Dock and Pak regulate olfactory axon pathfinding inDrosophila

Abstract

Experimental animals and transgenesAll fly lines were obtained from the Drosophila stock center except for the following. 981- Gal4 and SG18.1-Gal4 (Jhaveri et al., 2000) were kindly provided by V. Rodrigues. Or22a-Gal4, Or47a-Gal4 and Or47b-Gal4 (Vosshall et al., 2000) were gifts from L. Vosshall. MARCM (Lee and Luo, 1999) was carried out by heat-shocking thirdinstar larvae of the following genotypes: hs-FLP/+; dock Or47a-Gal4/dock (or +); FRT82 Gal80/FRT82 UAS-mCD8::GFP and hs-FLP/+; dock Or47b-Gal4/dock (or +); FRT82 Gal80/FRT82 UASmCD8::GFP at 37 o C for 40 minutes. Adult brains were dissected and processed as described below. GH146-Gal4 Clonal analysis ImmunohistochemistryAdult brains (from 1-to 2-day-old animals), pupal antennae and larval imaginal discs were dissected in phosphate buffered saline (PBS). Tissues were fixed in PLP (2% paraformaldehyde, 0.25% sodium periodate, 75 mM lysine-HCl and 37 mM sodium phosphate pH 7.4), washed with PBST (PBS with 0.5% Triton X-100) and subjected to antibody staining. nc82 mAb (1:20) (A. Hofbauer, PhD thesis, University of Wurzburg, 1991) was a gift from A. Hofbauer. Rabbit anti-Dock 1:500 (Clemens et al., 1996) was a gift from J. Dixon. Mouse 22C10 mAb (1:20) and rat anti-ELAV, 7E8A10 (1:20) were from Developmental Studies Hybridoma Bank. Rabbit anti-GFP polyclonal antibody (1:100) was from Clontech, and rat anti-CD8 α subunit mAb (1:100) was from Caltag. The secondary antibodies, FITC-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse and FITC-conjugated goat anti-rat, were purchased from Jackson Laboratories and used at 1:200 dilutions. Cuticle preparationsAdult antennae from animals expressing Or-Gal4/UAS-lacZ nuclear were fixed in 25% gluteraldehyde for 1 hour, washed in PBST and stained in 0.2% X-Gal solution (Ashburner, 1989). The antennae were then cleared in Faure's mountant (34% v/v chloral hydrate, 13% glycerol, 20 mg/ml gum Arabic) and photographed with the SPOT-RT cooled CCD camera. Sensilla on the third antennal segment were counted by projecting images on a video monitor.

Pak3 inhibits local actin filament formation to regulate global cell polarity

“…Our analysis also fits with the observation that Paks can act downstream of Cdc42 to modulate Rac activation, without their being absolutely required for lamellipodial formation (Cau and Hall, 2005), with observations suggesting that Cdc42 inhibits Pak activity (Weisz Hubsman et al, 2007), and by the presence of both Pak and Cdc42 in a single complex that regulates polarity in yeast (Kozubowski et al, 2008). The effects of Paks on changes in the direction of cell migration, e.g., during neural growth cone turning (Ang et al, 2003;Hing et al, 1999;Newsome et al, 2000), may reflect a similar function for Paks in damping local actin polymerization to facilitate the necessary redistribution of actin filaments.…”

Genetic Control of Wiring Specificity in the Fly Olfactory System

“…Several additional molecules have been shown to be required for the initial coarse targeting of ORN axons to specific glomeruli. For example, Dscam and two downstream effectors of Dscam, the SH2/SH3 adaptor Dock and the serine/threonine kinase Pak, are broadly expressed in the developing antennal lobe, and their loss-of-function mutants display ectopic targeting of ORN axons (Ang et al 2003;Hummel et al 2003). The Robo receptors are also involved in the coarse targeting of ORN axons, as removal of Robo receptors results in widespread mistargeting phenotypes (Jhaveri et al 2004).…”

DrosophilaFragile X Mental Retardation Protein and Metabotropic Glutamate Receptor A Convergently Regulate the Synaptic Ratio of Ionotropic Glutamate Receptor Subclasses

“…For example, the PDZ [postsynaptic density 95 (PSD-95)/Discs Large (DLG)/zona occludens-1]-domain scaffold DLG, a PSD-95 homolog, is involved in the localization of many synaptic proteins (Lahey et al, 1994;Budnik et al, 1996;Guan et al, 1996;Tejedor et al, 1997;Thomas et al, 1997Thomas et al, , 2000Mathew et al, 2002;Roche et al, 2002) but plays a specific role in B-class GluR regulation: GluRIIB abundance correlates with DLG level, but GluRIIA localization is unaffected in dlg mutants . Similarly, the Rho-type guanine nucleotide exchange factor dPix (the Drosophila homolog of the Pak interacting exchange factor) (Werner and Manseau, 1997;Hakeda-Suzuki et al, 2002), its interacting Drosophila p-21 activated kinase (dPak), a serine threonine kinase activated by GTPases Rac and cell division cycle 42 (Harden et al, 1996;Newsome et al, 2000;Mentzel and Raabe, 2005), and a dPak binding partner, the adaptor Dreadlocks (Dock; Nck homolog) (Rao and Zipursky, 1998;Buday et al, 2002;Ang et al, 2003;Rao, 2005), are all required to facilitate synaptic expression of A-class GluRs, but GluRIIB is reportedly not affected in mutants of this pathway (Parnas et al, 2001;Albin and Davis, 2004). Trafficking mechanisms likely involve GluR tethering to the cytoskeleton.…”