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First published online 30 January 2007
doi: 10.1242/jcs.03376

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IQGAP1 regulates cell motility by linking growth factor signaling to actin assembly

¹ Departments of Biology, University of Virginia, Charlottesville, VA 22904, USA
² Cell Biology, University of Virginia, Charlottesville, VA 22904, USA
³ Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA

View larger version (87K): [in this window] [in a new window]   Fig. 1. IQGAP1 is required for FGF2-stimulated migration of MDBK cells. (A) MDBK cells were transfected with IQGAP1-specific siRNA or scrambled RNA (scrRNA) using a Nucleofector. To allow direct visual comparison of IQGAP1 levels in the two samples by western blotting, a concentration series of each cell extract was analyzed at the indicated relative dilutions. Note that siRNA reduced the IQGAP1 level to 20% of normal, but had no effect on cellular actin content. (B) Confluent monolayers were then serum-starved for 8 hours, wounded with a micropipette tip, and 2 hours later were stimulated with 25 ng/ml FGF2. Note that movement of IQGAP1-depleted cells into the wound, as seen after 7 hours of FGF2 exposure, was severely impaired, and that broad wounds persisted for more than 24 hours after wounding in both scrRNA-treated and IQGAP1-depleted cells that were not stimulated with FGF2.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Reduced lamellipodial dynamics in IQGAP1-depleted cells. Individual cells in sparse cultures that were serum-starved for 18 hours were imaged by phase contrast, time-lapse microscopy for 5 minutes before FGF2 was added to a final concentration of 25 ng/ml, and for 10 minutes thereafter. (A) Kymographic images (right panels) obtained from the indicated regions of interest (roi) at the margins of individual cells (left panels) treated with scrRNA or siRNA. Note how dynamic and motile the cell margin was in the control, scrRNA-treated cells compared to the cell depleted of IQGAP1 with siRNA. (B) Comparative responses of scrRNA-treated control (–) and IQGAP1 siRNA-treated (+) cells to FGF2 stimulation. The three parameters of lamellipodial dynamics that were measured before and after FGF2 stimulation were frequency, velocity and persistence of protrusion. The raw data were obtained from 180 regions of interest (roi) in 19 scrRNA-treated control cells, and from 180 roi in 21 siRNA-treated cells. Error bars indicate s.e.m. Differences between groups were analyzed using a one-way ANOVA test. Statistically significant differences at =0.001 are indicated by * for control versus siRNA-treated cells after FGF2 exposure (see supplementary material Table S1 for detailed statistics, including post-hoc comparisons). The net conclusion is that control cells, but not IQGAP1-deficient cells, respond to FGF2 by making more dynamic lamellipodia.

View larger version (57K): [in this window] [in a new window]   Fig. 3. FGF2 stimulates recruitment of IQGAP1, N-WASP, Arp2/3 complex and FGFR1 to lamellipodia. (A) Low-density cultures of serum-starved MDBK cells were stimulated with FGF2, and lysed at various times thereafter. At each indicated time point, IQGAP1 was immunoprecipitated out of the lysates with monoclonal anti-IQGAP1, and the immunoprecipitates were analyzed by immunoblotting with antibodies to IQGAP1 (polyclonal), N-WASP, Arp3 and FGFR1. As a control for non-specific immunoprecipitation, the tau-1 monoclonal antibody to tau, which is not expressed in MDBK cells, was substituted for monoclonal anti-IQGAP1, 10 minutes after FGF2 stimulation. Note that the IQGAP1 immunoprecipitates contained time-dependent increases in the levels of coimmunoprecipitated N-WASP, Arp3 and FGFR1, and that none of the proteins assayed by western blotting were immunoprecipitated by anti-tau. (B) Glutathione-Sepharose 4B beads were loaded with a fusion protein of the FGFR1 cytoplasmic tail coupled to GST, or to GST alone, and the beads were then mixed with IQGAP1, N-WASP, or both, and finally immunoblotting was used to detect any IQGAP1 or N-WASP that may have bound to the beads. Immunoblotting demonstrated direct binding of IQGAP1 and indirect, IQGAP1-dependent association of N-WASP with GST-FGFR1 tail, but not with GST.

View larger version (64K): [in this window] [in a new window]   Fig. 4. IQGAP1-dependent recruitment of N-WASP, Arp3 and FGFR1 to the cell periphery after FGF2 stimulation. Immunofluorescence microscopy revealed co-recruitment and colocalization of IQGAP1, N-WASP, Arp3 and FGFR1 to lamellipodia after FGF2 stimulation for 10 minutes, of low-density cultures of serum-starved MDBK cells containing IQGAP1 (Control cells). FGF2 was unable to recruit N-WASP, Arp3 and FGFR1 to cell margins in IQGAP1-deficient, siRNA-treated cells. To improve visualization of cell margins and intracellular details in IQGAP1-depleted cells, micrograph exposures for the siRNA samples were twice as long in the TRITC channel and 4.5 times longer in the FITC channel as they were for the scrRNA samples.

View larger version (56K): [in this window] [in a new window]   Fig. 5. N-WASP is required for FGF2-stimulated migration of MDBK cells. (A) MDBK cells were transfected with N-WASP-specific siRNA or scrambled RNA (scrRNA) using a Nucleofector. To allow direct visual comparison of N-WASP levels in the two samples by western blotting, a concentration series of each cell extract was analyzed at the indicated relative dilutions. Note that siRNA reduced the N-WASP level to 1/3 of normal, but had no effect on cellular IQGAP1 content. (B) Confluent monolayers were then serum-starved for 8 hours, wounded with a micropipette tip, and 2 hours later were stimulated with 25 ng/ml FGF2. Note that movement of N-WASP-depleted cells into the wound after 9 or 24 hours of FGF2 exposure was severely impaired. (C) Percentage of wound closure was quantified by measuring the average width of eight randomly chosen regions of interest of each wound at 0, 9, and 24 hours after FGF2 addition. Data are expressed relative to the average wound widths at 0 hours. Error bars indicate standard deviations for three experiments, and asterisks (*), indicate significant differences between scrRNA controls and corresponding siRNA-treated cultures at <0.001.

View larger version (53K): [in this window] [in a new window]   Fig. 6. Stimulation of branched actin filament assembly by IQGAP1. (A) Effects of IQGAP1 on actin (5% labeled with pyrene) assembly in the presence of N-WASP and the Arp2/3 complex were monitored using a pyrene-actin fluorescence assay. Note that maximal stimulation of assembly was achieved at 30 nM IQGAP1, and that higher and lower concentrations stimulated less or not at all. (B) Actin assembly stimulation by IQGAP1 requires N-WASP plus Arp2/3 complex. (C) IQGAP1-stimulated assembly of branched actin filament networks observed directly by TIRF microscopy. (D) Total lengths of actin filaments observed by TIRF microscopy were measured as a function of time, for samples containing or lacking IQGAP1 or activated Cdc42, a previously described for the N-WASP activator (Rohatgi et al., 1999). Maximum rates of actin assembly were achieved in the IQGAP1 sample. (E) The same micrographs were used to determine the number of filament branch points per µm of actin filament as a function of time. Note that the IQGAP1 and Cdc42 samples quickly attained a filament branch density four- to fivefold greater than the control sample that contained neither IQGAP1 nor Cdc42.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Co-stimulation of actin assembly by IQGAP1 plus activated Cdc42, and inhibition of VCA-stimulated actin assembly by IQGAP1. (A) When used together, IQGAP1 and activated Cdc42 stimulated actin assembly rates additively, and virtually eliminated the lag period before peak assembly rates were reached using either IQGAP1 or activated Cdc42 alone. (B) In the presence of a GST-tagged, constitutively active, N-WASP VCA fragment, IQGAP1, but not IQGAP1NT, caused dose-dependent inhibition of assembly.

View larger version (41K): [in this window] [in a new window]   Fig. 8. IQGAP1 fragments stimulate actin assembly and bind N-WASP. (A) A schematic representation of functional domains present in recombinant full-length IQGAP1 and four fragments used for experiments documented here: CHD, F-actin-binding calponin homology domain; IR, IQGAP repeats that mediate homodimerization; WW, ERK2-binding WW domain; IQ, calmodulin-binding IQ motifs; GRD, GAP (GTPase activating protein)-related domain involved in binding activated Cdc42 and Rac1. All proteins were his-tagged at their N termini, and the diagrams indicate whether each protein is a monomer or homodimer in aqueous solution. (B) The pyrene actin assembly assay was used to evaluate each protein at several concentrations in the presence of 1.3 µM actin (5% pyrene-labeled), 50 nM N-WASP and 50 nM Arp2/3 complex. Shown here are the maximum velocities (Vmax) of actin assembly (upper panels) and lag times before Vmax was reached (lower panels). Note that optimal concentrations of all recombinant proteins, except IQGAP1NT, supported a Vmax approx. twofold higher than controls that contained only actin, N-WASP and Arp2/3 complex, but that the optimal concentration of full-length IQGAP1 (IQGAP1FL) reached Vmax at least twice as fast as the fragments. (C) N-WASP was mixed with nickel-agarose beads or nickel-agarose beads that were pre-loaded with recombinant, his-tagged IQGAP1FL, IQGAP1NT, IQGAP12-522, IQGAP12-210, IQGAP12-71, or tau as a negative control. Beads contained an approx. twofold molar excess of his-tagged proteins relative to N-WASP, and chemiluminescent immunoblotting was used to detect any N-WASP that may have bound to beads. Note the specific binding of N-WASP to all forms of IQGAP1 that were tested. The slightly increased electrophoretic mobility of N-WASP in the IQGAP12-522 pull-down assay probably represents a gel artefact caused by that fact N-WASP and IQGAP12-522 migrate nearly identically in SDS-PAGE.