Materials were obtained from the following companies: anti–α-tubulin from Sigma-Aldrich; phalloidin-rhodamine from Molecular Probes; anti-EB1 from Transduction Labs; anti-Dlg1 from Santa Cruz Biotechnology, Inc. and Upstate Biotechnology; and anti-pericentrin from BabCO. Two different anti-APC antibodies were used for this study; anti-APC (C-20), which was obtained from Santa Cruz Biotechnology, Inc., was used for Western blotting, whereas anti-APC, which was used for immunofluorescence, was a gift from I. Näthke (University of Dundee, Dundee, Scotland, UK). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, GF109203X was purchased from Calbiochem, and PKCζ pseudosubstrate was obtained from Biosource International. GTPases, Par6, and PKCζ constructs have been described previously (Etienne-Manneville and Hall, 2001
). APC-ΔMT was obtained from I. Näthke. Other APC constructs were generated by PCR of hAPC (provided by B.M. Gumbiner, Sloan-Kettering Institute, New York, NY) and were subcloned into pRK5-myc. EB1 constructs were generated by PCR of human EB1 and were subcloned into pEGFP. Dlg1 constructs were obtained from T. Akiyama (University of Tokyo, Tokyo, Japan; Matsumine et al., 1996
APC and Dlg1 siRNA
Four siRNA duplexes corresponding to rat APC starting at nt 3577 and 5199 (GenBank/EMBL/DDBJ accession no. D38629) and to rat Dlg1-SAP97 starting at nt 1060 and 2273 (GenBank/EMBL/DDBJ accession no. U14950) were obtained from Proligo. siRNA and pEGFP were introduced into cells by nucleofection according to the vendor's instructions (Amaxa GmbH). Cells were plated on polyornithine-coated plates or coverslips, and Dlg1 expression was examined at different times (Fig. S1). Centrosome reorientation was assessed 3 d later.
Cells were washed with ice-cold PBS containing 1 mM orthovanadate and were lysed at 4°C in Nonidet P-40 buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM orthovanadate, 1% Nonidet P-40, 2 mM PMSF, 5 mM EDTA, 20 μg/ml aprotinin, and 20 μg/ml leupeptin). Nuclei were discarded after centrifugation at 10,000 g for 10 min. Lysates were incubated for 2 h at 4°C with Dlg1 antibodies and protein G–Sepharose beads. Immunoprecipitates were collected and washed in Nonidet P-40 buffer. Immunoprecipitated proteins were eluted with SDS sample buffer and were analyzed by 8% SDS-PAGE.
Cell culture and scratch-induced migration
Primary rat astrocytes were prepared as described previously (Etienne-Manneville and Hall, 2001
). For scratch-induced assays, cells were seeded on poly-l
-ornithine–precoated coverslips or 90-mm diameter dishes and were grown in serum to confluence, and the medium was changed 16 h before scratching. Individual wounds (suitable for microinjection and immunofluorescence; ~300 μm wide) were made with a microinjection needle. Wound closure occurred ~16–24 h later. Multiple wounds (suitable for subsequent biochemical analysis) were made with an eight-channel pipette (0.1–2-μl tips) that was scratched several times across the 90-mm dish. Nuclear microinjections in the first row of wound edge cells were performed immediately after scratching. Expression vectors were used at 100–200 μg/ml, and cells were stained as described previously (Etienne-Manneville and Hall, 2001
). Conventional epifluorescence images of fixed cells mounted in Mowiol were obtained on a microscope (model DM6000; Leica) equipped with a 63× NA 1.32 objective and were recorded on a CCD camera (CoolSNAP HQ; Roper Scientific) using MetaMorph software (Universal Imaging Corp.).
Dual color TIRF and confocal microscopy
The TIRF microscope that was used in this study has been previously described in detail (Manneville et al., 2003
). In brief, TIRF (for review see Toomre and Manstein, 2001
) was achieved at the glass slide/culture medium interface using a trapezoidal glass prism. Experiments were performed at 37°C on an upright microscope (Axioplan, Carl Zeiss MicroImaging, Inc.) that was equipped with a 100× NA 1.0 water immersion objective (Carl Zeiss MicroImaging, Inc.) and an intensified CCD camera (Remote Head Darkstar, S25 Intensifier; Photonics Science). Fluorescence was excited by either an argon ion laser (λ
= 488 nm; 25 mW; Melles-Griot) or a Nd:YAG laser (λ
= 532 nm; 50 mW; CrystaLaser). The angle of incidence of the excitation light was fixed to 68–70° above the critical angle θc
= 61.5°. The calculated penetration depth for the argon ion laser was dP
= 75–85 nm, and for the Nd:YAG laser, it was dP
= 85–95 nm. TIRF images were acquired by using the image analysis software Optimas 6.5 (Media Cybernetics, LP). Confocal images of fixed cells that were mounted in Mowiol were taken on a scanning confocal microscope (model LSM510 Meta; Carl Zeiss MicroImaging, Inc.) with a 40× NA 1.3 oil immersion objective (Carl Zeiss MicroImaging, Inc.).
Colocalization of Dlg1 and APC puncta or Dlg1 and microtubules was quantified by using the measure colocalization function within the Metamorph software. Images were first filtered by using the flatten background function. Colocalization was quantified in a 5-μm–wide region that was drawn in different areas of the cells. Background colocalization was estimated by measuring colocalization in a region that was devoid of cells located in front of the wound edge.
Polarized microtubule anchoring at the plasma membrane was assessed by TIRF microscopy 8 h after wounding in cells that were stained with antitubulin antibody. Cells showing an increase in tubulin fluorescence specifically near the leading edge ( A) were defined as cells with polarized microtubule anchoring. Cells with microtubules randomly contacting the plasma membrane were scored as negative. At least 100 cells from three independent experiments were scored.
Centrosome reorientation was determined as described previously (Etienne-Manneville and Hall, 2001
). In brief, 8 h after wounding, astrocytes were fixed and stained with antipericentrin (centrosome), Hoechst (nucleus), and anti-myc when necessary. Cells in which the centrosome was within the quadrant facing the wound were scored as positive (polarized centrosome). Random orientation of the centrosome, therefore, corresponds to a value of 25% of correctly polarized cells. For each point, at least 300 cells from three independent experiments were examined.
Online supplemental material
Fig. S1 shows Western blot analysis of Dlg1 and APC expression after siRNA transfection in astrocytes. Fig. S2 shows the effects of APC and Dlg1 constructs on astrocyte migration. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200412172/DC1