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.
Shane
Nelson, Ph.D.
Post-doctoral Student
Myosin V Vesicle Transport
in COS-7 cells
My work focuses on Myosin Va (myoVa), which is
an intracellular cargo transporter. Based on in vitro experiments,
a single myoVa molecule could perform this function as it can
move processively along actin tracks in a “hand-over-hand”
fashion. However, this has yet to be demonstrated in vivo. The
cellular context presents numerous challenges to myoVa processivity
such as the dense cytoskeletal network, actin-binding proteins,
and other motors that share cargo transport duties with myoVa.
To study the in vivo motion and processivity of myoVa, I introduced
quantum dot (Qdot) labeled myoVa molecules into cultured fibroblast
(COS-7) cells by pinocytosis, and observed the motion of individual
motor molecules by TIRF microscopy. I have shown that individual
myoVa molecules undergo a random walk by making frequent turns
onto intersecting actin filaments in the densely packed subplasmalemmal
actin cortex. My current efforts are to begin to understand how
myoVa is targeted to its cargo and how multiple motor molecules
coordinate their activity to bring about effective cargo transport.
Jessica
Martel, Ph.D.
Post-doctoral Student
Class V myosin motors are responsible,
at least in part, for the transport of intracellular cargo (i.e.
vesicles, secretory granules, etc.) along the dense actin cortex
towards the cell membrane. The mechanical logistics behind this
process are not entirely understood. Over the past decade, investigators
have identified three myosin V isoforms (a, b and c) with differential
tissue expression and transport capabilities. My research investigates
the involvement of myosin Va, a processive class V myosin, in
insulin granule transport and exocytosis. We plan to introduce
quantum dot-labeled myosin Va into pancreatic beta cells that
express a fluorescently tagged insulin granule marker. In doing
this we expect to be able to determine when and where myosin Va
attaches to the insulin granule and carries it to the cell membrane,
with high real time and spatial resolution.
Abbey
Weith
Pre-doctoral 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.
My goal is to provide the tools for our lab to
do world class research in molecular physiology. Molecular reactions
require measurement of nanometer displacement, pico-newton force,
and single molecule fluorescence. Microscopy based instrumentation
using recent techniques in TIRFM, Laser Tweezers, Confocal detection,
and Photon counting are my focus. I am currently testing new instrumentation
allowing us to make real time measurements of ATP hydrolysis during
the actin-myosin interaction. In progress projects include single
molecule 3D fluorescence polarization measurements, and other
initiatives in nano-technology.
