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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