QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 January 2007
doi: 10.1242/jcs.03356

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, S. Articles by Kaibuchi, K. Search for Related Content PubMed PubMed Citation Articles by Wang, S. Articles by Kaibuchi, K.

IQGAP3, a novel effector of Rac1 and Cdc42, regulates neurite outgrowth

Shujie Wang¹, Takashi Watanabe¹, Jun Noritake¹, Masaki Fukata^1,*, Takeshi Yoshimura¹, Norimichi Itoh¹, Takumi Harada¹, Masato Nakagawa^1,, Yoshiharu Matsuura², Nariko Arimura¹ and Kozo Kaibuchi^1,

¹ Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, 466-8550, Japan
² Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Japan

View larger version (35K): [in this window] [in a new window]   Fig. 1. IQGAP3 is a novel member of the IQGAP family. (A) Schematic representation of the IQGAP family. The domain structures and amino acid homologies are indicated. CHD, calponin homology domain; IQ repeats, IQGAP-specific repeats; IQ motif, a calmodulin binding motif; GRD, RasGAP-related domain; RasGAP C, RasGAP C terminus. (B) Total extracts from the indicated EGFP-fused IQGAP-expressing COS7 cells were subjected to immunoblot analysis with each anti-IQGAP antibody. Each antibody detected tagged IQGAP in a specific manner. Note that EGFP-IQGAP3 migrated further than IQGAP2. The results shown are representative of four independent experiments. (C) The expression of the IQGAP family in various rat tissues. Actin was monitored as a loading control. Equal amounts of total proteins were subjected to immunoblot analysis, except for IQGAP1 from lung and spleen, for which only half amount of was loaded because of its high level expression. The results represent three independent experiments.

View larger version (57K): [in this window] [in a new window]   Fig. 2. IQGAP3 is an effector for Rac1 and Cdc42. (A) IQGAP3-V5Hisx6 was mixed with the affinity beads coated with the indicated GST-fused small GTPases. Bound IQGAP3-V5Hisx6 was co-eluted with GST-fused proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE, followed by immunoblot analysis with anti-His antibody (top) and silver staining (bottom). The results shown are representative of three independent experiments. (B) COS7 cells expressing EGFP-IQGAP3 were lysed, and the supernatants were incubated with the beads coated with the indicated GST-fused small GTPases. The eluates were subjected to SDS-PAGE, followed by immunoblot analysis with anti-GFP antibody (top) and silver staining (bottom). The results shown are representative of three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3. IQGAP3 interacts directly with actin filaments. (A) Schematic representation of GST-IQGAP3. The structure of IQGAP3 and its various deletion mutants are represented. (B) GST-IQGAP3-WT or GST-IQGAP1-WT was mixed with F-actin, followed by incubation at room temperature for 1 hour. After the incubation, 50 µl of each reaction mixture was layered onto a 100-µl 20% (w/v) sucrose barrier and centrifuged. Two fifteenths of the supernatants or the pellets was subjected to SDS-PAGE, followed by immunoblot analysis with anti-GST antibody (top) and anti-actin antibody (bottom). One twentieth of the total proteins was shown as `input'. GST-IQGAP1-WT and GST-IQGAP3-WT were approximately 70% purity. The results shown are representative of three independent experiments. (C) The cosedimentation assay of the indicated IQGAP3 mutants with F-actin was performed as described in B. Two fifths of each pellet sample was subjected to SDS-PAGE. GST-RhoGDI was used as a negative control. The purity of GST-IQGAP3-CHD, -NR, and -N was 90%, 80% and 80%, respectively. The results shown are representative of three independent experiments.

View larger version (48K): [in this window] [in a new window]   Fig. 4. Localizations of the IQGAP family in PC12 cells. (A) Total extracts from the indicated siRNA-transfected PC12 cells were collected after the treatment with NGF for 48 hours, and were subjected to immunoblot analysis with the indicated antibodies. The expression levels of IQGAP1 and IQGAP3 were decreased specifically. The results shown are representative of five independent experiments. (B) PC12 cells were treated with NGF for 48 hours, followed by immunostaining with the indicated anti-IQGAP antibodies and Texas Red-phalloidin. The regions in white boxes are shown magnified in the far right panels. The results shown are representative of five independent experiments. Bar, 20 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 5. IQGAP3 is necessary for neurite outgrowth in PC12 cells. (A) EGFP-GST and indicated siRNA were cotransfected into PC12 cells. The cells treated with NGF for 48 hours were labeled with anti-GFP antibody and Texas Red-phalloidin. Arrowheads indicate the cotransfected cells that were identified by anti-GFP antibody. The transfection of siIQGAP3 impaired neurite outgrowth, but that of scramble or siIQGAP1 did not. The results represent five independent experiments. Bar, 20 µm. (B) PC12 cells cotransfected with indicated RNAiR-IQGAPs and siRNA were treated with NGF for 48 hours. The cells were labeled with anti-GFP antibody and Texas Red-phalloidin. Arrowheads indicate the cotransfected cells which were identified by anti-GFP antibody. RNAiR-IQGAP3 specifically rescued the impaired neurite outgrowth by siIQGAP3, but RNAiR-IQGAP1 did not. The results shown are representative of five independent experiments. Bar, 20 µm. (C) Schematic diagram of the quantification of the total length of neurites per cell. For each transfected PC12 cell, we determined the total length of neurites for which the lengths exceeded twice of the diameter of its cell body. See also Materials and Methods. l, length; d, diameter of the cell body. (D) Quantification of the total length of neurites. The total length of neurites was measured in the cells with or without NGF. Data represent the means ± s.d. of five independent experiments. n>150. Single and double asterisks indicate the difference in the value of each of the control cells at P<0.01.

View larger version (73K): [in this window] [in a new window]   Fig. 6. IQGAPs localize at the tips of axons in hippocampal neurons. (A) Localization of the IQGAP family in stage 3 hippocampal neurons. The cells were labeled with each anti-IQGAP antibody (red), anti-unique -tubulin (TUJ1) antibody (green), and Alexa Flour 647-phalloidin (blue). The regions in white boxes are shown magnified (right). The results shown are representative of five independent experiments. Bar, 20 µm. (B) Detailed localization of the IQGAP family in the growth cones. The cells on PDL alone were labeled as described in A. The results shown are representative of six independent experiments. Bar, 5 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 7. IQGAP3 plays important roles for axon outgrowth in hippocampal neurons. (A) The neurons were cotransfected with Myc-GST and siRNA, followed by double staining with anti-Myc (green) and Tau-1 (red) antibodies at DIV3. Arrowheads indicate the cotransfected cells that were identified by anti-Myc antibody. Axon outgrowth was impaired in the neurons transfected with either siIQGAP2 or siIQGAP3. The results shown are representative of seven independent experiments. Bar, 20 µm. (B) Quantification of the length of the longest primary axon. The axon length was measured in the cells cotransfected with Myc-GST and the indicated siRNA. Data represent the means ± s.d. of five independent experiments. n>150. Asterisks indicate a significant difference from the value of the control cells at P<0.01.

View larger version (47K): [in this window] [in a new window]   Fig. 8. The expressions of RNAiR-IQGAP2 and - IQGAP3 rescue axon outgrowth. (A) The neurons were cotransfected with the indicated siRNA and RNAiR-IQGAP3, followed by immunostaining with anti-Myc (green) and Tau-1 (red) antibodies at DIV3. Arrowheads indicate the cotransfected cells that were identified by the anti-Myc antibody. RNAiR-IQGAP3 rescued the impaired axon outgrowth created by siIQGAP2 or siIQGAP3. The results represent five independent experiments. Scale bar equals 20 µm. (B) Quantification of the length of the longest primary axon. The axon length was measured in the cells cotransfected with the indicated combinations. Data represent the means ± s.d. of five independent experiments. n>150. Asterisks indicate a significant difference from the value of the control cells at P<0.01.