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. 2017 Dec 29;38(2):e00371-17.
doi: 10.1128/MCB.00371-17. Print 2018 Jan 15.

Sld5 Ensures Centrosomal Resistance to Congression Forces by Preserving Centriolar Satellites

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Free PMC article

Sld5 Ensures Centrosomal Resistance to Congression Forces by Preserving Centriolar Satellites

Manpreet Kaur et al. Mol Cell Biol. .
Free PMC article

Abstract

The migration of chromosomes during mitosis is mediated primarily by kinesins that bind to the chromosomes and move along the microtubules, exerting pulling and pushing forces on the centrosomes. We report that a DNA replication protein, Sld5, localizes to the centrosomes, resisting the microtubular pulling forces experienced during chromosome congression. In the absence of Sld5, centriolar satellites, which normally cluster around the centrosomes, are dissipated throughout the cytoplasm, resulting in the loss of their known function of recruiting the centrosomal protein, pericentrin. We observed that Sld5-deficient centrosomes lacking pericentrin were unable to endure the CENP-E- and Kid-mediated microtubular forces that converge on the centrosomes during chromosome congression, resulting in monocentriolar and acentriolar spindle poles. The minus-end-directed kinesin-14 motor protein, HSET, sustains the traction forces that mediate centrosomal fragmentation in Sld5-depleted cells. Thus, we report that a DNA replication protein has an as yet unknown function of ensuring spindle pole resistance to traction forces exerted during chromosome congression.

Keywords: GINS; centriolar satellites; chromosome congression; kinesin CENP-E; microtubule forces; multipolarity.

Figures

FIG 1
FIG 1
Sld5 colocalizes with γ-tubulin at centrosomes. Sld5 localization to centrosomes was confirmed by immunofluorescence assays with multiple antibodies. (A) His6-tagged Sld5 protein expressed in E. coli was injected into rabbits to produce anti-Sld5 antibody. (i) His6-tagged Sld5 (0.5 μg) and His6-tagged RPA32 (0.5 μg) purified on a nickel-NTA column and 15 μg HeLa cell lysate were resolved by SDS-PAGE and stained with Coomassie blue. (ii) Alternatively, they were probed with Ab2 anti-Sld5 antibody. (iii) Ab2 anti-Sld5 antibody was incubated with 5 ng/μl His6-Sld5 or control His6-RPA protein, and the blots were developed with the same exposure time. Preincubation with His6-Sld5 but not His6-RPA protein led to the loss of Sld5 immunoblot (IB) signal observed at 31 kDa. Note that the nonspecific bands did not significantly change due to preincubation with His-Sld5 protein. Due to the presence of bacterial protein, some nonspecific sticking occurred (bands marked by asterisks), which was absent in the initial Ab2 immunoblot. (iv) Specificity of Ab1 antibody was demonstrated as explained for blot iii. (B) HeLa cells were prepermeabilized to remove the nuclear fraction of Sld5, followed by coimmunofluorescence assays with rabbit anti-Sld5 (Ab1) and mouse anti-γ-tubulin antibodies, in combination with anti-rabbit Alexa Fluor 488- and anti-mouse Alexa Fluor 555-conjugated antibodies, respectively. DNA was stained with DAPI. The right column is a merge of Alexa Fluor 488, Alexa Fluor 555, and DAPI images. (i to v) Cells in interphase (i and ii) and different phases of mitosis (iii to v). (vi) Immunofluorescence assays carried out in the absence of anti-Sld5 antibody, with other conditions and antibodies remaining similar, ruled out nonspecificity of secondary Alexa Fluor 488-conjugated antibody, as well as bleed-through of the Alexa Fluor 555 signal. (C and D) HeLa cells in different phases of the cell cycle were prepermeabilized to remove the nuclear fraction of Sld5, followed by coimmunofluorescence assay with either Ab3 anti-Sld5 antibody (C) or Ab2 anti-Sld5 antibody (D). Centrosomes are marked by arrowheads. Scale bars, 10 μm.
FIG 2
FIG 2
Localization of Sld5 to centrosomes was confirmed by RNAi-mediated depletion. (A) Coimmunofluorescence assay of HeLa cells, as described in the legend to Fig. 1, was carried out in the presence of 5 ng/μl bacterially purified proteins. Preincubation with His6-Sld5 protein but not His6-RPA32 inhibited the localization of anti-Sld5 antibody to the centrosomes (red arrowheads). The top two rows demonstrate that after His6-Sld5 preincubation, the anti-Sld5 antibody was absent at the location where γ-tubulin stained, confirming that the antibody specifically binds to endogenous Sld5. (B) Coimmunofluorescence assay with Ab1 anti-Sld5 antibody without prepermeabilization showing nuclear localization of Sld5. The images were captured at different magnifications to show Sld5 localization in the entire field (top row) or individual cells (bottom row). (C) HeLa cells were transfected on three consecutive days with control GL2 or SLD5 siRNA, and the lysates were immunoblotted with anti-Sld5 antibody to confirm its specificity. LC, loading control showing equal protein loads in different lanes; the numbers indicate levels of Sld5 relative to control GL2 siRNA-transfected cells. (D) The decrease of SLD5 mRNA was confirmed by reverse transcriptase PCR. The numbers indicate the SLD5 mRNA levels following Sld5 depletion relative to control GL2 siRNA-transfected cells. Beta-2 microglobulin (BMG) served as the internal RNA-loading control. (E) HeLa cells were transfected on three consecutive days with control GL2 or SLD5 siRNA and costained for Sld5 (green), γ-tubulin (red), and DNA (blue) to confirm centrosomal localization of Sld5. (i and iii) Sld5 signal at mitotic centrosomes. (ii and iv) Sld5 signal at interphase centrosomes. Scale bars, 10 μm.
FIG 3
FIG 3
Depletion of Sld5 leads to accumulation in M phase. (A) HeLa cells were transfected on three consecutive days with control GL2, SLD5 (1), or SLD5 (2) (targeting a different region of Sld5) siRNA, and the lysates were immunoblotted with anti-Sld5 antibody to confirm the specificity of RNAi-mediated depletion. LC, loading control showing equal protein loads in different lanes; the numbers indicate levels of Sld5 relative to control GL2 siRNA-transfected cells. (B and C) Transfected cells were harvested 24 h after the last transfection, followed by staining with propidium iodide alone (B) or in combination with anti-phospho-histone H3 (Ser 10) antibody (C), which marks the mitotic cells. The percentages in panel B show cell cycle distribution, while panel C shows cells in M phase. (D) Sld5 depletion causes S phase delay. Transfected cells were pulsed with BrdU for 30 min, followed by staining with anti-BrdU antibody conjugated to fluorescein isothiocyanate (FITC), along with propidium iodide (PI). The dot plots show BrdU incorporation (y axes) and DNA content (x axes), and the percentages of cells incorporating BrdU. (E) Transfected cells were pulsed with BrdU for 20 min, followed by staining with anti-BrdU (red) antibody. The coimmunofluorescence images display BrdU incorporation in different samples, while DNA was stained with DAPI (blue). Scale bar, 10 μm.
FIG 4
FIG 4
Depletion of Sld5 at centrosomes leads to spindle pole defects. (A and B) HeLa cells transfected with control GL2 or SLD5 (1) siRNA were costained for α-tubulin (green) and γ-tubulin (red) and with DAPI for DNA (blue). GL2 siRNA-transfected cells in different mitotic phases displayed normal chromosome congression and spindle pole formation; however, Sld5-depleted cells displayed asymmetric mitotic spindles (red arrowhead in panel B, row i), chromosome congression failure (yellow arrowhead in panel B, row ii), multipolar spindles (red arrowhead in panel B, row iii), and decreased γ-tubulin staining at centrosomes (blue arrowhead in panel B, row iv). (C) Quantification of spindle pole and chromosome alignment defects due to Sld5 depletion (expressed as a percentage of total mitotic cells). The data are represented as means and SD; P values were calculated using a two-tailed t test and show that SLD5 (1) siRNA-transfected samples were significantly different from control GL2 siRNA-transfected samples (*, P < 0.05). (D) The γ-tubulin levels in the GL2 or SLD5 (1) siRNA-transfected cells were surveyed to identify the spindle poles with low levels of γ-tubulin staining, which was confirmed with NIS Elements imaging software (version 3.22.00) for multiple examples to be less than 50% of the mean intensity of the signal observed in the control cells. The data are represented as the means and SD of the results of three independent experiments, with more than 40 cells analyzed in each sample. Scale bar, 10 μm. *, P < 0.05.
FIG 5
FIG 5
Knockdown with an independent siRNA confirmed that Sld5 depletion leads to multiple mitotic aberrations. (A) Control GL2 or SLD5 (2) (targeting a different region of Sld5) siRNA-transfected HeLa cells were costained for α-tubulin (green) and γ-tubulin (red) and for DNA with DAPI (blue). Control GL2 siRNA-transfected cells in different mitotic phases are shown. Examples of Sld5-depleted cells display an asymmetric mitotic spindle (red arrowhead in row iv), chromosome congression failure (yellow arrowhead in row iii), and decreased γ-tubulin staining at centrosomes (blue arrowhead in row v). (B) Quantification of spindle pole defects due to Sld5 depletion by siRNAs targeting different regions of Sld5 (expressed as percentages of total mitotic cells). The data are represented as the means and SD of the results of two independent experiments, with more than 40 cells analyzed in each sample. The P values were calculated using a two-tailed t test and show that the SLD5 siRNA-transfected samples were significantly different from control GL2 siRNA-transfected samples. *, P < 0.05. Scale bar, 10 μm.
FIG 6
FIG 6
Sld5 depletion-induced centrosomal aberrations are not caused by DNA damage. (A and B) HeLa cells were transfected with siRNA duplexes targeting replication factor RFC2 or ORC3, and the lysates were immunoblotted with the indicated antibodies. The numbers indicate levels of RFC2 or ORC3 relative to control GL2 siRNA-transfected cells. (C and D) HeLa cells were transfected with siRNA duplexes targeting GINS subunit PSF2 or PSF3, and the decrease in mRNAs was confirmed by reverse transcriptase PCR. The numbers indicate the mRNA levels following specific siRNA depletion relative to control GL2-transfected cells. BMG served as the internal RNA-loading control. (E) Immunoblotting of control GL2, PSF2, or PSF3 siRNA-transfected samples with anti-Sld5 antibody confirmed that depletion of PSF2 or PSF3 did not significantly decrease Sld5 protein levels, contrary to what had been reported previously (36). The numbers indicate levels of Sld5 relative to control GL2 siRNA-transfected cells. (F) HeLa cells transfected with siRNA duplexes targeting replication factor RFC2, ORC3, PSF2, or PSF3 were costained for α-tubulin (green) and γ-tubulin (red) and for DNA with DAPI (blue). Merged images are shown. (G) Quantification of spindle pole defects due to RNAi depletion (expressed as a percentage of total mitotic cells). The data are represented as the means and SD of the results of three independent experiments, with more than 20 cells analyzed in each sample. The P values were calculated using a two-tailed t test and show that the SLD5 samples were significantly different from control GL2, RFC2, ORC3, PSF2, and PSF3 samples (*, P < 0.05). The single-factor ANOVA results showed that there was a significant difference in spindle pole defects between the different transfected samples [F(5, 12) = 13.0; ANOVA, P < 0.001]. (H) Sld5 depletion does not cause centrosome overduplication in interphase. HeLa cells were transfected with control GL2 or SLD5 siRNA and costained for α-tubulin (green), γ-tubulin (red), and DNA (blue); representative images of cells in interphase are shown. Centrosomes are marked by arrowheads. (I) Quantification of the experiment shown in panel H demonstrating that depletion of Sld5 did not cause an increase in the centrosome number in interphase cells (expressed as a percentage of total interphase cells). The data are represented as the means and SD of the results of two independent experiments, with more than 20 cells analyzed in each sample. The t test showed that the SLD5 samples were not significantly different from the GL2 samples (P = 0.25). (J and K) Immunoblotting of control GL2, SLD5 (1), SLD5 (2), or PCNA siRNA-transfected samples to detect phospho-Chk1 (Ser 345) or phospho-γ-H2AX (Ser 139) confirmed that the DNA damage checkpoint was not activated after Sld5 depletion. LC, loading control showing equal protein loads in different lanes; the numbers indicate levels of Sld5 relative to control GL2 siRNA-transfected cells. Scale bars, 10 μm.
FIG 7
FIG 7
Sld5-deficient centrosomes fragment during chromosome congression. (A) HeLa cells stably coexpressing a red chromatin marker (H2B-mCherry) and a marker for microtubules (mEGFP–α-tubulin) were transfected on three consecutive days with control GL2 or SLD5 siRNA, followed by fixation for visualization of spindle pole defects. Multiple fields of SLD5 siRNA-transfected cells are shown. (B) Immunoblotting confirmed depletion of Sld5 in cells coexpressing H2B-mCherry and mEGFP–α-tubulin. LC, loading control showing equal protein loads in different lanes; the numbers indicate levels of Sld5 relative to control GL2 siRNA-transfected cells. (C) Quantification of spindle pole defects observed in GL2 or SLD5 siRNA-transfected cells coexpressing H2B-mCherry and mEGFP–α-tubulin. The data are represented as the means and SD of the results of two independent experiments, with more than 20 cells analyzed in each sample (*, P ≤ 0.05). (D and E) HeLa cells stably coexpressing H2B-mCherry and mEGFP–α-tubulin were transfected on three consecutive days with control GL2 or SLD5 siRNA, followed by live-cell imaging for almost 4 h. Selected frames at the indicated time points are shown (live-cell image capture is shown in Movies S1 and S2 in the supplemental material). Note that control cells progressed from interphase to cytokinesis, whereas SLD5 siRNA-transfected cells were arrested in an abnormal prometaphase until the end of the imaging period. (F) Quantification of the time taken by GL2 or SLD5 siRNA-transfected cells to progress from prometaphase to cytokinesis. Each point represents a single cell, while the mean is shown as a horizontal bar. Live-cell imaging is presented up to 230 min, at which time the Sld5-depleted cells were alive but had not progressed to anaphase. (G) HeLa cells stably coexpressing H2B-mCherry and mEGFP–α-tubulin were transfected on three consecutive days with SLD5 siRNA, followed by live-cell imaging. The captured images show a cell (yellow arrowheads) that did not complete mitosis and developed chromosomal and spindle pole defects. A small fraction of the cells (red arrowheads) completed mitosis, albeit slowly (mitosis started at time point 3:15 and was complete by 6:30). Note that almost all GL2 siRNA-transfected cells completed mitosis without developing chromosomal and spindle pole defects, establishing that the mitotic defects were due to Sld5 depletion. Scale bars, 10 μm.
FIG 8
FIG 8
Sld5 depletion leads to dispersion of centriolar satellite protein PCM-1, causing a decrease in pericentrin at centrosomes. (A) HeLa cells were transfected with control GL2 or SLD5 siRNA and costained for pericentriolar protein PCM-1 (green), γ-tubulin (red), and DNA (blue). Note that control cells displayed a dense, granular localization of PCM-1 around centrosomes, whereas Sld5-depleted cells displayed a diffuse cytosolic signal of PCM-1 in interphase. For GL2 or SLD5 siRNA-transfected samples, representative images of the entire field (i and ii) or individual cells (iii and iv) are shown. (B) Sld5 depletion does not alter the levels of TACC3, a protein involved in microtubule nucleation and stabilization of γ-tubulin ring complex. HeLa cells were transfected with control GL2 or SLD5 siRNA and costained for TACC3 (green), α-tubulin (red), and DNA (blue). (C) HeLa cells were transfected with control GL2 or SLD5 siRNA, and a coimmunofluorescence assay was performed to display α-tubulin (green) and pericentrin (red). For GL2 or SLD5 siRNA-transfected samples, representative images of the entire field (i and ii) or individual cells (iii and iv) are shown. Note that interphase cells in SLD5 siRNA-transfected samples displayed significantly reduced levels of pericentrin at the centrosomes (arrowheads). (D) Quantification of the pericentrin levels observed in panel C. Each point represents the pericentrin signal of an individual cell, whereas the horizontal bars represent the means of pericentrin signal in GL2 or SLD5 siRNA-transfected samples. Variability within each sample is shown by the SD (error bars). More than 50 cells were analyzed in each sample (**, P < 0.001). Scale bars, 10 μm.
FIG 9
FIG 9
Sld5 depletion leads to centriole splitting and PCM fragmentation. (A) HeLa cells were transfected with control GL2 or SLD5 siRNA and costained for centrin-2 (green) and γ-tubulin (red) with specific antibodies, as well as for DNA with DAPI (blue). The centrosomes are numbered, and the insets show magnifications of the centrin-2 signal at the indicated poles. Note that in the control GL2 sample, one γ-tubulin spot coincides with a doublet of centrin-2 foci. Each centrin-2 focus marks one centriole and has been identified by staining with anti-centrin-2 antibody (i and ii). Examples of Sld5-depleted cells are shown in rows iii and iv. In rows iii and iv, centrosomes of mitotic cells (marked 1 to 4) show centrosome splitting and PCM fragmentation. (B) Quantification of the experiment shown in panel A. Spindle poles in GL2 or SLD5 siRNA-transfected samples were surveyed for the number of centrioles, as shown in the upper half of the bar graph. The lower half of the graph shows the percentages of monocentriolar and bicentriolar spindle poles that displayed low levels of centrin-2 staining in GL2 or SLD5 siRNA-transfected samples. The level of centrin-2 staining was surveyed at each monocentriolar and bicentriolar spindle pole to identify the poles with low levels of centrin-2 staining, which was confirmed by NIS Elements software for multiple examples to be less than 50% of the mean intensity of the signal observed in control cells. The data are represented as the means and SD of the results of two independent experiments, with more than 40 spindle poles analyzed in each sample (*, P < 0.05). (C) Centriole disengagement in HeLa cells stably coexpressing EGFP–centrin-2 and α-tubulin–m-RFP. Cells were transfected on three consecutive days with control GL2 or SLD5 siRNA, fixed, and imaged to capture EGFP, RFP, and DAPI signals. Centrioles, as identified by centrin-2 staining, are marked by arrowheads. Scale bar, 10 μm.
FIG 10
FIG 10
Spindle pole fragmentation due to Sld5 depletion is preceded by the presence of unaligned chromosomes. (A to D) HeLa cells were transfected with control GL2 or SLD5 siRNA and costained for two structural centrosomal proteins, Cep170 (A, green) or pericentrin (C, red), in combination with γ-tubulin (red) or α-tubulin (green), respectively, as indicated. GL2 or SLD5 siRNA-transfected cells were surveyed to identify spindle poles with low levels of Cep170 or pericentrin staining, which was confirmed by NIS Elements software for multiple examples to be less than 50% of the mean intensity of the signal observed in the control cells. Quantification of panels A and C is shown in panels B and D, respectively. The data are represented as the means and SD of the results of two independent experiments, with more than 20 cells analyzed in each sample for the levels of Cep170 and pericentrin signals (*, P < 0.05). (E and F) HeLa cells stably coexpressing H2B-mCherry and mEGFP–α-tubulin were transfected on three consecutive days with control GL2 or SLD5 siRNA, followed by live-cell imaging. Selected frames at the indicated time points are shown (live-cell capture is shown in Movies S3 and S4 in the supplemental material). In row iii of the control GL2 samples, symmetric spindle poles (red arrowhead) and chromosomes aligned at the metaphase plate (yellow arrowhead) that result in equivalent cytokinesis are visible. In row iii of the Sld5-depleted samples, lagging or unaligned chromosomes are visible (yellow arrowhead), though the spindle poles appear to be normal. In rows iv and v, the appearance of supernumerary spindle poles is marked by red arrowheads. (G and H) Live-cell imaging of GL2 or SLD5 siRNA-transfected cells stably coexpressing H2B-mCherry and mEGFP–α-tubulin obtained from an independent experiment (see Movies S5 and S6 in the supplemental material) different from that shown in panels E and F. In panel H, lagging chromosomes are visible, followed by the appearance of supernumerary spindle poles. (I) Spindle pole defects observed after monastrol treatment, demonstrating that centriole splitting occurs after centrosome separation at prophase. Shown are merged images of control GL2 or SLD5 siRNA-transfected cells costained for α-tubulin (green) and γ-tubulin (red) and for DNA with DAPI (blue) after incubation with monastrol for 4 h. (J) Quantification of the spindle pole defects observed in panel I. The data are represented as the means and SD of the results of three independent experiments, with more than 20 cells analyzed in each sample (*, P < 0.05). (K and L) Codepletion of separase does not prevent spindle pole fragmentation in Sld5-depleted cells. HeLa cells were transfected with control GL2, SLD5, or SEPARASE siRNA as indicated, with the combined concentration brought to 80 nM with GL2 siRNA. The cells were fixed and costained for α-tubulin (green), γ-tubulin (red), and DNA (blue). The quantification is shown in panel L. The data are represented as the means and SD of the results of two independent experiments, with more than 20 cells analyzed in each sample. The t test showed that SLD5-plus-SEPARASE samples were not significantly different from the SLD5 samples (P = 0.77). (M) Decrease of SEPARASE mRNA confirmed by reverse transcriptase PCR. The numbers indicate the mRNA levels following specific siRNA-mediated depletion relative to control GL2 siRNA-transfected cells. BMG served as the internal RNA-loading control. Scale bars, 10 μm.
FIG 11
FIG 11
Sld5-depleted centrosomes fragment due to CENP-E- and Kid-mediated forces. (A) HeLa cells transfected with control GL2 or CENP-E siRNA were immunoblotted with anti-CENP-E antibody to confirm RNAi depletion. The numbers indicate levels of CENP-E protein relative to control GL2 siRNA-transfected cells. (B and C) GL2 or CENP-E siRNA-transfected samples were costained for α-tubulin (green) and γ-tubulin (red), whereas TOTO-3 was used to stain the nucleus (pseudocolor blue). The arrowheads point to unaligned chromosomes observed in CENP-E-depleted samples. Quantification of the congression defects is shown in panel C. (D) HeLa cells were transfected with control GL2, SLD5, or CENP-E siRNA as indicated, with the combined concentration brought to 80 nM with GL2 siRNA. The cells were fixed and costained for α-tubulin (green) and γ-tubulin (red), whereas TOTO-3 was used to stain the nucleus (pseudocolor blue). (E) The transfected cells were immunoblotted with anti-Sld5 antibody to confirm RNAi depletion. The numbers indicate levels of Sld5 relative to control GL2 siRNA-transfected cells. (F) Decrease of CENP-E mRNA confirmed by reverse transcriptase PCR. The numbers indicate the CENP-E mRNA levels following siRNA-mediated depletion relative to control GL2 siRNA-transfected cells. BMG served as the internal RNA-loading control. (G and H) HeLa cells transfected with control GL2, SLD5, or CENP-E siRNA, as described for panel D, were stained with anti-CENP-E antibody to confirm RNAi depletion. Quantification of the spindle pole defects is shown in panel H. (I) HeLa cells transfected with different siRNAs, as indicated, were surveyed to identify mitotic cells with low levels of Cep170 or pericentrin staining, which was confirmed by NIS Elements software for multiple examples to be less than 50% of the mean intensity of the signal observed in control cells. (J and K) HeLa cells transfected with control GL2, SLD5, or KID siRNA, as indicated, with the combined concentration brought to 80 nM with GL2 siRNA. The cells were fixed and costained for α-tubulin (green) and γ-tubulin (red) and for DNA with DAPI (blue). Note that inhibition of KID activity resulted in multiple chromosome congression defects. Quantification of the spindle pole defects is shown in panel K. (L) The transfected samples were immunoblotted with anti-Sld5 antibody to confirm RNAi depletion. The numbers indicate levels of Sld5 protein in different samples relative to control GL2 siRNA-transfected cells. (M) Decrease of KID mRNA confirmed by reverse transcriptase PCR. The numbers indicate the KID mRNA levels following siRNA-mediated depletion relative to control GL2-transfected cells. BMG served as the internal RNA-loading control. Quantification of data is represented as the means and SD of the results of two independent experiments, with more than 20 cells analyzed in each sample. *, P < 0.05. LC, loading control showing equal protein loads in different lanes. Scale bars, 10 μm.
FIG 12
FIG 12
Inhibition of HSET prevents spindle pole fragmentation in Sld5-depleted cells. (A) HeLa cells transfected with control GL2, SLD5, or HSET siRNA, as indicated, with the combined concentration brought to 80 nM with GL2 siRNA. The cells were fixed and costained for α-tubulin (green) and γ-tubulin (red) and for DNA with DAPI (blue). Note that HSET depletion resulted in shortened or asymmetric spindle poles. Scale bar, 10 μm. (B) The transfected samples were immunoblotted with anti-Sld5 and anti-HSET antibodies to confirm RNAi depletion. LC, loading control showing equal protein loads in different lanes; the numbers indicate levels of Sld5 and HSET proteins in different samples relative to control GL2 siRNA-transfected cells. (C) Quantification of the spindle pole defects shown in panel A, represented as the means and SD of the results of three independent experiments, with more than 20 cells analyzed in each sample (*, P < 0.05). (D) Schematic representation of spindle pole fragmentation in the absence of Sld5. (i) Depletion of Sld5 leads to the dissipation of centriolar satellite protein PCM-1 in interphase cells, resulting in the loss of its known function of recruiting the centrosomal protein pericentrin. (ii and iii) During alignment of chromosomes in prometaphase, kinesin, CENP-E, and the chromokinesin Kid cause the unaligned chromosomes to migrate away from the spindle poles, exerting a significant pulling force on the centrosomes. While a wild-type centrosome (shown as the right pole) is able to resist this force, a Sld5-deficient centrosome (shown as the left pole) splits, forming monocentriolar or acentriolar spindle poles, as depicted in diagram iii. The minus-end-directed kinesin-14 motor protein, HSET, anchors the microtubules at the centrosomes.

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