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

2. Cadherins in development:cell adhesion, sorting and tissue morphogenesis Jennifer M. Halbleib and W. James Nelson, Genes and Development, 2006 2 Summary Although cadherins evolved to facilitate mechanical cell-cell adhesion, they play a very important role in tissue morphogenesis

3. Cadherins 3 Surface glycoprotein responsible for Ca 2+ 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] http://en.wikipedia.org/wiki/Cadherinshttp://en.wikipedia.org/wiki/Cadherins [1]

4. Classical cadherin 4 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 5 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.htmhttp://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 6 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 7 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 8 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 9 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 10 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 11 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 12 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 13 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 14 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 15 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

16. Atypical cadherins in vertebrate development 16 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 Complex Yamada et al., Cell 2005 17 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 18 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 19 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 20 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 21 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 22 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. 23 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

24. Disrupting actin organization does not affect cadherin or α-catenin dynamics 24 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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26. Conclusion 26 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 ? 27