Cytoskeleton and Cell Motility

• V.5.i The Microtubule Cytoskeleton
• V.5.ii The Mitotic Spindle Apparatus
• V.5.iii Organelle Transport by Motors
• V.5.iv Actin Networks

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V.5.iii Organelle Transport by Motors:

Another critical element of microtubule-based cytoplasmic organization is the transport of organelles by microtubule motor proteins. Sequence comparisons have identified two families of microtubule motors: the dyneins, and the kinesins. In vitro tests indicate that cytoplasmic dyneins, which participate in cytoplasmic transport, move exclusively toward microtubule minus ends. Most classes of kinesins that are capable of active movement are directed toward plus ends. However, one class, the NCD subfamily, moves toward minus ends (Goldstein and Philp, 1999). Biochemical and biophysical approaches have determined how some microtubule motors convert the chemical energy of ATP into force-producing conformation changes and movement (Vale and Milligan, 2000). Analysis of the structure and function of other microtubule motors is less extensive, but we know a good deal about their biochemical composition, their kinetic relationships with ATP and microtubules, and their abilities to move on or otherwise interact with microtubules (Goldstein and Philp, 1999).To understand transport mechanisms and hence cytoplasmic organization, we need to be able to dissect them in vivo. Research in this area has been challenging because of the complexity of cells. In many cell types, filament patterns and filament dynamics are difficult to decipher. The number of motors and cytoplasmic cargoes is large. Finally, cargoes can probably bind more than one type of motor at a time, so we need to consider the influence of proteins that regulate motor output (Gross et al., 2002).Overall, defining a transport mechanism requires knowing the pattern of the filament tracks, their dynamic behavior and their polarity, as well as the identity of the specific cargo and which motors move it. Further challenges are in understanding how motors link specifically to their cargoes and how regulatory systems modulate mechanochemical activity (force and velocity) and cargo linkage. Neuronal axons and blastoderm stage embryos in Drosophila are excellent models for quantitative analysis of organelle movements (Gross et al., 2002; Martin et al., 1999; Welte et al., 1998; Pilling and Saxton, in preparation). In both, microtubule organization and polarity are uniform, allowing us to focus specifically on the activities of microtubule motors. Using these systems, we can answer questions about the mechanisms of motor-driven organelle transport in the quantitative terms needed for modeling.

V.5.iv Actin Networks:

One essential aspect of biocomplexity at the cellular level is the assembly of a large number of proteins to generate force and movement. The dynamic assembly of the actin network triggered by signals through the membrane and facilitated by a number of accessory proteins including arp2/3 complex, cofilin/ADF, capping proteins, etc., provides an excellent setting to understand cellular machinery. Indeed, an essential set of four proteins in vitro can propel micron-sized beads coated with a trans-membrane protein called VASP (Loisel, 1999). We propose to use computer modeling, accounting for the ultrastructure of the cell leading edge, to predict force generation and its translation into movement. We will model the growth of the actin network as a stochastic process, using the experimentally determined kinetic rate constants for actin polymerization, end uncapping, branch formation induced by arps, as well as crosslinking by other actin binding proteins at the leading edge. Predictions will include the density of the actin mesh, filament length and branch distribution, the velocity of movement, and the force generated to compare to experiment.One of the most important physical attributes of the cytoskeleton is its interconnected character, which ensures physical links between distant intracellular compartments. Modeling of a cellular scale actin network or the entire cytoskeleton will require significant abstraction, which is beyond our current ability. We will investigate a simplified model of actin network organization, based on percolation, which can simultaneously accommodate local dynamic changes and preserve the global interconnections of the mesh.