khri final presentation_final

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Kresge Summer Research Internship – Final Presentation Keerthana Velappan Mentor: Dr. David Kohrman August 6 th , 2015

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Page 1: KHRI Final Presentation_FINAL

Kresge Summer Research Internship – Final Presentation

Keerthana VelappanMentor: Dr. David Kohrman

August 6th, 2015

Page 2: KHRI Final Presentation_FINAL

Introduction to Grxcr1 and Grxcr2

• Cochlear-specific genes required for stereocilia development and maintenance

• Grxcr1 spontaneous mutant: pirouette (pi)

• Grxcr2 targeted mutant

Page 3: KHRI Final Presentation_FINAL

Grxcr1/Grxcr2 Domain Structure

Page 4: KHRI Final Presentation_FINAL

Preliminary Correction

• Actions of Grxcr1 and Grxcr2 affect development and maintenance of stereocilia, likely through effects on actin filament architecture (structure and rigidity)

Page 5: KHRI Final Presentation_FINAL

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

Page 7: KHRI Final Presentation_FINAL

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

Page 8: KHRI Final Presentation_FINAL

Experimental Test

Grxcr1 GFP

GFP Grxcr2

Page 9: KHRI Final Presentation_FINAL

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)

Page 10: KHRI Final Presentation_FINAL

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

Page 11: KHRI Final Presentation_FINAL

Grxcr2: Site-Directed Mutagenesis

Page 12: KHRI Final Presentation_FINAL

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

Page 13: KHRI Final Presentation_FINAL

Grxcr2: Results

• Confirmed overall structure of mutant plasmid: BamH1-EcoRI restriction enzyme digest

Page 14: KHRI Final Presentation_FINAL

Grxcr2: Results (cont’d)

• Confirmed deletion is only in one place => sequencing (used SeqMan to align sequences)

Page 15: KHRI Final Presentation_FINAL

Grxcr1: Overlap PCR

056 170

171 172

227bp

923bp

1.1kb056

172

Page 16: KHRI Final Presentation_FINAL

056/171 and 170/172 (1 ng, 200 pg, 40 pg)

PRIMARY PCR REACTIONS

Page 17: KHRI Final Presentation_FINAL

SECONDARY PCR REACTIONS

Page 18: KHRI Final Presentation_FINAL

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

Page 19: KHRI Final Presentation_FINAL

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

Page 20: KHRI Final Presentation_FINAL

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!

Page 21: KHRI Final Presentation_FINAL

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

Page 22: KHRI Final Presentation_FINAL

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)

Page 23: KHRI Final Presentation_FINAL

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?

Page 24: KHRI Final Presentation_FINAL

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

Page 26: KHRI Final Presentation_FINAL

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)

Page 27: KHRI Final Presentation_FINAL

Acknowledgments

• Dr. Kohrman • Cathy, Catherine, Jessica• Kresge Hearing Research Institute (KHRI)• National Institute on Deafness and Other

Communication Disorders (NIDCD)