The Role of the Nectin-Afadin Cell Adhesion Complex in

Migrating Parietal Endoderm

Caroline Kuzoian and Anurag Ojha

Cell Migration is found in:◦ Wound healing◦ Embryonic Development◦ Cancer (metastasis)

PE (Parietal Endoderm) and F9 cells were used in the experiment

PE cells are made from F9 cells The interaction with each other helps the cells

migrate properly to form the yolk sac surrounding the embryo

Cell Migration

To determine the presence and function of the Nectin-Afadin complex.

Objective

Model System

Afadin – intracellular component of nectin-afadin complex, binding to the cytoskeleton

Nectin – extracellular component of the nectin-afadin complex, creates homophilic bonds

Partitioning Defective 3 (Par-3) – binds to nectin and recruits afadin to create the nectin-afadin complex

Actin – is a major component of the cytoskeleton and is involved in cell-to-cell interactions.

Proteins of Interest

Par-3 binds to nectin, nectin binds to afadin, which binds to actin◦ This causes actin to reorganize and recruit inactive E-

Cadherins to the binding site.

Nectin-Afadin Complex

Cell One

Cell Two

Primary Secondary

◦ Tags Horseradish Peroxidase – Western blotting Alexa fluor – immunofluorescence

Antibodies

A process to determine presence of specific proteins within a cell lysate.◦ Used to compare presence of the same proteins

within different cell types Identification of proteins by molecular

weight

Western Blotting

•Lyse=burst• Isolate proteinsCell Lysis

•Find unknown protein concentration

Bradford Assay

•Run current through gel

•Separate proteins by size

Loading Gel

•Run current•Proteins pulled onto membrane

Semi-Dry Blotting

• Incubate overnight

•WashPrimary Antibody

• Incubate one hour

•Wash

Secondary

Antibody

•Substrate produces color

OPTI-4CN Stain

WB:ProcessProtein

Western Blots

Protein Molecular Weight (kDa)

Nectin 87

Afadin 200

Par-3 180, 150, 100

Actin 42

Si Treatment Afadin 7/17

Si Treatment Actin 7/17

Control Lipofectamine Si Scramble Actin Par-3 Control

Control Lipofectamine Si Scramble Actin Par-3 Control

Add biotin to the cells when they are whole Labels proteins on cell surface

Bradford Assay- Protein Concentrations Antibodies over night to isolate desired protein Run Gel Transfer to membrane Streptavidin: Binds to biotin to show whether the protein

was cell surface bound Used OPTI4CN to develop a blot Biotin can be directly searched for

Immunoprecipitation

Immunofluorescence

Immunofluorescence

A technique used to decrease the translation of a targeted protein. ◦ Results in double stranded mRNA which cannot be

translated completely.

siRNA Mediated Knockdown

Immunofluorescence staining of siRNA treated cells to determine functional changes of the nectin-afadin complex

Western blotting to determine changes of levels in par3 and nectin after siRNA treatment

Future Directions

Kathy Martin Jim Mulrooney Theo Szurlo David Artus Nate Aponte Alex Pokorski

Acknowledgements

Kudra, Randy, 2011, The Role of Cell to Cell Adhesions in Parietal Endoderm Migration, Central Connecticut State University, 1-34.

Science Direct. 2012 Elsevier B.V., 22 Sept. 2003. Web. 19 July 2012. <http://www.sciencedirect.com/science/article/pii/S0959438803001247>.

The University of Queensland. 2012 The University of Queensland, n.d. Web. 20 July 2012.

References