KHRI Final Presentation_FINAL

Kresge Summer Research Internship – Final Presentation

Keerthana VelappanMentor: Dr. David Kohrman

August 6th, 2015

Introduction to Grxcr1 and Grxcr2

• Cochlear-specific genes required for stereocilia development and maintenance

• Grxcr1 spontaneous mutant: pirouette (pi)

• Grxcr2 targeted mutant

Grxcr1/Grxcr2 Domain Structure

Preliminary Correction

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

How does Grxcr1 and Grxcr2 work?

Putative PP1 Binding Sites

Grxcr1 interacts with/inhibits PP1 phosphatase

Hendricks, A., et al. (2009). Chemistry and Biology, 16:365-371.

RKVRF

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

Experimental Test

Grxcr1 GFP

GFP Grxcr2

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)

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

Grxcr2: Site-Directed Mutagenesis

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

Grxcr2: Results

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

Grxcr2: Results (cont’d)

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

Grxcr1: Overlap PCR

056 170

171 172

227bp

923bp

1.1kb056

172

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

PRIMARY PCR REACTIONS

SECONDARY PCR REACTIONS

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

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

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!

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

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)

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?

Results: Mutant less microvilli enrichment

CL4 C024 (wild type)

CL4 C026 (mutant)

eGFP parent plasmid

Results: PP1 similar in WT/mutant

C024 GFP C026 GFP eGFP GFP

C026 PP1C024 PP1 eGFP PP1

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)

Acknowledgments

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

Communication Disorders (NIDCD)