Drosophila auditory transducers:
function and homeostasis
Nicholas Boyd-Gibbins, Ryan G. Kavlie and Joerg T. Albert
UCL Ear Institute
Abstract
Here we demonstrate how to dissect the molecular basis of mechanotransduction in the fruit fly’s Johnston’s Organ. Specifically, we use Laser Doppler Vibrometry (LDV) in coordination with electrostatic actuation of the antennal
sound receiver and extracellular electrophysiology (via sharp tungsten electrodes) from the antennal nerve. This combined approach allows very sensitive functional assessments of the fly’s hearing apparatus.
A dual pharmacogenetic approach, mainly focusing on the investigation of distinct isoforms of the putative Drosophila transducer channel, NompC, suggests that different isoforms may be required for full transducer function in
wildtypes. Using temperature-modulated conditional expression of both NompC and a reported trafficking protein, King Tuby (also known as dTulp), we have found that a continual turnover of NompC is required to maintain
functional transducer modules in adult flies. Consistent with this finding, experiments using Fluorescence Recovery After Photobleaching (FRAP) of the sensory neuron dendrites (thought to be the site of mechanotransduction)
show recovery of GFP-tagged NompC after photobleaching.
Biomechanical and Electrophysiological dissection of
Drosophila Hearing:
Fly image taken from exploratorium.edu
-
Images from Zanini et al (2013) and Nadrowski et al (2008)
We are interested in the molecular dissection of the
mechanosensory transducer complexes mediating the
Drosophila senses of hearing, wind and gravity.
Due to the likelihood of many molecular interactions with
associated structures, an in vivo approach currently offers
the most powerful insight.
From the biomechanics data we can estimate populationspecific values for various channel properties like singlechannel gating forces, transducer channel numbers or
gating spring stiffness.
nompC isoforms (α, β and γ) contribute differently to antennal mechanics.
The TRP channel NompC is one of the strong
mechanotransduction channel candidates.
Ion channel homeostasis:
The temperature sensitive conditional
expression system, Gal80, is used to
control expression of NompC in adult
flies.
Development
20°
NompC
Adult
NompC
NompC
NompC
NompC
NompC
NompC
Testing
NompC
30°
Time
We carried out
experiments comparing
the difference between
flies raised with and
without the supply of
NompC and flies only
given the supply of
NompC as an adult.
Results:
Our results show (A and B) the effect of adultspecific expression of NompC (red) in a nompC
loss-of-function mutant background (blue).
Mechanical response (A) to force steps shows
restoration of the increased compliance at small
peak displacements in experimental flies (red). The
electrical response (B) shows a left shift and
increased compound action potential (CAP)
amplitudes, indicating restoration of nerve
responses.
The bottom two plots (C and D) show the
mechanical and electrical response of control flies
(blue) and those with adult-specific knock down of
King Tubby (via Gal80ts-mediated, temperaturecontrolled gene expression). Mechanical responses
(C) show a decreased compliance at small peak
displacements in experimental flies (red) as well as
decreased CAP amplitudes, indicating loss of
nerve responses (D). This is consistent with a loss
of mechanotransducer modules.
FRAP:
Fluorescence Recovery After
Photo-bleaching (FRAP) analysis
shows turnover of auditory
transducer modules and relocalisation to dendritic tips,
considered
the
site
of
transduction. The flies shown here
expressed a (functional) NompCL-GFP in a loss-of-function
mutant background (nompC3).
Future Work and Questions
- Do other mechanotransduction proteins show turnover
or other signs of homeostasis?
- Do these findings apply to humans and, if so, is the
turnover disrupted in auditory disease or age?
- Could this turnover process be a future target for
restorative interventions?