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First published online 12 August 2008
doi: 10.1242/jcs.030353

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P-Rex1 – a multidomain protein that regulates neurite differentiation

Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Victoria, Australia

View larger version (60K): [in this window] [in a new window]   Fig. 1. P-Rex1 localises to the shaft and distal tip of PC12 neurite growth cones. (A) Domain structure of P-Rex1 showing the Dbl homology (DH), pleckstrin homology (PH), Dishevelled, EGL-10, pleckstrin homology (DEP), post-synaptic density, disc-large, ZO-1 homology (PDZ), PEST sequences (P) and 4-phosphatase homology (4ptase) domains. The peptide sequence used as an immunogen to generate the P-Rex1-specific antibody is indicated. (B) Cell lysates from mouse brain, rat E18 hippocampal neurons and undifferentiated PC12 cells (30 µg) were immunoblotted with affinity-purified P-Rex1 antibodies. Immunoreactive endogenous P-Rex1 is indicated by the arrow. (C) PC12 cells were left undifferentiated, or NGF-stimulated as indicated, then immunostained with P-Rex1-specific antibodies (green) and Texas Red-conjugated phalloidin (red). Merged images are shown in the third column and enlarged images of the boxed areas of growth cones of immature (1) or mature (2) neurites are shown in the bottom row. The growth cone peripheral (P) and central (C) zones are indicated in the bottom row. P-Rex1 localisation at the distal tip of filopodia and lamellipodia is indicated by arrows and at growth cones by arrowheads. Undifferentiated PC12 cells were also stained with P-Rex1 peptide-adsorbed antibody as control (right-most panel). Scale bars: 10 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 2. P-Rex1 distribution during hippocampal differentiation. (A) One day in vitro (d.i.v.) embryonic rat hippocampal neurons were immunostained with P-Rex1-specific antibodies (green), Rac antibodies (red), Texas Red-conjugated phalloidin (red) or β-tubulin antibodies (red) as indicated. Higher-magnification images of the boxed area outlining the growth cones are shown in the fourth column. Arrows indicate endogenous P-Rex1, Rac or F-actin at tips of growth cones. Co-localisation in merged images appears yellow. (B,C) Three (B) and 4-7 (C) d.i.v. embryonic rat hippocampal neurons were co-stained with P-Rex1-specific antibodies (green), Texas Red-conjugated phalloidin (red), β-tubulin (red), Tau1 (red) or MAP2 (red) antibodies. The relative intensity of P-Rex1 antibody staining is shown in the left-hand column by `glowover' images, in which blue indicates high-intensity staining (see scale beneath B). Merged images in the right-hand column demonstrate co-localisation by yellow staining. Open arrows indicate primary neurites. Arrowhead indicates P-Rex1 co-localisation with β-tubulin. White arrows in C indicate Tau1-positive or MAP2-negative processes. Scale bars: 10 µm in A; 20 µm in B,C.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Ectopic expression of P-Rex1 inhibits NGF-mediated neurite differentiation and is dependent on both the Rac-GEF activity and the 4-phosphatase domain. (A) Schematic of P-Rex1 deletion mutant constructs. PC12 cells were transiently transfected with P-Rex1 mutant constructs and cell lysates were immunoblotted with HA or myc antibodies, as shown beneath. 1, HA-vector; 2, HA-P-Rex1; 3, HA-P-Rex1N; 4, HA-P-Rex14P; 5, myc-P-Rex1; 6, myc-P-Rex1GEFdead. To the right, neurite outgrowth, neurite F-actin and initiation are summarised for each P-Rex1 mutant protein. For neurite outgrowth: +++, as vector control; +, reduced neurite outgrowth; –, no outgrowth. For F-actin: +, as vector control; +++, increased. For initiation: +, initiation as vector control. (B,C) PC12 cells were transiently transfected with wild-type or mutant P-Rex1 (5 µg, unless otherwise indicated) and NGF-stimulated for 3 days in the presence or absence of 1 µM cytochalasin D. (B) Cells were co-stained with HA or myc antibodies (green) and Texas Red-conjugated phalloidin (red or grey). Merged images are shown (lower row) with neurites or actin-rich projections indicated by arrows. Scale bar: 10 µm. (C) Cells containing actin-rich projections were imaged and the length of the neurite/projection and the cell body diameter determined. Bars indicate the mean ± s.e.m. of cells bearing neurites longer than one cell body diameter. 100 cells were scored for each construct for three independent transfections. *P<0.05; **P<0.01; ***P<0.001.

View larger version (40K): [in this window] [in a new window]   Fig. 4. P-Rex1 co-localises with, and promotes the activation of, HA-Rac3 in NGF-stimulated PC12 cells. (A) PC12 cells were co-transfected with myc-Rac1 or HA-Rac3 and vector control, P-Rex1 or P-Rex1GEFdead, and left serum starved or stimulated with NGF for 3 minutes. Duplicate samples of cell lysates were subjected to colorimetric Rac1,2,3 G-LISA Activation Assays and the average Rac activation was calculated relative to serum-starved HA-vector-only controls. Equivalent expression of Rac constructs was confirmed by immunoblot analysis using a pan-Rac antibody (not shown). Bars indicate mean ± s.e.m. of relative Rac activation for four independent experiments (*P<0.05). (B) PC12 cells were transfected with HA-P-Rex1 (upper row) or co-transfected with HA-P-Rex1 and myc-Rac1 (middle row) or myc-P-Rex1 and HA-Rac3 (bottom row). Cells were stimulated with NGF for 3 minutes. P-Rex1 (green) and Myc-Rac1 or HA-Rac3 (red) were detected using antibodies to each tag. Co-localisation is indicated by yellow staining in the merged images (right-hand column). Arrowheads indicate P-Rex1/Rac1 membrane co-localisation, with P-Rex1/Rac3 perinuclear co-staining indicated by the arrow. Scale bars: 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Hippocampal neurons ectopically expressing P-Rex1 show enlarged growth cones with prominent F-actin accumulation. Embryonic rat hippocampal cells were transiently transfected at 7 d.i.v. with plasmids encoding HA-P-Rex1. At 9 d.i.v., cells were co-stained with HA antibodies (red or green) and either (A) Alexa Fluor 594-conjugated phalloidin (green) or (B) Tau1 (red) antibodies. (A) Representative images of differentiated neurons (upper row), with higher-magnification images of the boxed areas (1, 2) shown in the two lower rows; growth cones are outlined. The relative intensity of F-actin staining is shown on the left in glowover images, in which blue indicates high-intensity staining (see scale). Scale bars: 10 µm. (B) Co-localisation of HA-P-Rex1 and Tau1. In the merged image, axon is indicated by an arrowhead. Scale bar: 20 µm. (C) The fold increase in growth cone area in HA-P-Rex1 or HA-P-Rex1N relative to mock-transfected cells. Axonal and dendritic growth cones were identified by Tau1 and MAP2 staining, respectively. Thirty cells were scored for each construct for two independent transfections. Bars indicate the mean ± s.e.m. of the growth cone area; *P<0.05.

View larger version (62K): [in this window] [in a new window]   Fig. 6. Targeted depletion of P-Rex1 promotes spontaneous outgrowth of β-tubulin-rich projections (A) The location of the unique nucleotide sequence used to generate P-Rex1 RNAi clones is indicated in the P-Rex1 structure. (B) Cells were stably transfected with a plasmid encoding P-Rex1 RNAi sequence, or a scrambled RNAi control. Cell lysates (40 µg) were immunoblotted with P-Rex1 or actin antibodies. Relevant samples loaded on the same, representative immunoblot are shown. The relative P-Rex1 expression in P-Rex1 clones (1 and 5) was determined by densitometry of P-Rex1-immunoreactive polypeptides from five independent immunoblots, standardised to the actin loading control and expressed as a percentage of that detected in scrambled RNAi clones (3 and 4 combined). The bars indicate the mean ± s.e.m. of five independent experiments. (C) RT-PCR analysis of P-Rex1 RNAi clones. RT-PCR was performed on mRNA extracted from scrambled and P-Rex1 RNAi clones using Gapdh as a control. P-Rex1 expression was calculated and expressed relative to that from Scram(3), which was designated as one. Bars indicate the mean ± s.e.m. of three independent experiments. (D) Cells stably transfected with P-Rex1 RNAi or scrambled RNAi control were left unstimulated (a,b,c) or NGF-stimulated for 10 minutes (d). Cells were stained with Texas Red-conjugated phalloidin (red or grey) or β-tubulin antibody (green) to image cell morphology. Representative images of unstimulated P-Rex1 RNAi clones (1 and 5) and scrambled RNAi control (4) are shown (a,b) with merged magnified images of boxed regions (c). β-tubulin-containing projections are indicated (b, arrows). Merged images of cells treated with NGF for 10 minutes are shown (d) with areas of extensive peripheral F-actin indicated by arrowheads. Scale bars: 5 µm. (E) RNAi-mediated depletion of P-Rex1 in PC12 cells reduces Rac3 activation in response to NGF stimulation. Scrambled RNAi or P-Rex1 PC12 RNAi clones were transiently transfected with myc-Rac1 or HA-Rac3, serum starved and NGF-stimulated (3 minutes). Duplicate samples of cell lysates were subjected to colorimetric Rac1,2,3 G-LISA Activation Assay and the average Rac activation was calculated relative to serum-starved scrambled control cells. Equivalent expression of Rac constructs was confirmed by immunoblot analysis using a pan-Rac antibody (not shown). Bars indicate mean ± s.e.m. of relative Rac activation for four independent experiments. **P<0.005; ***P<0.001.

View larger version (50K): [in this window] [in a new window]   Fig. 7. P-Rex1 regulates NGF-stimulated neurite elongation. Cells stably transfected with P-Rex1 RNAi, or a scrambled control RNAi, were NGF-stimulated for 3 days and stained with Texas Red-conjugated phalloidin and β-tubulin antibodies to image neurite morphology. (A) Representative images of NGF-differentiated P-Rex1-depleted or control PC12 cells. Scale bars: 10 µm. (B) The numbers of differentiated PC12 cells with short neurites (length less than two cell body diameters) and long neurites (length greater than three cell body diameters) were expressed as a percentage of all differentiated neurites. Bars indicate mean ± s.e.m. of at least 100 cells scored for each of three independent differentiation experiments. (C) Neurite length was expressed as the fold increase (mean ± s.e.m.) of the length of the longest neurite for P-Rex1 clones 1 and 5 relative to that of the scrambled control (4). At least 60 neurites were scored for each of three independent differentiation experiments. (D) P-Rex1 RNAi clone (5) was transiently transfected with HA empty vector or with plasmids containing HA-P-Rex1 (1, 2 or 5 µg of DNA), myc-P-Rex1GEFdead (5 µg), HA-P-Rex1N (5 µg) or HA-P-Rex14P (5 µg) and NGF-differentiated for 3 days. Cells were stained with Texas Red-conjugated phalloidin and HA or myc antibodies. The number of cells bearing neurites longer than two cell body diameters was calculated and standardised relative to that of the P-Rex1 RNAi clone (5). Knockdown cells transfected with HA empty vector were scored as 100%. Bars indicate the mean ± s.e.m. for at least 50 cells scored per indicated construct for each of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

View larger version (52K): [in this window] [in a new window]   Fig. 8. P-Rex1-depleted neurites exhibit small growth cones with decreased F-actin. (A) Embryonic rat hippocampal cells (1 d.i.v.) were transiently transfected with plasmids encoding P-Rex1 RNAi or a control RNAi. Cells were co-transfected with a low concentration of pEGFP-C2 vector (ratio of 0.5 µg eGFP:1.5 µg RNAi plasmid) to allow for identification of RNAi P-Rex1-depleted neurons. At 3 d.i.v., cells were stained with Texas Red-conjugated phalloidin to visualise F-actin. Representative images are shown in the left column, with magnified images of the boxed growth cones (1-4) shown in the middle and right-hand columns. Scale bars: 10 µm. (B-D) The indicated scrambled RNAi and P-Rex1 PC12 RNAi clones were NGF-differentiated for 3 days, co-stained with Alexa Fluor 488-conjugated phalloidin to visualise F-actin and Alexa Fluor 594-conjugated DNaseI to visualise G-actin and analysed using confocal microscopy. (B) Representative images of growth cone F-actin. Lamellipodial veils are indicated by arrows. Scale bar: 5 µm. (C) The mean fluorescence intensity of F-actin relative to G-actin at the growth cone was determined. Bars indicate the mean ± s.e.m. of the F-actin:G-actin ratio for at least ten growth cones per RNAi clone for each of three independent differentiation experiments. (D) Growth clones were scored for the presence of lamellipodial veils by F-actin morphology. Bars indicate the mean ± s.e.m. for the percentage of cells containing growth cone lamellipodial veils for at least ten growth cones per RNAi clone for each of three independent differentiation experiments. *P<0.05.

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