The murine microglia cell line Ra2 (licensed by the Japan Science and Technology Agency, Patent ID US6.673,6,5; JP3410738; EP10/602,234) was maintained in MEM with 10% FCS, 1 ng/ml GM-CSF (Peprotech, UK), and 5 μg/ml bovine insulin . FMLP, PMA, luminol, latrunculin A, and HRP-II were purchased from Sigma (St. Louis, MO, USA) and jasplakinolide from Calbiochem (Darmstadt, Germany). Rabbit anti-LIMK1 antibodies were from Transduction Laboratories, and rabbit polyclonal anti-ser3(P)-cofilin antibodies from Cell Signaling Technology (Danvers, MA, USA). Alexa-conjugated phalloidin and secondary antibodies for immunofluorescence were all from Molecular Probes (Carlsbad, CA, USA).
The human cDNA's coding for amino acids 4-647 of wild type LIMK1 (LIMK1-WT) and kinase dead LIMK1-D406A (LIMK1-DN), and β-actin-YFP (Clontech #6902-1; Mountain View, CA., USA) were inserted into the tetracycline-responsive lentiviral vector pLOX TW , and used to superinfect Ra2 045 cells expressing the tetracycline-responsive transactivator protein. For shRNA knock-down of mouse cofilin two DNA sequences containing the target sequences GGAGGACCTGGTGTTCATC (cofilin shRNA 1) and GGTGTTCAATGACATGAAG (cofilin shRNA 2) with loop sequence ACTCGAGA were synthesized, annealed, and cloned MluI/ClaI into lentivector pLVTHM-DsRed . The vector was then used for superinfection of Ra2 tTR-KRAB cells expressing a tetracycline-responsive KRAB repressor protein, which in the absence of doxycycline represses transcription from polymerase I, II and III . Expression of cofilin shRNA in Ra2 cells was induced 4-5 days at 50 ng/ml doxycycline before cells were used for experiments.
Measurement of Superoxide production
FMLP or PMA-induced superoxide release was measured by luminol-enhanced chemiluminescence (luminol E-CL). Ra2 cells in HBSS buffer with 62.5 μM luminol and 2 U/ml HRP II were warmed two minutes in a 37°C water bath before distribution into Wallac isoplate wells (100.000 cells/well). Subsequently, superoxide production was measured as chemiluminescence before and after stimulation with 4 μM FMLP or 100 ng/ml PMA, respectively, delivered through the injector module of a thermostated Synergy HT microplate reader. With the luminol E-CL method used here, ca. 20-25% of the luminal E-CL signal probably derives from the formation of peroxynitrite via reaction of superoxide with NO, as discussed further in .
Immunofluorescence and FRAP studies
For immunofluorescence Ra2 cells were fixed with 2% paraformaldehyde in phosphate-buffer pH 7.2, washed in PBS, and then incubated with polyclonal rabbit anti-LIMK1 antibodies, anti-Ser3(P)cofilin antibodies, and/or Alexa 488-conjugated phalloidin in blocking buffer consisting of PBS with 5% goat serum and 0.2% saponin (Sigma). Primary antibodies were detected with Alexa488 or Alexa568-conjugated goat-anti mouse or rabbit antibodies (Invitrogen). Images were acquired with a Zeiss LSM510 confocal microscope equipped with a C-Apochromat X63, 1.2 oil immersion objective. Confocal sections (1.0-1.5 μm) were collected and saved as 512 × 512-pixel images at 8-bit resolution before import into Adobe Photoshop for compilation. The same instrument was used for FRAP experiments. Here Ra2 cells contained on thin glass-bottomed 8-chamber slides were allowed to attach for 60 minutes in medium before incubation in HEPES-buffered salt solution at 37°C in atmospheric air for FRAP analysis. A circular ROI of 43 × 43 pixels (diameter 6 μm) was chosen corresponding to either plasma membrane or podosomal F-actin. After obtaining 2 baseline images bleaching of β-actin-YFP was performed with 60 iterations at full power of the krypton/argon laser (514 nm line), and subsequently images (depth 0.8 μm) were collected at 5 second intervals for 2-3 minutes at low laser emission. The fluorescence intensity in the photobleached region was normalized to the fluorescence intensity measured in a non-bleached region at the same post-bleaching time point to correct for bleaching during time lapse (usually less than 1-2%). Data were fitted to a fluorescence recovery curve with formula F(t) = (F0+(Finf * t/t1/2))/(1 + (t/t1/2))), where F0 is the fluorescence intensity immediately after bleaching and Finf is the intensity at infinite times of recovery, to obtain the half-time of recovery t1/2.
F/G- actin ratio measurements
Ra2 cells were collected in PBS without Ca2+ and Mg2+ and then seeded in full growth medium in tissue culture dishes for one hour at 37°C. Subsequently, cells were washed once in ice-cold PBS before lysis with actin stabilization buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 1 mM MgSO4, 1 mM EGTA, 1% TX-100, 1 mM ATP, and protease inhibitor) on ice for 10 minutes. Cells were dislodged by scraping and the entire extract centrifuged at 4°C for 75 minutes in a tabletop centrifuge at 16.000 g. The supernatant containing G-actin was recovered, and the pellet containing F-actin was solubilised with actin depolymerization buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO4, 10 mM CaCl2, and 5 μM cytochalasin D). Aliquots of supernatant and pellet fractions were separated on 12% SDS-PAGE gels and then western blotted with monoclonal anti-β-actin antibody. Signal was detected by ECL in a digital dark room and integrated optical band density was used to estimate the cellular F/G-actin ratio.