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Cell structure

        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.

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