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
* Author for correspondence (e-mail: alan.hall{at}ucl.ac.uk)
Accepted 16 March 2005
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Summary |
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Key words: Cdc42, Cell migration, Microtubules, Actin
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Introduction |
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There is growing evidence that another small GTPase, Cdc42, is a key regulator of directional sensing. It was first implicated in the establishment of cell polarity in Saccharomyces cerevisiae, where it is required both for polarized bud formation and the formation of polarized mating projections in response to pheromone gradients (see Etienne-Manneville, 2004; Nelson, 2003
). In leukocytes, Cdc42 is essential for directional migration, but not for random migration, suggesting that it is required to spatially localize actin polymerization in response to an external cue (Allen et al., 1998
; Srinivasan et al., 2003
). Using a monolayer of primary fibroblasts in a scratch-induced migration assay, Cdc42 is also required for spatially localizing membrane protrusions at their leading edge, as well as for promoting reorientation of the Golgi/centrosome to face the direction of migration (Nobes and Hall, 1999
). Further analysis of centrosome reorientation using primary astrocytes in a similar assay has revealed that Cdc42 mediates its effects through a complex containing Par6, a scaffold protein, and PKC
, an atypical protein kinase C (aPKC) (Etienne-Manneville and Hall, 2001
). Par6 is a direct target of Cdc42 and interestingly the same complex is involved in numerous other polarity-establishing processes, such as asymmetric cell division, epithelial cell morphogenesis and axon formation in neurons (Etienne-Manneville and Hall, 2003b
; Henrique and Schweisguth, 2003
; Shi et al., 2003
).
We now show that the Cdc42/Par6/aPKC pathway is also required for centrosome/Golgi reorientation in fibroblasts, but that this pathway does not control the spatial localization of protrusive activity. The latter is instead controlled by a Cdc42-dependent activation of the Ser/Thr kinase Pak, which controls the localization of Rac activity through recruitment of its associated ßPIX Rac-GEF to the front of the cell. We conclude that Cdc42 controls the polarity of the actin and microtubule cytoskeletons during cell migration through two distinct signal transduction pathways.
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Materials and Methods |
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Immunofluorescence and microscopy
Cells were washed once in BRB80 (2 mM EGTA, 2 mM MgCl2, 80 mM K-Pipes pH 6.8), and fixed in 4% paraformaldehyde (PFA) in BRB80 4.5 hours after scratching. 0.2% saponin was added to the wash and fixative to reduce background during staining for endogenous PIX with the panPIX antibody. Unreacted PFA was quenched, cells were permeabilized and non-specific binding blocked by incubating cells for 1 hour in 2% BSA 0.2% saponin in BRB80. This buffer was further used for all washings and incubations. Images were taken with a MRC1024 (Bio-Rad) confocal OptiphotII (Nikon) microscope using a 60x planapochromatic objective (NA 1.4) and a Kr/Ar laser (Fig. 1A, Fig. 2A and Fig. 3C). Otherwise, quadruple channel images were taken with a Axioplan microscope using a 63x planapochromatic oil immersion objective (NA 1.4) and Zeiss FS02 (Ex G365 Dichroic FT395 Em LP420), FS10 (Ex BP450-490 Dichroic FT510 Em BP515-565), FS43 (BP545/25 Dichroic FT570 Em BP605/70) and FS26 (Ex BP575-625 Dichroic FT645 Em BP660-710) filter sets. The same range of fluorochrome intensity was empirically set-up to minimize bleed-through of RFP (red fluorescent protein), when used, into the far-red channel due to unequal intensities. Acquisition was performed with an ORCA-ER (Hamamatsu) camera driven by Openlab software (Improvision). For video-microscopy, an average of five cells were imaged on an inverted fully motorized Axiovert 200M microscope fitted with a temperature-controlled Plexiglas box and an anti-vibration table (Improvision). Images were acquired using a 40x oil immersion planapochromatic objective (NA 1.35), Zeiss FS37 (Ex BP450/50 Dichroic FT480 Em BP510/50) and FS00 (Ex BP530-585 Dichroic FT600 Em LP615), Ludl transmission- and in-built fluorescence shutters, an ORCA-ER camera (Hamamatsu), and the Openlab acquisition software. Images were analysed and processed for presentation with Metamorph (UIC).
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RNAi experiments
All siRNAs used were from Ambion, obtained at standard purity. Cells were trypsinized, resuspended in 10% FCS/DMEM (Gibco) and centrifuged. Pellets were then washed with PBS at room temperature, and cells were centrifuged again to remove any traces of DMEM. 106 cells were resuspended in 100 µl of nucleofection solution V (Amaxa). 360 pmol siRNA against mock (GFP-22 siRNA, Qiagen) or the Rac guanine nucleotide exchange factor ßPIX (GGGUUCGAUACGACUGCCAtt) were added and cells electroporated with program G-09. Another siRNA for ßPIX gave the same results (UCUAUCAGGAUGAUAAUCCtc). Cells were rapidly resuspended in 10% FCS/DMEM + 10 mM Hepes pH 7.2 (Gibco) and left in suspension for 1 hour at 37°C. Cells were then centrifuged, resuspended in 10% FCS/DMEM and seeded at 4-5x104 cells/cm2 and left for 4 days, rather than the usual 3 days, to allow maximal depletion. Cells were then either lyzed in immunoprecipitation lysis buffer (IPLB) [10 mM Tris pH 7.5, 140 mM NaCl, 5 mM EDTA, 1% IGEPA®, 1 mM sodium orthovanadate supplemented with 2 mM phenyl methyl sulfonyl fluoride (PMSF) and one Roche complete tablet/50 ml] or scratched and microinjected. For rescue experiments, cells were nucleofected with ßPIX siRNA and injected 4 days later with pXJ40-HA::ratßPIX wt.
Immunoprecipitations and western blots
Cell lysates were prepared from confluent monolayers (in 92 cm2 dishes) that were scratched 200 times with a multi-tip Pipetman fitted with P2 tips, (approx. 1 mm wide srcatches). Cells were washed with PBS and lyzed in 18 µl/cm2 of cold IPLB. 100 µg protein were used for immunoprecipitation with protein G-Sepharose beads and 1 µl Pak1 antibody. Beads were further washed with cold IPLB (10 mM Tris pH 7.5, 140 mM NaCl, 5 mM EDTA, 1% IGEPAL, 1 mM sodium orthovanadate supplemented with 2 mM PMSF and one Roche complete tablet/50 ml). 20-25 µg were used for western blotting of total extracts. Extracts were processed using standard SDS-PAGE and western blotting procedures.
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Results |
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Pak controls the localization of Rac-dependent protrusions, but has no effect on Golgi reorientation
p21-activated kinase (Pak), one of the first targets of Cdc42 and Rac to be identified, has been implicated in regulating the actin cytoskeleton. In particular, it influences persistence during the random migration of fibroblasts (Sells et al., 1999). Thus, to determine whether Pak plays a role in polarization during cell migration, we microinjected leading edge cells with Pak constructs. Kinase dead (Pak1 KD), but not wild type (Pak1wt) constructs, dramatically inhibit localized protrusions (Fig. 3A,B), with 83% of injected cells now showing delocalized protrusions all around the cell periphery. Neither construct had any effect on Golgi reorientation (Fig. 3A,B). Similar results were obtained with Pak2 constructs (data not shown). Pak1 KD is still able to interact with a large number of proteins, and, therefore, to determine specifically whether its kinase activity is required, we expressed an auto-inhibitory domain derived from Pak1 (PakAID), which has been shown to interact with and inhibit the kinase activity of Paks 1, 2 and 3 (Zenke et al., 1999
; Zhao et al., 1998
). PakAID also dramatically increased the number of cells with delocalized protrusions (Fig. 3A,B, and Movie 3 in supplementary material). A control construct containing a point mutation that blocks its activity had no effect (L107F, Fig. 3B). Moreover, dominant negative Rac blocked the formation of delocalized protrusions seen after expression of PakAID (Fig. 3C). We conclude that Pak controls the localization of Rac activity at the front of the cell, but that neither Pak kinase activity nor Cdc42 are required for Rac activation, during scratch-induced cell migration.
Pak is activated by Cdc42 at the front of the cell
To examine the localization of Pak during scratch-induced migration, we used antibodies to visualize active, auto-phosphorylated Pak. As previously reported with overexpressed constructs (Sells et al., 2000), phosphorylation of endogenous Pak1/2 on S199-S204/S192-S197 increases upon wounding (see Fig. S1 in supplementary material) and phosphorylated wild-type Pak1 is found concentrated in discrete puncta at the leading edge of migrating cells (Fig. 4). The fluorescence signal disappears after expression of either PakAID (see Movie S4 in supplementary material), or WASp-CRIB (Fig. 4), showing that it is dependent on both Pak and Cdc42 activities. Expression of dominant negative Rac, blocked the formation of protrusions, but it did not affect Pak activation (see Fig. S2 in supplementary material), showing that Pak does not act downstream of Rac in this system. We conclude from this experiment that Pak is activated at the front of the cell by Cdc42.
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To analyse further the role of endogenous ßPIX during scratch-induced cell migration, we used an RNAi approach. Western blot analysis (Fig. 5C, bottom-left panel) reveals an 80-90% reduction in ßPIX protein after nucleofection. Immunofluorescence analysis after nucleofection reveals that the monolayer is heterogenous, with some cells lacking any detectable ßPIX, while others still contain ßPIX, albeit at reduced levels. When control monolayers were scratched 4 days after nucleofection with mock siRNA, only 10% of front row cells showed no detectable protrusions (Fig. 5C, bottom right panel). However, after nucleofection with ßPIX siRNA, 42% of front row cells showed no detectable protrusions, but instead had elongated, thick filopodia-like extensions (Fig. 5C top panel and small open arrows in bottom-right panel). To analyse protrusion formation specifically in cells lacking ßPIX, we examined ßPIX levels in individual cells by immunofluorescence. We found that over 95% of the cells that completely lack detectable ßPIX have no detectable protrusions. Importantly, siRNA nucleofection had no effect on Golgi reorientation, irrespective of the ability of cells to form protrusions (data not shown). We conclude that ßPIX, is the major Rac-GEF required for Rac-dependent formation of protrusions.
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Discussion |
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ßPIX is usually found in central focal adhesions and peripheral focal complexes (Manser et al., 1998). We think it is probable that immediately after scratching the monolayer, ßPIX is activated and targeted to the cell periphery in an unpolarized fashion. Localized activation of Cdc42 and Pak then promotes the accumulation or retention of active ßPIX at the front of the cell. The relationship between Pak and PIX with respect to localization is complex and contradictory. On one hand, PIX complexed with GIT (a member of the adhesions-bound Arf-GAP protein family) has been reported to target Pak to focal complexes (Manabe Ri et al., 2002
; Manser et al., 1998
; Stofega et al., 2004
; Turner et al., 1999
). On the other hand, it has been found that localization of Pak to focal complexes is mediated by Nck or paxillin, i.e. independently of PIX (Bokoch et al., 1996
; Hashimoto et al., 2001
; Zhao et al., 2000a
), and that Pak then localizes PIX/GIT complexes (Zegers et al., 2003
; Zhao et al., 2000b
). Such Pak-induced sequestration of ßPIX has previously been documented in MDCK cells (Zegers et al., 2003
). The identity of the Pak substrate involved in localization of ßPIX in these scratched fibroblast monolayers is not clear. ßPIX is itself a substrate, although the significance of this is unknown phosphorylation on S525 and T526 for example is not thought to affect its Rac-GEF activity (Koh et al., 2001
; Shin et al., 2002
). Paxillin and GIT are also substrates of Pak (Bokoch, 2003
) and it is possible that ßPIX, GIT and/or paxillin phosphorylation by Pak modulates ßPIX interactions with other molecules, affecting localization to the membrane and incorporation into leading edge focal complexes. These different possibilities are currently being examined.
Pak has been implicated in cell migration of many cell types in Drosophila and mammals (Adam et al., 2000; Harden et al., 1996
; Kiosses et al., 1999
; Kiosses et al., 2002
; Sells et al., 1999
; Vadlamudi et al., 2000
) and there have been numerous reports linking Pak to the formation of protrusions, but in many cases the relationship has been unclear (Bokoch, 2003
). For example, Pak affects the persistence of protrusions in cells randomly migrating (i.e. in the absence of any directional cue), but these protrusions were reported to be Rac-independent (Sells et al., 1999
). Studies in Dictyostelium (Chung and Firtel, 1999
; Labruyere et al., 2003
) show that Paks are crucial for polarization of the actin cytoskeleton during directed migration, but it is not known how they themselves are polarized in the first place. Recently, Pak1 has been shown to be required for directional sensing in leukocytes (Li et al., 2003
). In that case however, Pak1 acts as a scaffold protein required for the activation of Cdc42 by the GEF
PIX and by Gß. The role of its kinase activity, if any, was not explored in this assay.
PIX is found mainly in haematopoietic cells and is not detectable in REFs (our unpublished results) and furthermore, RNAi knock-down of ßPIX in REFs, or inhibition of Pak activity does not affect Golgi reorientation, suggesting that Cdc42 is still activated in cells lacking PIX or Pak kinase activity. We cannot rule out, however, the interesting possibility that Pak may play two roles in directional sensing; first as a scaffold in the activation of Cdc42 (kinase independent) and second as a mediator for polarization of Rac activity (kinase dependent). Further experiments will be required to address this issue.
In conclusion, Cdc42 orchestrates polarization of the actin and microtubule cytoskeletons through two different signal transduction pathways. A direct interaction with Par6 leads to activation of the associated atypical PKC and polarization of microtubules, while a direct interaction with Pak activates this kinase, leading to the localization of active ßPIX/Rac and polarized actin polymerization.
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Acknowledgments |
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Footnotes |
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