uconn hsrap 2012 - pp oral presentation
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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.
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