S2 cell culture
S2 cells stably transfected with an EGFP–α-tubulin construct (under control of the copper-inducible pMT promoter or the constitutive pAc5.1 promoter [Invitrogen]) or with an EB1–EGFP construct (controlled by the pMT promoter) were a gift from R. Vale (University of California, San Francisco, San Francisco, CA). Cells were cultured according to published methods (Rogers et al., 2002
Sequences to be used for RNAi were selected by alignment of mRNAs to identify 500–600-bp regions for each protein that displayed minimal homology with other proteins in the FlyBase database. Selected sequences are as follows: Dm-Kat60 (CG10229), nucleotides 1885–2448 (in 3′ UTR of NM_080258); Dm-Spastin (CG5977), 2831–3389 (in 3′ UTR of NM_170115); Dm-Fidgetin (CG3326), 150–671 (in CDS of NM_134919); Asp (CG6875), 4207–4872 (in CDS of NM_079764). DNA templates for RNA synthesis were obtained by PCR of D. melanogaster ESTs (Drosophila Genomics Research Center) or S2 cell cDNA using the primers listed in the following paragraph. dsRNA was generated using commercial transcription kits (Megascript T7 [Ambion] or Ribomax T7 [Promega]) according to the manufacturers' instructions. For RNAi, S2 cells were treated on day 0, 2, and 4 by incubating for 1 h in 1 ml serum-free Schneider cell medium (Invitrogen) with 20 μg dsRNA, followed by addition of 1 ml Schneider medium containing 20% heat-inactivated FBS. Cells were replated and analyzed on day 5.
Each primer for generating dsRNA was preceded with T7 sequence (taatacgactcactataggg). The gene-specific sequences used for primers are as follows (listed as 5′ to 3′ for both forward/reverse primers): control, atggataagttgtcgatcg/accaggttcacatgcttgcg (template = pBluescript SK+; Stratagene); Asp, ctacatctgcgcgaggttacc/agcccttcgcttcatctcg; Dm-Fidgetin, tgctgcgctcaaggatcac/ttcgagctcacagttcgcttg; Dm-Kat60, gaatggctagcgattgtagg/atctctgcctgcactaaactatg; Dm-Spastin, cgttgtttaac caccatgtcc/acaccagatccatacgcacc.
GST and maltose binding protein fusions of the N-terminal regions of each protein displayed in Fig. S1 were bacterially expressed and purified using glutathione–Sepharose or amylose resins. Polyclonal antibodies were generated against the GST fusions (ProteinTech). Antibodies were affinity purified from sera using their respective maltose binding protein fusion proteins coupled to Affigel resin (Bio-Rad Laboratories). In addition, affinity-purified antibodies were preabsorbed with resin-bound fusions of N-terminal regions of the other AAA proteins to eliminate cross-reactivity.
After RNAi, S2 cells were plated on concanavalin A–coated coverslips to stimulate cell spreading for microscopy (Rogers et al., 2002
). Cells were fixed in −20°C methanol for 20 min and blocked with 5% normal goat serum in PBS containing 0.1% Triton X-100. Primary antibodies (against the Dm-AAA proteins or α-tubulin [DM1a; Sigma-Aldrich], γ-tubulin [GTU-88; Sigma-Aldrich], phospho-histone H3 [Upstate Biotechnology], or centromere marker Cid [a gift from G. Karpen, University of California, Berkeley, Berkeley, CA]) were applied at 1–20 μg ml−1
final concentrations in blocking buffer. Fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) were used at 7.5 μg ml−1
. DNA was stained with 1 μg ml−1
propidium iodide, 5 μM Draq5, or 0.3 μg ml−1
Hoechst 33258. Specimens were imaged using an Ultraview spinning-disk confocal system (PerkinElmer) mounted on an inverted microscope (Eclipse TE 300; Nikon) with a 100×, 1.4 NA objective and captured with a digital camera (Orca ER; Hamamatsu). Most images are displayed as maximum intensity projections of the captured z stacks. For experiments requiring MT disruption, cells were treated with 30 μM colchicine for 16 h just before fixation.
In vivo severing assay
Two similar approaches were used to assay severing activity. For , S2 cells (constitutively expressing mRFP–α-tubulin) were transiently transfected with a copper-inducible gene encoding full-length Dm-AAA protein fused to EGFP. Transfected cells were induced with 500 μM CuSO4 for 8–12 h and then imaged with the system described. Images of live cells were captured digitally with identical system settings (exposure time, gain, etc.). Using ImageJ (NIH), fluorescence intensities for both EGFP and mRFP were measured from entire cells expressing Dm-AAA-EGFP and neighboring control cells not visibly expressing EGFP.
For , wild-type S2 cells were transiently transfected with plasmids encoding EGFP or full-length Fidgetin-EGFP. After induction, cells were fixed (4% paraformaldehyde, 0.14% glutaraldehyde, 1 μM taxol, 0.1% Triton X-100, 1 mM MgCl2, 1 mM EGTA, and 80 mM Pipes, pH 6.8; 15 min, 24°C) and immunostained with DM1a to visualize MTs, and digital micrographs were captured as described. Polymer fluorescence intensity was calculated by subtracting the mean cytosolic fluorescence intensity of a cell (calculated from measurements made in several cytoplasmic regions devoid of MTs) from its total mean fluorescence intensity (measured from the entire cell).
Measurement of flux and anaphase chromatid-to-pole rates
Two techniques were used to measure poleward flux rates of preanaphase spindles: (1) fluorescence speckle microscopy and (2) the tracking of marks photobleached onto uniformly fluorescent spindle MTs. Because the flux rates produced by each technique were in good agreement for several experiments, the data were pooled.
To measure flux rates by fluorescence speckle microscopy, RNAi-treated cells expressing EGFP–α-tubulin (under control of the inducible pMT promoter) were plated on concanavalin A–coated microwell dishes as described. The leaky basal expression of the pMT promoter sometimes produces a low EGFP–α-tubulin titer necessary for speckling of spindle MTs. Speckled MTs were visualized with the Ultraview spinning-disk confocal system, and images were captured at 2- or 5-s intervals as single optical sections. Only cells with spindle orientations near perpendicular to the light path were analyzed. Using MetaMorph (Universal Imaging Corp.), images were processed with the high-sharpen and low-pass functions, and kymographs were generated from prominent speckles in each half-spindle. Each kymograph included the spindle pole, which served as a fiduciary point relative to which the rates of fluxing speckles were measured. Flux rates were calculated from the angles between the tracks of pole and speckles measured by MetaMorph.
Poleward MT flux rates were also measured on spindles of S2 cells constitutively expressing EGFP–α-tubulin (under control of the pAc5.1 promoter). Narrow rectangular regions were photobleached across the fluorescent bipolar spindles of RNAi-treated S2 cells using a confocal system (TCS SP2; Leica) on an inverted microscope (DMIRE2 [Leica]; Plan Apo 63× objective, 1.4 NA). Time-lapse videos of the photobleached spindles were captured with 3–6-s frame intervals. The movement of the bleach mark through the spindle was measured with the MetaMorph calipers tool, and flux rate was calculated from the change of distance between bleach mark and spindle pole as a function of time.
Chromatid-to-pole rates were measured in RNAi-treated anaphase S2 cells constitutively expressing EGFP–α-tubulin and vital stained with Hoechst 33258. Images of the Hoechst-stained chromosomes on fluorescently tagged spindle MTs were captured at 3–5-s intervals, and the chromatid anaphase rates were measured from the translocation distance through time. Alternatively, anaphase chromatids were tracked in EGFP– α-tubulin expressing cells by their negatively stained profiles at the ends of prominent kinetochore fibers.
To measure α-tubulin turnover at MT ends, rectangular regions at the pole (MT minus ends) and spindle equator (MT plus ends) of RNAi-treated S2 cells stably expressing EGFP–α-tubulin were photobleached using the confocal system described in the previous section, and time-lapse videos of the bleached cells were immediately recorded. To measure γ-tubulin turnover at centrosomes, the two large fluorescent spots of S2 cells stably expressing γ-tubulin–EGFP were photobleached and their subsequent recoveries recorded. The half-time for fluorescence recovery (t1/2) of each bleached region was measured from the plots of fluorescence recovery (corrected for postbleach fluorescence loss because of imaging). For those cases when the percentage of fluorescence recovery was <25%, the corresponding t1/2 values were not included in calculating the mean t1/2 for a treatment. Photobleaching was not observed to adversely affect cells; for example, photobleached cells were sometimes observed to proceed to anaphase.
Measurement of EB1-EGFP densities and tubulin polymer/monomer fluorescence ratios
Densities of comets of a fluorescently tagged +Tip protein, EB1-EGFP, were measured within metaphase spindles of RNAi-treated S2 cells by capturing each spindle as a z series of 1-μm optical sections with a spinning-disk confocal microscope (see Immunofluorescence microscopy), and images were processed by sequentially applying the “convolve” and “smooth” functions of ImageJ. Two spindle pole regions (each extending 1.25 μm from the tip of a pole toward the metaphase plate) and a single spindle equator region (extending 1.25 μm to each side of the metaphase plate) were delimited in each spindle. Fluorescent puncta of EB1-EGFP were counted within each z section of each region and then totaled. (Comet totals for the two pole regions were combined.) Densities were calculated by dividing EB1 comet totals by the region volumes (calculated by multiplying region areas [obtained from ImageJ] by the number of sections).
Total fluorescence of EGFP–α-tubulin within spindles and total fluorescence of EGFP–α-tubulin within the remainder of the cytoplasm were measured from projections of z series of confocal digital images of metaphase S2 cells using ImageJ. Ratios of these values were calculated and used to estimate proportions of tubulin distributed between polymer and soluble fractions. This estimation assumes that spindle fluorescence is primarily due to MT polymer and that fluorescence outside the spindle is primarily due to soluble tubulin.
Image processing and data analysis
Datasets were saved as stacks of TIFF files, and time-lapse series were saved as AVI videos. Datasets were processed and analyzed with MetaMorph or ImageJ as described. When fluorescence intensities were to be quantitated (), the digital images were recorded with identical settings of microscope and Ultraview software. The statistical differences between treatments were analyzed using either a one-way nonparametric analysis of variance (Kruskal-Wallis) for multiple group comparisons or a nonparametric t test (Mann-Whitney) for two group comparisons (SigmaStat, Systat Software, or GraphPad Prism; GraphPad Software). Measurement means were taken to be statistically different if P < 0.05.
Online supplemental material
Supplemental figures show production of mono-specific antibodies against the AAA proteins (Fig. S1), verification of target AAA protein knockdown after RNAi (Fig. S2), immunolocalization of AAA proteins after colchicine treatment (Fig. S3), analysis of spindle phenotypes after AAA RNAi (Fig. S4), and representative fluorescence recovery curves of photobleached α-tubulin or γ-tubulin of RNAi-treated spindles (Fig. S5). Videos 1–4 are recordings of live EGFP–α-tubulin expressing, anaphase S2 cells after RNAi with control, Dm-Katanin, Dm-Spastin, or Dm-Fidgetin dsRNA, respectively. Videos 5–7 are recordings of live EGFP–α-tubulin expressing, anaphase S2 cells after RNAi to knock down control, Dm-Katanin, or Dm-Spastin, respectively. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200612011/DC1