The D10.G4 cell line was maintained in RPMI-1640 supplemented with 10% FCS, l
-glutamine, penicillin, streptomycin, β-mercaptoethanol, and 10 U/ml human interleukin-2. As D10.G4 cells were used only for studies of motility and not activation, they were not periodically restimulated, as has been previously described (Tooley et al., 2009
). Cells were maintained at a density <106
cells/ml. A subclone of the D10.G4 line was generated by electroporating cells with the SEPT6-GFP plasmid described in the next section, sorting cells using a cell sorter (MoFlo; Dako), and maintaining in the media previously described supplemented with 50 µg/ml Geneticin for selection.
Plasmids and transfections
All transfections were performed by electroporation using Gene Pulser (Bio-Rad Laboratories). shRNA against Sept7 and control shRNA plasmids were used as previously described (Tooley et al., 2009
). Cells transfected with knockdown plasmids for Sept7
were cotransfected with EGFP-N1 (Takara Bio Inc.) as a marker. SEPT6-GFP consists of the mouse Sept6
sequence with a 22–amino acid linker sequence added by PCR and cloned into EGFP-C1 (Takara Bio Inc.). mCherry-GPI was a gift from G.S. Baron (Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT; Speare et al., 2010
). LifeAct-Ruby consists of 17 amino acids from Saccharomyces cerevisiae
Abp140 (Riedl et al., 2008
) subcloned into pCMV-RFP-Ruby-N1 vector (Takara Bio Inc.) via EcoRI–BamHI sites.
For observations of crawling cells, cells were allowed to adhere to poly-l-lysine or 0.2 µg/ml ICAM-1–coated coverslips for 30 min before fixation. For volume-change experiments, chambered coverslips (Thermo Fisher Scientific) were coated with 0.5 µg/ml anti-CD44 antibody (clone IM7) for 60 min and then washed three times. Next, cells were suspended in 150 µl of serum-deficient RPMI-1640 and allowed to adhere to coated coverslips for 30 min. 300 µl of either 37°C deionized water or 37°C RPMI-1640 was added to each chamber (for hypotonic and isotonic conditions, respectively). After these preparations, warmed 16% PFA (Electron Microscopy Sciences) was added to each chamber for fixation at a final concentration of 4% for 10 min. After thorough washing with PBS, fixed cells were blocked with 2% donkey serum and 2% FCS and permeabilized with 0.2% saponin (Sigma-Aldrich) in PBS for 30 min. Cells were incubated with rabbit α-SEPT7 antibody (IBL-America, Inc.) for 120 min in permeabilization buffer, washed, and then stained with secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) in permeabilization buffer for 60 min and washed again. Samples were imaged immediately or stored at 4°C overnight for subsequent imaging.
Widefield images were acquired on a modified microscope (Axiovert 200M; Carl Zeiss) with a Plan-Neofluar 63× objective (Carl Zeiss). The microscope was fitted with dual excitation and emission filter wheels and a camera (CoolSNAP HQ; Roper Scientific). Image acquisition was performed using MetaMorph imaging software (Molecular Devices). For motility and mitosis imaging, 0.25% low–melting point agarose was included in the media to minimize drift. Time-lapse images were acquired every 30 s for mitosis and hypotonic stress experiments and every 1 or 2.5 s for motility experiments. Live cells were maintained at 37°C during imaging, and fixed samples were imaged at room temperature. For confocal imaging, cells were placed onto Lab-Tek II 8 chamber dishes (Thermo Fisher Scientific) with no. 1.5 coverslip bottoms. Spinning-disk confocal images were acquired using a microscope (Axiovert 200M) with a 63×/1.4 NA oil objective, electron-multiplying charge-coupled device camera (iXon 887; Andor Technology), ProScan III stage (Prior Scientific), and a spinning disk (CSU10; Yokogawa Electric Corporation) with 488- and 561-nm laser lines modulated by an acousto-optical tunable filter (NEOS Technologies) assembled by Solamere Technology Group. Cells were kept at 37°C using an objective and sample heater (Carl Zeiss). The microscope was controlled by MetaMorph software.
Nocodazole (Sigma-Aldrich) was used at 5 µM for flow cytometry experiments and 13 µM for cell cycle synchronization. Latrunculin B (Sigma-Aldrich) was used at 10 µM. Blebbistatin (racemic) was used at 100 µM. Jasplakinolide was used at 100 nM. Dynasore (Sigma-Aldrich) was used at 80 µM. Charybdotoxin (EMD) was used at 25 nM, and NPPB (EMD) was used at 100 µM. Nocodazole, taxol, latrunculin B, blebbistatin, jasplakinolide, and dynasore stock solutions were in DMSO. Stock solutions of charybdotoxin and NPPB were in water.
Flow cytometry volume-change assay
Cells were suspended in serum-free RPMI-1640 at a concentration of 2e6/ml containing 10 nM SYTOX red stain (Invitrogen) for dead cell exclusion and incubated at 37°C with or without inhibitors at the concentrations indicated for at least 60 min before the assay. To monitor the response to osmotic stress, forward scatter values were recorded over time, and after 1 min of recording, cells were transferred to a hypotonic solution of 100 mOsm sucrose, which contained SYTOX red and inhibitors, as necessary. Samples were kept warm with a water jacket for the length of the 6-min time course. For analysis, no forward or side scatter gates were used. Rather, gating was on SYTOX red–negative and, when necessary, GFP-positive cells. Slopes were computed during the linear phase of contraction between 150 and 300 s from the start of the experiment. Approximate cell size was calculated by using 4.5–45-µm microspheres (Polysciences, Inc.) to create a standard curve and fitting forward scatter measurements of cells to this curve (Fig. S2
). All experiments were performed on a FACSCalibur flow cytometer (BD).
Cell cycle synchronization
D10 cells were incubated with 13 µM nocodazole for 16–20 h and were then washed in 15 ml of media four times to remove the drug. They were then resuspended in a small volume and incubated at 37°C in chambered coverslips for 30 min before imaging.
Cell sorting was performed on a cell sorter (MoFlow) or special order research products (FACSAria II; BD) using 70-µm nozzles.
Analysis and statistics
All flow cytometry analysis was performed in FlowJo (Tree Star), and the FlowJo kinetics platform was used for osmotic volume-change measurements. Images were analyzed in MetaMorph (Molecular Devices) or Imaris (Bitplane) software depending on the type of analysis. MetaMorph was used for 3D reconstructions. The mitotic blebbing index consists of the number of blebs observed divided by the product of the time followed (in seconds) and the circumference of the cell (in micrometers). All statistical analyses were performed in Prism software (GraphPad Software), and individual tests are named in the legends.
Online supplemental material
Fig. S1 shows protein levels of a panel of septins after SEPT7 knockdown. Fig. S2 shows the standard curve used for calibrating cell size in the flow cytometry volume-change assay. Video 1 shows the location of membrane protrusions relative to SEPT6-GFP in a crawling cell. Video 2 demonstrates cortical morphology during mitosis of control and SEPT7KD cells, sequentially. Video 3 shows high-resolution time-lapse images of the formation of septin rings during RVD. Video 4 shows a 3D reconstruction of cells with septin rings. Video 5 shows septin rings appearing during RVD in a field of cells. Video 6 shows invaginations in the plasma membrane during RVD. Video 7 shows SEPT6-GFP distribution in cells treated with jasplakinolide. Video 8 shows septin accumulation at the base of actin-containing protrusions during RVD. Video 9 shows recruitment of SEPT6-GFP to blebs in a crawling dynasore-treated cell. Video 10 shows recruitment of LifeAct-Ruby to a bleb preceding SEPT6-GFP recruitment. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201105127/DC1