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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