Handout 15 - Cell-Cell Adhesion - Cell-cell/ECM...

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Cell-cell/ECM interactions, Cell-cell adhesion Background (experimental embryology) Cell adhesion molecules Cadherins - Identification - Functional analysis Cadherin-associated proteins
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Early experiments: cell adhesion H.V. Wilson (1907)- sponge cell adhesion http://www.mbl.edu/animals/images/microciona.3.jpg pass through sieve (mechanical dissociation) individual cells (not to scale) reaggregate
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pass through sieve (mechanical dissociation) mix none Use two different species: sort sort
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Early Embryogenesis Red = ectoderm Blue = Neural Green = mesoderm Yellow = endoderm Alberts 22-75
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Reaggregation of amphibian embryonic cells (Townes and Holtfreter, 1955) http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=dbio.figgrp.379
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Sorting in amphibian embryonic cell aggregates http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=dbio.figgrp.380 Epidermis + Mesoderm Mesoderm + Endoderm Neural plate + Epidermis Neural plate + Mesoderm + Epidermis Epidermis + Mesoderm + Endoderm
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Cell sorting in embryonic organs http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=dbio.figgrp.384 1. Dissociate embryonic organs 2. Combine 3. Cells reaggregate 4. Sort according to organ type 5. Specific patterns of aggregation Differential adhesion hypothesis (Steinberg): Cells sort due to differences in adhesive properties (could be quantitative or qualitative)
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Three ways in which surface molecules can mediate adhesion Alberts, Fig. 19-26 Aggregation experiments defined two types of adhesions: Classes: Ca 2+ -dependent and Ca 2+ -independent
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How can one identify adhesion molecules? First, need an assay: in vitro aggregation. dissociated cells in Petri dish rotate slowly for 1 hour aggregate Where are the molecules that mediate adhesion localized? So, look for antibodies against surface proteins that can block aggregation + antibodies don’t aggregate
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Adhesion blocking antibodies were used to identify the Ca 2+ -dependent adhesion molecules How can antibodies be used to identify the gene encoding a protein of interest? - Screen cDNA expression library - Clone cDNA to vector with bacterial promoter and shine delgarno site, transcribe, translate. Each bacteria will make a different protein. - The cDNA encodes a putative cell-adhesion molecules (CAM); how do you convince yourself that it really is a CAM? - Introduce blocking antibody to see which bacteria type has the proper encoding cDNA transformed.
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Experimental demonstration: knowing you have the CAM Express CAM ectopically in cells that lack Ca 2+ -dependent adhesion. Take clone and transfect them into cells that don’t normally adhere to each other
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This note was uploaded on 06/12/2010 for the course MCB 252 taught by Professor Prasanth during the Fall '08 term at University of Illinois at Urbana–Champaign.

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Handout 15 - Cell-Cell Adhesion - Cell-cell/ECM...

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