We used the following lines obtained from the Bloomington Drosophila Stock Center and the community: sn28
, upstream activation sequence (UAS)-GFP-Moesin (Dutta et al., 2002
), UAS-mCherry-Moesin (Millard and Martin, 2008
), UAS-GFP-fascin (Zanet et al., 2009
), and nanos-Gal4VP16. The percentage of the rescue of fertility was calculated by counting the number of pupae generated by one female sn28
expressing the different GFP-fascin transgenes in nurse cells under the nanos-Gal4 driver in comparison with a wild-type fly.
Plasmids and transgenesis
UAS-fascin lines were generated by inserting a full-length singed
cDNA (RH62992) into the pUASp vector. GFP-fascin and mCherry-fascin fusions were obtained by fusing GFP or mCherry sequences to the N terminus of full-length singed
cDNA, and site-directed mutagenesis on serine S289 was performed by PCR. The LifeAct-GFP and LifeAct-TagRFP lines were generated by inserting the cDNA sequence of the LifeAct (Riedl et al., 2008
) tagged with the GFP or the TagRFP (Evrogen) in the C terminus of the pUASp vector.
Singed (sn)-Gal4 lines were obtained by cloning the genomic region (X chromosome position: 7,867,099–7,867,592) of the singed gene, which controls Drosophila macrophage expression of fascin, upstream of the Gal4 sequence. Genomic DNA was amplified by PCR by using the following primers: 5′-CTCCATTAAATTGAAGAACCTC-3′ and 5′-CAGTACGTTGGGCATTCC-3′. This fragment was subcloned in the pGEM-T Easy (Promega) vector then cloned in the pPTGal4 plasmid to obtain the driver singed-Gal4. Transgenic flies were generated by P element insertion. For each individual construct, we established and tested a minimum of three independent lines.
For hfascin knockdown, the target shRNA, 5′-GCCTGAAGAAGAAGCAGAT-3′, was cloned into the pLLT3.7 vector (gift from J. Adams, University of Bristol, Bristol, England, UK). shRNA-resistant hfascin cDNA (in which the shRNA target sequence was changed to 5′-GCCTGAAA
CAGAT-3′ [bold letters indicate changes]), and its wild-type and mutant forms were generated and tagged in the N-terminal site with EGFP (pEGFP vector; Takara Bio Inc.) or mRFP into pcDNA3.1 (Invitrogen). GFP-LifeAct and Ruby-LifeAct constructs were a gift from R. Wedlich-Soldner (Max Planck Institute, Munich, Germany; Riedl et al., 2008
). For recombinant fascin purification, the cDNA encoding hfascin and mutants were cloned between NotI and XhoI restriction sites in the pET-30a(+) vector (EMD).
Cell culture and transfections
MDA-MB-231 (human breast carcinoma) and SW480 (human colon carcinoma) cells were cultured in DME supplemented with 10% FBS. For fascin localization and filopodia formation analysis, MDA-MB-231 fascin knockdown cells were established by lentiviral infection. These cells were transiently cotransfected with the mRFP-tagged shRNA-resistant fascin constructs and GFP-LifeAct using Lipofectamine 2000 reagent (Invitrogen). After transfection, cells were plated on 10 µg/ml fibronectin-coated coverslips (Millipore) and, 24 h later, sealed in imaging chambers and visualized as described in Live imaging.
UAS transgenic constructs encoding fluorescent proteins (GFP or mCherry) were expressed in macrophages using the singed
-Gal4 driver line. Live embryos were mounted as previously described (Wood et al., 2006
). In brief, stage 15 embryos were dechorionated in bleach and mounted under coverslips on hydrophobic Lumox dishes (Sarstedt) in Voltalef oil. Embryos were then imaged using a confocal microscope (TCS SP5 [Leica] or UltraVIEW VoX [PerkinElmer] spinning disk with a 63× N.A. 1.4 objective). For colocalization experiments of GFP- and mCherry-tagged proteins, images were acquired sequentially in the red and green channels; inverting the order of acquisition had no effect on the localization. For filopodia formation analysis in human carcinoma cells, MDA-MB-231 cells were visualized using a confocal microscope (A1R; Nikon). Filopodia number and length were measured from three independent experiments using ImageJ software (National Institutes of Health), and statistical significance of differences between groups was assessed by analysis of variance (ANOVA).
Immunoprecipitation and Pro-Q Diamond staining
Cells were transfected with the different hfascin constructs (GFP-fascin wild type, GFP-fascin S39A, GFP-fascin S274A, and GFP-fascin S39AS274A) and then lysed in radioimmunoprecipitation assay buffer supplemented with antiphosphatase inhibitor cocktails (EMD). Drosophila fascin (UAS-mCherry-fascin and UAS-mCherry-fascin S289A) was expressed in embryos with the ubiquitous daughterless-Gal4 driver. Wild-type and mutant fascins were immunoprecipitated with GFP-Trap or RFP-Trap beads (Chromotek). Proteins were blotted on polyvinylidene fluoride membrane and stained with Pro-Q Diamond (Invitrogen). Total fascin protein amounts were detected on the same blot with Coomassie or the antifascin antibody (sn7C; Developmental Studies Hybridoma Bank). Pro-Q Diamond signal and protein levels were quantified with ImageJ.
For metabolic incorporation of radioactive orthophosphate, cells were transfected with the different hfascin constructs (GFP-fascin S39A, GFP-fascin S274A, and GFP-fascin S39AS274A). Cells were starved for 4 h in DME depleted in phosphate and containing 0.5% dialyzed FBS (Invitrogen). This media were subsequently replaced by DME depleted in phosphate containing 10% dialyzed FBS and 0.5 mCi/ml of radiolabeled P32-orthophosphate (PerkinElmer) for 1 h. Cells were lysed and immunoprecipitated as described in the previous paragraph. Proteins were separated in an SDS-PAGE gel and analyzed using a phosphoimager (FLA-3000; Fujifilm).
The LifeAct-TagRFP and the GFP-fascin (wild-type and mutated forms) were coexpressed in the fly germline using the driver nanos-Gal4VP16. Ovaries were dissected in PBS and fixed in 4% paraformaldehyde for 25 min. Ovaries were rinsed in PBS and quenched for nonspecific fluorescence in PBS containing sodium borohydride (Sigma-Aldrich) at 1 mg/ml for 20 min. Ovaries were extensively rinsed in PBS/0.1% Tween 20 and then mounted in Vectashield mounting medium (Vector Laboratories). For FRET/FLIM cell culture experiments, wild-type MDA-MB-231 and human colon carcinoma SW480 cells were transiently cotransfected with GFP-tagged fascin (wild type and mutants) and the LifeAct-Ruby construct. Time domain FLIM was performed with a multiphoton microscope system (with TE2000 microscope; Nikon) described in detail previously (Parsons and Adams, 2008
). In brief, cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% (wt/vol) Triton X-100 in PBS. After quenching with 1 mg/ml sodium borohydride in PBS for 10 min at RT, cells were washed in PBS and mounted in Mowiol containing 2.5% Dabco (Sigma-Aldrich). Fluorescence lifetime imaging capability was provided by time-correlated single-photon counting electronics (SPC-700; Becker & Hickl). A 40× objective was used throughout (Plan Fluor NA 1.3; CFI 60; Nikon), and data were collected at 500 ± 20 nm through a band pass filter (35–5040; Coherent, Inc.). Acquisition times of the order of 300 s at a low 890-nm excitation power were used to achieve sufficient photon statistics for fitting, while avoiding either pulse pile up or significant photobleaching. Histogram data are plotted as mean FRET efficiency from >10 cells per sample over three independent experiments or from six egg chambers per sample. ANOVA was used to test statistical significance between different populations of data. Lifetime images of example cells are presented using a pseudocolor scale, whereby blue depicts normal GFP lifetime (no FRET) and red depicts lower GFP lifetime (areas of FRET).
Recombinant fascin expression was induced in the BL21 Escherichia coli strain with 1 mM IPTG (Sigma-Aldrich) for 5 h at 25°C. The bacterial pellet was resuspended in 300 mM NaCl, 10 mM imidazole, and 50 mM Na2HPO4, pH 8, sonicated, cleared, and purified using Ni–nitriloacetic acid beads (QIAGEN). Proteins were dialyzed against a buffer containing 150 mM NaCl, 10 mM imidazole, and 50 mM Na2HPO4, pH 8, at 4°C. Actin from rabbit muscle was purchased from Sigma-Aldrich.
30 µM actin was polymerized in the following buffer for 2 h at 4°C: 50 mM KCl, 2 mM MgCl2, and 1 mM ATP. F-actin was then pelleted for 1.5 h at 50,000 g at 4°C and dialyzed overnight at 4°C in G buffer (2 mM Tris, pH 7.4, 0.2 mM CaCl2, 0.2 mM ATP, and 0.5 mM DTT). The different forms of purified fascin proteins were first centrifuged at 80,000 g for 30 min to remove potential aggregates, and actin was centrifuged at 13,000 rpm five times at RT. Fascin and actin at a final concentration of 15 µM each were incubated for 1 h at RT in KME buffer (50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole, pH 7). Samples were centrifuged for 30 min at two different speeds, 13,000 g (for bundling experiments) and 80,000 g (for binding experiments). Equal volumes of supernatant and pellet were analyzed by 8% SDS-PAGE, and proteins were stained with the Imperial Protein Stain (Thermo Fisher Scientific).
EM analysis of fascin and actin bundles
For the ultrastructural analysis of fascin-actin bundles, actin-binding experiments were initially performed as described in the previous section. Before centrifugation steps, samples were diluted 1:10 in KME buffer and prepared as previously described (Jansen et al., 2011
). In brief, 3 µl of the diluted sample was left to adsorb onto Formvar- and carbon-coated 200-mesh copper grids for 30 s, excess solution was removed, and the sample was negatively stained with uranyl acetate 1% for 1 min. The remaining uranyl acetate solution was removed, and the grids were dried at RT. The EM study was performed in a transmission electron microscope (F20; Tecnai) at 25,000× magnification with an accelerating voltage of 200 kV.
Online supplemental material
Fig. S1 shows blots of the knockdown of fascin in MDA-MB-231 and the reexpression of fascin transgenes. Fig. S2 shows FRET/FLIM data of different forms of fascin interacting with actin in human cells and Drosophila
nurse cells. Video 1 shows time-lapse imaging of wild-type fascin, fascin S289A, and fascin S289D in migrating macrophages in embryos. Video 2 shows coexpression of wild-type mCherry-fascin and GFP-fascinS289A in macrophages in sn28
mutant embryos. Video 3 shows high magnification time-lapse sequence of a macrophage coexpressing wild-type mCherry-fascin and GFP-fascinS289A in sn28
mutant embryos. Videos 4 and 5 show time-lapse imaging of mCherry-fascin and mCherry-fascinS289A, respectively, and LifeAct-GFP in migrating macrophages. Video 6 shows time-lapse imaging of filopodia from human cells which express the different transgenes of hfascin. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201110135/DC1