Cell Biology Lecture No. 14: Actin Filaments – Microfilaments III
Monday March 4 , 2013
Classes Of Myosin:
-There are three common classes of myosin, which all move toward the plus-end of actin filaments.
Myosin I consists of a head domain with a variable number of light chains associated with the neck
domain. Members of the myosin I class are the only myosins to have a single head domain and associate
directly with membranes through lipid interactions (like in endocytosis). Myosin II proteins have two
head domains and two light chains per neck and are the only class that can assemble into bipolar
filaments. Myosin V proteins have two head domains and six light chains per neck. They bind to specific
receptors on organelles, which they transport (also involved in transport of endocytic vesicles).
-A sliding filament assay can be used to detect myosin-powered movement after myosin molecules are
absorbed onto the surface of a glass coverslip and the excess unbound myosin is removed. The coverslip
then is placed myosin-side down on a glass slide to form a chamber through which a solution of actin
filaments (made visible and stabilized by rhodamine-labelled phalloidin) can flow. In the presence of
ATP, the myosin heads walk toward the plus-end of the filaments, but because myosin tails are
immobilized, walking of the heads toward the plus-ends causes sliding of the filaments, which appear to
be moving with their minus-ends leading the way. The length of the myosin II neck domain determines
the rate of movement (the longer the lever arm, the faster the myosin moved).
-Myosin V has a step size of 36 nm, yet each head moves in 72-nm steps, so it moves hand over hand.
Two models for myosin V movement down a filament have been suggested: The hand-over-hand model
and the inchworm model. In the hand-over-hand model, one head binds an actin filament, and the other
then swings around and binds a site 72 nm ahead. In the inchworm model, the leading head moves 36
nm, then the lagging head moves up behind it, allowing the leading head to take another 36-nm step.
Depending on the step size taken by a fluorescently-labelled two-headed myosin V, it will walk by either
of these two methods.
Conformational Changes Of ATP-Driven Myosin Movement:
-In the absence of ATP, the myosin head is firmly attached to the actin filament. Although this state is
very short-lived in living muscle, it is the state responsible for muscle stiffness in death (rigor mortis). 1)
On binding ATP, the myosin head releases from the actin filament. 2) The head hydrolyzes the ATP to
ADP and P, which induces a rotation in the head with respect to the neck. This cocked state stores the
energy released by ATP hydrolysis as elastic energy, like a stretched spring. 3) Myosin in the "cocked"
state binds actin. 4) When it is bound to actin, the myosin head couples release of P witi the release of
elastic energy to move the actin filament. This is known as the power stroke, as it involves moving the
actin filament with respect to the end of the myosin neck domain. 6) The head remains tightly bound to
the filament as ADP is released and before fresh ATP is bound by the head (back in the rigor state). Skeletal Muscle Sarcomeres & The Sliding Filament Model:
-Skeletal muscles consist of muscle fibres made of bundles of multinucleated cells. Each cell contains a
bundle of myofibrils, which consist of thousands of repeating contractile structures called sarcomeres.
Sarcomeres are repeating structures of stable thick filaments (shaft of myosin II proteins arranged in a
cylinder) bound to actin and interdigitated with Z-disks. On either side of the Z-disks are the lightly
stained I bands, composed entirely of actin thin filaments (made of actin). These thin filaments extend
from both sides of the Z disk to interdigitate with the dark-stained myosin thick filaments in the A band.
Important to remember are that the A band is where thick filaments are (no actin) and the I band is
where thin filaments are (no myosin II).
-The sliding-filament model of contraction in skeletal muscles (type of striated muscle tissue) begins with
the arrangement of thick myosin and thin actin filaments in the relaxed state. In the presence of ATP
and Ca , the myosin heads extending from the thick filaments walk toward the plus-ends of the thin
filaments. Because the thin filaments are anchored at the Z-disks, movement of myosin pulls the actin
filaments toward the center of the sarcomere, shortening its length in the contracted state. Note th