Ned Debold, Ph.D.
Post-doctoral Associate
The mechanism through which muscle is able to
produce force and generate movement is a physiological phenomenon
still poorly understood at a molecular level. While it is generally
accepted that the mechanical events of muscle myosin are powered
by the chemical energy derived from ATP, the nature of coupling
between the structural changes in myosin with the steps of ATP
hydrolysis is less clear. I employ a two-pronged approach in an
effort to more fully understand this process. One approach takes
advantage of mutations in cardiac myosin, present in cardiomyopathies,
to determine the effect of specific amino acid substitutions on
myosin’s ability to produce force and generate motion. These
properties are quantified using the in vitro motility assay, and
unitary measures of myosin’s displacement and dwell time
using the three bead laser-trap assay. In another approach I examine
the effects of altering the level of free energy available from
ATP hydrolysis on myosin’s ability to translocate actin at
both the ensemble and single molecule level. Through these projects
I hope to both enhance our understanding of muscular contraction
and elucidate the molecular changes which cause compromised cardiac
function observed in diseases such as genetically linked cardiomyopathies.
Md.
Yusuf Ali, Ph.D.
Post-doctoral Associate
Myosin, a motor protein, captures the energy
from ATP hydrolysis to generate force and motion through its interactions
with actin filaments in both muscle and non-muscle cells. However,
it remains unclear how myosin converts chemical energy into mechanical
movement. To address this question, I am studying a two-headed,
processive myosin V molecular motor that moves along an actin
filament over long distances. To understand the mechanism of force
generation at a single molecule level, I am using quantum dots
which are bound to each of the two heads of myosin V through a
biotin-streptavidin linkage. I observe the movement of myosin
V at different conditions using a TIRF microscope that provides
both high spatial and time resolution. I am mainly interested
in characterizing myosin V’s walking mechanism and conformational
changes during its movement along an actin filament. Myosin V
is implicated in organelle transport in neurons and must move
within a dense intracellular cytoskeletal network that is composed
of overlapping and crisscrossing actin, intermediate, and microtubular
filaments. The microtubule network is believed to be used for
long-range cargo transport through kinesins and dyneins while
the actin filaments are used for short range transport through
myosin motors. But the mechanism by which this transport is coordinated
is still unclear and is the present focus of my research.
Sam
Walcott, Ph.D.
Post-doctoral Associate
Smooth Muscle Myosin
Head-Head Communication
Shane
Nelson, Ph.D.
Post-doctoral Associate
Myosin V Vesicle Transport
in COS-7 cells
Chong
Zhang
Graduate Student
Myosin V is a molecular motor that converts the
energy from ATP hydrolysis to long distance movement along actin
filament. It moves processively in a “hand over hand”
fashion and transport organelles in the cytoskeleton network.
This processivity requires the coordination between the two heads
which is believed to be strain-dependent. My current study is
to use a combination of TIRF and laser trap technique to study
the load dependent stepping behavior of myosin V and the coordination
between the two heads.
Abbey
Weith
Graduate Student
Actin is a key filamentous cytoskeletal
protein involved in many processes, including muscle contraction,
cellular motility, and vesicle trafficking. One of an actin filament’s
fundamental properties is its flexural rigidity, or the stiffness
with which it bends. In my research I digitize images of actin
moving under Brownian motion, and then use a computer program
to find the flexural rigidity through Fourier transforms. Once
this technique is reliable, the method will be applied to several
actin mutations that cause heart failure to see if the mutants
have a different flexural rigidity than wild-type actin.
Samantha
Beck
Laboratory Technician
I am involved in a project that investigates
the effects of single point mutations in human skeletal and
cardiac muscle myosin found in patients with familial hypertrophic
cardiomyopathy (FHC). In order to study the effects of these
mutations, we purify myosin from human muscle biopsies and use
in vitro motility assays to measure actin velocity. The actin
velocities of the mutant myosins allow us to compare their mechanics
and biochemistry to that of normal myosin, which may lead us
to a better knowledge of FHC.
