Studies of Conventional
and Unconventional Myosin Motor and Actin
major aim of my research program is to understand how molecular
motors produce force and motion, and how the motor’s activity
is regulated. We are also focusing on the structure and function
myosin V is a processive motor that moves cargo along actin for
long distances without dissociating. Single molecule and biochemical
techniques are being used to understand features of the molecule
that enhance its processivity, including its interaction with the
microtubule-based motor kinesin. The two class V myosins in budding
yeast, which transport mRNA and membranous organelles, are being
studied with complementary in vitro and in vivo approaches.
run lengths of kinesin are enhanced by myosin V bound to the same
cargo. Kinesin returns the favor when myosin V transports cargo
on actin. The non-working motor acts as an electrostatic tether
to prevent cargo dissociation. Ali,
M.Y., Lu, H., Bookwalter, C.S., Warshaw, D.M., and Trybus, K.M.
(2008). Myosin V and Kinesin act as tethers to enhance each others'
processivity. PNAS 105, 4691-4696.
V surprisingly undergoes a one-dimensional diffusion on microtubules!
This potentially allows it to hook up with
cargo being transported by microtubule-based motors. Myosin V
is also adept at maneuvering through actin intersections, a feature
necessary for transport through the dense actin meshwork. Ali,
M.Y., Krementsova, E.B., Kennedy, G.G., Mahaffy, R., Pollard,
T.D., Trybus, K.M., and Warshaw, D.M. (2007). Myosin Va maneuvers
through actin intersections and diffuses along microtubules. PNAS
that Myo4p, a class V myosin in budding yeast, is single-headed
by virtue of its tight association with the adapter protein She3p.
She3p binds to the rod portion of Myo4p, and potentially forms
a hetero-coiled coil with it. Having the adapter protein become
a subunit of the motor makes biological sense given that it is
required for binding to all of Myo4p’s cargo, namely mRNA
and cortical ER. Hodges,
A.R., Krementsova, E.B., and Trybus, K.M. (2008). She3p binds
to the rod of yeast myosin V and prevents it from dimerizing,
forming a single-headed motor complex. J Biol Chem 283, 6906-6914.
V, when not bound to cargo, forms an inhibited folded conformation
in which the motor domain binds to the globular tail. This allows
the motor to not wastefully use ATP unless it is actively transporting
cargo. Cargo-binding unfolds and activates the molecule. Liu,
J., Taylor, D.W., Krementsova, E.B., Trybus, K.M., and Taylor,
K.A. (2006). Three-dimensional structure of the myosin V inhibited
state by cryoelectron tomography. Nature 442, 208-211.
muscle myosin is regulated by light chain phosphorylation The
structure of the inhibited, dephosphorylated double-headed subfragment
of smooth muscle myosin was obtained from 2-D
crystals formed on lipid monolayers. An unexpected, asymmetric
structure was observed that allowed us to propose a model by which
the ATPase activity of both heads could be inhibited, albeit by
different mechanisms. This structure has recently been shown to
also occur in striated muscle myosins. (Wendt,
T., Taylor, D., Trybus, K.M., and Taylor, K.A. (2001) 3-D image
recons truction of dephosphorylated smooth muscle heavy meromyosin
reveals asymmetry in the interaction between myosin heads and
the placement of subfragment 2. Proc. Natl. Acad. Sci. USA,
are pursuing the mechanism by which regulatory light chain phosphorylation
activates the molecule via mutational approaches, coupled with
enzymatic and motility assays. Ongoing crystallographic studies
of the alpha-helical coiled-coil rod of myosin are being used
to elucidate why this domain is critical for regulation.