Current Research
Cells are highly structured, and much of this order depends on
transport that uses directed polymers as roads, along which enzymes
called molecular motors move. The polymers structures, called
microtubules, are typically quite stiff, approximately 25 nm in
diameter, and a number of microns in length, and extend from the cell
center (their ‘minus-end’) the the cell periphery (their ‘plus-end’).
There are two families of molecular motors—kinesin and dynein—both of
which hydrolyze ATP into ADP, and convert some of the energy from the
phosphate bond into mechanical work, moving in a directed fashion along
the microtubules. The kinesin family of motors moves toward the
plus-ends of microtubules, while the dynein motors move towards the cell
center. Because these motors are able to attach to and move a wide range
of cargoes (e.g. vesicles containing proteins or other small molecules,
cytoskeletal components, cell nuclei, or even chromosomes during
mitosis) the activity of these motors is crucial in many cellular
processes.
Many of the cargos moved by these motors are transported to
different places in the cell at different times. For instance, the
ability of some fish to change color occurs through redistribution of
pigment granules in melanophore cells, which can either disperse
throughout the cell (and thus turn the cell the color of the pigment) or
aggregate to the cell center (making the cell much less light
absorbent). How is the cell able to regulate the transport of cargos, so
that they can be selectively transported to particular locations?
Because kinesin and dynein move in opposite directions along the
microtubule, the obvious solution is merely to attach only one class of
motor at a time, moving a cargo either to the plus or the minus-end of
the microtubule. While this simple regulatory scheme is the case for
some cargos, others are observed to move in a salutatory,
bi-directional fashion. These cargoes move back and forth on
microtubules, using both motors either simultaneously or in rapid
succession. Nonetheless, this apparent random walk can be biased to
either the plus- or minus-end of the microtubule, allowing control of
average direction of cargo transport. Some neurotropic viruses move in
this manner. See, for example, our recent paper on herpes virus motion
in cultured neurons (
Download PDF
here).
My research is focused on understanding the details of this
bi-directional transport: to what extent is the activity of plus and
minus-end motors coordinated? What physical properties of motion are
regulated to switch transport from net plus to net minus-end motion?
Does regulation occur at the level of individual motors, the number of
motors on a cargo, or perhaps alteration of the way the plus-and
minus-end motors interact? Finally, if there is some higher level of
organization allowing multiple opposite-polarity motors to work together
in a coordinated fashion, how is this organization achieved, and what
are the components of the organization complex? In order to investigate
these questions, new tools are necessary: we must develop a system where
we can combine biochemical and genetic manipulations with the ability to
measure the properties of motion of individual transported cargoes in
vivo. My post-doctoral work in the labs of Drs. Steven Block and Eric
Wieschaus was devoted to the development of such a system: the transport
of lipid droplets in early embryos of the fruit fly Drosophila. During
early development, the distribution of these half-micron solid lipid
droplets shifts twice: first (in early cycle 14) droplets are
transported from the embryo’s periphery into the center, and
approximately an hour later (in late cycle 14) the droplets are
transported in reverse, to the embryo’s periphery. I developed
biophysical tools and an embryo preparation procedure that allowed me to
make high precision measurements to quantify the motion of individual
moving droplets: laser tweezers to measure the forces generated by the
molecular motors moving the droplets, and video-enhanced single particle
tracking and analysis to determine the particles location with
few-nanometer precision, at 30 Hz.The analysis of the forces required to
stop individual moving particles suggested that multiple motors function
together to move individual droplets (
PDF:
Cell, 1998), and that the number of active motors was regulated
developmentally. The motion analysis showed that changes in net
direction of transport were achieved by changing the transport
properties in only one direction: to achieve plus-end transport the
average plus-end travel distance was increased, while net minus-end
transport occurred when the average plus-end travel distance was
decreased. Studying the changes in motion and stalling forces caused by
mutations in a novel protein, Klarsicht, suggested that there may be
coordination of the plus and minus-end motor activity, in part mediated
by Klarsicht.A second paper (
PDF:
JCB, 2000) showed that there was probably some organization of
motors that allowed coordinated regulation of plus- and minus-end motor
activity, since plus-end motors became active simultaneous with
inactivation of minus-end motors, and vice-versa. This type of
regulation is unlikely to occur in the absence of a mechanism
controlling the plus-minus motor interactions.
Future work in my lab will continue to investigate
bi-directional motor transport, again combining biophysical tools (laser
tweezers and particle tracking) with genetic and biochemical ones. One
set of experiments will build on out previous work on Klarsicht. We will
biophysically study the phenotype of motion in different Klarsicht
mutant backgrounds (we already have the mutants), in order to determine
which portions of this large protein are important for its roles in
lipid-droplet transport. Once we have identified the key regions of the
protein, we can biochemically look for interactions between these
domains and other proteins, in order to better understand how Klarsicht
functions.
A second set of experiments will look at the physical role of
other proteins that play a role in transport processes. Such studies
have a dual role: they both elucidate the role of the protein studied,
but the changes in overall motion caused by their loss or impairment may
tell us about the overall transport machinery: how closely is plus and
minus-end transport coordinated? Does a specific type of impairment of
minus-end motion always affect plus-end motion in the same way? If we
were to see different subclasses of effects, perhaps we could define
subsets of the transport machinery, where all the proteins in a
particular subset were involved in the same aspects of motion. I have
already done preliminary experiments, and identified a number of
proteins in which mutations alter the lipid droplet motion. We will
fully characterize the effects of loss of these proteins to better
understand the proteins’ physical role. Later studies will look at
specific lesions in the proteins to better understand how the proteins
are able to function as they do. Because impairment of these proteins
seems to alter transport in both directions, that the full
characterization of motion should not only clarify the minus-end role of
these proteins, but also further our understanding of plus-minus
interactions, and perhaps the function of these proteins in the
regulation of such interactions. While our understanding is still quite
limited, and this set of questions may be difficult, combining
biochemical, genetic, and biophysical measurements should give us the
tools to make significant progress in understanding these important
processes.
A final set of experiments involves a collaboration with the
laboratory of Dr. Vladimir Gelfand, applying the biophysical tools I
have developed to study the bi-directional transport of pigment granules
in melanophores. We would like to determine the extent of similarity
between the Drosophila and Melanophore transport systems, to help us
what aspects of the systems are generic and which are unique.