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Understanding deafness in the Usher syndrome:
A possible interaction between Grxcr1 and the zebrafish
Usher complex
Jennifer M. Panlilio
Westerfield Lab
Roadmap I. Introduction to the Usher Syndrome and
mechanosensation
II. Grxcr1 as a possible protein partner of the Usher complex
III. Results: Characterization of Grxcr1 depletion phenotype in zebrafish
V. Future Directions
Overview of Usher Syndrome•Most common form of hereditary deafblindness in humans
•Symptoms include issues with hearing, balance and vision
•Three clinical subtypes:
Usher Type I Usher Type II Usher Type III
How Hearing Works: A look into the inner ear hair cells
Sound
MechanoreceptorStereocilia +Kinocilium
Cell body
Nerve fibers
Actin
Usher protein complex: Protein
Myosin VIIa
Harmonin
Cadherin 23
Protocadherin 15
Sans
Usherin
VLGR1b
Whirlin
Clarin-1
Hars-1
Finding proteins interacting with the Usher protein complex:
How are the Usher proteins transported?
How is the complex is assembled?
Grxcr1 as a candidate Glutaredoxin cysteine-rich 1 in Inner ear of mouse in p5
Frolenkov et al. Nature Reviews, 2004 Odeh et al., Journal of Human Genetics ,2010
Grxcr1: green Actin: red
Grxcr1 is present in the stereocilia
Karolyi et al. Human Molecular Genetics, 2003
There are phenotypic similarities in the inner ear between Grxcr1 mutant and the Usher mutants
Grxcr1 mouse mutant
Wild type mouse
Usher mutant (Myo7a) mouse
Grxcr1 as a candidate
Strategy
Characterization of grxcr1 expression pattern
Depletion of Grxcr1 to determine whether it is required in mechanoreceptor development
Effects of depletion of Grxcr1 on the Usher protein complex
grxcr1 expression patternWhere is grxcr1 expressed?
grxcr1 is expressed in the ears, eyes, brain and liver of 5 dpf larvae
Brain
Eyes
Inner Ear
Liver
Loss of Grxcr1 function
Exon 1 2 3 4
Exon 1 2 3 4Wt mRNA
Pre-mRNA
Splice blocker MO1-grxcr1
MO1-grxcr1 protein
Wt protein
Exon 1 2 3 4MO1-grxcr1 mRNA
Loss of Grxcr1 functionSplice blocker MO1-grxcr1
Exon 1 3 4
Exon 1 2 3 4Wt mRNA
2pre-RNA
Exon 1 2 3 4MO2-grxcr1 mRNA
Loss of Grxcr1 functionSplice blocker MO2-grxcr1
MO2-grxcr1 protein
Wt protein
Loss of Grxcr1 functionSplice blocker MO2-grxcr1
Non injected
MO-2 grxcr1
Defects in mechanoreceptor development following Grxcr1
depletion
Wild type MO2-grxcr1
MO2-grxcr1
Actin Staining
Usher protein complex: Protein
Myosin VIIa
Harmonin
Cadherin 23
Protocadherin 15
Sans
Usherin
VLGR1b
Whirlin
Clarin-1
Hars-1
Depletion of Grxcr1 on the Usher complex in 5 dpf larvae
Myo 7aa
Myo7aa seems to be less concentrated apically in morphants
Harmonin
Harmonin seems to be depleted in morphants
Wild type MO2- grxcr1
\
Conclusionsgrxcr1 is expressed in the eyes and inner ear like usher genes
Grxcr1 may play a role in mechanoreceptor development
Grxcr1 effects the localization of some Usher proteins
Future Directions
How does Grxcr1 affect the Usher complex?-via study of changes of localization with other known Usher proteins
Does Grxcr1 interact with the complex?-via Colocalization
-via Coimmunoprecipitation
AcknowledgementsMonte WesterfieldAurélie ClémentBerde Blanco-SanchezJudy PeirceJeremy WegnerSabrina ToroJennifer PhillipsRachel Rae Drake Marcel RockwellPeter O’DayAdam UngerNIHCD