The nexin–dynein regulatory complex (N-DRC) is implicated in the control of dynein activity as a structural component of the nexin link. This study identifies several new subunits of the N-DRC and demonstrates for the first time that it forms a discrete biochemical complex that maintains outer doublet integrity and regulates microtubule sliding.
Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.
To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120--defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.
The transport of materials to and from the cell body and tips of eukaryotic flagella and cilia is carried out by a process called intraflagellar transport, or IFT. This process is essential for the assembly and maintenance of cilia and flagella: in the absence of IFT, cilia cannot assemble and, if IFT is arrested in ciliated cells, the cilia disassemble. The major IFT complex proteins and the major motor proteins, kinesin-2 and osm-3 (which transport particles from the cell body to ciliary tips) and cytoplasmic dynein 1b (which transports particles from ciliary tips to the cell body) have been identified. However, we have little understanding of the structure of the IFT particles, the cargo that these particles carry, how cargo is loaded and unloaded from the particles, or how the motor proteins are regulated. The focus of this chapter is to provide methods to observe and quantify the movements of IFT particles in Chlamydomonas flagella. IFT movements can be visualized in paralyzed or partially arrested flagella using either differential interference contrast (IFT) microscopy or, in cells with fluorescently tagged IFT components, with fluorescence microscopy. Methods for recording IFT movements and analyzing movements using kymograms are described.
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