Introduction to MCB 252 Topic 27Integrating Cells into Tissues Part 1 Cadherins and Cell Adhesion Prof David Rivier MCB 252 Spring 2015 MCB 252 Topic 27 Reading Lodish 20 0 20 2 Integrating Cells into Tissues Cell Cell Adhesion Cell ECM Adhesion Types of Tissues Multicellular Organisms Sorting of cells Discovery of Cell Adhesion Molecules Cadherins Structure and mechanism of cadherin mediated adhesion Types of Tissue Epithelial Tissue Connective Tissue Muscle Tissue Nervous Tissue Epithelia Simple Keratinized vs soft Epithelia Epithelial Tissue Absorption Exchange Secretion Protection Waterproofing Connective Tissue Dense Connective Tissue Epithelia Connective Tissue Connective Tissue Connective Tissue Connective Tissue Anchor Support Connect Cushion Adipose Padding p rotection Insulation Muscle Nervous Tissue Plus glial cells and other specialized cells Integrating Cells into Tissues Types of Tissues Multicellular Organisms Discovery of Cell Adhesion Molecules Cadherins Identification Functional analysis Cadherin associated proteins Other Classes of CAMs Some Major transitions in evolution 3 5 billion years ago unicellular life emerges photosynthetic bacteria begin to release oxygen 2 longer ago than 2 7 BYA Eukaryotes emerge 555 million years ago Multicellular organisms common in the sea Single cell organisms multicellular organisms predates this time 500 million years ago vertebrate fish 450 million years ago Arthropods move onto land Descendants become spiders mites scorpions and millipedes 420 MYA Land plants evolve emerge 360 MYA 4 limbed vertebrates move onto land as seed plants and large forests emerge Philosophical or life style differences between unicellular organisms and multicellular organisms C elegans Development follows an Invariant Lineage Integrating Cells into Tissues Cell Cell Adhesion Cell ECM Adhesion Types of Tissues Multicellular Organisms Sorting of cells Discovery of Cell Adhesion Molecules Cadherins Structure and mechanism of cadherin mediated adhesion Early experiments to understand cell adhesion H V Wilson 1907 sponge cell adhesion pass through sieve mechanical dissociation reaggregate individual cells not to scale http www mbl edu animals images microciona 3 jpg Use two different species pass through sieve mechanical dissociation mix Use two different species sort pass through sieve mechanical dissociation mix sort Embryogenesis Alberts 22 75 Red ectoderm Green mesoderm Blue Neural Yellow endoderm Reaggregation of amphibian embryonic cells Townes and Holtfreter 1955 http www ncbi nlm nih gov books bv fcgi rid dbio figgrp 379 Sorting in amphibian embryonic cell aggregates Epidermis Mesoderm Mesoderm Endoderm Epidermis Mesoderm Endoderm Neural plate Neural plate Mesoderm Epidermis Epidermis http www ncbi nlm nih gov books bv fcgi rid dbio figgrp 380 Cell sorting in embryonic organs 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 http www ncbi nlm nih gov books bv fcgi rid dbio figgrp 384 Discovery of cadherin a k a Superman my hero novel antibody that disrupts cell cell adhesion C a C a Cell aggregation in calcium non immune control Ab Antibody recognizes a band on cells trypsinized in the presence of calcium but not in the absence of calcium Takeichi J Cell Biol 1977 Yoshida and Takeichi 1982 How can one identify the hypothetical adhesion molecules First need an assay in vitro aggregation rotate slowly for 1 hour aggregate dissociated cells in Petri dish antibodies Where are the molecules that mediate adhesion localized So look for antibodies against surface proteins that can block aggregation don t aggregate 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 The cDNA encodes a putative cell adhesion molecules CAM how do you convince yourself that it really is a CAM Genomic Library Libraries are almost always made in E coli Libraries are collections of a mixture of clones Plating for Single Cells Plate Single cells Grow to colony Rich media Transformed E coli cells All Cells in a Colony are Identical Each Colony Contains a Different Clone Genomic Library Libraries are almost always made in E coli Libraries are collections of a mixture of clones Libraries can refer to bacterial colonies or to DNA purified from pooled bacterial colonies Genomic Library To find the clone you are interested in within the library you need a way to search through all the clones in the library for the rare clone that is the one you are interested in You need a way to screen the library cDNA Library Lodish 5 15 Expression Library Pr SD cDNA library Term Each bacterial colony within the library will produce the human protein corresponding to the particular cDNA clone in that colony Screening a Library To find the clone you are interested in within the library you need a way to search through all the clones in the library for the rare clone that is the one you are interested in You need a way to screen the library How would you identify a protein a band on a gel in the genomic era Use antibody to precipitate the protein of interest or to identify which band on a gel is your protein Cut out band on gel or take precipitated protein and subject it to mass spectrometry Identify protein based on protein composition and mass compared to a data based of information on each and every protein in the genome Adhesion blocking antibodies were used to identify the Ca2 dependent adhesion molecules How can antibodies be used to identify the gene encoding a protein of interest Screen cDNA expression library The cDNA encodes a putative cell adhesion molecules CAM how do you convince yourself that it really is a CAM Expressing cadherins in a fibroblast confers cell cell adhesive properties Transfecting these cells with a cDNA encoding for E cadherin confers the property of calcium dependent cell cell adhesion Particles at T Particles at T0 Fibroblasts and certain cancer cells show very little cell cell adhesion even in the presence of Ca2 Control E cadherin Time An Experiment Demonstrating that Cadherins are Necessary for Strong Cell Cell Adhesion Cells inside this embryo which has been depleted of the major cadherin expressed at this time C