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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Our other model studies the transport of lipid storage vesicles in blastoderm embryos (Welte et al., 1998). The vesicles move along short channels formed between closely packed nuclei. Microtubules in those channels are reasonably parallel and of uniform polarity (Welte et al., 1998), with the minus ends at the embryo periphery. Thus initial vesicle transport towards the embryo's center is plus-end directed, and the later reversal of transport toward the periphery is minus-end directed. The lipid storage vesicles are refractile, allowing imaging by differential interference microscopy with excellent spatial (8 nm) and temporal resolution (30 frames/sec). We have developed automated tracking and analysis of droplet motion (Gross et al., 2000), producing large, high-quality data sets for analysis of transport behavior. We will use this tracking and data analysis software as a starting point for developing new software for fluorescent organelles in the axonal transport studies above. We have also used in vivo optical trapping to investigate motor function and coordination between dynein and kinesin-family motors (Gross et al., 2002). These studies have shown that cytoplasmic dynein drives minus-end transport (Gross et al., 2000). They also indicate that coordination between dynein and the plus-end motor is critical for control of the net direction of vesicle transport (Welte et al., 1998). Two proteins, klar and P150/glued, help create and maintain this coordination. Future work will focus on a) identifying other components involved in coordination, b) clarifying at a molecular level how coordination comes about, and c) determining how coordination itself is regulated to control the net direction of droplet transport.

Organelle transport is complicated, with numerous interacting factors determining the resultant motion. Because of this complexity, quantitative models will be critical to a full understanding of transport mechanisms. The axonal organelle and lipid vesicle studies are an ideal starting point for such models, because they are synergistic: both are in Drosophila, so mutants we identify in one can be studied in the other. This cross-referencing will be important in identifying common features of transport mechanisms and process specific features. Already, we are applying some of the theoretical framework for motor coordination that was developed for lipid vesicles to explain findings in axons. We propose to develop-and experimentally test-theoretical models that apply to both systems.

We envision two classes of models. First, heuristic models suggesting specific roles for proteins or groups of proteins engaged in transport. Cross-validation will help strengthen and refine such models. Second, quantitative models that predict measurable parameters of motion such as average cargo flux, instantaneous number of a particular class of cargo moving in each direction, etc. These quantitative models could be entirely analytic, or could be a set of coupled differential equations best analyzed numerically. Initial models will be largely phenomenological, but as we discover more components of the transport pathways and clarify their roles, our models will become more complete.