile strength keratin filaments) of partnering cells
• Common in skin and cells needing mechanical strength
• Spot junctions
• Connect intermediate filaments from cell to cell
• Bridged by another type of cadherins

The synaptic junction is a specialized junction using protocadherins. Cadherins may link, but keep apart, pre-
and post-synaptic membranes. Protocadherins (evolutionarily older than cadherins) may provide specificity of
axon with correct post-synaptic cell (i.e., roles in forming neural circuits).
Occluding (tight) junctions are crucial in the kidney and intestines
to separate the fluid part of one epithelial cell from the fluid part of
another. These junctions are like gaskets used to keep water out
of the other junctions. Therefore, they form closest to the apical
surface of the cells.

Tight junctions encircle the apical surface of some epithelia.

Shown right is the epithelial layer of the intestine. The tight
junctions are what form closest to the surface of this layer
(exposed of the lumen of the intestine). They keep substances
from diffusing between cells and entering the blood.

Tight junctions seal off body cavities and restrict

diffusion of membrane component
• Tight junctions (TJ) form a selectively
permeable junction barrier across epithelial
sheets
• Shown right, when a black dye is placed in the
lumen of the intestine, the dye is able to enter
between villi, but stops just at the tight junction
between the cells. Similarly, when black dye is
placed on the bottom end of the layer of cells, it
makes its way upward and stops before the tight junction.
• Occludin and claudin family are transmembrane proteins that self-associate with related particles in
neighboring cells
• Open/close regulated by extracellular stimuli (hormones, ATP depletion, toxins)
Glucose is kept low in the lumen of the
intestine and in the capillary. A
Na+/glucose symporter forces glucose up
its concentration gradient (sodium going
down concentration gradient) into the cell.
A facilitated transporter allows it to flow
down its concentration gradient into the
capillaries.
The symporter must be kept on the lumen
of the intestine and the facilitated
transporter must be kept on the bottom of
the cell.
The symporter can diffuse all along the
apical surface of the cell, but the tight
junction keeps it
from reaching the
bottom end of the
cell.

Communicating Junctions (Gap junctions) allow for the exchange of molecules.

• Passage of chemical or electrical signals between animal cells
• “pipeline” allows free movement of small molecules (amino acids, nucleotides,
water, cAMP) between cells
• Gated: can “close” in the presence of high Ca++ (damaged cell will rupture)
 Shown left, only molecules around 100-1000 daltons in molecular weight are able to
travel across gap junctions. Larger molecules are too big to readily travel across the
junction.

Structure of a gap junction:

Six connexins, transmembrane proteins, form hydrophilic cylindrical channels
called connexons.

1) Pass ions: electrochemical gradient between cells (e.g., electrical

synapses in cardiac and smooth muscle). On a dish, two separated
cardiomyocytes will beat separately. When they touch, their beating
becomes synchronized.
2) Pass second messengers: establish common network of information
(e.g., movement of Ca++ and cAMP can coordinate hormonal
stimulation)
3) Pass metabolites: sharing of resources

Experiment using neurons showed that when a fluorescent dye is injected into
one neuron, open gap junctions (no dopamine) made the dye radiate to all
neighboring neurons. After treatment with dopamine, closed gap junctions will prevent the dye from radiating.

Summary – be familiar with the order of junctions from the apical surface to the basal lamina.

Exam on Thursday, May 22nd from 1:45 P.M. to 3:45 P.M.