Myelinating SC-DRG cocultures
DRG neurons were isolated from embryonic day 16 rat spinal cords and either trypsinized or directly plated as explants on collagen-coated coverslips (BD Biosciences). Cultures were maintained in serum-free neurobasal medium (NB medium; 2% B27 supplement, 2 mM l-glutamine, 0.4% glucose, and 50 ng/ml 2.5S NGF). Nonneuronal cells were removed by feeding the cultures with NB medium containing 5-fluorodeoxyuridine and uridine. SC were isolated from postnatal day 2 sciatic nerves and expanded for ~3 wk in D media (DME, 10% FBS, and 2 mM l-glutamine) supplemented with 4 μM forskolin and 5 ng/ml of the EGF domain of rhNRG-1-β1 (R&D Systems). Myelinating cocultures were established by seeding purified DRG neuron cultures with 100,000 SC in C media (MEM, 10% FBS, 2 mM l-glutamine, 0.4% glucose, and 50 ng/ml 2.5S NGF). After 3 d, cocultures were changed to media supplemented with 50 μg/ml ascorbic acid to initiate basal lamina formation and myelination. For studies of the effects of myosin II inhibition on myelination, cocultures were switched to myelin-promoting media containing blebbistatin (EMD). Control and treated cultures were allowed to myelinate for 2–3 wk, with fresh media provided every 2–3 d. To determine the extent of myelination in SC-DRG cocultures, the total number and length of MBP+ segments were counted in micrographs from 10–12 random low-power fields per coverslip using ImageJ version 1.38 (total of 20–24 fields per condition per experiment; total of 2–3 experiments). Statistical tests (t test and ANOVA) were performed using GraphPad Prism (GraphPad Software).
Purified OPC cultures and OPC-DRG myelinating cocultures
Primary OL precursors (OPC and A2B5+
) were purified by immunopanning from mixed glial cultures of postnatal day 1 rat cerebral cortices as described previously (Taveggia et al., 2008
). Purified OPC were seeded onto poly-lysine–coated glass coverslips and maintained in Sato media (DME, 100 μg/ml transferrin, 100 μg/ml BSA, 20 nM progesterone, 100 μM putrescine, 30 nM sodium selenite, 2 mM l
-glutamine, 5 μg/ml insulin, 60 μg/ml N-acetyl cysteine, and 10 μM forskolin) with 10 ng/ml PDGF and 10 ng/ml bFGF for proliferation or induced to differentiate in Sato containing T3 (30 ng/ml). DRG explants were isolated from rat embryonic day 16 spinal cords and plated directly onto glass coverslips coated with Matrigel (BD Biosciences). Explants were kept in NB medium and treated with antimitotic agents to eliminate nonneuronal cells. Explants were allowed to extend a dense neurite network for at least 2–3 wk before their use in coculture experiments. OPC-DRG cocultures were established as described previously (Chan et al., 2004
). In brief, freshly purified OPC (50,000–100,000 cells) were seeded directly onto DRG explants in MEM media containing 10% FBS, 2 mM l
-glutamine, and 0.4% glucose. Myelination was induced the following day by addition of 1 μg/ml TrkA-Fc (R&D Systems). For studies of the effects of myosin II inhibition on myelination, blebbistatin (EMD) was also added to the media at this point and maintained throughout the experiment. Cultures were allowed to myelinate up to 18 d, with fresh media provided every 2–3 d. Myelin segments were identified by MBP and neurofilament staining, then counted in micrographs from 8–10 random low-power fields per coverslip using ImageJ version 1.38 (total of 16–20 fields per condition per experiment; total of 2–3 experiments). Statistical tests (t
test and ANOVA) were performed using GraphPad Prism software.
RNAi of myosin II in purified glia and myelinating cocultures
To generate shRNAs, we used the pLentiLox (pLL3.7) vector (provided by L. Van Parijs, Massachusetts Institute of Technology, Cambridge, MA; Rubinson et al., 2003
). Two 19-nucleotide shRNAs (MLC1, 5′-GCACGGAGCGAAAGACAAA-3′; and MLC2, 5′-GAGGCCTTCAACATGATCG-3′) targeting the regulatory (light) chain of rat myosin II (MLC) were designed using Easy siRNA (ProteinLounge). The 5′-phosphorylated PAGE-purified oligonucleotides were annealed and subcloned into HpaI–XhoI sites of pLL3.7. The lentiviral vector was transfected into 293FT cells together with packaging plasmids Δ8.9 and pCMV-VSVG (provided by J. Milbrandt, Washington University, St. Louis, MO) using Lipofectamine 2000 (Invitrogen). As control, we used the empty pLL3.7 vector or a vector encoding shRNA to a nontargeting (scrambled) sequence. Viral supernatants were collected 72 h after transfection, centrifuged at 3,000 rpm for 15 min, aliquoted for one-time use, and frozen at −80°C. Freshly plated SC were incubated for 3 d with viruses at a 2:3 dilution (vol/vol) in D media (DME, 10% FBS, and 2 mM l
-glutamine) supplemented with forskolin and rhNRG-β1 (EGF domain). Cells were expanded for an additional week and maintained for 3 d in D media before use for myelination experiments. Protein knockdowns were confirmed by Western blotting. For shRNA knockdown experiments in OPC-DRG cocultures, cultures were incubated with lentivirus supernatant at 1:3 dilution for 2 d. The virus was removed and cultures were allowed to myelinate for an additional 2 wk. This protocol resulted in the effective infection of OPC over neurons. As a control, we used established DRG cultures that were infected with the virus before adding the OPC and inducing myelination.
Antibodies used in these studies included those reactive to: MBP (SMI-94) and neurofilament (SMI-31 and SMI-32; Sternberger Monoclonals); myosin IIA and IIB (Covance), cofilin, phosphorylated cofilin, MLC2, phosphorylated MLC2, phosphorylated FAK, Arp2, and vinculin (Cell Signaling Technology); phalloidin-FITC, phalloidin-coumarin, rabbit anti-EHS laminin, anti–caspase 3, and anti-actin (Sigma-Aldrich); anti-GFP, WAVE-1, and Olig2 (Millipore); anti–N-WASP (H100; Santa Cruz Biotechnology, Inc.); anti–N-cadherin and anti-Tau (BD Biosciences). Polyclonal antibodies to Krox-20 and Oct-6 (provided by D. Meijer, Erasmus University, Rotterdam, Netherlands), P0 (M. Filbin, Hunter College, City University of New York, New York, NY), laminin α-2 chain (P. Yurchenco, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ) and myelin-associated glycoprotein were also used. Mouse A2B5 and Ran-2 hybridomas were obtained from the American Type Culture Collection. Mouse O4 was a gift from M. Chao (New York University, New York, NY). Secondary antibodies conjugated to rhodamine, fluorescein, coumarin, or cyanin 5 were obtained from Jackson ImmunoResearch Laboratories.
Cultures (SC, OPC, and myelinating cocultures) were fixed in 4% PFA and processed for immunocytochemistry as described previously (Melendez-Vasquez et al., 2004
). For surface antigen staining (A2B5 and O4), cells were blocked with 1% donkey serum and 5% BSA in PBS for 30 min, then incubated with primary antibody as described previously (Zhang et al., 2006
). Cultures were examined by epifluorescence or confocal microscopy (see the following paragraph).
Image acquisition and analysis
Epifluorescence images were acquired using a microscope (DMI 4000B; Leica) with LAS 1.7.0 software equipped with a digital camera (DFC350FX; Leica) and the following objectives: N PLAN 10×/0.25 NA; N PLAN L 40×/0.55 NA; and HCX PL APO CS 63×/1.4 NA oil UV (all from Leica). Confocal images were acquired with a laser scanning microscope (LSM 510; Carl Zeiss, Inc.) using Plan-Apochromat 20×/0.75 NA or Neofluor 40×/1.3 NA oil objectives and LSM software (all from Carl Zeiss, Inc.). Image processing and quantitation (including fractal analysis) was performed using ImageJ version 1.38 and Photoshop CS8 (Adobe). Adjustment of image brightness or contrast was performed in some cases but without misrepresenting data.
Videos were acquired with a DMI 4000B microscope system (see the previous section) fitted with a stage incubator and a temperature and CO2 digital controller (CTI Controller 37000; Pecon). Cells were plated on poly-lysine–coated glass-bottomed 35-mm tissue culture dishes (MatTek) on D media (SC) or proliferating OPC media. Phase images from SC and OPC cultures were acquired for a period of 15–30 min using an N PLAN L 40×/0.55 NA objective. Cultures were maintained at 37°C and 5% CO2 throughout the observation period.
Proliferation and survival assays
To investigate the effect of myosin II inhibition on the proliferation and cell survival of SC and OPC, BrdU incorporation (Boehringer Mannheim Gmbh) and TUNEL assays (Promega) were performed according to manufacturers' instructions as described previously (Melendez-Vasquez et al., 2004
; Zhang et al., 2006
Cell extracts and Western blotting
Lysates from rat tissues (brain and nerves), cultures of SC, OPC, and myelinating cocultures were prepared as described previously (Melendez-Vasquez et al., 2004
), subjected to SDS-PAGE, and blotted onto nitrocellulose. Appropriate regions of the blots were cut and incubated with specific antibodies and developed using chemiluminescent substrate (Thermo Fisher Scientific).
Control and treated myelinating cocultures were rinsed in PBS, fixed overnight at 4°C in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, and processed further as described previously (Einheber et al., 1995
). Specimens were examined on an electron microscope (CM10; Philips).
Online supplemental material
Fig. S1 shows changes in SC morphology after blebbistatin treatment in cocultures. Fig. S2 shows an example of SC forming multiple short internodes in blebbistatin-treated cultures. Fig. S3 shows preferential infection of OPC by lentivirus in cocultures. Fig. S4 shows expression of phosphorylated MLC during OPC differentiation in vitro. Table S1 shows the quantitation of BrdU incorporation in PNS and CNS myelinating cocultures. Video 1 shows formation of ruffling lamellipodia in SC after blebbistatin treatment. Videos 2 and 3 show actin-protrusive activity of an OPC before and after treatment with blebbistatin. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200802091/DC1