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In the developing forebrain neuronal polarisation is a stepwise and initially reversible process that underlies correct migration and axon specification. Many aspects of cytoskeletal changes that accompany polarisation are currently molecularly undefined and thus poorly understood. Here we reveal that the Pak1 kinase is essential for the specification of an axon and dendrites. In hippocampal neurones activation of Pak1 is spatially restricted to the immature axon despite its uniform presence in all neurites. Hyperactivation of Pak1 at the membrane of all neurites or loss of Pak1 expression disrupt both neuronal morphology and the distinction between an axon and dendrites. We reveal that Pak1 acts on polarity in a kinase dependent manner, by affecting the F-actin and microtubule cytoskeleton at least in part through Rac1 and cofilin. Our data are the first to demonstrate the importance of localised Pak1 kinase activation for neuronal polarisation and differentiation.
Polarisation of neurones is essential for their correct migration and subsequent specification of axons and dendrites. In the neocortex, hippocampus and cerebellum establishment of polarity correlates with the positioning of the centrosome and Golgi to the base of the leading process of a migrating neurone. An axon and dendrites are commonly specified by the location of the leading and trailing processes, suggesting that neuronal polarity is set at the time of migration (Noctor et al., 2004; Tsai and Gleeson, 2005; Solecki et al., 2006).
The molecular pathways of axonal specification are predominantly analysed in vitro where differentiation of hippocampal neurones is morphologically classified. Stage 2 neurones extend several uniform neurites. During stage 3 only one neurite becomes an axon which matures together with dendrites in stages 4-5 (Dotti et al., 1988). The microtubule plus end motor protein, kinesin-1, transiently localises to neurite terminals in a non-polarised neurone but defines the emerging axon by stably accumulating in its tip (Jacobson et al., 2006). PI3 kinase activation, subsequent enhancement of Akt and suppression of GSK3β kinases are required for axonal establishment and rapid outgrowth, while GSK3β remains active in the immature dendrites (Jiang et al., 2005; Wiggin et al., 2005). Cell surface proteins DOCK7, IGFR and PMGS regulate the polarised activities of the Rho GTPases, Cdc42, Rac1 and RhoA (Da Silva et al., 2005; Sosa et al., 2006; Watabe-Uchida et al., 2006). Cdc42 controls the activity of JNK kinases and a polarity complex consisting of Par3, Par6 and atypical protein kinase C (Wiggin et al., 2005; Oliva et al., 2006). Inhibition of RhoA reduces recruitment of ROCK kinase and profilin IIa to the tip of the future axon, promoting its formation by destabilising F-actin (Da Silva et al., 2005). Activation of Rac1 by the exchange factor TIAM1 or DOCK7 promotes axon formation in part by changing the activity of the microtubule severing protein stathmin/Op18 (Watabe-Uchida et al., 2006). Neuronal polarisation retains a degree of flexibility after its establishment due to the precise function of key proteins such as CRMP2, which controls axonal elongation by regulating microtubule assembly, reorganisation of F-actin filaments and endocytosis. CRMP2 loss-of-function causes the elaboration only of dendrites (Inagaki et al., 2001; Arimura and Kaibuchi, 2005, 2007).
The p21 activated kinase (Pak1) is a Cdc42 and Rac1 effector and a member of a family of six serine/threonine kinases classed into Groups I (Paks1-3) and II (Paks 4-6) (Jaffer and Chernoff, 2002; Bokoch, 2003). Pak1 activation requires GTPase dependent or independent conformational changes leading to multiple phosphorylations that commonly occur at the plasma membrane. In S.cerevisiae, C.neoformans and Dictyostelium Pak kinases are essential for polarisation (Ottilie et al., 1995; Holly and Blumer, 1999; Lee et al., 2004; Nichols et al., 2004). Mammalian Pak1 can facilitate directed migration of cultured fibroblast and neutrophils (Dharmawardhane et al., 1999; Sells et al., 1999; Cau and Hall, 2005). We now reveal that localised activation of Pak1 is pivotal for neuronal polarity by affecting the function of proteins that regulate the F-actin and microtubule cytoskeleton.
Pak1 activation is accompanied by sequential phosphorylation catalysed by Akt/PKB, PDK1 and Pak1 (Bokoch, 2003). Phosphorylation of S199/204 and T423 were shown to strongly correlate with an increase in Pak1 catalytic activity and are widely used as markers of its activation (King et al., 2000; Sells et al., 2000; Chong et al., 2001; Loo et al., 2004; Zhao et al., 2005). We examined the activation profile of Pak1 by following the levels of its phosphorylation on S199/204 (Pak1(P)) during differentiation of cultured cortical neurones. Western blots revealed high levels of Pak1(P) from 1 to 7 days in culture with a subsequent decrease in both total and phosphorylated Pak1 (Fig. 1 A). Since the phospho-specific antibody can also detect Pak2 and possibly Pak3 it is interesting to note that we only observed one band on our western blots which corresponded to Pak1, suggesting that in these cultures it is the predominant Group I Pak kinase. We compared the subcellular distribution of total and S199/204 phosphorylated Pak1 in cultured cortical and hippocampal neurones. Western blots revealed a transient enrichment of Pak1(P) in membrane fractions derived from cortical cultures that peaked at 2-4 days in vitro (div) and dramatically dropped at 7 div (Fig. 1 B). The timing of high membrane Pak1(P) coincided with the transition of most neurones from a non-polarised to a polarised state (stages 2 to 3). In contrast, membrane levels of total Pak1 increased with time in culture, remaining high after 4 div. At stage 1 when hippocampal neurones have not yet elaborated neurites, Pak1 was asymmetrically enriched and activated in an area encompassing the Golgi (data not shown). In stage 2 neurones that have extended multiple neurites but not yet polarised, Pak1 was uniformly distributed and activated. Interestingly, on average 16% ± 1.5% stage 2 neurones had higher levels of Pak1(P) in a single neurite (Fig. 1 C). By stage 3, when neurones undergo specification of a future axon, active Pak1 was evident only in the cell soma and the distal half of the longest neurite, despite continued uniform distribution of total Pak1 (Fig. 1 C). These results reveal that polarisation of hippocampal and cortical neurones is accompanied by asymmetric activation of Pak1.
To determine whether Pak1 expression and phosphorylation accompany neuronal differentiation in vivo, we examined the developing cerebral cortex and hippocampus at mouse embryonic days 14 and 16 (E14 and E16) and at birth (P0). In confirmation with our previous findings, Pak1 levels were low in the proliferating ventricular zones (VZ) and high in the axon rich intermediate zones (IZ) of the cerebral cortex and hippocampus in all examined ages (Fig. 2) (Zhong et al., 2003). Development of the cortex was marked by progressive enrichment of Pak1 in the cortical plate (CP) from E14 to P0 (Fig. 2 A, C and E), while high levels were already present in the hippocampal plate (HP) at E14 (Fig. 2 G). In embryonic stages the distribution of phosphorylated Pak1 was similar but not as extensive as that of total Pak1. Small foci of concentrated Pak1(P) were seen in the E14 cortex and hippocampus primarily in the IZ, CP and HP (Fig. B and H). At E16 Pak1(P) accumulated more broadly in axonal tracts of the IZ and neurones of the CP and HP (Fig. 2 D and J). Phosphorylated Pak1 remained high in the CP and HP but was reduced in the IZ at birth (Fig. 2F and L). These results were consistent with the segregation of Pak1(P) to the distal portion of the axon in polarised hippocampal neurones and its subsequent downregulation observed in vitro (Fig. 1C). At all stages lowest levels of Pak1(P) were seen in the VZ.
Pak1 exists in the cytoplasm in an inactive homodimerised state and can be activated by recruitment to the membrane (Lei et al., 2000; Bokoch, 2003). Since membrane enrichment of activated Pak1 coincides with the timing of axon specification, we examined the consequences of Pak1 hyperactivation using a protein that has a Ras prenilation sequence (Caax box) at its C terminal domain (Pak1Caax) which renders it constitutively active (Daniels et al., 1998). This also provided us with an ideal tool to disrupt the asymmetry of endogenously activated Pak1. To verify that in neurones fusion of Pak1 to the Caax box results in its activation at the membrane we compared the levels of phosphorylated Pak1 in membrane fractions derived from Pak1Caax and EGFP expressing cortical neurones. Clear activation of Pak1Caax was evident, confirming previous reports (Supplemental Fig. 1A) (Daniels et al., 1998). We also examined the levels and activities of Pak2 and Pak3, observing no detectable changes after Pak1Caax expression (Supplemental Fig. 1B). Hippocampal neurones were transfected with Pak1Caax or its inactive mutant, Pak1R299Caax, and examined after 1, 2 or 3 days. Neurones expressing Pak1Caax displayed increased lamellipodia and a star like morphology with all neurites of apparently similar lengths, a phenotype not observed following Pak1R299Caax or EGFP expression (Fig. 3 A, B). Overexpression of Pak1Caax reduced outgrowth of the longest neurite (which was considered the most likely future axon), while the remaining neurites (considered future dendrites) extended further than the EGFP controls (Fig. 3 D, E). Expression of the Pak1R299Caax mutant had transient and minor consequences on neurite outgrowth and was otherwise indistinguishable from EGFP controls (Fig. 3 C, D, E). Our data suggest that in hippocampal neurones localised activation of Pak1 in a single neurite is necessary for correct axon and dendrite morphology.
In mature, polarised hippocampal neurones Pak1(P) was only present in axons where it co-segregated with the axonal marker, Tau-1 (Fig. 4 A). In contrast total Pak1 remained high in both dendrites and axons (Fig. 4 A). Since activation of Pak1 at the membrane differentially affected neurite outgrowth and morphology, we examined the consequences of its expression on the specification of axons and dendrites. Transfected neurones were cultured for 3 or 7 days prior to fixation when axons and dendrites were identified by expression of Tau-1 and Map2, respectively. At 3 div hyperactivation of Pak1 caused on average 35.2% ± 0.6% neurones to elaborate more than 1 Tau-1 positive neurite, in contrast to 17.4% ± 0.3% of EGFP controls (Supplemental Fig. 2). This phenotype became increasingly apparent with time in culture, thus at 7 div 60.8% ±1.5% of Pak1Caax expressing neurones had more than one Tau-1 positive neurite, in contrast to an average of 21.7% ± 1.8% EGFP controls and 28.2% ± 0.2% of catalytically inactive Pak1R299Caax (Fig. 4 B, D). To investigate whether the effects of Pak1 on polarisation depended on its membrane localisation, we overexpressed a constitutively active Pak1T423E mutant. Neurones expressing Pak1T423E exhibited increased lamellipodia but normal polarity with only 16.6% ± 2% elaborating multiple Tau-1 positive neurites, confirming that Pak1 affects polarity when at the membrane (Fig. 4 C, D).
In EGFP expressing controls the segregation of Map2 to dendrites and absence from axons was evident at 3 div and highly apparent at 7 div. In contrast, in many Pak1Caax expressing neurones Map2 extended further down all neurites at 3 and 7 div (Fig. 5 A). We measured the area of a cell that contained Map2, revealing that after 3 div on average 59.6% ± 6.9% of a Pak1Caax expressing neurone had Map2 in contrast to 38.7% ± 6.7% of an EGFP expressing control. The differences remained apparent at 7 div when on average 25.6% ± 0.2% of a control cell had Map2, in contrast to 40.8% ± 2.8% of a Pak1Caax expressing neurone (Fig. 5 B). Due to the extended presence of Map2 in axon-like neurites many of which were Tau-1 positive, we utilised a later axonal marker, Map1b phosphorylated on T1265 (Map1b(P)), a known GSK3β target site (Trivedi et al., 2005). In 81.9% ± 0.9% of EGFP expressing controls this marker only labelled a single axon (Fig. 5 C). In 24.2% ± 4.9% Pak1Caax expressing neurones, Map1b(P) labelled multiple long neurites suggesting the formation of several axon-like neurites (Fig. 5 D). Unexpectedly, in 32.9% ± 3.8% of Pak1Caax expressing neurones Map1b(P) was not detectable in any neurites. These neurones always had stage 2-like morphologies suggesting that they had not transferred to stage 3. Together our results suggest that many neurones respond to Pak1Caax overexpression by failing to specify an axon and dendrites. Pak1Caax can also cause neurones to elaborate multiple Tau-1, Map1b(P) positive axon-like neurites. This variation of consequences may reflect the levels and timing of Pak1Caax expression in relation to the differentiation status of the targeted neurone.
To examine the role of endogenous Pak1 we utilised two shRNA expression vectors, one of which was previously used to demonstrate Pak1 importance for neutrophil chemotaxis (Li et al., 2003). The effectiveness of the shRNA vectors was verified by transfection into Pak1 expressing Cos7 cells or cortical neurones which were analysed by western blotting (Fig. 6 A, B). We also verified that downregulation of Pak1 activity had no significant effect on the expression levels and activities of Pak2 or Pak3 (Supplemental Fig. 1B). In hippocampal neurones expression of either Pak1 shRNA caused the appearance of aberrant F-actin rich areas of cell spreading around the soma and along the neurites (Fig. 6 C). The somal protrusions in particular contained an abundance of curved microtubules, many of which were acetylated and thus stabilised (Fig. 6 D). The formation of extensive lamellipodia was not observed in EGFP or control shRNA expressing neurones. After 3 div 38% ± 3.6% of Pak1 shRNA expressing neurones had disorganised neurites that were both Tau-1 and Map2 positive, in contrast to the control shRNA neurones of which only 15.3% ± 3.3% had not polarised (Fig. 7). At 3 div Pak1 shRNA expression caused a 13% reduction in total neurite outgrowth when compared to control shRNA. Unexpectedly, at 7 div expression of Pak1 shRNA caused neuronal lethality (Supplemental Fig. 3). Comparing the numbers of live targeted cells at 3 and 7 div revealed a 8.3% ± 3.3% viability after Pak1 shRNA expression, in contrast to 80.6% ± 6.1% seen in the shRNA control. Some neurones that remained viable at 7 div were had no visible neurites, while others had extended one or a few highly disorganised processes. Lamellipodia rich in F-actin and microtubules remained evident particularly in large somal extensions (Supplemental Fig. 3). The loss of neuronal viability coupled with the severely altered morphologies prevented us from scoring the number of polarised neurones.
Neuronal polarisation is characterised by local destabilisation of F-actin in the growth cone of one neurite which will subsequently become the axon. Local application of low doses of the F-actin destabilising drug cytochalasin D (CD) can induce a neurite to become an axon (Bradke and Dotti, 1999, 2000). To examine Pak1 activation in response to the induction of multiple axons we exposed cultured hippocampal neurones to low doses (1μM) of CD a few hours after plating when all neurones had stably attached to the substratum. After 7 days in culture CD treatment resulted in the appearance of hippocampal neurones elaborating multiple axons that were positive for Tau-1 and Map1b(P) (Fig. 8 A and data not shown). Interestingly all of the Tau-1 positive neurites also had high levels of Pak1(P), suggesting that Pak1 activation is responsive to changes in the F-actin cytoskeleton and accompanies axon formation.
Since CD can cause Pak1 activation, we attempted to rescue the effects of Pak1Caax expression by exposure to low doses of the F-actin stabilising drug jasplakinolide. Hippocampal neurones transfected with EGFP alone or with Pak1Caax were treated a few hours after plating with 5nM jasplakinolide and maintained in culture for 7 days. Long term exposure to jasplakinolide caused an increase in disorganised lamellipodial protrusions of both EGFP and Pak1Caax expressing neurones (Fig. 8 B). Jasplakinolide partially rescued the Pak1Caax phenotype, reducing the percentage of neurones with multiple Tau-1 positive neurites from 60.8%±1.5% to 41.6%±1.4% (Fig. 8 C). No significant effect on polarity was observed in the EGFP controls. Together these data suggest that a dynamic interplay exists between F-actin organisation and Pak1 activity which is required for axon formation in hippocampal neurones.
It is well established that both Cdc42 and Rac1 can activate the Group I Pak kinases (Bokoch, 2003). It has also transpired that Pak1 can induce Rac1 activation through the association with PIX/Cool exchange factors or inhibitory phosphorylation of the Rac dissociation inhibitor RhoGDI (Manser et al., 1998; Obermeier et al., 1998; DerMardirossian et al., 2004). Importantly, association of Pak1 and the exchange factors PIX/Cool was recently shown to be responsible for the formation of polarised lamellipodia in epithelial cells (Cau and Hall, 2005). To investigate how Pak1 regulates neuronal morphology and polarity we attempted to rescue the Pak1Caax phenotype by co-expression with a dominant negative mutant of Rac1, Rac1N17. At 7 div co-expression of RacN17 reduced the percentage of neurones extending multiple axons from 60.8%±1.5% (Pak1Caax) to 33.4%±5.3% (Pak1Caax + RacN17) (Fig. 9 A, C). However, RacN17 did not rescue the effects of Pak1Caax on neuronal morphology and lamellipodia extension. Expression of RacN17 alone decreased the number of neurones elaborating multiple axons from 21.7% ± 1.8% in the EGFP control group to 13% ± 0.5% (Supplemental Fig. 4). In contrast, inhibition of Cdc42 by co-expression of the Cdc42N17 mutant with Pak1Caax had no significant effect on the number of Tau-1 and Map2 positive neurites (Fig. 9 A and Supplemental Fig. 4). To further examine whether Rac1 acts upstream and downstream from Pak1Caax, we compared the levels of Pak1Caax activation in cortical neurones in the presence or absence of co-expressed RacN17, revealing no significant differences (Supplemental Fig 1A). These data suggest that Rac1 lies downstream from the polarising effects of Pak1.
Pak1 is known to affect F-actin organisation by phosphorylating and activating the LIM-1 kinase (LIMK-1) (Edwards et al., 1999). This results in an increase in LIMK-1 activity towards a family of F-actin depolymerising and severing proteins, cofilin/ADF. Phosphorylation of cofilin on S3 is inhibitory, reducing its binding affinity towards F-actin (Sarmiere and Bamburg, 2004). To determine whether Pak1Caax overexpression in forebrain neurones results in cofilin inactivation we examined the levels of S3 phosphorylated cofilin, revealing a ~3 fold increase in lysates obtained from Pak1Caax transfected neurones when compared to the EGFP expressing controls (Fig. 9 B). This is most likely underestimated as the transfection efficiency ranged between 40% and 60%. To determine whether cofilin phosphorylation is required for the effects of Pak1 on neuronal polarity and morphology, we co-expressed a non-phosphorylatable, constitutively active mutant (cofilinS3A) with Pak1Caax in hippocampal neurones. Constitutively active cofilin did not reduce the effects of Pak1Caax on axonal outgrowth and branching (data not shown). However, the percentage of neurones with multiple, Tau-1 immunoreactive axons decreased from 60.8%±1.5% (Pak1Caax) to 39.3%±2.7% (Pak1Caax + cofilinS3A) (Fig. 9 A and C). These data further confirm that Pak1 acts on neuronal polarity by affecting F-actin organisation and identify cofilin as its likely target.
Postnatal inhibition of Paks 1-3 causes a morphological change and reduced number of dendritic spines which in mice results in impaired memory consolidation (Hayashi et al., 2004) In vitro Pak1 can act on spine morphogenesis by phosphorylating the myosin II regulatory light chain and as a downstream target of ephrinB1/EphB receptor signalling (Penzes et al., 2003; Zhang et al., 2005). Therefore, once the forebrain is formed, Pak1 has a major postsynaptic role, however its importance for early stages of neuronal development have not been elucidated. Our study is the first to demonstrate a pivotal role for Pak1 for the specification and outgrowth of an axon and dendrites.
Hippocampal neurones provide a well established model for studying neuronal differentiation in vitro. We demonstrate that asymmetric activation of Pak1 occurs as a hippocampal neurone transits from a non-polarised to an axon specified stage. Enrichment of active Pak1 continues to predominate in the axon suggesting that it is subsequently required for its outgrowth and function. Interestingly, all of the multiple axons that formed as a consequence of exposure to CD had increased Pak1(P), confirming that Pak1 activation is an integral aspect of axon formation and growth. The fact that F-actin destabilisation caused Pak1 activation also suggests the existence of feedback mechanisms from actin to its regulatory proteins which ultimately controls the balance between its polymerisation and depolymerisation in a temporally and spatially regulated manner.
The consequences of Pak1 loss-of-function on neuronal differentiation are striking. If Pak1 was only required for axonal specification we would have predicted that its downregulation would induce the formation only of dendrites, in a similar manner to CRMP2 loss-of-function (Inagaki et al., 2001). However, expression of two Pak1 shRNAs affected the segregation of Tau-1 and Map2, indicating its requirement for a neurone to transit from a non-polarised to a polarised stage. Loss of Pak1 also inhibited outgrowth of all neurites and caused neuronal death, indicating it’s essential role during neuronal differentiation. These consequences ranged in severity which likely reflects the differentiation status of individual neurones at the time of dissociation from the hippocampus, the levels of shRNA expression and the half-life of endogenous Pak1 protein that had been expressed prior to mRNA degradation. Our findings, together with published literature suggest that in neurones Pak1 initially acts to promote the specification and rapid outgrowth of an axon. Subsequently, Pak1 is necessary to promote dendrite elaboration where it maintains its postsynaptic function through adulthood by regulating spine morphology and density (Hayashi et al., 2002; Penzes et al., 2003; Zhang et al., 2005). A similar functional switch from axon to dendrites has been reported for the small GTPase Rab8 (Huber et al., 1993; Huber et al., 1995).
Pak1Caax expressing neurones assumed a star-like morphology due to the reduced outgrowth of the future axon and extension of the remaining neurites which contained both Tau-1 and Map2. Interestingly a similar morphology and distribution of Map2 and Tau-1 was reported following loss of the neuronal microtubule regulating SAD-A and SAD-B kinases (Kishi et al., 2005). To date there are no known links between SAD and Pak1 kinase functions and a clear role for Pak1 as a regulator of microtubules has not been established. The massive accumulation of stabilised, looped microtubules around the cell soma of Pak1 shRNA expressing neurones may be a direct consequence of its loss or a secondary effect caused by the appearance of extensive lamellipodia. However, the similarity between Pak1 gain-of-function and SAD loss-of-function is intriguing and suggests their mutually antagonising relationship.
Membrane localisation of Pak1 can induce neurite outgrowth in PC12 cells irrespective of its kinase activity (Daniels et al., 1998). We reveal that the effect of Pak1 on the polarisation of hippocampal neurones requires its kinase activity and membrane localisation. The fact that PC12 cells do not polarise may account for the observed differences, suggesting that in neurones polarity and outgrowth have a molecular link, part of which may involve the regulated activation of Pak1. It is striking that the highly active Pak1T423E mutant did not alter axonal specification despite a clear induction of actin polymerisation, further confirming that polarisation requires activation of Pak1 at the membrane. In cortical neurones Pak1 can promote elaboration of primary dendrites as a major downstream target of Cdc42 and Rac1 (Hayashi et al., 2002). We did not observe an increase in the number of primary neurites following Pak1 gain-of-function, although Pak1Caax did induce an increase in dendritic outgrowth. Hayashi and colleagues examined neurones that had been electroporated in utero with cytoplasmic Pak1 and subsequently analysed after dissociation from mouse cortices. These experimental differences may account for our non-overlapping results. We did, however find that long term loss of Pak1 severely affected dendritic growth, thus supporting the proposal that Pak1 is required for dendrite formation and function.
A Cdc42 mutant that autonomously cycles between a GTP and GDP bound state (Cdc42L28) can induce multiple axons downstream of the small GTPase Rap1b (Schwamborn and Puschel, 2004). Further upstream signals include PI3K and the insulin-like growth factor receptor-1, the loss of which prevent axon formation (Arimura and Kaibuchi, 2005; Wiggin et al., 2005; Sosa et al., 2006). It is therefore likely that Pak1 controls neuronal polarity, at least in part, as a Cdc42 effector. The fact that RacN17 can rescue the Pak1Caax induced defects in axon specification suggests that it lies downstream, rather than upstream of Pak1 during neuronal polarisation. DOCK7 was recently identified as the activator of Rac1 required for the inhibition of stathmin/Op18 and the consequent stabilisation of microtubules during the formation of an axon (Watabe-Uchida et al., 2006). Pak1 is a well known inhibitor of stathmin/Op18 and thus a likely regulator in polarising neurones (Fig. 10) (Wittmann et al., 2003). However, Watabe-Uchida et al. concluded that Pak1 is not required for the establishment of neuronal polarity as expression of its autoinhibitory domain (AID) did not rescue the effects of DOCK7 overexpression (Watabe-Uchida et al., 2006). Activation of Pak1 is required before the AID can bind and the Pak1 AID may also associate with Pak2 and Pak3, which can reduce its effectiveness in neurones. Consequently, we utilised the shRNA approach ensuring specific targeting of Pak1 mRNA, thus preventing the bulk of its protein synthesis. The fact that two Pak1 shRNA construct caused similar consequences, while a nonspecific shRNA was indistinguishable from an EGFP expressing control, further supports the effectiveness of our approach (Li et al., 2003). Our data suggest that Pak1 promotes the stage 2 to stage 3 transition in polarising neurones. Interestingly, the JNK kinases were recently identified as essential regulators of axon specification and neuronal transition from stage 2 to 3 and thus may be neuronal Pak1 effectors (Oliva et al., 2006).
In fibroblast and epithelial cells localised activation of cofilin can increase the number of free barbed ends and induce polarised F-actin polymerisation (Dawe et al., 2003; Ghosh et al., 2004). In rat cortical neurones cofilin activation has been associated with increased outgrowth of the longest neurite (Kuhn et al., 2000; Meberg and Bamburg, 2000). Interestingly, a constitutively active cofilin mutant was less effective at promoting neurite outgrowth than its wild type form (Meberg and Bamburg, 2000). Furthermore, phosphorylation and thus inactivation of cofilin was recently shown to accompany VEGF induced neurite outgrowth in rat cortical neurones (Jin et al., 2006). Together these findings suggest that the biological consequences of cofilin activity can vary depending on the extracellular signalling factors. They also indicate that regulated cofilin function is important during neuronal differentiation. We propose that Pak1 is responsible for controlling cofilin activity during the transition of a neurone from stage 2 to stage 3. Overexpression of Pak1Caax in a stage 2 neurone therefore results in uniform and constant inactivation of endogenous cofilin. The consequential effects on the F-actin cytoskeleton can be rescued by co-expression of a cofilin mutant that is not subject to Pak1Caax regulation. The restored asymmetry in the F-actin cytoskeleton facilitates axon formation. The effects of Pak1Caax were also rescued by jasplakinolide. In vitro jasplakinolide induces F-actin polymerisation, however in living cells it can also disrupt regulated actin filament elongation by inducing spontaneous aggregates of G-actin (Bubb et al., 2000). Long term exposure of neurones to low doses of jasplakinolide may protect from the effects of Pak1Caax overexpression by changing the rate of F-actin turnover and its availability for regulation by Pak1.
Together our data suggest that localised activation of Pak1 is required to generate asymmetric consequences on the F-actin cytoskeleton of a stage 2 neurone. This causes changes in microtubule organisation which is required for the establishment and outgrowth of an axon and dendrites (Fig. 10). We also reveal that Pak1 is essential for neuronal viability.
Catalytically active Pak1 (Pak1Caax) and inactive mutant (Pak1R299Caax) were subcloned into pCAG-IRES-EGFP vector using ClaI and BamHI. EGFP, GST-RacN17, GST-Cdc42N17, myc-Pak1T423E, pSuper Pak1 shRNA, cofilin and its constitutively active mutant (cofilinS3A) were described previously (Nikolic et al., 1998; Kawauchi et al., 2003, 2006).
To generate shRNA expression vectors an oligonucleotide targeting the coding region of Pak1 (sh1412: 5′-TTTGAGCCTTGTACCTCATTGCTTCAAGAGAGCAATGAGGTACAAGGCTCTTTTT-3′) or control sequence (control shRNA: 5′-TTTGATGGATCGATATAGTGAGTTCAAGAGACTCACTATATCGATCCATCTTTTT-3′) and their complementary sequences were inserted into BbsI and XbaI restriction sites of the mU6pro vector. All DNA preparations were made using plasmid purifications kits from Qiagen.
The following antibodies were used in this study. Anti-Pak1 (N-20), anti-Pak2 (V-19) (Santa Cruz Biotechnology); anti-phospho Ser199/204 Pak1/Pak2, anti-Pak3 (Upstate Biotechnology); anti-cofilin (Cell Signalling); anti-phospho Ser3 Cofilin, anti-dephospho Tau (Tau-1), anti-actin (Chemicon); anti-βIII-tubulin (TUJ1) (Babco); anti-Map2 (AP20), anti-vinculin, anti-acetylated tubulin (Sigma-Aldrich); anti-tyrosinated tubulin (YL1/2) (Oxford Biotechnology); anti-SV2 (Developmental Studies Hybridoma Bank); anti-phospho Thr 1265 Map1b (superBUGS) (Trivedi et al., 2005), anti-GFP, anti-mouse Alexa 488 or 568, anti-rabbit Alexa 488, 568 or 633 (Molecular Probes). Jasplakinolide and cytochalasin D (Calbiochem) were applied at 5 nM and 1 μM concentrations, respectively. All other chemicals were obtained from Sigma and VWR.
Dissociated cortical or hippocampal l neurones from embryonic (E17-19) rat brains were prepared as described previously (Nikolic et al., 1998) and plated on poly-D-lysine (16 μg/ml) and laminin (5 μg/ml, Sigma) coated glass or tissue culture plates. Neurones were transfected using Amaxa’s rat neuron nucleofector kit (Amaxa Biosystems) according to the manufacturer’s protocol and cultured in Neurobasal media supplemented with factor B27, 2mM L-glutamine, 0.06 mg/ml cysteine, 1mM sodium pyruvate, penicillin and streptomycin (Invitrogen) at 37°C and 5% CO2.
Neurones were fixed in 4 % paraformaldehyde for 20mins, permeabilised in 0.2 % TritonX100, blocked in 0.2 % fish skin gelatine and immunostained as previously described (Nikolic et al., 1998). Images were captured using a Leica TCS SP/UV confocal microscope and processed using Leica software.
Cells were lysed in 25 mM Tris pH7.5, 150 mM NaCl, 5mM EDTA, 1 %Triton X-100 and 10 % Glycerol with Complete protease inhibitors (Roche), 1 mM PMSF, 10 mM NaF and 1 mM Na3VO4. Lysates were cleared by centrifugation at 20K for 10min. Membrane preparations were made as described previously (Nikolic et al., 1998). All lysates were separated by SDS PAGE, western blotted onto PVDF membranes and probed as previously described (Nikolic et al., 1998) using horse radish peroxidase conjugated secondary antibodies (Vector laboratories) followed by incubation with ECL (Amersham). Densitometric analysis of scanned films was used to compare the intensities of individual protein bands.
Cells were lysed in 25 mM Tris pH7.5, 150 mM NaCl, 5mM EDTA, 1 %Triton X-100 and 10 % Glycerol with Complete protease inhibitors (Roche), 1 mM PMSF, 10 mM NaF and 1 mM Na3VO4. Lysates were cleared by centrifugation at 20K for 10min. 100-200 μgs of lysate were immunoprecipitated with appropriate primary antibody and protein A-sepharose beads and subsequently washed multiple times in kinase buffer (50mM HEPES, pH7.5, 10mM MgCl2)(as described previously, (Nikolic et al., 1998; Rashid et al., 2001). Kinase assays were performed in the presence of 50μM ATP, 1μCi [32Pγ-ATP] (GE Healthcare), 2μg histone H4 (Roche), 1mM DTT and kinase buffer for 20mins at 30°C. The reactions were stopped with 2x sample buffer, resolved by SDS PAGE, dried and exposed to autoradiography.
Neurones were examined using a Nikon TE2000 microscope and Plan Fluor 20x/0.45 objective, images were captured with a Hamamatsu Orca camera and Openlabs software (Improvision). Neurite lengths were measured using Volocity 4.0 software (Improvision) with any length less than 10 μm discounted. The effects of Pak1Caax and EGFP on neuronal morphology after 1-3 days in culture were scored from over 120 neurones (n=3). Data for EGFP and Pak1Caax expressing neurones after 7 div were pooled from all experiments (n=14) and are therefore based on the scoring of over 1,000 neurones. For comparisons between outgrowth at 3 and 7 div data were collected from over 200 neurones (n=2). The effects of Pak1R299Caax, Pak1T423E and Cdc42N17 were analysed from over 200 neurones (n=2), the consequences of cofilinS3A and RacN17 were analysed in over 400 neurones (n=4) and the effects of jasplakinolide examined in 300 neurones (n=3). To determine the distribution of Pak1(P) images of over 100 neurones (n=3) were captured using constant exposure settings and analysed with Openlabs software. To measure the distribution of Map2 images of over 140 neurones (n=3) were captured using constant exposure setting and analysed with Openlabs software
We thank John Parnavelas and Sonja Rakić for the generous use of confocal microscopes; Phillip Gordon-Weeks for kindly providing anti-Map1b superBUGS antibody; Gary Bokoch and Jonathan Chernoff for pCMV6M-Pak1T423E, Ed Manser for phospho-Pak1 antibodies; Dan Wu for pSuper-Pak1 shRNA; David Turner for the mU6pro vector; Takeshi Kawauchi for technical advice; Junichi Miyazaki and Takeshi Kawauchi for pCAG-IRES-EGFP. FC is a recipient of a long term EMBO fellowship. The research was funded by a Wellcome Trust grant to MN.