Transcript Slide 1
Cadherin-Catenin-Actin Complex
Piyush Bajaj
BIOE 506
April 29th, 2008
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Cadherins in development:cell adhesion, sorting
and tissue morphogenesis
Jennifer M. Halbleib and W. James Nelson, Genes
and Development , 2006
Summary
Although cadherins evolved to facilitate mechanical cell-cell
adhesion, they play a very important role in tissue morphogenesis
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Cadherins
Surface glycoprotein responsible for Ca2+
dependent cell-cell adhesion
Greater than 100 family members have been
identified with diverse protein structures but
with same extracellular cadherin repeats
(ECs)
Important to vertebrates, insects, nematodes
and even unicellular organisms.
Important in the formation and maintenance
of diverse tissues and organs
Defects will lead to different types of diseases
3 different types of cadherin and their roles
in development
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[1] http://en.wikipedia.org/wiki/Cadherins
[1]
Classical cadherin
First type of cadherin family to be identified
These are subdivided into Type 1 and Type 2 each of which have 5 ECs in
the extracellular domain
Type 1 mediate strong cell-cell adhesion and have a conserved HAV
tripeptide motif in the most distal EC1
Type 2 cadherin lacks this motif
EC domains interact with different binding partners
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Classical cadherin
The cytoplasmic domain is highly conserved
in different types of classical cadherin and
binds to several proteins
However, recent study Dress et al., 2005
showed that α-catenin acts in an allosteric
manner with β-catenin and actin
[1]
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[1] http://calcium.uhnres.utoronto.ca/cadherin/pub_pages/general/intro_cadherins.htm
[2] Dress et al., α-catenin is a molecular swiitch that binds E—cadherin -β-catenin and regulates actin filament assembly. Cell 123: 903-915
Regulation of cadherin activity
Regulation happens at many levels including gene
expression, transport and protein turnover at the
cell surface
Methylation and repression of the promoter
activity
During carcinogenesis, methylation of the E-
cadherin promoter reduces its expression and
leads to disease progression and metastasis
Decreased E-cadherin gene transcription results
in a loss of cell-cell adhesion and increased cell
migration
Newly synthesized E-cadherin at the plasma
membrane requires binding of β-catenin and this
process is regulated by phosphorylation,
proteolysis, etc.
E-cadherin is actively endocytosed via clathrin
coated vesicles which can result in rapid loss of
cell-cell adhesion
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Classical cadherins in cell sorting
Each type of classical cadherin
tends to be expressed at the
highest level in distinct tissues
during development
E-cadherin is expresses in
expressed in all epithelial tissue
and is important for cell
polarity
N-cadherin is expressed in
neural tissue and muscle
R-cadherin is expressed in
forebrain and muscle
The role of cadherin subtypes
in mediating cell sorting has
been shown in tissue culture
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Classical cadherins in cell sorting
The specificity of adhesion by the EC1 domain provides one
mechanism to explain how cells segregate from each other
within complex cell mixtures
Each type of cadherin might activate tissue specific
intracellular signaling pathway by using the conserved
binding partners of the cytoplasmic domain
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Cadherin subtype switching in
development
Subtype switching is a prominent physiological feature of
cadherin morphogenetic function during development
Conversion from E-cadherin to N-cadherin is observed during
neurulation in chick embryos
Cells loose their previous epithelial morphology and get converted to a
fibroblastic shape by a process known as epithelial mesenchymal transition
During tumor progression, E-cadherin is down regulated and
concomitantly N-cadherin is upregulated
N-cadherin activates MAPK signaling which then regulates mitosis,
differentiation and cell apoptosis
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Classic cadherins – nervous system
The development and maintenance of the nervous system are
major areas of focus
Different cadherins are expressed in different cells and layers of
the nervous system
Layers that receive information VS that send
Dynamic cadherin adhesion is important in neurite outgrowth
and guidance and synapse formation
Cadherin 11 promotes axon elongation while cadherin 13 acts as a
repellant cue for growth cones
Cadherins regulate synaptic plasticity
LTP
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Protocadherin
They are primarily expressed in the nervous system although
have important development expressions in no-neuronal
tissues.
Present in vertebrates and certain sea sponges but not found in
Drosophila or C. elegans
Work on understanding protocadherin function is still in its
infancy compared with classical cadherin
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Structural organization and gene
structure
Protocadherins are type 1 transmembrane proteins like
classical cadherins.
However, they have six to seven EC domains
They have weak adhesive properties
The cytoplasmic domain of protocadherins is structurally
diverse in contrast to classic cadherins
Majority of protocadherin can be classified into three clusters
(α,β,γ) each with a unique gene structure that encode
constant and variable domains
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Protocadherin function in cell
organization
Pcdh 10 although mainly expressed in the nervous system is
also present in somites and facilitates their segregation
Pcdh are present during embryogenesis and gradually
become enriched at synapses and their expression decreases
after the neurons mature and become myelinated
However, deletion of the entire cluster of Pcdh- γ genes in
mice resulted in no general defects in neuronal survival,
migration etc.
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Protocadherin function in cell signaling
The primary function of protocadherins is to relay a signal to
the cytoplasm in response to cell recognition and not
maintain physical interactions between cells
Pcdh-α proteins in mice have a RGD motif that can facilitate
interactions with integrins in vitro
Protocadherins play a crucial role during embryogenesis,
particularly in the CNS
These functions require activation of intracellular signaling in
response to engagement of cell-cell interactions
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Atypical cadherins and PCP
PCP refers to polarized orientation of epithelial cells along the
long axis of the cell monolayer
Large atypical cadherins Dachsous (Ds), Fat, and Flamingo (Fmi)
are involved in PCP signaling
Ds, Fat, Fmi have 27, 34 and 9 ECs instead of 5 in the classic
cadherins
The cytoplasmic domains of Ds and Fat have sequence homology with
the β-catenin binding site of classic cadherins
Loss of Fat function leads to hyperproliferation of Drosophila
imaginal discs
However, only the cytoplasmic tail of cadherin is required for this
effect
Therefore, atypical cadherins mediate cell-cell adhesion and
thereby regulate tissue size and polarity cues
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Atypical cadherins in vertebrate
development
In vertebrate development, PCP components function in
convergence and extension movements
Organization of hair cell in the stereocilia within the inner
ear because of the cadherin interaction in the vertebrates
Involved in mechanotransduction
Also, have roles in cell recognition and participate in
complex, highly conserved signaling pathway
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Deconstructing the Cadherin-CateninActin Complex
Yamada et al., Cell 2005
Summary
The prevailing dogma is that cadherins are linked to the actin
cytoskeleton through β-catenin and α-catenin, however, the authors
show that this quaternary complex does not happen
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Introduction
The spatial and functional organization of cells in tissues is
determined by cell-cell adhesion
Disruption of this activity is a common occurence in metastatic
cancer
The cadherin cytoplasmic domain forms a high affinity, 1:1
complex with β-catenin, and β-catenin binds with lower affinity
to α-catenin
Several studies (12) show that α-catenin interacts with actin
cytoskeleton
However, no experiment has shown the formation of quarternary
complex in solution or in cell membranes
These are mutually exclusive events
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Binding of α-catenin to actin and βcatenin is mutually exclusive
Actin-filament pelleting assay
α-catenin pelleted with actin filaments in
the presence of increasing concentrations
of E-cadherin-β-catenin complex
However, E-cadherin- β-catenin did not pellet
above the background level
Result
The chimera failed to bind actin in the
pelleting assay
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Reconstitution of β and α-catenin
assembly on membrane patches
A – Unroofing of MDCK
cells
B – After sonication, a
patchwork of ventral
membranes attached to
cadherin substratum
C - Reconstitute the actin
catenin binding, GnHcl was
used
β-catenin addition to the
patches reached about 80%
of the prestripped level
while only 25% for αcatenin
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Actin filaments do not assemble on
reconstituted membranes
Actin binding was not
detected on stripped
membrane patches which
were preincubated with αcatenin-β-catenin complex
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Measurement of the complex at
mature cell-cell contacts
E-cadherin, α-catenin, β-catenin were tagged with GFP
The level of exogenous protein expression in stable cell lines was less than that
of the endogenous protein
Protein dynamics were measured by FRAP
The recovery time and mobile fraction for E-cadherin-GFP (0.54 min, 22.9%),
α-catenin (0.43 min, 33.7%), β-catenin (0.66, 34.2%) were similar
Mutants of E-cadherin (lacking the cytomplasmic domain) and α-catenin
(lacking the actin binding domain) were expressed
Both mutant E-cadherin and α-catenin had mobility rate similar to those of full
length of these species
Therefore, cadherin-catenin complex and actin cytoskeleton did not affect the
dynamics of this complex
The mobile fraction for GFP-actin was almost complete (90%) and rapid (0.16
min) in contrast to more immobile E-cadherin, α-catenin, β-catenin
Rhod-actin had recovery kinetics similar to that of GFP-actin (recovery – 0.21
min)
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Contd.
Thus actin associated
with cell-cell contacts
is unusually dynamic
compared to that
associated with cell
substrate adhesion
Therefore, it is a
mutually exclusive
event
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GFP
Endogenous
Disrupting actin organization does not
affect cadherin or α-catenin dynamics
Cytochalasin D was used to disrupt the actin dynamics at cell-cell
contacts and jasplakinolide was used to stabalize it
After 1 hr treatment with CD, the actin dynamics were
redistributed and aggregated in the cytoplasm
A small fraction remained associated with intact cell-cell contacts
After photobleaching, the recovery rate and mobile fraction of
actin was much lower than the control
The recovery rate and mobile fraction of E-cadherin-GFP and α–
catenin-GFP remained the same as control
Vice versa for jasplakinolide
Together these results show that mobility of cadherin-catenin
complex at cell-cell contacts is independent of actin organization
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Conclusion
A general assumption has been that binding of a given protein
to two distinct partners means that all the three are in the
same complex
The authors show that this is not the case
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Questions ?
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