khri final presentation_final
TRANSCRIPT
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Kresge Summer Research Internship – Final Presentation
Keerthana VelappanMentor: Dr. David Kohrman
August 6th, 2015
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Introduction to Grxcr1 and Grxcr2
• Cochlear-specific genes required for stereocilia development and maintenance
• Grxcr1 spontaneous mutant: pirouette (pi)
• Grxcr2 targeted mutant
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Grxcr1/Grxcr2 Domain Structure
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Preliminary Correction
• Actions of Grxcr1 and Grxcr2 affect development and maintenance of stereocilia, likely through effects on actin filament architecture (structure and rigidity)
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How does Grxcr1 and Grxcr2 work?
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Putative PP1 Binding Sites
Grxcr1 interacts with/inhibits PP1 phosphatase
Hendricks, A., et al. (2009). Chemistry and Biology, 16:365-371.
RKVRF
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Hypothesis/Experimental Test
• Hypothesis– Grxcr1 (and/or Grxcr2) exerts effects on
stereocilia by control of phosphorylation via interaction with/inhibition of PP1
• Experimental Test– Delete 5 a.a. (15bp) predicted PP1 interaction site
from Grxcr1 and Grxcr2• Express wild type and mutant proteins from peGFP-N1
hybrid plasmid vector
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Experimental Test
Grxcr1 GFP
GFP Grxcr2
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Predictions for Hypothesis
• If hypothesis is correct…– Cells expressing mutant proteins (relative to wild
type) expected to exhibit alterations in PP1 localization and/or phosphorylation activity
– Mutant proteins expected to lack ability to ‘correct’ stereocilia defects in explant cultures (wild type expected to correct those defects)
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Initial Work
• Generation of Grxcr1 and Grxcr2 mutant plasmids (without 5 a.a./15bp PP1 site)
• Methods for Grxcr2– Site-directed mutagenesis– Bacterial transformation
• Methods for Grxcr1– Overlap PCR
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Grxcr2: Site-Directed Mutagenesis
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Site-Directed Mutagenesis (cont’d)
• Add WT plasmid/mutant oligos – Complementary genes (oligos)
• Denature both components• DNA polymerase extends oligos during temperature
cycling, incorporates mutation– Stem-loop structure (15 nucleotide deletion)– High-fidelity low error rate polymerase (Pfu) – Extend 3’ end of oligos– Product treated with DpnI/nicked DNA is transformed
• WT template is methylated=> DpnI-sensitive fragments (cuts/circle)
• Mutated molecule unmethylated=> DpnI-resistant
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Grxcr2: Results
• Confirmed overall structure of mutant plasmid: BamH1-EcoRI restriction enzyme digest
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Grxcr2: Results (cont’d)
• Confirmed deletion is only in one place => sequencing (used SeqMan to align sequences)
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Grxcr1: Overlap PCR
056 170
171 172
227bp
923bp
1.1kb056
172
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056/171 and 170/172 (1 ng, 200 pg, 40 pg)
PRIMARY PCR REACTIONS
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SECONDARY PCR REACTIONS
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Grxcr1: Results
• Grxd1 #4 replaced faulty wild type Grxcr1/C023 clone
• Considerable attempts to improve the specificity/yield of the products:– Altered annealing temperatures and cycling
conditions during PCR– Different amounts of templates– Purification of initial smaller products
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Grxcr1: Results (most recent)
• Third PCR amplification with DK177/178: initial gel with this indicated likely expressed product – Used PrimerSelect to order new primers that fit
certain conditions
177
178
~1.0kb
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Grxcr1: Results (repeatability!)
• New starting point from 227 and 923 insert sizes (1:20)–Standard dilutions
(initial product above from 1:10000 dilution)
–Ran under 60° annealing temp/35x cycles
1:1000 generated the right size product!
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Mutant Expression in Eukaryotic Cells
• COS7: fibroblasts from African Green monkeys – Grxcr1 and Grxcr2 know to localize to actin
filament-rich filopodia
• CL4: epithelial cells derived from pig kidneys– Fibroblasts to epithelial cells (tight junctions)– Grxcr1 and Grxcr2 know to localize to actin
filament-rich microvilli on top
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Mutant Expression: Methods
• Plate cells on glass coverslips• Transfection of plasmid DNA with LTX reagent• Fix/permeabilize transfected cells
– Incubate with anti-GFP primary antibody (mouse) and anti-PP1 primary antibody (rabbit)
– Identify location of protein-antibody complexes with secondary antibodies:
• Anti-mouse IgG-Alex488 (green for GFP complexes)• Anti-rabbit IgG-Alex594 (red for PP1 complexes)
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Mutant Expression: Results
• Expected:– Localization of wild type Grxcr2-GFP (and Grxcr1-
GFP) to microvilli of CL4/filopodia of Cos7 cells • Experimental Questions:
– Localization of mutant proteins?– Altered localization of PP1 in mutant expressing
cells?
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Results: Mutant less microvilli enrichment
CL4 C024 (wild type)
CL4 C026 (mutant)
eGFP parent plasmid
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Results: PP1 similar in WT/mutant
C024 GFP C026 GFP eGFP GFP
C026 PP1C024 PP1 eGFP PP1
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In Conclusion/Future Work
• Grxcr2 mutant sequence confirmed• Grxcr1 mutant clone yet to be generated
• Continue to experiment with Grxcr1 mutant to replicate initial overlap
• Continue to follow up on transfected cells (i.e. laser scope microscope for fine sections)– Top of cell vs. inside of cell – (Small region of N-terminus needed for localization)
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Acknowledgments
• Dr. Kohrman • Cathy, Catherine, Jessica• Kresge Hearing Research Institute (KHRI)• National Institute on Deafness and Other
Communication Disorders (NIDCD)