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Unique Muscle Structure
Typically there is a trade of between
muscle force and speed. Fast muscles
have narrow myofibrils (that allow
rapid calcium diffusion into and out of
the myofibrils) along with extensive
sarcoplasmic reticulum (for rapid
calcium release and reuptake). Slower
muscles can pack in thicker myofibrils
with less sarcoplasmic reticulum at the
expense of contraction speed. Insect
flight muscle fibers have a unique
structure that seems to break these
rules. Myofibrils are cylindrical
shaped with a large diameter of ~1.5
um. Within the myofibrils the characteristic banding pattern of striated
muscle can be seen with sarcomeres that range from 3.0-3.4 um. Although
IFM contract rapidly (~ 200 Hz), the sarcoplasmic reticulum of this
muscle type is scarce. In its place are numerous mitochondria. The stretch
activation response allows the high muscle contraction frequencies
independent of direct nervous control.
The Drosophila thoracic muscular
system.
Wing movement in Drosophila is powered by two
antagonistically oriented muscle fibers that are
attached to the thoracic cuticle from which the
wings extend. There are 14 dorsal ventral muscle
fibers (DVM) that are located toward the outsides
of the thorax. The dorsal longitudinal muscles
(DLM) can be seen flanking the mid-saggital
plane. The tergal depressor of trochanter (TDT),
or "jump" muscle is shown lateral to the the
DVMs.
Wing Movement
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The Moore Laboratory Drosophila Page Page
The wing-thorax-flight muscle system of Dipterans is capable of highly
tuned resonance. It is believed that contraction of the jump muscle
deforms the thoracic cuticle in such a way that causes the both the wings
to move through their upstroke and to stretch the DLMs. Stretching of the
DLMs activates them and their contraction deforms the cuticle the
opposing way and stretches the DVMs. Stretching of the DVMs leads to
their contraction which stretches the DLMs. If the timing between the
delay in activation matches the characteristic frequency of the flight
system a resonating oscillator will be formed.
Stretch Activation
It is important to note that the IFM
are activated by intracellular
calcium via sporadic nervous
stimulation. Only with this
fluctuating (but sufficiently high)
calcium level does the muscle
contract alternately in response to a
stretch produced by the other. In
isolated IFM the response to stretch
consists of a rapid, spring-like,
increase in tension followed by a
decay that is eventually overcome by a significant increase in tension (the
stretch activation response). Stieger (1977) and Pringle (1978) observed
that the delayed tension rise was a property of all muscles that happens to
be enhanced in muscles that drive oscillating systems like cardiac and
indirect flight muscle. Basically, if the delay matches the timing of the
muscle extension and shortening then the muscle will perform oscillatory
work and thus feed the resonating system. Several models have been
proposed to explain stretch activation. Click here for more information.
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The Moore Laboratory Techniques
We use a variety of techniques to study the biochemical and
mechanical properties of molecular motors, the cytoskeleton and
biopolymers.
Unloaded in vitro motility
assay
The unloaded in vitro motility
assay has all the advantages of
solution biochemistry (i.e., temp,
ionic conditions, substrate
concentrations etc. are easily
manipulated); however,
movement, one of the fundamental properties of molecular motor
function, can also be quantified. In this assay myosin is adsorbed to
a silanized coverslip surface. Fluorescently labeled actin filaments
are then allowed to bind to the surface-bound myosin. Movement
is initiated by the addition of ATP and recorded with an ICCD
camera.
Frictional
loading in vitro
motility assay
The unloaded
motility assay
gives important
information about
the motion
generating
capacity of molecular motors. However, motor molecules also
produce force. We assess the effects of myosin or thin filament
regulatory protein mutations on relative (to wild-type) force
generation. In this assay, load is applied to the myosin motor by
applying it to the surface along with small amounts of non motile
actin binding proteins (e.g., alpha-actinin). The alpha-actinin will
introduce a frictional load that resists filament motion. The amount
of alpha-actinin required to stop filament motion is an indicator of
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The Moore Laboratory Techniques
myosin force generating capability. Since the load is applied to all
of the filaments in a field of view, this assay is useful for rapidly
screening a variety of experimental conditions (e. g.: regulated
actin, unregulated actin, submaximal calcium activation, etc.).
Optical trap
Optical traps use photon momentum to
capture and hold small transparent
particles with a higher refractive index
than the medium. As a trapped particle
moves away form the laser focus there is
a restoring force that is proportional to
the distance the particle moves. Therefore, an optical trap can be
used as a very sensitive force tranducer that can measure forces in
the piconewton range.
Laser trap loading assay
We use both optical trap and
frictional loading based
assays to measure the
inherent force generating
capacity of mutant myosin
molecules. Frictional loading
assays provide an indication
of relative force while optical
trap based assays measure the absolute isometric force generating
capability of the myosin mutants one filament at a time.
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