Piyush Bajaj

1
Cadherin-Catenin-Actin Complex

• Piyush Bajaj
• BIOE 506
• April 29th, 2008

2
Cadherins in developmentcell adhesion, sorting
and tissue morphogenesisJennifer 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

3
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

1
1 http//en.wikipedia.org/wiki/Cadherins
4
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

5
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 a-catenin acts in an allosteric manner with
  ß-catenin and actin

1
1 http//calcium.uhnres.utoronto.ca/cadherin/pub
_pages/general/intro_cadherins.htm 2 Dress et
al., a-catenin is a molecular swiitch that binds
Ecadherin -ß-catenin and regulates actin
filament assembly. Cell 123 903-915
6
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 ß-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

7
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

8
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

9
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

10
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

11
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

12
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 (a,ß,?) each with a unique gene
  structure that encode constant and variable
  domains

13
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-
  ? genes in mice resulted in no general defects
  in neuronal survival, migration etc.

14
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-a 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

15
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 ß-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

16
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

17
Deconstructing the Cadherin-Catenin-Actin
ComplexYamada et al., Cell 2005

• Summary
• The prevailing dogma is that cadherins are linked
  to the actin cytoskeleton through ß-catenin and
  a-catenin, however, the authors show that this
  quaternary complex does not happen

18
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, 11 complex with ß-catenin, and
  ß-catenin binds with lower affinity to a-catenin
• Several studies (12) show that a-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

19
Binding of a-catenin to actin and ß-catenin is
mutually exclusive

• Actin-filament pelleting assay
• a-catenin pelleted with actin filaments in the
  presence of increasing concentrations of
  E-cadherin-ß-catenin complex
• However, E-cadherin- ß-catenin did not pellet
  above the background level
• Result
• The chimera failed to bind actin in the pelleting
  assay

20
Reconstitution of ß and a-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
• ß-catenin addition to the patches reached about
  80 of the prestripped level while only 25 for
  a-catenin

21
Actin filaments do not assemble on reconstituted
membranes

• Actin binding was not detected on stripped
  membrane patches which were preincubated with
  a-catenin-ß-catenin complex

22
Measurement of the complex at mature cell-cell
contacts

• E-cadherin, a-catenin, ß-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), a-catenin (0.43
  min, 33.7), ß-catenin (0.66, 34.2) were similar
• Mutants of E-cadherin (lacking the cytomplasmic
  domain) and a-catenin (lacking the actin binding
  domain) were expressed
• Both mutant E-cadherin and a-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, a-catenin, ß-catenin
  
• Rhod-actin had recovery kinetics similar to that
  of GFP-actin (recovery 0.21 min)

23
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

24
Disrupting actin organization does not affect
cadherin or a-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 acatenin-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

25
(No Transcript)
26
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

27
Questions ?