SSRP1 colocalizes with spindle and midbody MTs during mitosis. (A) Specificity of the polyclonal anti-SSRP1 antibody. Portions (10 μg [lane 1] and 30 μg [lane 2]) of HeLa whole-cell lysate (WCL) were subjected to SDS-PAGE for WB with polyclonal anti-SSRP1 antibodies. An asterisk indicates a truncated SSRP1 fragment. (B) The monoclonal anti-SSRP1 antibody 5B10 specifically detects a single band of SSRP1. Then, 50 μg of HeLa NE was used for WB analysis with affinity-purified 5B10 antibodies. (C) SSRP1 colocalizes with MTs during mitosis in HeLa cells. HeLa cells were synchronized by double-thymidine blocking and stained by IF at interphase and the different phases of mitosis, using polyclonal anti-SSRP1 (red) or anti-tubulin (green) antibodies and DAPI (blue) for DNA. Colocalization of SSRP1 with tubulins was also observed in H1299 and 293 cells using polyclonal anti-SSRP1 antibodies (data not shown and Fig. 4; see also Fig. S2 and S5 in the supplemental material). Bar, 5 μm. (D) Monoclonal anti-SSRP1 antibodies also detect the colocalization of SSRP1 with spindle and midbody MTs during mitosis. Synchronized H1299 cells that harbor-inducible SSRP1 siRNA in the absence of doxycycline was stained with 5B10 (red) and polyclonal anti-tubulin (green) antibodies and DAPI (blue) for DNA. This panel is also a control for Fig. 4B. Bar is 5 μm.

SSRP1 binds to tubulins in cells and in vitro. (A) α-Tubulin is found in a cytoplasmic Flag-SSRP1-associated protein complex. The 0.3 M KCl fractions (2 mg) of NE and S100 samples from phosphocellulose chromatography were incubated in an immunoprecipitation reaction with anti-Flag, and the immunoprecipitates were eluted from the beads, subjected to SDS-PAGE, and stained with colloidal blue. The polypeptide bands were sequenced by mass spectrometry. The SPT16 in lane 1 was also identified by mass spectrometry, as indicated. (B) SSRP1 cofractionates with MTs. Microtubules were purified from HeLa S100 fractions using alternate steps of polymerization at 37°C and depolymerization at 4°C as described in the supplemental material. Fractions (lanes 1 to 3, 50 μg; lanes 4 to 5, 30 μg; lanes 6 to 7, 10 μg) were subjected to SDS-PAGE and transferred for WB analysis. (C) SSRP1 exists in a high-molecular-weight complex with polymerized MTs. A partially purified tubulin fraction (700 μg) was incubated at 37°C to polymerize MTs, loaded onto a 12.5 to 25% glycerol gradient, and centrifuged. Forty fractions were collected in all. The “L” denotes the loading input of the samples. Fractions were subjected to SDS-PAGE for WB using polyclonal antibodies against the indicated proteins. (D) SSRP1 associates with MTs in vitro. GST-SSRP1 fusion proteins (2 μg) were incubated with S100 from HEK 293 cells (500 μg of total proteins) at 30°C. Bound proteins were detected by WB using anti-α-, β-, and γ-tubulin antibodies. (E) Schematic presentation of tubulin- and DNA-binding domains of SSRP1. (F) SSRP1 binds to MTs in vitro. Purified His-SSRP1, as shown in Fig. 3A, was incubated alone or with polymerized MTs at 25°C for 30 min. Mixtures were subjected to sucrose cushion centrifugation assays. Proteins in supernatants/low-molecular-weight fractions (S) or pellets/high-molecular-weight fractions (P) were subjected to SDS-PAGE for WB. Blots were probed with anti-SSRP1 antibodies (5B10) at 1:500 and antitubulin antibodies at 1:2,000.

SSRP1 facilitates MT growth and bundling in vitro. (A) Coomassie brilliant blue staining of purified proteins used for the assays in Fig. 3B to H and S4. A total of 300 ng of His-SSRP1, 1.0 μg of tubulins, 1.0 μg of His-C-SSRP1, or 1.0 μg of bovine serum albumin (BSA) was loaded onto an SDS gel and stained. (B) SSRP1 promotes tubulin polymerization in vitro in a dose-dependent fashion. In vitro tubulin polymerization assays were conducted as described in Materials and Methods. Reaction mixtures containing the reagents GTP plus tubulin plus 2 μM SSRP1 (▴), GTP plus tubulin plus 1 μM SSRP1 (▪), GTP plus tubulin (⧫), or 2 μM SSRP1 () were incubated at 37°C for different time points for measuring the absorbance at 340 nm as indicated. Then, 20 μM tubulin was used in each reaction, and the same was true to the reactions in panels C and D below. (C) All of the reactions in panel B were repeated in the presence of 20 μM Taxol. (D) Tubulin polymerization enhanced by WT SSRP1, but not the deletion mutant, in vitro is inhibited by nocodazole. The same tubulin polymerization assays as that in panel B were conducted using the reagents GTP plus tubulin plus 1 μM SSRP1 (▪), GTP plus tubulin plus 1 μM BSA (▴), GTP plus tubulin plus 1 μM His-C-terminal SSRP1 (⧫), GTP plus tubulin (), or GTP plus tubulin plus 1 μM SSRP1 plus 5 μM nocodazole (□). The results in panels B to D have been repeated three times. (E) SSRP1 promotes tubulin polymerization at 5 μM tubulin. The tubulin polymerization assay was performed as that in panels B to D except that 5 μM tubulin was incubated with 5 μM (▴) or 0 μM (⧫) of SSRP1, and the latter was supplemented with 5 μM BSA. (F) IF analyses of polymerized tubulins in vitro. Aliquots from the reactions at 15 min in panel D were deposited on glass slides for IF analyses with monoclonal anti-tubulin (green) and polyclonal anti-SSRP1 (red) antibodies. Representative IF images are shown. These IF results were also confirmed with polyclonal anti-tubulin and monoclonal anti-SSRP1 antibodies (see Fig. S4A in the supplemental material). Bar, 5 μm. (G) SSRP1 facilitates MT bundling in vitro. Tubulins (20 μM) were incubated with 20 μM Taxol at 37°C for 1 h. Tubulins from the reaction cocktail were incubated with purified His-SSRP1 at the concentration as indicated for an additional 30 min. Proteins in the reactions were stained with antibodies as described for panel F. Bar, 5 μm. (H) EM analysis of in vitro-polymerized MTs. The same reactions as those described in panels B to D were used for EM analysis. Representative images are shown here, as indicated, using the samples from the reactions at 15 min, as shown in panel D (except the first column for tubulin alone, which was taken from the reaction after 30 min of incubation for better images of polymerized MTs). The images in the top row are 26.5 by 26.5 μm; the images in the bottom row are 0.54 by 0.54 μm. (I) Quantification of the number of MTs per bundle as a percentage. The numbers of MTs per bundle was counted for 202 MT bundles formed in the absence of SSRP1 and for 600 MT bundles formed in the presence of SSRP1 based on the EM images of panel H, as well as those not shown, respectively. The percentages of bundles with two or more MTs, three or more MTs, and four or more MTs were calculated as indicated in the graph. Note that the percentage for two or more MTs per bundle includes that for three or more, and the percentage for three or more MTs per bundle includes four or more. (J) Kinetics of MT bundling mediated by SSRP1. Rhodamine-conjugated tubulins (20 μM final concentrations of tubulin dimmer mixtures containing a 1:5 ratio of rhodamine tubulins to tubulins) were incubated with 1 mM GTP and 20 μM Taxol at 37°C for 30 min and then mixed with 8 μM His-SSRP1 or BSA, and the reactions were stopped by the addition of 2 reaction volumes of antifade at different time points, as indicated at the top, for fluorescence microscopic analysis. Bar, 5 μm.

SSRP1 is required for the normal progression of mitosis. (A) Establishment of an inducible SSRP1 siRNA expression cell line. H1299 cells that harbor a Tet-inducible SSRP1-siRNA vector were established and were treated with or without doxycycline (Dox + or Dox −) to induce siRNA expression for 2 days. Cell lysates (50 μg) were harvested for WB (six upper panels) and reverse transcription-PCR (RT-PCR) (three lower panels) analyses with antibodies and DNA primers as indicated on the right. RNAs were detected by 23 cycles of RT-PCR and stained with ethidium bromide. Of note, the slight change of α-tubulin levels was not reproducible. (B) Ablation of SSRP1 by siRNA results in shortened mitotic MTs and small and disorganized spindles during mitosis. H1299 cells harboring the inducible SSRP1-siRNA vector were treated as described in panel A. Cells were fixed for IF staining with antibodies against α-tubulin (white or green) or SSRP1 (red) as indicated on top. DNA was stained with DAPI. The normal controls in the absence of doxycycline are also shown in Fig. 1D, and a set of representative images with normal spindle and midbody structures [Dox(−)] are also shown on the left of this panel, as side-by-side controls for prophase (Pro), prometaphase (Prom)/metaphase (Meta), anaphase (Ana), and telophase (Telo) MTs, respectively. Bar, 5 μm. (C) Ablation of SSRP1 by siRNA remarkably reduces the number and size of K-fibers in mitotic spindles, as well as the number and intensity of the bundled MT in midbodies. H1299 cells were synchronized under the same conditions as that described in panels A and B and incubated in ice water for 15 min before permeabilization with cold PTEMF and fixation with 100% methanol (−20°C). Cells were stained with the anti-α-tubulin antibody and DAPI. Images were taken under a ×60 zoom lens. Defects of K-fibers and midbody fibers were detected in most of the SSRP1-ablated cells. Representative images are shown here. Bar, 10 μm. (D) Percentages of abnormal mitotic cells after ablation of SSRP1 by siRNA. Mitotic cells from mock or siRNA-induced samples were counted (200 in all) to determine the percentages of cells that displayed defects in mitotic spindles, as shown in panel B and Fig. S5B in the supplemental material. (E) Distribution of cells in each phase of mitosis after siRNA induction. Cells displaying normal (Fig. 1D) or abnormal mitotic structures as shown in panel B and as counted in panel C were grouped into prophase, metaphase, anaphase, and telophase. The data from total 200 mitotic cells counted are summarized in a graph, as shown here. Of note, because some SSRP1 knocked-down cells showed abnormal spindle structures with less condensed and misaligned chromosomes due to severe spindle defects, it was hard to distinguish prometaphase from metaphase and thus we put them into the same group. (F) Distribution of defective mitotic cells in each phase of mitosis among total cells counted. From the data of panel C, cells displaying mitotic defects at each specific phase were regrouped to determine at which phase more defective cells are detected. The percentage of defective cells was represented here as a column. Values on top of each column indicate the number of total defective cells among 200 mitotic cells counted. These results were also repeated using scramble or SSRP1 siRNA in H1299 cells independently, as described for Fig. 5.

Knockdown of SSRP1 by siRNA delays chromosome condensation and prevents chromosome segregation. (A) Knockdown of SSRP1 by siRNA markedly delays chromosome condensation and prevents chromosome segregation. GFP-H2B stable HeLa cells were transfected with scrambled or SSRP1 siRNA (the siRNA sequence against SSRP1 was 5′-AAGAAUGGCCAUGUCUACAAG-3′) as indicated. Transfected cells were monitored under a fluorescence microscope as described in Materials and Methods. Live mitotic cells were followed, and their images were taken at different time points after initiation of mitosis as indicated on top until completion of a division (scrambled) or 6 h (SSRP1 siRNA). This panel is only a representative result for a number of SSRP1-siRNA transfected cells displaying a similar phenotype, which was not seen in scrambled siRNA cells. Bar, 5 μm. (B) Knockdown of SSRP1 by siRNA in GFP-H2B HeLa cells used for panel A. Immediately after the completion of image collection, cells were harvested for WB analysis (50 μg of cell lysates per lane), using antibodies as indicated. (C) Knockdown of SSRP1 by siRNA in H1299 RFP-H2B stable cells used for panels D and E. WB was carried out as described in the above panel B. GFP-si-scramble and GFP-si-SSRP1 indicate the vectors for expressing GFP-scramble siRNA and GFP-SSRP1 siRNA, respectively. (D and E) Knockdown of SSRP1 by siRNA in H1299 RFP-H2B cells delays chromosome condensation and prevents segregation. The H1299 RFP-H2B stable cell line was transiently transfected with either the GFP-scramble siRNA expression vector (D) or the GFP-SSRP1-siRNA expression vector (E). All of the cells that expressed GFP should also express siRNAs against SSRP1 or scramble siRNAs since they are in the same vector (see panel C for WB). Live cells were monitored as described in the above panel A. (Note that all of the H1299 GFP-SSRP1-siRNA/RFP-H2B cells [10 in all observed], but none of the H1299 GFP-scramble siRNA/RFP-H2B cells [20 in all observed], underwent abnormal mitosis similar to the representative result in panel E.) Bar, 5 μm.

SSRP1 is required for centrosomal MT growth in vivo. (A) Depletion of SSRP1 by siRNA reduces the plus-end growth of MTs in cells to 20%. Tet-inducible SSRP1 or scramble siRNA H1299 cells, established as shown in Fig. 4A, were treated with doxycycline for 72 h and treated with 8 μM nocodazole for an additional 4 h. The cells were fixed for IF staining 6 min after being washed with PBS with anti-EB1 and anti-γ-tubulin antibodies. Arrows point to the centrosomes as stained with anti-γ-tubulin antibodies. Bar, 5 μm. (B) Knockdown of SSRP1 by siRNA inhibits the time-dependent growth of centrosomal MTs. IF analyses of the same cells as those used and treated for panel A with monoclonal anti-EB1 and polyclonal anti-SSRP1 antibodies, except for that cells were fixed 2 and 6 min, as indicated on the right, after washing with PBS as indicated. Bar, 5 μm. (C) WB analysis of SSRP1 levels in H1299 SSRP1 or scramble siRNA cells used for the assays in panels A and B. (D) WB analysis of SSRP1 levels in H1299 Tet-inducible SSRP1 or scramble siRNA cells used for the assays in panels E and F. Two independent SSRP1 siRNA expression cell clones were used here as indicated as SSRP1-siRNA (I) and SSRP1-siRNA (II). (E and F) Knockdown of SSRP1 by siRNA markedly inhibits the growth of MTs from centrosomes. The same regrowth assay as that in panel B was conducted using two H199 SSRP1-siRNA expression clones (I and II) in comparison with H1299 scramble siRNA expression cells. Cells were fixed 2 and 6 min after regrowth and stained with antibodies against α-tubulin (white) and DAPI (purple).