Cytoskeleton and Cell Motility

• V.6.iii Subproject 3 - What Mechanisms Drive the Movement and Positioning of Subcellular Elements?
• V.6.iii.a Fast Axonal Transport
  • V.6.iii.a.1 Introduction
  • V.6.iii.a.2 Methodology
    • V.6.iii.a.2.i Forward and reverse genetics
    • V.6.iii.a.2.ii Functional analysis in vivo
• V.6.iii.b Vesicle Transport in Embryos
• V.6.iii.c Modeling Transport Mechanisms

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V.6.iii Subproject 3 - What Mechanisms Drive the Movement and Positioning of Subcellular Elements?

Our research into organelle transport mechanisms requires mathematical modeling to explain how antagonistic and cooperative motor activities result in directed transport and specific localizations. The ideal set of information for accurate predictive models would include the identities of all the proteins and other molecules that participate, and the function of each defined in quantitative terms. We have developed a system in Drosophila that could identify many components of the machinery that drives organelle transport in the axons of neurons, and can define their transport functions quantitatively. We easily recognize the consequences of disrupted axonal transport from changes in larval crawling behavior. Larvae become uncoordinated and paralytic as neuron function declines (Saxton et al., 1991). Forward and reverse genetic screens for mutations that cause this behavior have identified genes for kinesin heavy chain, kinesin light chain, Unc104/Kif1A, kinesin II, dynein heavy chain, P150glued, Arp2/3, Jip1/2, JSAP/Sunday Driver, APPL and other proteins (Bowman et al., 2000; Gindhart et al., 1998; Martin et al., 1999; Saxton et al., 1991; Torroja et al., 1999, Barkus et al., unpublished).

We are conducting direct and genetic enhancer screens to identify as many new components of the axonal transport machinery as possible. The current focus is on proteins that interact with conventional kinesin and Unc104. Both are well-characterized microtubule motors that have distinct functions in axonal transport. One of the more exciting proteins that we discovered in our first interaction screen is a homologue of a Map kinase scaffolding protein, Jip1/2. We are now designing screens for interactors with Jip1/2, in the hope that we can continue stepping away from the motor to find more components of the conventional kinesin transport machinery. We have recently started screening all the lethal transposon insertion lines (Bloomington Stock Center) for those that interact genetically with kinesin heavy chain, Unc104, and Jip1/2. This effort should produce a number of new transport components. We also expect the C. elegans motor interactome project at IUB to identify new candidate axonal transport components whose functions we can pursue by reverse genetics and RNAi.