Cadherin-Catenin-Actin Complex

1. Cadherin-Catenin-Actin Complex Piyush Bajaj BIOE 506 April 29th, 2008

2. Cadherins in development:cell 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 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

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 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

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 (α,β,γ) 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- γ 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-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

15. Atypical cadherins and PCP • PCP refers to polarized orientation of epithelial cells along the long axis of the cell monolayer • Large atypical cadherinsDachsous (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

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 β-catenin and α-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, 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

19. Binding of α-catenin to actin and β-catenin 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

20. 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 α-catenin

21. Actin filaments do not assemble on reconstituted membranes • Actin binding was not detected on stripped membrane patches which were preincubated with α-catenin-β-catenin complex

22. 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)

23. Contd. GFP Endogenous • 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 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

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 ?