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First published online 31 May 2005
doi: 10.1242/jcs.02385

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Cdc42 controls the polarity of the actin and microtubule cytoskeletons through two distinct signal transduction pathways

¹ MRC Laboratory for Molecular Cell Biology, Cancer Research UK Oncogene and Signal Transduction Group, University College London, Gower Street, London, WC1E 6BT, UK
² Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (73K): [in a new window]   Fig. 1. Cdc42 controls Golgi reorientation and the polarized localization of Rac-dependent protrusions. (A) A representative cell injected with pcDNA3::GFP-ß-actin (Actin) and pRFP-F (Membrane) along with either control pRK5-MYC or pRK5-MYC::WASp CRIB. Expression of MYC-WASp-CRIB was confirmed by immunofluorescence (not shown). The front edge of the scratched monolayer is visualized with phalloidin staining (not shown) and is indicated on the merged image as a white line. Protrusions, identified by their typical lamellipodium-like, smooth convex shape, are normally found at the front (open arrows), but upon inhibition of Cdc42 by WASp-CRIB expression, protrusions are depolarized and can now be seen at the back (filled arrows). Insets are magnified (2x) views of the boxed areas. (B) The effects of various constructs on Golgi reorientation (black bars) or protrusion polarization (white bars). Error bars represent standard deviation. Asterisks mark significant (*P<0.01) and highly significant (***P<0.0001) differences compared with control. (C) Representative cell co-injected with pEGFP::WASp-CRIB, pRFP-F and pRK5-MYC::RacN17. The cell shows no typical protrusions. Bars, 20 µm.

View larger version (83K): [in a new window]   Fig. 2. The Par6/aPKC pathway controls Golgi reorientation but not localization of protrusions. (A) A representative cell injected with pEGFP-F (membrane) and either pMT21::PKC kinase dead (KD) or pRK5-MYC::Par6 Nt. Expression was checked by immunofluorescence (not shown). Protrusions are mainly found at the front (open arrows), while Golgi (asterisk) reorientation into the 120° sector facing the wound is inhibited. (B) Effects of various constructs on Golgi reorientation (black bars) or protrusion polarization (white bars). Error bars represent standard deviation. Asterisks mark significant (*P<0.01) and highly significant (**P<0.001) differences compared with control. Bars, 20 µm.

View larger version (53K): [in a new window]   Fig. 3. Pak kinase is required for polarization of protrusions but not Golgi reorientation. (A) Pak kinase activity controls the spatial localization of protrusions. Representative cells injected with pEGFP-F (membrane) along with a control plasmid, pRFP::Pak1 K299A (Pak KD), or pRFP::Pak1 AID (PakAID). Expression of constructs was confirmed by immunofluorescence (not shown). Protrusions are found at the front (open arrows) in control cells, but are delocalised in cells in which Pak is inhibited (filled arrows). None of the constructs inhibit Golgi reorientation (asterisk). (B) The effects of various constructs on Golgi reorientation (black bars) or protrusion polarization (white bars). Error bars represent standard deviation. Asterisks mark highly significant (***P<0.0001) differences compared with control. (C) The delocalized protrusions, induced after Pak inhibition are dependent on Rac. Representative cell injected with pEGFP-F (Membrane), pRFP::Pak1 AID and pRK5-MYC::RacN17. Bars, 20 µm.

View larger version (58K): [in a new window]   Fig. 4. Cdc42-activated Pak is concentrated at the leading edge. Active Pak is localized at the front of the cell upon wounding. Phospho-Pak (PPak) is specifically detected at the front of cells microinjected with a wild-type pRK5-MYC::Pak1 construct (not shown), in discrete foci that are distinct from thick ruffled areas of the cell. Upon inhibition of Cdc42 by co-injection of pRK5-HA::WASp CRIB (not shown), Pak is no longer activated (expression level of pRK5-MYC::Pak1 wt is comparable to control cells, not shown). Bars, 20 µm.

View larger version (56K): [in a new window]   Fig. 5. ßPIX accumulates at the front of the cell to confer localized Rac activation. (A) ßPIX controls the formation of protrusions. Representative cells injected with pEGFP-F (Membrane) along with pXJ40-HA::wt ßPIX. Expression of constructs was confirmed by immunofluorescence (not shown). Protrusions are found at the front (open arrows) and also at the back (filled arrows). Expression of wt ßPIX has no effect on Golgi reorientation (asterisk). (B) Interfering with the endogenous Pak/ßPIX interaction blocks polarization of protrusions. Effects of various GFP-fusion constructs on Golgi reorientation (black bars) or protrusion polarization (white bars). (C) Down-regulation of ßPIX affects formation of the protrusions. 4 days after nucleofection with siRNA, monolayers were scratched and front row cells injected with pGFP-F to determine the localization of protrusions. siRNA-mediated down-regulation of ßPIX was visualized by western blotting (bottom left). The representative control cell extends protrusions at the front (open arrows) and has ßPIX localized in front focal-complex-like puncta (arrowheads). Down-regulation of ßPIX increases the proportion of cells having no detectable protrusions (bottom panel). Instead, cells have elongated, thick filopodia-like extensions (small open arrows). In A and B, error bars represent standard deviation and asterisks mark a highly significant (***P<0.0001) difference compared with control. Bars, 20 µm.

View larger version (70K): [in a new window]   Fig. 6. Pak activity controls the specific localization of ßPIX to the leading edge. Pak activity is needed to sequester ßPIX at the front. Endogenous PIX is normally found in focal-complex-like puncta (filled arrowheads) located at the front in control-injected cells (top panel). Inhibition of Pak by injection of pEGFP::Pak1 AID (PakAID) delocalizes PIX throughout the cell [bottom panel; at the front (filled arrowheads) and also at the back (open arrowheads)]. Bars, 20 µm.

View larger version (10K): [in a new window]   Fig. 7. Cdc42 controls two distinct polarity pathways in migrating cells. Microtubule cytoskeleton polarization occurs through activation of Par6/aPKC and subsequent inhibition of GSK-3, whereas Pak controls the localization, though not the activity, of the Rac-GEF ßPIX, allowing the polarization of actin-rich, Rac-dependent protrusions.