MCB 252 Topic 28Integrating Cells into Tissues Part 2 Cadherins and other CAMs Prof David Rivier MCB 252 Spring 2015 MCB 252 Topic 28 Reading Lodish 20 0 20 2 Topic 28 Integrating Cells into Tissues Cadherins and other CAMS Outline Cadherin cadherin interactions Sorting by a Single Type of Cadherin Role of Cytoplasmic Domain Attachment to Actin Cytoskeleton Other CAMs and Wiring the Brain CAMs and Cell Movement Cadherins in Cancer Different cadherins are expressed in different cell types E cadherin epithelia N cadherin neurons heart skeletal muscle P cadherin placenta breast epithelium VE cadherin endothelial cells How does cadherin mediated adhesion work Express E cadherin in L cells label w green fluorescence Express N cadherin in L cells label w red fluorescence sort out Qualitative differences in adhesion molecules can mediate cell cell interactions Three ways in which surface molecules can mediate adhesion Aggregation experiments defined two types of adhesions Ca2 dependent and Ca2 independent Alberts Fig 19 26 Cell sorting can be driven by differential adhesive strength Lower level of N Cadherin in red Higher level of N cadherin in green Cell sorting may be due to both different types of cadherins and different levels of cadherins Other levels of regulation such as PTM These Models Based Analysis of the Extracellular Domain Alone Cell 1 Cell 2 Lodish Fig 6 2 Alberts Fig 19 24 Does the Cytoplasmic Domain Contribute to Adhesion Does Cadherin s Cytoplasmic Domain Contribute to Adhesion Cytoplasmic Domain is Conserved Experimental Strategy seems odd but see results in 2 slides Start with Epithelia cells in culture Overexpress Cytoplasmic Domain of N Cadherin in those cells Overexpression of a cadherin cytoplasmic tail disrupts cell cell adhesion Wild type Cells Overexpressed Cytoplasmic Domain Phase contract microscopy Ab to E cadherin Ab to N cadherin Overexpression of a cadherin cytoplasmic tail disrupts cell cell adhesion A Dominant Negative Effect How might overexpression of cytoplasmic domain disrupt adhesion Hypothesis Cytoplasmic domain sequesters or binds a component that is necessary for adhesion in such that the component is not available to to the wild type cadherin Prediction 1 Cytoplasmic domain binds something required for adhesion and 2 The cytoplasmic domain itself is required for or contributes to adhesion Is Cadherin s Conserved Intracellular Domain Required for Adhesion Delete different portions of the intracellular domain EC TM intracellular domain adhesion activity Intracellular domain is required for adhesion What does Cadherin s Intracellular Domain Interact with Immuno precipitation and co immuno precipitation 1 Add antibodies that recognize protein of interest Cell extract in test tube 3 2 Isolate beads analyze to see if other proteins are bound Add beads containing antibody binding protein e g protein A What does Cadherin s Intracellular Domain Interact with Co Immuno precipitation Co IP Use anti cadherin antibodies to immuno precipitate Cadherin pull down three other proteins that bind Cadherin alpha beta gamma catenin from catena Latin for chain These proteins link the IC domain of cadherin to the actin cytoskeleton Proteins that link Cadherin intracellular domain to the actin cytoskeleton Lodish Fig 20 13 Cadherins are Sometimes Normally Clustered with Actin Filaments Inside Cells Tang and Brieher J Cell Biol 2012 Punctate staining Clustering Co localization with Actin Role of Cadherin s Conserved Intracellular Domain Delete different portions of the intracellular domain EC TM intracellular domain adhesion activity cytoskeleton attachment Intracellular domain is required for adhesion Topic 28 Integrating Cells into Tissues Cadherins and other CAMS Outline Cadherin interactions Sorting by a Single Type of Cadherin Role of Cytoplasmic Domain Attachment to Actin Cytoskeleton Other CAMs and Wiring the Brain CAMs and Cell Movement Cadherins in Cancer Major Families of CAMs an ECM molecule Ig immunoglobulin Immunoglobulin Ig superfamily Ca 2 independent adhesion Neural Cell Adhesion Molecule NCAM Lodish 19 2 Like Cadherin originally identified by antibody inhibition of aggregation mediates homophilic interaction At least 20 forms generated by alternative splicing Carries large amount of negatively charged sugar poly sialic acid PSA NCAM PSA aggregates less well than NCAM PSA amount of PSA decreases during development What does this mean Intercellular Adhesion Molecules ICAMs leukocyte movement into tissues Ig superfamily In general play important roles in proper targeting of neurons Hundreds of family members 750 in human genome Why so many Complexity of human brain 100 x 109 neurons 1000 connections each 100 x 1012 connections Differential cadherin expression demarcates regions of the developing brain Takeichi and others think that the diversity of cadherin family members might help explain complex segregation of different cell types Different cadherins for example are expressed in discrete regions of the brain Topic 28 Integrating Cells into Tissues Cadherins and other CAMS Outline Cadherin interactions Sorting by a Single Type of Cadherin Role of Cytoplasmic Domain Attachment to Actin Cytoskeleton Other CAMs and Wiring the Brain CAMs and Cell Movement Cadherins in Cancer Cadherins in Developing Nervous System Alberts 19 12c Loss of Reduced Adhesion Needed for Movement Cadherins and Cancer Metastasis Loss of E cadherin correlates with metastasis Reexpressing E cadherin in a highly metastatic epithelial cancer cell suppresses metastasis Mix a normal heart fragment H with normal epithelial cells Epithelial cells encapsulate the heart fragment Mix a normal heart fragment H with metastatic epithelial cells Epithelial cells invade the heart fragment Force expression of E cadherin in the metastatic epithelial cells Epithelial cells once again encapsulate the heart fragment K Vleminckx et al Cell 1991