CURRENT RESEARCH AND FUTURE DIRECTIONS

Our research continues to focus on the machinery for transport of organelles and RNAs, but with a renewed emphasis on the development and use of advanced instrumentation for light microscopy.

Molecular motors. We are continuing to elucidate the cargoes and cargo-motor linkers for kinesins, particularly conventional kinesin (also called kinesin I or KIF5 family) and members of the monomeric or KIF1 family.With respect to conventional kinesin, our research is addressing how the same motor carries multiple signal transduction cargoes (e.g. APP and a JNK signaling cascade), how signaling through these cascades feeds back on the motor, and how the binding of cargo molecules activates kinesin motor activity. These studies employ biochemistry, molecular genetics and advanced microscopic techniques (e.g. multi-color deconvolution microscopy and total internal reflection microscopy). We anticipate that this line of investigation will 1) clarify the interface of signal transduction and transport by molecular motors and 2) shed light on how cargoes activate kinesins "on demand" by linking binding of the cargo molecule to activation of motor activity (i.e. walking along the microtubule).

With respect to KIF1 kinesins, these motors play a fundamental role in organizing the extensive array of signaling complexes at synapses. Over the years we have cloned many members of this family, and are now identifying their protein-interaction domains. Using these domains, we are searching for the binding partners and thereby the regulators and cargoes. Our research on these motors employs genome wide molecular genetic and functional screens. We anticipate that this line of investigation will shed light on the assembly and operation of signal transduction cascades at synapses, and on the role of transport in signaling between synapse and cell body.

RNA localization. We are pursuing several lines of investigation in both Xenopus and Drosophila to resolve how Vera/ZBP protein localizes specific mRNAs. The highly conserved nature of Vera/ZBP structure and expression - from flies to mammals implies that this protein will be a keystone toward resolving the biochemical mechanism of RNA localization. We anticipate moving between flies and frogs, exploiting the advantages of each system. The Drosophila studies are in collaboration with the lab of Dr. Daniel St. Johnston in Cambridge, supported by a Human Frontiers Grant that also includes Dr. Michael Keibler (the PI of our network) whose lab is in Tubingen. We have recently determined that Vera/ZBP has an interesting localization pattern in fly embryos, consistent with a role in localizing the well-known maternal RNAs that play commanding roles in the establishment of this organism's body plan. Using a number of approaches (biochemical, genetic, histological) in both flies and frogs, we are now trying to determine which of the fly RNAs are localized via Vera/ZBP. Eventually, we hope to use genetic and live cell imaging approaches in flies to address the function of Vera/ZBP. Another avenue we are interested in exploring is the biochemical reconstitution of RNA transport in vitro - using Xenopus and fly oocyte components and extracts.

Microscopy. The development of advanced light microscopic methods has played a central role in the research of this laboratory. During the last five years, we have continued to incorporate advanced microscopy into aspects of our work, but our problems demanded primarily biochemical and molecular genetic approaches. Our projects are now at the point where we need to be more innovative with respect to microscopy. To this end, we have been building a state of the art total internal reflection microscope to visualize in real time the dynamics of single proteins or RNAs tagged with fluorophores. We will use this equipment to develop in vitro motility assays for RNA transport, using fluorescent RNAs, putative trans-acting factors, and microtubules. In other studies, we will image within cells the interactions between single molecular motors, their cargoes, and the scaffolds that link motors to cargoes. This type of approach will be needed to understand how cargoes release their plus-end motors, and acquire minus-end motors, upon reaching their destinations.