The Role of the Nectin-Afadin Cell Adhesion Complex in Migrating Parietal Endoderm UCHC High School Reasearch

Apprenticeship Program

Caroline Kuzoian and Anurag Ojha

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

• PE (Parietal Endoderm) cells• First migratory embryonic cells • Derived by F9 cells

• Interaction helps cells migrate properly to form yolk sac surrounding embryo

Objective• To determine the presence and function of the

Nectin-Afadin complex

F9 Model SystemF9 Primitive Endoderm PE/VE

Proteins of Interest• Nectin – trans-membrane protein, extracellular

component of the Nectin-Afadin complex• Afadin – intracellular component of Nectin-

Afadin complex, binding to the cytoskeleton• Partitioning Defective 3 (Par-3) – binds to

nectin and recruits afadin to create the Nectin-Afadin complex

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

Nectin-Afadin Complex• 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• E-Cadherins create a strong bond between cells in the

adherens junction

siRNA Mediated Knockdown• Small interfering • A technique used to decrease the translation of a

targeted protein• Results in double stranded mRNA which cannot be

translated completely

Antibodies• Primary• Secondary• Tags• Horseradish Peroxidase – Western blotting• Alexa fluor – Immunofluorescence• Beads coated in A/G protein – Immunoprecipitation

Western Blotting• A process to determine presence of specific

proteins within a cell lysate• Used to compare presence of same proteins within

different cell types• Proteins are identified by their molecular weight

WB:Process• 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• Wash

Primary Antibod

y

• Incubate one hour

• Wash

Secondary

Antibody

• Substrate produces color

OPTI-4CN Stain

Protein

Protein

Western Blots

Protein Molecular Weight (kDa)

Nectin 87

Afadin 200

Par-3 180, 150, 100

Actin 42

Probed by afadin antibody 7/17

Probed by actin antibody 7/17

Control Lipofectamine siScramble siAfadin siPar-3 Control

200

kDa

42 kDa

Control Lipofectamine siScramble siAfadin siPar-3 Control

Biotin Immunoprecipitation• Biotinylation – process in which biotin, a

coenzyme, attaches to all the proteins present on the cell surface

• Immunoprecipitation - Primary and secondary antibodies bind to target protein and pull down the protein of interest with the help of a bead coated in an A/G protein

Immunofluorescence

Immunofluorescence

Future Directions• More immunofluorescence staining of siRNA

treated cells to determine functional changes of the Nectin-Afadin complex

• More western blotting to determine changes of levels in par3 and nectin after siRNA treatment

AcknowledgementsWe would like to acknowledge the support of:• The Department of Health Career Opportunities• Aetna Health Professions Partnership Initiative.• Theo Szmurlo• David Artus• Alexander “The Great” Pokorski• Nathaniel Aponte• Dr. Kathy Martin PhD.• Dr. Jim Mulrooney PhD.