A domain from RalGDS selectively binds RAP1-GTP; a domain from c-Raf binds Let-60-GTP (de Rooij and Bos, 1997 and Franke et al., 1997). RAP-1-GDP and LET-60-GDP are not bound. GSH-Sepharose 4B beads (Pharmacia) containing 5 μg of bound GST-RalGDS-RBD or GST-Raf-RBD were added to clarified lysates. After incubation at 4°C for 3 hr,

beads were isolated by centrifugation at 10,000 × g for 10 min. After 4 washes in Ral buffer, isolated proteins were analyzed by western immunoblot assays. Assays were performed on 10 cm Petri plates (Bargmann and Horvitz, 1991). Attractant (1 μl) and 1 μl of ethanol (neutral control) were applied to the agar at opposite ends of the plate (0.5 cm from the edge). NaN3 (1 mM) was added to attractant and ethanol to immobilize animals that reached the reservoirs. Animals (150) were placed at the center of the plate. After LDK378 datasheet 2 hr at 20°C, numbers of animals clustered at attractant (A) and ethanol (C) reservoirs were counted. A chemotaxis selleck chemical index (CI) was calculated: CI = (A − C)/(A + C + worms elsewhere). The maximum chemotaxis value is +1.0. CI values were measured on triplicate plates and averaged. Experiments

performed with BZ, BU, or IAA yielded similar results. Representative data, obtained using one, two, or all three attractants, are presented. Characterization of RGEF-1a and RGEF-1b cDNAs; preparation of transgenes, expression vectors, and transgenic animals; mutagenesis, DNA, protein, and qR-PCR analyses; characterization of an rgef-1 gene deletion; antibody production, intracellular targeting of RGEF-1b-GFP; immunofluorescence microscopy; and the MPK-1 activation assay are described in Supplemental Experimental Procedures. This work was supported by NIH grants GM080615 (C.S.R.) and T32 HL007675 (L.C.). We thank Erik Snapp, Dave Hall, and Zeynep Altun for reagents, discussions, and advice. “
“The delivery, removal, and recycling of surface Mephenoxalone membrane proteins through cytoskeletal transport regulates a variety of cellular processes including cell adhesion and cellular signaling in various cell types (Hirokawa and Takemura, 2005 and Soldati and Schliwa,

2006). Because of their polar and excitable nature, neurons represent cells with special requirements for transport. For instance, the rapid turnover of neurotransmitter receptors to and from postsynaptic membranes controls synaptic plasticity, the ability of individual synapses to change in strength (Kennedy and Ehlers, 2006 and Nicoll and Schmitz, 2005). Cytoskeletal transport is powered by molecular motor complexes that shuttle cargoes to specific subcellular compartments. A growing number of transport complexes have been functionally described in neurons (Caviston and Holzbaur, 2006, Hirokawa and Takemura, 2005 and Soldati and Schliwa, 2006). However, the question of how cargo is guided across different cytoskeletal tracks to reach distinct subcellular destinations remains unanswered.