Lecture 2 - Adherence Junctions
January 7, 2016:45 PM
There are two classes of cells. Epithelium and
mesynchymal. Think that they decided to get
together and form a multicellular organism. That
requires amazing genetic engineering
coordination. How do they recognize each other
and bind to each other. When you think about the
evolution of orgnaisms that all create receptors
that selectively binds to each other, you are still
gping to end up with a blob of cell. So the
coincidence of the adhesion moeldules is the
extracellular matrix moelcules that gives your
There is no such thing in the body that has a
We are starting today from a simple level and looking at the cell-cell single biological function. Most of them have a
adhesion. context dependent activity. It might inhibit the
With respect to the starts of cell-cell adhesion and transmembrane
same event later. If you think about the
proteins that are Ca dependent and called cadherin. They start very cadherence it helps binds a population of cells.
early in embryogensis. Cadherence in addition to binding cells together
communicate with the actin cytoskeleton and
What happens when we interfere them? affects the actin dynamics. This has a lot of
complex regulations invovling growth factor
signaling, cell- cell adhesions and cross talking
between cells. If you communicate to the actin
cytoskeltin then you are also indirectly
communicating with the other side if the ----
elements tubulin and intermediate filaments. You
start off by just binding cells together that
recognizes each other. And that rapidly translated
the inside of the cell into actin dynamics. Whats
the major properties of the actin filament in terms
of activiites in addition to cell shape? What do
they do? They pull. Mechanical forces comes into
play. That’s why its called cellular dynamics during
CSB331 Page 1 EMT vs MET: Epithelial to Mesynchymal
transtition. And vice verca.
This is a highly polarized cell it has a apical, basal
and lateral domain.
This cartoon doenst have the apical distribution of
the receptors on the surface, so this image is less
polarized. But it is polar.
CSB331 Page 2 You have a series of junctions. These are called as
junctions because they were seen trying to dense
stage. As a tremendous aggregation of proteins at
that junction. This is a generic organization.
- tight junctions --> This creates a barrier
between environments. Allows the diffusion
of the nutrients in the proper direction.
Cell cell anchoring junctions: creates the major
contribution towards the adhesion interaction of
Cell-cell adhesion in a epithelium we also have
What is so special about the adherence junctions?
- Adherence junctions are made from
What kind of instrument are we going to use?
From our naked eye we can see things about
200nm. But many cells are smaller than that. So
we need a microscope to see them. When you use
the light microscopy you can get into a few
Light mic is limited. So to look beyond the cell,
you use the electron microscopy. There isa
differnce in magnification between the electron
mic and light mic and naked eye.
Don’t memorize them. Just appreciate the
CSB331 Page 3 Adherence junction.
- One of the transmembrane adherence
proteins are Cadherins. Calcium dependent
adhesions. That’s how the name comes.
- Classical cadherins: are the ones that are
abundant, and the ones that were identified
- Connects actin filaments.
- Connecting between the actin filaments and
the recptor cell is fine tunesd by lot of
adaptor proteins. This is a very dynamic state,
and we focus only on some components
- These are made from a dozen of different
proteins. There are lot of dynamics involved
and fine tuning regulations.
- Are linked to intermediate filaments
Classical cadherins: The proteins best made by
embryos are cadherence. What they call the
CSB331 Page 4 Cadherin family is expanded. In blue shows some
of the signature motifs all the cadherin's and
these EC domains and immunoglobin like
structures. So The questions is when you see
something like that , you see the desmosome
cadherins and you see that there is a sequence
similarity in the molecular design. There is a EC
domain ig like structures.
there is a sequence similarity more than a 3D
orgnaization of ig.
Why am I putting planar polarity here? Think
about the epithelia having a multiple dimensions.
So you have apical lateral and basal. But you also
can have the planar polarity. Think about The hair
in the back of insects that is pointing in one
direction. That is also another kind of level in
polarity. So you have some of the cadherins, like
the Flamingo plays a central role in regulating
another level of polarity.
So where do you think the functional specificity
Its going to be in the endo domain. There are
regions that are sitting into the cytoplasm
Members of the Fat and Dachsous cadherin subfamilies. Drosophilaas two Fat cadherins (Fat and Fat-like) and one Dachsous cadherin. In vertebrates,
four Fat cadherins (Fat-J, Fat1, Fat2 and Fat3) and two Dachsous cadherins (Dachsous 1 and Dachsous 2) have been reported. Fat-J is probably the
counterpart of Drosophilaat. Fat1 and Fat3 resemble each other in their overall sequences, and they show weak similarity to Drosophilaat-like. The
precise length of Dachsous 2 has not been determined. The rectangular boxes depict the intracellular domains of the various Fats. The regions
between the last cadherin repeat and the first EGF motif or the laminin A-G motif (for Fat2) of Fat cadherins show weak similarities to the Flamingo
box, which is conserved in Flamingo cadherins ( Usui et al., 1999
So what's so special about the classical cadherins
and what about the complexes in the slide we
• Not a memorization slide. Only to remember
- E cadherin: found in most epithelia
- N cadherin: mostly assocaited with neurons
- But these extend to other cadherins as well.
P cadherin - placenta cadherin.
VE : vascular endothelial cadherin.
These were named on the place where they were
A cell cant express different combination of
cadherins depending ont the seqquence.
CSB331 Page 5 This suggestion here is that if youa re going to
EMT --> you loss of E cadherin. If you reverse is
you are bringing in E cadherin.
State if been an epithelia is triggered by the E
Its not clearly on E cadherin..? If I don’t lose that I
don’t lsoe the epithlium character. What triggers
the intial polarized state is much more complexly
If you selectively knock down E cadherin you will
lose epithelium character. But it is deceiving…
The signature feature of MOST cadherins is its
Heterophilic means: 2 different types binds.
Classical cadherins have homophilic interactions.
And hetero doesn’t happen.
E with E. wont form interactions with N.
CSB331 Page 6 Video:
To give a perspective on dynamics.
This is GFP tagged cadherins.
If you look closely and follow a cell, you can see
blobs, in real life .
Any kind of itneraction in cell is dynamic. There
are cues to promoite and inhibit interactions. Ther
are lot more dynamic stages going on in the cell.
Ectodomain: whats sticking out of the cell.
Selectivity and binding comes from this domain.
Endodomain: what is in the cytoplasm. Functional
There are 5 EC1-EC5 Ig like domains. There are
little linker regions where Ca2+ ions bind.
You need Ca2+ for this to work. There is a very
special function on the animals, The N terminal
domain is the more globular like domain is called
the EC domain that is involved in the leading
specificity. The rest enhances the interaction
between the cells.
CSB331 Page 7 Closer look:
EC domain have ig like folds. They both are E
cadherins, and similar.
They have homophilic interactions.
The distance here, when they a re sticking like this
in the end, is like 40nm apart. But this collapses
down to like 20nm when you have full maximum
What's so special is, This shows what Ig like folds
You have a loop domain with 2 antiparallel beta
plated sheets looking like this .
the protruding loops is what gives you specificity.
Small AA acid changes in the loop domain have a
major impact on what molecular interactions they
can form. In some instances they have beta
sheets, you can have beta stranded strings that
augments the interactions. But that’s not …?
No need to memorize the molecular design of the
Ig. Just to show that you can stabilize them with
disulfide bridges. There is an antigen bindings
domain and the loop domain.
CSB331 Page 8 How do they get together?
Light integrin's binds to their ligands. They are
weak forces. They are not like the typical growth
factor binding to its cognate receptor. What
cognate means is two partners.
What it looks like form molecular studies is that it
starts with monomers. This is two opposing cells,
and youa re looking at the PM. And there is a the
cadherin and this forms a cis dimer. These
homodimers forms a trans interaction and zippers
down to 20nm. So from 40 --> 20nm. So they lock
in nicely at the end.
Models for the
of cadherin and
This is a strategy for each other. There is like a bell
curve Like arrangement. They don’t have lot of
affinity to each other, but when it starts to
combining in aggregates it ends up been __ in
100s of moelcules tat are visible at the ___ where
it is becomes really tight
The models for this forms, where the zipper like
binding or forming the parallel like binding
assembly is not 100% resolved, but msot
literature would point to the zipper like
arrangement. But to all purposes its not 100%
Models for the trans-interactions of nectins and cadherins and for
those among nectins, nectin-like molecules (Necls), and other
immunoglobulin (Ig)-like molecules. (a) Two nectin molecules of the
same plasma membrane first form cis-dimers and then form a trans-
interaction by the cis-dimers on apposing cells through each first Ig-
like loop or in a zipper-like fashion with all three Ig-like loops (upper
panel). Similar to nectins, two cadherin molecules of the same plasma
membrane first formcis-dimers, followed by a trans-interaction by
the cis-dimers on apposing cells through each EC1 ectodomain or in a
zipper-like fashion with all five ectodomains (middle panel). A recent
study proposed that this trans-interaction may be formed by a
parallel-like multiply bonded system with four ectodomains (lower
CSB331 Page 9 EC1 domain:
What we see in the knob like way, what you can
see highlighted ebtween 2 EC1 domians, te same
type of cadherins, is there is a Ca ions gone into
the defelxible hinge regions to promote the
interaction. There is a knob here which is actually
a tryptophan residue which promotes the
exchange. So you will see within the knob
structure the tryptophan going into the knob
opposing going into the region here exchanging
the tryptophan interacting with the gap/loop. So
that’s the specificity.
But what promtoes it?
As opposed to the pockers, this is important for
the stabilization .
What actually happens:
Once you add Cal to the hinge region you create
an unstable change. How you resolve this is,
(stabilize it in terms of energy is) go to undergo
conformational change now you can exchange the
In this you can see that in the hydrophobic
pocket, containing in the same cadherin you add
Ca and you get an exchange, you put the
trytophan in the opposing cadherin EC1 domain
and vice versa. That stabilizes the interaction.
Theya re cis tryptophan residues.
Cadherins are cell adhesion proteins that are important for tissue formation and integrity. Cell–cell adhesion occurs through the
formation of the strand-crossover dimer between identical cadherins on the surface of neighboring cells. The strand-crossover
dimer forms exclusively between their EC1 domains via swapping of the βA sheet by undocking the conserved tryptophan 2, W2,
from its own hydrophobic pocket and docking it into the hydrophobic pocket of its adhesive partner. An interesting aspect of the
system is the fact that critical noncovalent interactions in the monomer re-form in the dimer. Thus, as these noncovalent
interactions are conserved, what drives the formation of dimer? Moreover, why is dimer formation calcium-dependent? Thus, to
probe the structural and energetic effects of calcium on the noncovalent interactions that are necessary for dimer formation, we
performed spectroscopic, stability, and assembly studies of wild-type and two mutants, W2A and E89A, of neural (N-) cadherin. We
find that while the ionic interaction involving E89 has a minimal effect on the general stability of the closed conformation of the βA
sheet, the hydrophobic interaction involving W2 is the source of the calcium requirement for adhesive dimer formation. The binding
of calcium creates strain in the W2–hydrophbic pocket interaction through direct connection of E11 at the C-terminus of the βA
sheet to calcium. To overcome this unfavorable condition in the monomer, N-cadherin forms a dimer. Taken together, our data
provide a thermodynamic basis for the calcium dependence of strand-crossover dimer formation in N-cadherin.
CSB331 Page 10 How can we make a newly made embryo fall
The idea is that in the EC space there is 1mM of
Ca. so if we can use a chealor, You can use EDTA,
but there is a better chealtor called EGTA. . What
B)Thee xtracellulapra rto f each is the differencve between something that binds
polypeptidec onsistso f a and a chelator?
serieso f compactd omainsc
alledc adherin Chealator means high affnity.
repeatsjo, inedb y flexibleh If you remove, go down to the  of Ca allmost at
inger egionsC. a2+b indsi n the
the same level of wahts inside the cell, what
neighborhood actually happens to Ca is that it comes away from
of eachh inge,p reventingi t the hinge regions --> the cadherence falls apart,
from flexing.I n the absenceo f
and loses the association. And in actual fact this
Ca2+t,h e cadherence is now very close to be broken down
moleculeb ecomesfl oppya nd by proteases. There is a special type of proteases
that are embedded in the membrane that can
shed the molecules off the surface and that’s a
major mechanism of turning things over. In the
rod like arrangement there is a strong
degradation by proteases. In the floppy state
without Ca it is much more prone for it.
Taking a look at this, cell 1 and cell 2. and the
This is self explantory in terms of the itneractions
we talked about. Now we are talking about the
intracellular proteins associated with it.
C)A t a typicaljunction,
manyc adherinm oleculeas
rea rrayedin parallelf,u
nctioningli keV elcroto
holdc ellst ogetherC.
adherinosn the samec ella
ret houghtt o be coupled
teractionbse tweent heirN -
terminahl eadr egionsa, ndv
the attachmentosf theiri
ntracellulatar ilst o a mat of
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CSB331 Page 11 E cadherin interactions: are alpha and beta
catenin and so on.
Todays focus: p 120 catenin and beta catenin.
Beta catenin binds to the end of the tail of
- This is a complex mixture of proteins. (p120)
This is a very dynamic state of regulation.
Whats missing from this is, there is another group
of molecules called polartity complex molecules.
- There is a bunch of adaptor molecules that
help anchor the actin filaments here at this
moments, and beta catenin is the moelcule
with interacts with alpha catenin which
directly interacts with actin filament.
CSB331 Page 12 You can see that it gets a little bit more
We have p120, alpha and beta catenin. An there is
a whole bunch of different molecules depenidng
on what epithlia you are looking at and what cell
stage, and if it undergoes EMT. Don’t memorize
Factors required to polarize an epithelium. (A) E-Cadherin can
dimerize and form trans-homophilic interactions to form cadherin
clusters. Ca 2+ ions are required to stiffen the extracellular domain
and are essential to form homophilic interactions. The E-cadherin
intracellular domain contains binding sites for the catenins p120
and β-catenin, thereby forming the cadherin–catenin complex.
p120 catenin links cadherin to microtubules and is also important
to prevent cadherin endocytosis and degradation. β-Catenin binds
α-catenin, which in turn binds actin and several actin-associated
proteins, including α-actinin, vinculin, and formin-1. The cadherin–
catenin complex also binds many other proteins, including
signaling proteins, and cell surface receptors and forms a hub for
Whats so special about beta catenin>
- It is important for promoting and stabilizng
It is also capable of doing
- Helps communication of actin dynamics.
- Importantly, it
Beta cadherin: it is an improtant thing to
promoting and stabilizing the structure.
CSB331 Page 13 Beta catenin: you can see that adherence
moelcules was also seen in the nucleus. The
problem with nucleur staiing it can be highly
unspecific, so it was ignored for a while
thinking it was an antibody. Because antibodies
are pathological liars. Lol.
They found it and part of it was in the Wnt
signaling pathway (don’t memorize yet). The
idea here is that a subset was it coming from a
membrane or from a separate pool of catenin?
But the idea is that if you don’t have Wnt
signaling, this complex which involves the
_____-synthases (check book), kinase will
phsophorylate beta catening and have
One in Wnt signal you end up taking a subset
of beta catenin and goes to the nucleus and
acts in combination of other transcription
factors and you regulate the gene expression.
That’s why we need beta catenin in the context
of adherence juctions. Specifically with the
calssical Wnt signaling pathway. This is also
called canonical signaling poathway ebcayse
this was the first one found with Wnt signaling.
Don’t memorize pathway. Key points: berta
catenin has a dual function in regulating the
communication of adherene to the the actin
cytoskelton and also as a TF.
How is it kept on the membrane?
One way to do that is:
If you don’t want to destroy molecules, and keep
them there is a few strategies..
We have the classical E cadherin. It is promoted
one way that use a lot to maintain the junctions,
not only for the cadherin, because remember if
you brigning in the cadherin complex for
endodomain to the other molecule, you are also
going to internalize the adaptor molecules. Re
think about that region that between all the
adaptor molecules are concentrated like sibling
So if you are bringing in E cadherin, I am talking
about cell cell adhesion as focus, your own sibling
effect that is going on inside cells.
So cadherin do promote differntiation.
But threre are 2 choices
CSB331 Page 14 Theres 2 choices.
Taking in by endosomes can go to late endosomes
and be degraded. One way of silencing
Or you can bring it to the recycling endosomes
and bring it back to the cell surface.
Phosphorylation events are part of that.
There's also this vision of neighbor bonds, small
monomeric GTPases of RAS super family.
Keeping the dynamic state is all promoted by the
endosomes recycling, or if you want to silence the
event you can degrade it by proteasomes. But if
you want to go in the proteasome direction you
will want to silence the expression of the gene. so
you have the expression of repressors that will
block the expression of E cadherin.
What will be another way of doing this?
One level of regulation:
2. Or bring it back to the target…
If i asked will a cell alter its Ca level in the
That doesn’t occur because the Ca  In the
extracellular space is so large so either you are
going to turn over? And one of the primary
actions is by recycle the endocytic pathway either
for degradation or recycling.
CSB331 Page 15 This p120 catenin actually serves to stabilize the
interactions here by preventing the ubiquitination
of beta catenin.
This covers the sensitive region
P120 catenin primarily serves to stabilize the
interactions by preventing the ubiquitination of
the beta catenin. What it actually does is that, it
covers the sensitive region which you will have
the ubiquitinin monomer and prevents the
targeting of degradation.
If you remove p120 it tends to target beta