Research Goals

1
Nanoelectromechanical Transduction in Auditory
Hair Cells
Robert M. Raphael TN Law Assistant Professor
Dept. of Bioengineering Rice University Houston,
TX
2
Dedication
James T. Cushing (1937-2002) Professor of Physics
and Philosophy, University of Notre
Dame Scientist, Mentor, Friend
3
Helmholtz Theory of the Auditory System

• there must be different parts of the ear which
  are set in vibration by different pitch and which
  receive the sensation of tones.
• Helmholtz the first bioengineer Murray Sachs,
  Chairman of Biomedical Engineering, Johns Hopkins
  University

4
Anatomy of Hearing
Comptons Interactive Encyclopedia
5
Cross Section of Cochlea
Organ of Corti
Pv
Pt
Frequency increase
Basilar membrane
6
Traveling Wave
amplitude
Distance along cochlea
frequency dependent peak
but passive traveling waves are not the whole
story
7
Passive
Mechanical models
Filtering
8
Innervation of Organ of Corti
Supporting cell
Afferent Fibers
Efferent Fibers
9
Outer Hair Cells as MEMs
Electromotility
Mechanism based in plasma membrane Requires
turgor pressure (1-2 kPa) Maximal gains of
gt15 nm/mV and gt 50pN/mV. Independent
of calcium and ATP
salicylate
Whole cell voltage clamp/photodiode
Data of Kakehata and Santos-Sacchi, 1996
10
Effect of Electromotility onVibration of
Cochlear Partition
active OHC contraction p/2 out of phase
passive outer hair cells
http//www.boystown.org/Btnrh/cel/cochmech.htm
11
Prestin The Motor Protein ?
Identified by differential subtraction IHC/OHC
library 744 AA, MW 81.4 kD, extremely hydrophobic
(13 TM helices) Homology to sulfate transporter
proteins in the anion transporter Slc26A
superfamily Voltage-induced shape changes can be
elicited in cultured human kidney cells that
express prestin
12
Prestin Knockout MiceNature, Aug. 29th, 2002
13
Unresolved Questions on Mechanism
of Electromotility

• How does prestin work ?
• Membrane area motor model (piezoelectricity)
• operates optimally in a flat membrane
  (adherents of this model may be members of the
  Flat Earth society)
• Moreover prestin operates at acoustic
  frequencies
• How is force transmitted to the cytoskeleton ?

14
Doth the Answer lie in Mechanics ? I never
satisfy myself until I can make a mechanical
model of a thing. If I can make a mechanical
model I can understand it. As long as I cannot
make a mechanical model all the way through I
cannot understand it and that is why I cannot
get the electromagnetic theory. -- Lord
Kelvin, 1884
15
Nanoscale Structure of Lateral Wall
Spectrin -- thin elastic filament 40 nm
long Actin -- thick filament, circumferential Pil
lar -- protein of unknown composition 30 nm long
connecting plasma membrane to cytoskeleton
Plasma Membrane
Pillar
Actin
Spectrin
16
Dimpled SurfacesNanoscale Membrane Bending
Force balance on pressurized cylinder supported
by circumferential rings (Timoshenko)
17
Stretching Elastic Membrane over Pressurized
Structure
Interior of the Hindenburg (or is it the cortical
lattice of the OHC ?)
The Hindenburg
18
Biomembranes as Liquid Crystals

• intermediate state of matter
• display orientational but not positional order
• composed of dipoles free to rotate in an applied
  field

• Protein and lipid molecules comprising
  biomembranes possess dipole moments

19
Do electrically-induced effects on membrane
curvature exist ?

• Expected from liquid-crystal treatment of the
  membrane
• Differential surface tension
• But would fluidity counteract it ?

You cant get these kinds of effects. Its not
the way God created the membrane. -- Evan
Evans
20
When a distinguished but elderly scientist states
that something is possible, he is almost
certainly right. When he states that something is
impossible, he is very probably
wrong. -- Arthur C. Clarke
21
Flexoelectricity

• curvature-induced polarization
• primary mode of electromechancial coupling in
  nematic liquid crystals
• distinguished from piezoelectric effect in solid
  crystals

f flexoelectric coefficient cl 1/30 nm,
cj 1/4500 nm
As electric field is increased, dipoles rotate to
align with the field increasing the polarization
of the membrane
22
The Langevin Function

• specifies fraction of dipoles oriented with the
  applied field
• statistical mechanics
• continuum of states

polarization
23

Nano-Electro-Mechanical Flexion Motor
The regular array of pillar structures gives the
membrane the opportunity to be curved at the
nanoscale. The pressurization of the cell from
the inside also predicts the existence of
nanoscale curvature in the membrane.
A voltage-induced nanoscale deformation in each
motile unit sums to a mircoscale cell deformation.
DV
DP
pillar
Spectrin
24
Energetics of Nanoscale Bending
Bending Energy
Spectrin Energy
kc bending stiffness cl
longitudinal curvature co spontaneous
curvature
ks spectrin stiffness nsp spectrin
density xo force-free length
Combine by writing the spring length as a
function of curvature where a and b are
constants The energy is redefined in terms of the
equilibrium curvature ce
25
Membrane NanomechanicsTether Formation
Axial View
Pipet D 8 mm
Vesicle
Tether
Bead
Rt 20 nm
f 30 pN
kc 1.2 x 10-19 J
f mg
Raphael and Waugh, 1996
26
Constitutive Equations of Orientational Motor
Model
Moment Resultant
Electric Displacement
h membrane thickness
27
Electromotility Predictions
It is more important to have beauty in ones
equations than to have them fit experiment.
Paul Dirac
Free parameters of fit Flexoelectric
coefficient f Number of dipoles N Dipole
Moment p0
f 10-19 C N 6000/mm2 po 125 Debye --
protein
salicylate
Data of Kakehata and Santos-Sacchi, 1996
Nf number of membrane folds
28
Intracellular Pressure Alters Nonlinear
Capacitance
Kakehata and Santos-Sacchi, 1996
Gale and Ashmore, 1994
Existing models had no explanation for this data
29
The Internal Field

• In patch clamp experiments an external field, E,
  is applied. What is important, however, is the
  local field in the membrane seen by the dipoles,
  Ep. How are these two quantities related ?
• Continuum Model

30
Sensitivity of Nonlinear Capacitance to Strain

magnitude
center point

• This equation predicts two very interesting
  features
• the voltage at peak capacitance Vo depends on the
  strain applied to the cell (i.e. changing
  internal pressure changes the curvature of each
  ripple)
• 2) the magnitude of the capacitance will decrease
  as the coupling between the internal and
  external field decreases

31
Predicted Shift and Decrease in Peak Capacitance
with Pressure
These predictions are in agreement with the
experimental data (Gale and Ashmore, 1994
Kakehata and Santos-Sacchi, 1996)
32
Summary

• We can account for the electromotility data and
  the shift and reduction in the nonlinear
  capacitance of the cell with pressure by
• Explicitly including effect of internal electric
  field
• And/or interpreting the strain-induced
  polarization in terms of nanoscale membrane
  bending

Remaining Questions Do we have evidence that
internal polarization is changing with voltage
and pressure application ? Do we have evidence
for nanoscale curvature ?
33
AFM Measurements

• AFM observation of voltage-induced membrane
  movement
• Ionic effects can be fit to Lippmann equation
  describing coupling between voltage and surface
  tension

Zhang, Keleshian and Sachs, Nature, 2001
34
Electrical potential changes ? Lippmann mercury
voltmeter
G. Lippmann, Ann. Phys. 149 (1873)
DC Grahame (1947)
35
Prestin and Anions

• Prestin-induced movements sensitive to internal
  anion concentration
• Follows Hofmeister series (like the membrane
  dipole potential, Clarke and Lupfert, Biophys. J.
  1999)

Olivier et al., Science, 2002
36
Fluorescence Recovery After Photobleaching
Oghalai, Tran, Raphael, Nakagawa, Brownell,
Hearing Research, 1999
37
Diffusion Results Confirm Liquid Crystal Nature
of the Outer Hair Cell Membrane
Diffusion in Liquid Crystal as function of
applied field
Diffusion in OHC as function of applied field
Yun and Fredrickson, 1970
Oghalai et al., 2000
38
Internal Polarization Models
a is related to dielectric anisotropy
39
Liquid Crystal Disorder-Order Transitions
Depolarization or increased pressure
Hyperpolarization or low pressure
40
Salicylate and Hair Cells

• Salicylate increases hearing
  thresholds causes reversible
  high frequency hearing loss
• Action on hair cells does not involve
  COX/prostaglandins
• - high Kd (4 mM)/rapid recovery
• Hypothesis SAL interacts with the lipid
  component of the membrane and changes membrane
  mechanics

Schkunecht,1993
41
Forming Tethers with Salicylate

• Salicylate added to giant vesicles causes
  spontaneous formation of nanotubes of diameter
  300 600 nm
• Interfacial chemistry exerts thermodynamic
  force that causes shape changes

42
Measurement of Membrane Compressibility
Micropipette aspiration
Membrane Tension
Fractional Area Expansion (areal strain)

• change in projection length in pipette measured
  as a function of applied pressure
• Geometric relationships
• Pressure membrane tension
• Projection length areal strain

• Ka
• where K elastic compressibility modulus
  of the membrane

43
Vesicle Aspiration Experiment Results

• reduction in compressibility modulus usually
  correlated with reduction in membrane elastic
  strength
• areal expansion a at which vesicle lysed
  indicator of membrane elastic strength
• salicylate decreases membrane compressibility
  and lysis tension significantly from control

t
44
Controlled Nanotube Formation with Optical
Tweezers The Area Motor vs. Bending Motor
45
The grand aim of all science is to cover the
greatest number of empirical facts by logical
deduction from the smallest number of hypotheses
or axioms. Albert Einstein
46
Outer membrane ripples on motile cells
Coincidence or functional roles?
OHC - Dieler et al. 1991
Oscillatoria - Adams et al. 1999
Flexibacter BH3 - Dickson et al. 1980
47
Flexoelectricity and the Design of Synthetic MEMs
Actin polymerize in equally spaced rings
Lipid bilayer ( prestin)
Control sensitivity range by pressure
difference Control magnitude of response by
surface charge
48
Making the NanomachineMotility of Cationic
Liposomes

• By altering the ionic composition and surface
  charge, we have made vesicles move

49
BioInterfacial Interactions
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(No Transcript)
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Thanks to the Raphael Lab
Pascal Phares, Emily Glassinger, Nathan Spencer,
Yong Zhou, Jonathan Lee, David Tran