1. Volume 51, Issue 6, Pages 727-739 (September 2006)
The Rac Activator DOCK7 Regulates Neuronal Polarity through Local Phosphorylation of Stathmin/Op18 
Mitsuko Watabe-Uchida, Keisha A. John, Justyna A. Janas, Sarah E. Newey, Linda Van Aelst 
Neuron 
Volume 51, Issue 6, Pages (September 2006)
DOI: /j.neuron
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 DOCK7 Associates with and Activates Rac
(A) YTH interactions between Rac and DOCK7-C. The L40 yeast strain was transformed with plasmids expressing DOCK7-C fused to the GAL4 activation domain (GAD), and the indicated Rac mutants, or Lamin control, fused to the LexA DNA-binding domain (LBD) and tested for growth on medium lacking histidine.
(B) Schematic representations of DOCK7 and DOCK7-C.
(C) Selective binding of DOCK7 to nucleotide-free Rac, but not Cdc42 or RhoA. GTPγS-loaded (GTP), GDPβS-loaded (GDP), or nucleotide-free (−) Rac1, Rac3, Cdc42, and RhoA-GST fusion proteins were incubated with extracts from N1E-115 cells. Bound DOCK7 was detected by immunoblotting with anti-DOCK7 antibody (Ab). Input lane shows 1/15 of the amount of extract used for the assay. Purified GST-Rac1, Rac3, Cdc42, or RhoA fusion proteins were detected by Coomassie blue staining.
(D) DOCK7 associates with Rac in mammalian cells. HEK293 cells were transfected with the indicated plasmids. Cell extracts containing 5 mM EDTA were immunoprecipitated (IP) with anti-Flag antibody and analyzed by Western blotting (WB) with anti-Flag and anti-Myc antibodies.
(E and F) Activation of Rac by DOCK7 measured with (E) PBD pull-down assays and (F) GDP release assays. (E) HEK293 cells were transfected with the indicated plasmids, and GTP-bound Rac1 was precipitated from detergent extracts with GST-PBD. GST-PBD-bound Myc-Rac1-GTP and total Myc-Rac1 in cell lysates were detected by immunoblotting with anti-Myc antibody and quantified by direct imaging of chemiluminescent signals. DOCK180 wt was included as a positive control. The relative amount of Myc-Rac1-GTP in extracts (compared to vector control) is indicated in the bottom panel. (F) [3H] GDP-loaded Rac1, Cdc42, and RhoA were incubated with extracts from HEK293T cells expressing DOCK7-C, DOCK180-DHR-2, or empty vector. At the indicated time points, the amount of [3H] GDP bound to the GTPases was measured using a filter-binding assay, and the results were presented relative to the 0 time point, which was defined as 1. Values are means of triplicate samples ± SD. Similar results were obtained in two other experiments.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2 DOCK7 Is Highly Expressed in the Developing Brain
(A) Multiple-tissue Western blot from a postnatal day 5 (P5) rat probed with anti-DOCK7 antibody. B, brain; SM, skeletal muscle; H, heart; Li, liver; Lu, lung; K, kidney; S, spleen.
(B) DOCK7 immunoblot of P5 rat brain regions. Ce, cerebellum; Hi, hippocampus; Th, thalamus; Co, sensory cortex; Fl, frontal lobes; Olf, olfactory bulb.
(C) DOCK7 immunoblot of embryonic day 18 (E18), P2, P6, P10, P30, and adult (>8 weeks old) rat brains.
(D–F) Immunoblots of rat hippocampal neurons cultured in vitro for 6 hr, 1 day, 2 days, 3 days, 7 days, and 21 days probed with anti-DOCK7 antibody (D), anti-DOCK180 antibody (E), or anti-oligophrenin-1 as a loading control (F).
(G) Parasagittal brain section through the hippocampus from a P2 rat was double-immunolabeled with anti-DOCK7 antibody (green) and an antibody against NeuN, which predominantly labels nuclei (red). Scale bar, 100 μm. Lower panel shows an enlarged view of the boxed area in the upper panel. Scale bar, 20 μm.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3 Polarized Distribution of DOCK7 in Hippocampal Neurons
(A) Neurons at stages 1, 2, and 3 of development were costained with anti-DOCK7 (green) and anti-α-tubulin, phalloidin, anti-Tau-1, or anti-MAP2 (red) antibodies. Arrowheads indicate enrichment of DOCK7 in a single neurite of unpolarized stage 2 neurons. Scale bar, 20 μm.
(B and C) High levels of DOCK7 expression correlate with localized reduction of F-actin staining in a single neurite of a stage 2 neuron. (B) Left panel shows a stage 2 neuron (from panel [A]) double-immunostained with anti-DOCK7 antibody (green) and phalloidin (red). The right panels show the profiles of DOCK7 and actin fluorescence intensities in each of the numbered processes of the depicted stage 2 neuron (from the proximal to the distal end of the processes). The data shown is representative of more than 40 stage 2 neurons. (C) DOCK7 and actin fluorescence intensities at a region of interest (ROI) at the distal end of each neurite shaft and at the tip of each neurite, respectively, were quantitated for 42 neurons. The mean fluorescence intensity of DOCK7 in neurites with lowest actin staining (left), and of actin in neurites with highest DOCK7 staining (right), versus all other neurites is shown (∗p < 0.005, t test). AU, arbitrary unit. Error bars indicate SEM.
(D) Distribution of DOCK7, MTs, and F-actin in the growth cone of stage 3 hippocampal neurons, visualized with antibodies specific for DOCK7, α-tubulin, and by phalloidin staining, respectively. Scale bar, 10 μm.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4
Ectopic Expression of DOCK7 Induces the Formation of Multiple Axons through Rac Activation
(A) Neurons coexpressing GFP and T7-DOCK7, or a control vector, were plated on laminin-coated coverslips and stained 45–52 hr later with anti-Tau-1 (blue) and anti-MAP2 (red) antibodies. Scale bar, 20 μm.
(B) Length of axons/neurites from DOCK7 and vector control expressing neurons after culturing them for 45–52 hr (n = 30 for each condition). The x axis indicates rank order of the processes by length, from long to short. Error bars indicate SD.
(C) Percentage of polarized neurons is shown. DOCK7 overexpression induces polarity defects (∗p < 0.005, χ2 test; n = 140 neurons for control vector and 139 for DOCK7).
(D) Quantification of the number of processes per cell. DOCK7 does not alter the total number of processes formed per cell (p = 0.48, t test; n = 140 neurons for control vector and 139 for DOCK7).
(E) Representative images of neurons cotransfected with vector expressing GFP and empty control vector, T7-DOCK7, Myc-Rac3N17, or a combination of T7-DOCK7 and Myc-Rac3N17 (left), and GFP and T7-DOCK7ΔDHR-2 (right). Overlay of GFP (green) and Tau-1 (red) staining is shown. Rac3N17 prevents the induction of multiple axons triggered by ectopic expression of DOCK7 (left panel). DOCK7ΔDHR-2 fails to induce multiple axons; either one or no axon is formed (right panel). GFP-positive cells were also tested for Myc- and/or T7-tag immunoreactivity (data not shown). Scale bar, 20 μm.
(F) Percentage of neurons with more that one axon is shown. Only cells expressing T7-DOCK7 have multiple axons (∗p < 0.005, χ2 test; n = 50 neurons for each condition).
(G) Quantification of the number of processes per cell. Expression of T7-DOCK7, T7-DOCK7ΔDHR-2, or Myc-Rac3N17 does not alter the total number of processes per cell (p > 0.5, t test, n = 50 neurons for each condition). Similar results were obtained for Rac1N17 (data not shown).
Error bars in (C), (D), (F), and (G) indicate SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 DOCK7 Knockdown Suppresses Axon Formation
(A) Neurons were cotransfected with plasmids expressing GFP and Dock7#2 hairpin (hp), or a scrambled (control) hp. GFP (green) and Tau-1 (red) stainings are shown. Scale bar, 20 μm.
(B) DOCK7 expression levels in neurons transfected with empty vector or the indicated hp RNA constructs were detected with anti-DOCK7 antibody. Erk2 was used as a loading control.
(C) Percentage of polarized neurons is shown. Dock7#1 hp and Dock7#2 hp, but not the control hp, perturb neuronal polarity (∗p < for both Dock7 hp constructs compared to vector, whereas p = 0.27 for control hp compared to vector, χ2 test; n = 133 neurons for vector, 153 for control hp, 183 for Dock7#1 hp, and 123 for Dock7#2 hp).
(D and E) Quantification of the number of processes per cell (D) and the total length of minor neurites per cell (E). DOCK7 knockdown does not affect the length of minor neurites or the total number of processes (p > 0.4 for all hp constructs when compared to empty vector, t test; n = 133 neurons for vector, 152 for control hp, 183 for Dock7#1 hp, 123 for Dock7#2 hp).
(F and G) DOCK7 expression rescues the loss of axon phenotype in Dock7#2 hp-expressing neurons. (F) Neurons were cotransfected with combinations of vectors expressing GFP, control hp, Dock7#2 hp, and T7-DOCK7. Overlay of GFP (green) and Tau-1 (red) staining is shown. Scale bar, 20 μm. (G) Percentage of polarized neurons is shown. The percentage of neurons with normal polarity is restored upon coexpression of DOCK7 with Dock7#2 hp (plasmid concentration: 2 to 1) (∗p < between control hp + vector and Dock7#2 hp + vector, whereas p = 0.3 between control hp + vector and Dock7#2 hp + DOCK7, χ2 test; n = 55 neurons for control hp, 64 for Dock7#2 hp, and 70 for Dock7#2 hp + DOCK7).
Error bars indicate SEM for (C)–(E) and (G).
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6 DOCK7 Mediates Laminin-Induced Op18-S16 Phosphorylation
(A) Detergent extracts from hippocampal neurons cultured for 45–60 hr in dishes coated with or without laminin were probed with anti-Op18-S16-P antibody and anti-Op18 Ab.
(B and C) DOCK7 knockdown reduces Op18-S16 phosphorylation. (B) Neurons plated on laminin were transduced with pTRIP lentiviral vectors expressing Dock7#1, Dock7#2, control, or no (vector) hp RNA. Detergent extracts were probed with anti-DOCK7, anti-Op18-S16-P, anti-Op18, and anti-Erk2 Abs. (C) Fold decrease in Op18-S16-P levels. The data were normalized to total Op18 levels and then to a value of 1.0 for control hp (∗p < 0.03 for Dock7#1 or Dock7#2 hp compared to control hp, t test; n = 3).
(D and E) DOCK7 overexpression increases Op18-S16 phosphorylation. (D) Neurons plated on laminin were infected with recombinant adenoviruses expressing GFP (vector), T7-DOCK7 or T7-DOCK7ΔDHR-2. Blots were probed with anti-Op18-S16-P, anti-Op18, anti-β-tubulin, and anti-T7 antibodies. (E) Fold increase in Op18-S16-P levels. The data were normalized to total Op18 levels and then to a value of 1.0 for the control virus (∗p < 0.03 for DOCK7 compared to control, t test; n = 3).
Error bars indicate SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7 Op18-S16 Phosphorylation Is Important for Axon Formation
(A) Higher levels of Op18-S16-P are present in axons than in future dendrites. Stage 3 neurons plated on laminin were incubated with Cell Tracker Green CMFDA (lower panel) and then immunostained with anti-Op18-S16-P antibody or Op18 antibody. Scale bar, 20 μm. The histograms show the profiles of Op18-S16-P (left) and Op18 (right) immunofluorescence intensities in each of the numbered processes of the depicted stage 3 neurons (from the proximal to the distal end of the process) by ratio imaging against the cytoplasmic volume. The data shown are representative of more than 40 stage 2 neurons.
(B) Percentage of polarized neurons ectopically expressing wt Op18, Op18-S16A mutant, or a control vector. Op18-S16A induces more severe polarity defects than wt Op18 (∗p < between Op18-S16A and vector, whereas p = 0.1 between wt Op18 and vector, χ2 test; n = 115 neurons for control vector, 112 for Op18-S16A, and 107 for wt Op18).
(C) Op18-S16A inhibits the formation of multiple axons induced upon DOCK7 expression. Percentage of neurons with more that one axon is shown. (∗p < 0.001, χ2 test; n = 55 neurons for each condition).
Error bars in (B) and (C) indicate SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions