Localized activation of p21-activated kinase controls neuronal polarity and morphology - PubMed (original) (raw)

Figure 1.

Figure 1.

Polarized activation of Pak1. A, B, Cortical neurons were cultured for increasing lengths of time and examined for total and phosphorylated Pak1 by Western blot. A, The levels of Pak1(P) were highest during early stages of neuronal differentiation, paralleling the timing of axon specification and enhanced outgrowth. B, Pak1(P) was most evident in membranes between 1 and 4 d in vitro. Membrane enrichment was confirmed with SV2. C, Pak1 was uniformly distributed in all neurites of stage 2 (ST2) and 3 (ST3) hippocampal neurons. Pak1(P) showed overlapping distribution with total Pak1 in stage 2; however, at stage 3 it was exclusive to the longest neurite. A proportion of stage 2 neurons (16%) increased Pak1(P) in one neurite, suggesting that they were at the boundary between stages 2 and 3. The identity of all neurons was confirmed using the neuronal specific marker β-III tubulin (TUJ1), whereas 4′,6′-diamidino-2-phenylindole (DAPI) staining revealed the nuclei. Scale bars, 25 μm.

Figure 2.

Figure 2.

The developmental pattern of Pak1 expression and activation. A–L, Immunodetection of Pak1 (A, C, E, G, I, K) and Pak1 phosphorylated on S199/204 (B, D, F, H, J, L) in the developing cortex (A–F) and hippocampus (G–L); 4′,6′-diamidino-2-phenylindole (DAPI; blue) revealed nuclei of all cells. A, A′, In E14 mouse, cerebral cortex Pak1 is highly enriched in the axons of the IZ and is also evident in the forming CP. The VZ has nearly undetectable expression of Pak1. B, B′, At E14, phosphorylated Pak1 is enriched in subcellular clusters most evident in the IZ and CP. C–D′, The pattern of Pak1 expression and phosphorylation at E16 is similar to E14 with increased accumulation of Pak1(P) in the IZ. Shown are higher magnifications, showing accumulation of Pak1 and Pak1(P) in axonal fibers of the IZ (arrows) and neurons of the CP. E, E′, At P0, increased levels of Pak1 are evident in the CP and IZ but remain low in the VZ. F, F′, Pak1(P) is downregulated at birth, when highest levels are seen in the CP. G–H′, The distribution of Pak1 and Pak1(P) in the E14 hippocampus is very similar to that seen in the cortex. Highest levels of Pak1 are evident in the IZ and HP, where Pak1(P) is seen in clusters. Low levels of both characterize the VZ. I–J′, At E16, the pattern of Pak1 and Pak1(P) resemble that seen at E14 with highest levels in the IZ, HP, and marginal zone (MZ). K, L, Pak1 remains high at birth in the IZ, HP, and MZ, whereas Pak1(P) is downregulated, with highest levels evident in the MZ and a relatively uniform distribution in the IZ and HP. Scale bars: B′ (for A–B′), D′ (for C–D′), F′ (for E–F′), H′ (for G–H′), L′ (for K–L′), 45 μm; I, J, 30 μm.

Figure 3.

Figure 3.

Overexpression of catalytically active Pak1 alters neuronal morphology. A, Neurons expressing Pak1Caax after 3 div had more lamellipodia than the EGFP-expressing controls. Constitutive activation and membrane localization of Pak1Caax was confirmed using the anti-S199/204 Pak1(P) antibody. Scale bars, 25 μm. B, Morphological comparison of EGFP- and Pak1Caax-expressing neurons. Note that Pak1Caax-positive neurons did not have a distinguishable longest neurite after 1 or 3 div. Scale bar, 50 μm. C, Total neurite outgrowth was normalized to the EGFP 1 div control. No significant differences were evident between expression of EGFP and Pak1Caax, whereas a 33% increased outgrowth was evident in Pak1R299Caax-positive neurons only at 1 div. D, The longest neurite was measured revealing a significant reduction in length after Pak1Caax expression. Pak1R299Caax enhanced outgrowth by 25% only at 1 div. E, Remaining neurites were significantly longer in Pak1Caax neurons when compared with EGFP controls at 1 and 3 div. Pak1R229Caax enhanced outgrowth only at 1 div. Error bars depict SEM. § p < 0.01 and *p < 0.001 using Student's t test.

Figure 4.

Figure 4.

Membrane localization of catalytically active Pak1 induces multiple axons. A, The subcellular distribution of S199/204 and total Pak1 were compared in polarized hippocampal neurons after 7 div. Known markers confirmed the identity of axons (Tau-1) and dendrites (Map2). Pak1(P) was detected only in axons despite uniform presence of Pak1 in axons and dendrites. Nuclei were visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining. B, C, After 7 div, expression of Pak1Caax alters the distribution of Tau-1 and Map2. In contrast, neurons expressing Pak1R299, EGFP, or Pak1T423E had segregated Tau-1 and Map2 to an axon and dendrites, respectively. Increased F-actin-rich lamellipodia were seen after Pak1T423E expression. D, Pak1Caax-expressing neurons elaborated multiple axons in 60.8 ± 1.5% of cases in contrast to Pak1R299Caax (28.2 ± 0.2%), EGFP (21.7 ± 1.8%), and Pak1T423E (16.6 ± 2%). Scale bars, 50 μm. Error bars represent SEM. *p < 0.001 using Student's t test.

Figure 5.

Figure 5.

Pak1 hyperactivation affects the distribution of microtubule-associated proteins. A, Pak1Caax-expressing neurons have a broader distribution of Map2 at 3 and 7 div than EGFP controls. Arrows mark the extent of Map2 presence in individual neurites. B, The average (av.) percentage of a cell area that contains Map2 is greater after Pak1Caax expression than in EGFP controls. C, Two representative examples show Pak1Caax-induced absence of Map1b(P) or widespread distribution of Map1b(P) in all neurites. D, Neurons were scored for the absence of Map1b(P) or its presence in one or multiple neurites. Scale bars, 50 μm. Error bars represent SEM. *p < 0.001 using Student's t test.

Figure 6.

Figure 6.

Loss of Pak1 expression affects the neuronal cytoskeleton. A, The levels of Pak1 in transfected Cos7 cells were reduced after coexpression with Pak1 shRNA but remained unchanged in the presence of a control shRNA. B, Cortical cultures transfected with empty vector (control) or Pak1 shRNA were examined by Western blot after 3 div, revealing a reduction in Pak1 protein levels. C, After 3 div, hippocampal neurons expressing Pak1 shRNA displayed extensive somal lamellipodia rich in F-actin. In some cases, no neurites were evident. This phenotype was not observed after expression of control shRNA. D, At 3 div, expression of Pak1 shRNA caused the presence of many looped microtubules, particularly in the somal lamellipodia. Many of the microtubules were stable as judged by the presence of acetylated tubulin (acT). An antibody to tyrosinated tubulin (Ytub) revealed the presence of newly formed microtubules in both control and Pak1 shRNA-expressing neurons. Scale bars, 50 μm.

Figure 7.

Figure 7.

Inhibition of Pak1 affects neuronal polarization. After 3 div, neurons expressing Pak1 shRNA revealed a widespread distribution of Tau-1 and Map2 in contrast to EGFP or control shRNA-transfected neurons, in which the markers segregated to axons and dendrites, respectively. Scale bar, 50 μm.

Figure 8.

Figure 8.

Pak1 induces axon formation by affecting F-actin organization. A, Hippocampal neurons were exposed to low doses (1 μ

m

) of CD or control DMSO and maintained for 7 div. CD caused the formation of multiple Tau-1-positive axons, all of which were also enriched with Pak1(P). B, Hippocampal neurons transfected with EGFP or Pak1Caax were exposed to 5 n

m

jasplakinolide (JP) or control DMSO after attachment to the substrate and cultured for 7 d. Neurons with multiple Tau-1-positive axons were common in Pak1Caax-expressing cultures but rarer after treatment with jasplakinolide or in EGFP-expressing controls. Scale bars, 50 μm. C, Quantification of the effects of jasplakinolide (JSP). Error bars represent SEM. *p < 0.001 using Student's t test. DAPI, 4′,6′-Diamidino-2-phenylindole; NS, not significant.

Figure 9.

Figure 9.

Pak1 induces multiple axons through Rac1 activation and cofilin inhibition. A, The consequences of coexpression of RacN17, Cdc42N17, or cofilinS3A mutants with Pak1Caax were examined in hippocampal neurons at 7 div. Both RacN17 and cofilinS3A increased the incidence of neurons elaborating a single axon, which was not observed after Cdc42N17 expression. Scale bar, 50 μm. B, Cortical neurons expressing EGFP or Pak1Caax were cultured for 7 d and compared by Western blotting. An approximately threefold increase in S3 phosphorylated cofilin was consistently observed in Pak1Caax-expressing neurons, whereas total cofilin levels remained unchanged. The bottom shows the presence of S199/204 phosphorylated Pak1Caax expression and endogenous Pak1, confirming increased catalytic activity. C, Quantification of RacN17 or cofilinS3A (CofS3A) effects. Scale bar, 50 μm. Error bars represent SEM. *p < 0.001 using Student's t test.

Figure 10.

Figure 10.

Model illustrating the role of Pak1 during neuronal polarization. Based on published literature, upstream signals that are most likely activators of Pak1 in polarizing neurons include PI3K. PI3K can activate Cdc42 via other GTPases, including Rap1b, all of which have been shown to affect axonal formation (Arimura and Kaibuchi, 2007). Cdc42 is an established direct activator of Pak1, which activates LIMK-1 and thus inhibits cofilin (Bokoch, 2003). Pak1 can also regulate Rac1 activation and directly inhibit the microtubule-severing protein Op18/stathmin (Obermeier et al., 1998; Wittmann et al., 2003; ten Klooster et al., 2006). It remains a question whether Pak1 affects microtubule organization in polarizing neurons by altering the function of Op18/stathmin directly (depicted by dotted arrow) or via Rac1 and its currently unidentified downstream targets. The correct specification of an axon and subsequent outgrowth of axons and dendrites require regulated and dynamic turnover of F-actin and microtubules, which are facilitated at least in part by Pak1 and its downstream targets.