Piyush Bajaj BIOE 506 April 29 th, 2008 1 Cadherin-Catenin-Actin Complex.
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Transcript of Piyush Bajaj BIOE 506 April 29 th, 2008 1 Cadherin-Catenin-Actin Complex.
Piyush BajajBIOE 506
April 29th, 2008
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Cadherin-Catenin-Actin Complex
Cadherins in development:cell adhesion, sorting and tissue morphogenesis
Jennifer M. Halbleib and W. James Nelson, Genes and Development , 2006
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SummaryAlthough cadherins evolved to facilitate
mechanical cell-cell adhesion, they play a very important role in tissue morphogenesis
Cadherins
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Surface glycoprotein responsible for Ca2+ dependent cell-cell adhesionGreater 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
[1] http://en.wikipedia.org/wiki/Cadherins
[1]
Classical cadherin
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First type of cadherin family to be identifiedThese 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 motifEC domains interact with different binding partners
Classical cadherin
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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]
[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
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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
Classical cadherins in cell sorting
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Each type of classical cadherin tends to be expressed at the highest level in distinct tissues during developmentE-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
Classical cadherins in cell sorting
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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
Cadherin subtype switching in development
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Subtype switching is a prominent physiological feature of cadherin morphogenetic function during developmentConversion from E-cadherin to N-cadherin is
observed during neurulation in chick embryosCells 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 upregulatedN-cadherin activates MAPK signaling which then
regulates mitosis, differentiation and cell apoptosis
Classic cadherins – nervous system
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The development and maintenance of the nervous system are major areas of focusDifferent cadherins are expressed in different
cells and layers of the nervous systemLayers that receive information VS that send
Dynamic cadherin adhesion is important in neurite outgrowth and guidance and synapse formationCadherin 11 promotes axon elongation while
cadherin 13 acts as a repellant cue for growth conesCadherins regulate synaptic plasticity
LTP
Protocadherin
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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
Structural organization and gene structure
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Protocadherins are type 1 transmembrane proteins like classical cadherins.However, they have six to seven EC domains
They have weak adhesive propertiesThe 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
Protocadherin function in cell organization
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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.
Protocadherin function in cell signaling
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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 CNSThese functions require activation of
intracellular signaling in response to engagement of cell-cell interactions
Atypical cadherins and PCP
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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 signalingDs, 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 discsHowever, only the cytoplasmic tail of cadherin is
required for this effectTherefore, atypical cadherins mediate cell-cell
adhesion and thereby regulate tissue size and polarity cues
Atypical cadherins in vertebrate development
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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 mechanotransductionAlso, have roles in cell recognition and
participate in complex, highly conserved signaling pathway
Deconstructing the Cadherin-Catenin-Actin Complex
Yamada et al., Cell 2005
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SummaryThe 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
Introduction
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The spatial and functional organization of cells in tissues is determined by cell-cell adhesionDisruption of this activity is a common occurence
in metastatic cancerThe 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 membranesThese are mutually exclusive events
Binding of α-catenin to actin and β-catenin is mutually exclusive
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Actin-filament pelleting assayα-catenin pelleted with actin
filaments in the presence of increasing concentrations of E-cadherin-β-catenin complexHowever, E-cadherin- β-catenin
did not pellet above the background level
Result
The chimera failed to bind actin in the pelleting assay
Reconstitution of β and α-catenin assembly on membrane patches
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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 α-catenin
Actin filaments do not assemble on reconstituted membranes
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Actin binding was not detected on stripped membrane patches which were preincubated with α-catenin-β-catenin complex
Measurement of the complex at mature cell-cell contacts
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E-cadherin, α-catenin, β-catenin were tagged with GFPThe 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 expressedBoth 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)
Contd.
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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
GFP
Endogenous
Disrupting actin organization does not affect cadherin or α-catenin dynamics
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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
contactsAfter 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 controlVice 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
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A general assumption has been that binding of a given protein to two distinct partners means that all the three are in the same complexThe authors show that this is not the case
Questions ?
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