Discussion paper 8 Flashcards
What proteins make up the tip link?
Protocadherin15 (PCDH15) and cadherin23 (CDH23)
What kind of protein is harmonin?
Scaffolding protein
Is harmonin localized at the UTLD or the LTLD?
UTLD
How many forms of harmonin are there?
3 (a b and c)
Which form of harmonin is found in hair cells?
Harmonin-b
What is H3?
An antibody which binds to the PDZ 3 domain of harmonin. This domain is found in B and C variants
Why is H3 used in this experiment?
Immunolabelling tool to see where harmonin is localized
Where is harmonin located in the stereocilia?
In the UTLD which is below the tips of the stereocilia
What was the purpose of using gold labelled EM in this experiment?
Confirm the immunolabelling data: was used to visualize the location of harmonin in the stereocilia – it was confirmed that harmonin is located at the UTLD
Which protein on the tip link does harmonin associate with?
CDH23
Which domain of harmonin does CDH23 interact with?
PDZ 2 domain
What is the key binding sequence of harmonin’s PDZ2 domain which binds to CDH23?
GLG
What is the Pdz^aaa mutant?
A mutant form of harmonin which has a triple alanine (AAA) instead of GLG
Describe the immunoprecipitation experiment
Bind cytoplasmic region of CDH23 (the part that interacts with harmonin) to IL2 (which we have an antibody for) and wait for this protein construct to interact with harmonin, magnetically separate the IL2 protein lysate and run a WB: if harmonin is in the WB, then we know CDH23 binds to harmonin
Can harmonin bind to the AAA-Pdz2?
No
Why did Pdz-AAA mice display head tossing behaviour?
Their hair cells in their vestibular system were defective
How were Pdz-AAA mutant mice different from wt mice in terms of harmonin localization?
Harmonin was diffuse: did not have punctate expression
Describe the hearing of Pdz-AAA mice
Had very limited hearing, even in response to loud sounds
Which region of harmonin is thought to bind to the cytoskeleton?
CC1-CC2-PST domain
What are deaf circler mice?
Mice which have a missing actin-binding domain of harmonin
Do deaf circler mice have harmonin which is capable of binding to CDH23?
Yes, because they have wild type PDZ2
CC1/CC2 domain of harmonin is also called the…
Coil-coil domain
Do deaf circler mice have normal tip links?
Yes, but totally lack UTLD
Why is it important that the mice studied in this paper still have a tip link?
There are so many components that make up the tip link that if you just get rid of it all together it won’t lead to any useful information on how it functions: need to mutate the elements to see how they change rather than just getting rid of them
Why does the study use so many different kinds of mice?
The different mice all have different mutations, this better allowed the researchers to understand the distinct relationships of each component to the mechanotransduction cascade
When mechanotransduction currents were studied in dfcr mice, what results were obtained?
The mice need a stronger stimulus to produce the same amount of current as a wild type mouse
Do dfcr mice have fewer mechanotransduction channels than wild type mice?
No, they have the same number of channels: only difference is in harmonin-actin binding
Describe 2 properties of the mechanotransduction currents in OHC of dfcr (other than requiring a stronger stimulus strength to produce the same result)
- Slower channel activation
- Slower recovery/adaptation to deflection
What is the proposed mechanism of adaptation of hair cells involving harmonin?
Harmonin binds to a form of myosin which can climb up and down the hair cell to provide tension: if harmonin is present this interaction cannot occur very well: takes more deflection to get a hair cell to react
