Cytoskeleton and Cell Motility

V.4 SPECIFIC RESEARCH OBJECTIVES:

We are engaged in six collaborative research projects:

1. Subproject 1 - The Goodson and Walczak groups employ biochemistry, structure-function analyses, protein depletion, and digital microscopy in vitro. These approaches are used in Xenopus oocyte extracts, and in cultured vertebrate cells to study interactions between microtubules and proteins that alter microtubule dynamics, specifically focusing on proteins that interact with microtubule plus ends. Quantitative data include biochemical kinetics and measurements of the dynamic polymerization behavior of microtubule plus ends. We will model the effects of these microtubule binding proteins on polymer behavior.

2. Subproject 2 - The Strome, Saxton, and White groups will collaboratively study how microtubule plus ends interact with proteins at the cell cortex to position and orient the mitotic spindle and hence to position cell division cleavage planes in early C. elegans embryos. We are particularly interested in cortically anchored microtubule motor protein complexes. Methodologies include genetics, protein localization/depletion experiments, and in vivo fluorescence microscopy. Quantitative data will include plus-end residence times at various positions on the embryonic cortex and position vs. time measurements for centrosomes, chromosomes, and the cleavage furrow. We will model the spindle and cleavage plane positioning-orientation mechanisms.

3. Subproject 3 - The Saxton and Gross groups will collaborate in a study of microtubule motor-driven cytoplasmic organelle transport/positioning in Drosophila. We will apply genetics, protein localization, in vivo fluorescence and DIC microscopy, and optical trapping to intact nerves, embryos, and cultured neurons. Quantitative data include organelle position vs. time measurements, organelle flux rates, and force of motion. We will model of the mechanisms of organelle movement.

4. Subproject 4 - The Tang, Glazier and Alber groups will use existing and experimentally determined kinetic rate constants for actin polymerization, end uncapping, branch formation by arps, and crosslinking by other actin binding proteins at the leading edge to model the growth of the actin network as a stochastic process. Comparisons of predictions from simulations and experimental measurements include the density of the actin mesh, filament length, branch distribution, the velocity of movement, and the force generated.

5. Subproject 5 - The Tang and Forgacs groups will model the global organization of the actin cytoskeleton to elucidate the cytoskeleton's role in intracellular signal and mechano transduction, using a "minimal" model of the microfilament network, which we will gradually embellish to be consistent with the work in the other Subprojects.

6. Subproject 6 - In conjunction with the Subproject 4 study of mechanical properties of isolated cross-linked actin networks, the Forgacs group will study intracellular viscoelastic properties using magnetic tweezers to provide critical quantitative data for stochastic modeling of the combined actin and microtubule networks.

V.4.i Levels of Detail:

In many areas of complexity, we study the same phenomenon at several levels of detail simultaneously. We aim to develop a hierarchical view where coarse graining at each level leads to a description at a higher level of abstraction. Section VI.10 discusses this process as part of our project to integrate cellular and sub-cellular models. The cytoskeleton is a microcosm that illustrates this concept, with our initial actin model operating at a greater level of detail than our tubulin model. At this stage of our understanding, we need to explore the consequences of different choices of level of abstraction. Our approach is to build models at each level and compare them both with experiment and with the results of simulations at a greater level of detail. This iterative process is at the heart of our methodology and is common in fields such as chemistry and condensed matter physics. We will use the Grid technology described in the Bioinformatics Core of Section VII to integrate these simulations. The cytoskeleton integration will provide a relativity simple case to test the fully integrated simulations Section VI.8 proposes. Such cross-scale simulations (one part simulated with a greater level of fidelity than others) are essential, as simulating a large system with all parts modeled in the finest detail is typically unnecessary, inefficient or impractical (in terms of computer time) and often produces unstable or uninterpretable results. The Grid integration technology supports such hybrid simulations.