HAIR BUNDLE MYSOIN ISOZYMES

Project: Research project

Description

Hearing loss affects tens of millions of Americans. For many of these
individuals, the deficit arises from damage to hair cells, the sensory
cells of the internal ear. By transducing acoustic and mechanical energy
into electrical signals that propagate to the brain, hair cells are
essential for our appreciation of sound and sense of balance. Damage to
hair cells can result in irreversible loss of hearing or vestibular
function. By determining fundamental properties of mechanoelectrical
transduction, the proposed experiments aim to ascertain what cellular
constituents are required to produce an intact, functioning hair cell.
A better understanding of a hair cell's normal functions should
facilitate design of rational strategies for amelioration of hearing
deficits. Experiments proposed in this application are designed to characterize
motor molecules utilized by hair cells. An active motor controls the
responsiveness of a hair cell, allowing the cell to adapt to sustained
stimuli. This motor could also play an important role in the assembly
of a hair bundle during development. The hair bundle's adaptation motor
is thus an essential component of the mechanoelectrical transduction
apparatus. Myosin molecules are excellent candidates for the active component of
this adaptation motor; indeed, a myosin isozyme resides in the hair
cell's mechanically sensitive organelle, the hair bundle, and appears to
be properly situated to carry out adaptation. We will address four
principal issues related to hair-bundle myosin molecules. First, how
many myosin isozymes are there in a hair bundle, and where are they
located? We expect that determination of the subcellular location of a
myosin molecule will suggest its role in the cell. Second, what hair-
bundle proteins bind myosin molecules? At lest two important molecules
should bind an adaptation motor: a Ca2+-binding element, perhaps
calmodulin, and a crosslinking protein that ties together enough motor
molecules to produce the force necessary for adaptation. Third, how are
myosin molecules regulated? Adaptation is controlled by intracellular
Ca2+, and may also be regulated by protein phosphorylation. We will
determine whether hair-bundle myosin molecules are phosphorylated, then
measure the modulation of myosin's ATPase activity by phosphorylation and
Ca2+. The final set of experiments will test whether a specific myosin isozyme
carries out adaptation. We will first develop an assay for the
adaptation motor that relies on Ca2+-modulated hair-bundle movement in
permeabilized hair cells. We will then allow dye-labeled antibodies to
enter the permeabilized bundles and to bind to their target myosin
molecules. Directing intense laser irradiation upon the bundle, we will
then specifically denature those antibodies and any bound myosin
molecules. Elimination of Ca2+-dependent bundle movement by this
treatment would confirm myosin's role in adaptation.
StatusFinished
Effective start/end date7/1/946/30/18

Funding

  • National Institutes of Health: $409,905.00
  • National Institutes of Health: $189,174.00
  • National Institutes of Health: $20,000.00
  • National Institutes of Health
  • National Institutes of Health: $474,087.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $397,967.00
  • National Institutes of Health: $281,531.00
  • National Institutes of Health: $466,341.00
  • National Institutes of Health: $406,910.00
  • National Institutes of Health: $424,648.00
  • National Institutes of Health: $422,204.00
  • National Institutes of Health: $183,580.00
  • National Institutes of Health: $307,345.00
  • National Institutes of Health: $289,976.00

Fingerprint

Myosins
Isoenzymes
Hair
Proteins
Hearing Disorders
Deafness
Fluorescence Recovery After Photobleaching
Regeneration
Hearing Loss
Carrier Proteins
Myosin Type I
Head Movements
Calmodulin
Molecular Cloning
Inner Ear
Gene Library
Usher Syndromes
Antibodies
Biochemistry
Protein Kinases

ASJC

  • Medicine(all)
  • Neuroscience(all)