Lecture #2
Finish Cell-substratum interactions
start cell-cell interactions
•Axiom #2: Unlike with human behavior,
polarity in cells is perfectly normal!
Hemidesmosomes (HD)
• a6b4 integrin is a major
component of the HD, and can
transduce signals from the
ECM to the interior of the
cell.
• This signal modulates the
cytoskeleton, differentiation,
apoptosis, and proliferation.
Hemidesmosomal proteins
• BP 230 and Plectin: link to Ifs
•
a6b4 integrin: binds laminin in
ECM and plectin in cytoplasm
• BP180: links integrin to BP230, may
bind laminin.
• Absence or defects in
hemidesmosome proteins results in
severe blistering of the skin.
Molecules that mediate cell-cell adhesion
•
•
•
•
Cadherins
Immunoglobulin members
Integrins (lecture #1)
Selectins
SELECTINS
Leukocytes
Endothelial
• The selectins are cell surface lectins
that mediate adhesion of white blood
cells to endothelial cells and
platelets.
• 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, ischemiareperfusion injury, inflammatory
diseases, and metastatic spreading of
some cancers
Recently, 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.
See next slide
. acute inflammation and
Cell adhesion is important in
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)
Fig. 7.22 Cell-adhesion molecule of the
immunoglobulin (Ig) superfamily
• Most Igs involved in immune function
Interaction of two L-1 molecules • Some mediate Ca2+-independent cell-cell
adhesion
with 6 immunoglobulin domains
Examples
• NCAM: Neural Cell Adhesion Molecule
• L1: also common in neurons
• VCAM: Vascular cell adhesion molecule
• Nectin: found at AJs.
• VCAM binds integrin, which links cells
to cells and to ECM
Fig 7.23
Cadherins: Mediate Ca2+-dependent cell
adhesion
1.
5 extracellular domain repeats
2.
Terminal domains form an interdigitating zipper with
cadherins on neighboring cells
Cadherins mediate signal transduction from
ECM to cytoplasm
Cell-cell junctional complexes
Components of a junctional complex:
Tight junction (zonula occludens)
Adherens junction (zonula adherens):
Desmosome (macula adherens)
Gap Junctions/Nexus Junctions: (GJIC)
Junctional complexes between cells
Fig. 7.25
Tight junction
Adherens junction
Desmosome
Hemidesmosome
Gap junctions
TJ
Vensus Says: “Why should you care about
junctional complexes?”
• LAD: Leukocyte adhesion deficiency: Afflicted individuals are unable to
produce b2 subunit of integrin. Leukocytes are unable to adhere to endothelial
layer of blood vessels. Bacterial infections can be life threatening
• Blood-Brain Barrier: The principal anatomical component of the blood-brain
barrier is the endothelial tight junction which opens in glioma microvessels. A common
property of brain tumors is their ability to cause edema in the surrounding brain. Edema
forms as a result of a leaky blood-tumor barrier and persists when the brain fails to clear
the excess fluid. It is a significant source of morbidity and mortality
• Cirrhosis: Tight junctions (TJ) of biliary epithelial cells and hepatocytes
prevent bile regurgitation from the biliary tract. Alterations in these TJs result
in chronic cholestatic liver diseases such as primary biliary cirrhosis (PBC)
and primary sclerosing cholangitis (PSC).
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.
Fig. 7.30 TEM of tight junction
Tight junction = zonulae occludens (ZO)
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.
Cell 1
Cell 2
139037
apical
Tight junctions Fig. 7.30
• 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 and Occludins
Claudin family (1-20). Small integral membrane proteins 20-24 kDa.
Might create the selective paracellular properties
Claudins 1-8 bind ZO-1,-2 and -3.
Occludin: an integral membrane protein that copolymerizes
with the clauding polymers
Tight Junction Proteins
• ZO-1, ZO-2, and ZO-3: all three bind occludin
• 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-95, Dlg-A, ZO-1.
SH3 domain: Src homology 3. Mediates protein interactions
GK domain: homologous to enzyme that catalyzes GMP to GDP.
MDCK cells stained for ZO-1 and claudin 4
• Figure 2. Immunofluorescent
colocalization of ZO-1 and
claudin-4 in tet-off claudin-4–
transfected MDCK cells.
• ZO-1 (a and c) and claudin-4 (b
and d). ZO-1 is narrowly
focused at the apical junction,
while claudin-4 in both
uninduced (b) and induced (d)
MDCK cells is concentrated at
cell-cell borders.
Regulated expression of claudin-4 decreases paracellular conductance through a
selective decrease in sodium permeability
Christina Van Itallie
1
1,
Christoph Rahner1 and James Melvin Anderson1,2
Department of Internal Medicine, and 2 Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA
J Clin Invest, May 2001, Volume 107, Number 10, 1319-1327
Copyright ©2001 by the American Society for Clinical Investigation
Components of a junctional complex:
Tight junction (zonula occludens)
Adherens junction (zonula adherens)
Desmosome (macula adherens)
Gap Junctions/Nexus Junctions: (GJIC)
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.
Adherens junctions
Major role in folding and bending of epithelial cell sheets
.
• Adhesive function
• Two types
1) Belts: Encircle cells
(Zonulae Adherens)
2) Spots: adhesion plaques
• Ca2+-sensitive
• Transmit EC signals to
cytoskeleton
Adherens junction
Fig 7.26a
Often referred to as cadherin-based AJs.
•Transmit extracellular signals to cytoskeleton : cadherin linked to
catenin which is linked to actin filaments
Cadherins: role in metastasis?
• Switch from E-cadherin to N-cadherin in
metastatic melanocytes.
Adherens Junctions: Zonulae Adherens

b-Catenin binds:
N, E-Cadherins
Transcription factors (Lef/Tcffamily)
Tumor supressor gene APC
2. The cadherin-catenin complex mediates
Ca2+-dependent cell-cell adhesion.
3.
g-Catenin binds cadherins and acatenin. Also mediates interaction
between desomsomal cadherins and
Intermediate filaments.
4. g Catenin: plakoglobin
b-Catenin Controls Hair Follicle Morphogenesis and Stem Cell
Differentiation in the Skin
•
•
•
•
Keratin 14-Cre-Induced Deletion of the catenin Gene Abrogates Hair Follicle
Morphogenesis and Maintenance of Hair
(a–c) Fur of K14-Cre(neo); floxed -catenin
mice at P8, P16, and P30, respectively.
(d) Hematoxylin/eosin-stained transversal
sections of mutant back skin at P8; the blue
line marks the area lacking hair follicles.
(e) Expression of -catenin in wild-type and
(f) in mutant skin at E15.
Immunofluorescence analysis (red) was
performed using an antibody against an
epitope of the C terminus of -catenin, DAPIstained nuclei are shown in blue, a white
dotted line marks the basement membrane
between epidermis and dermis. Bars: (d) 200
µm; (e and f, shown in [f]) 10
Cell, Vol 105, 533-545, May 2001
Joerg Huelsken1, Regina Vogel1, Bettina Erdmann1, George Cotsarelis2, and Walter Birchmeier1
B-catenin in involved in Wnt signalling pathway.
•
•
Diagram of Possible Ways to
Effect an Epithelial
Mesenchymal Transition
(EMT)
Evidence has been collected
from tissue culture
experiments for each of the
steps outlined. There are
some good correlations
between the expression of
Snail and EMTs in vertebrate
embryos. The possibility that
Wnt signaling has a similar
effect in vivo remains to be
explored
Take Home Messages
•
b –Catenin can be in the cytoplasm or the nucleus.
– Cytoplasm: located in adherens junctions where it binds cadherins through the armadillo
repeats and establishes a link to the cytoskeleton via N terminally bound b -catenin
– Nucleus: interacts with members of the LEF/TCF family of transcription factors and
activates gene expression.

•
b –Catenin is activated by the Wnt signaling pathway.
- Mutations of b-catenin in the N-terminal phosphorylation sites, interfere with
degradation. These mutations result in accumulation of cytoplasmic and nuclear bcatenin, and in constitutive signaling and gene activation. Such mutations are
causally involved in tumorigenesis, and are frequently observed in human tumors
Components of a junctional
:
complex
Tight junction (zonula occludens)
Adherens junction (zonula adherens)
Desmosome (macula adherens)
Gap Junctions/Nexus Junctions: (GJIC)
Desmosomes (Macula Adherens)
• Adhesive function
(cadherin family
members)
• Abundant in skin
• Link to intermediate
filaments
Fig 7.27 Structure of the desmosome
139034
Model showing molecular architecture of a desmosome
Fig. 7.27 b
• Cadherin family
members :
Desmoglein
Desmocollin
• Desmoplakin: link to IFs
Important for prevention of invasion and metastasis.
Components of a junctional complex
Tight junction (zonula occludens)
Adherens junction (zonula adherens)
Desmosome (macula adherens)
Gap Junctions/Nexus Junctions: (GJIC)
Gap Junctions
Intercellular communication
•Aside from ions,
important examples of
molecules that readily
pass include cyclic AMP
(329 Da), glucose-6phosphate (259 Da) and
nucleotides (250-300
Da).
The gap junction’s major physiological roles include
restricting cell proliferation , allowing differentiation and
synchronizing electrical and metabolic (Ca2+)
communication between cells
Gap junctions are seen in virtually all cells that contact other cells in tissues
. Some representative examples of their importance in physiology include:
•
Electrical coupling: Gap junctions are
abundant in cardiac and smooth muscle.
Depolarization of one group of muscle cells
rapidly spreads to adjacent cells, leading to
well-coordinated contractions of those muscles.
•
Metabolic coupling: Many hormones act by
elevating intracellular concentrations of cyclic
AMP, which initiates a signalling pathway
inside the cell. Cyclic AMP readily passes
through gap junctions and thus, hormonal
stimulation of one cell can lead to signal
propagation to a cluster of cells.
Fig. 7.32 Proteins of gap junctions
• Connexins
• Connexin 43 and connexin 45 interact with ZO-1
Gap junctions are dynamic
• Gap junctions are dynamic
structures because connexons
are able to open and close.
• Elevated intracellular calcium
and low intracellular pH are
established stimuli for rapid
closing of connexons.
• In general, the upper limit for
passage through gap junctions
is roughly 1000 daltons (Da).
Do bacteria have gap junctions?
No. It appears that single celled organisms which
survive by proliferation do not have either the
gene or the need for gap junction proteins.
Gap Junctions and cancer
• Michael Stoker showed that the growth of certain
transformed cell lines (cancer cells) could be suppressed
when grown in direct contact with normal cells. This
phenomenon was subsequently shown to correlate with
the ability of the two cell lines to form gap junctions
with each other.
•
Since then it has been shown that the growth of many
cells in vitro shows a strong negative correlation with
the extent of cell communication within the cell
population.
Think about gene therapy
•Transfection of cDNA encoding a gap junction protein or
connexin into several different communication deficient cell
lines leads to the formation of functional gap junctions and the
inhibition of cell growth.
•The importance of Cx43, and gap junctions in general, in the
social control of cell growth in vivo remains controversial.
For Thursday, read chapter 9.
END