FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle - PubMed (original) (raw)

FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle

Hui Zhu et al. J Cell Biol. 2008.

Abstract

Microtubules (MTs) are nucleated from centrosomes and chromatin. In addition, MTs can be generated from preexiting MTs in a gamma-tubulin-dependent manner in yeast, plant, and Drosophila cells, although the underlying mechanism remains unknown. Here we show the spindle-associated protein FAM29A promotes MT-dependent MT amplification and is required for efficient chromosome congression and segregation in mammalian cells. Depletion of FAM29A reduces spindle MT density. FAM29A is not involved in the nucleation of MTs from centrosomes and chromatin, but is required for a subsequent increase in MT mass in cells released from nocodazole. FAM29A interacts with the NEDD1-gamma-tubulin complex and recruits this complex to the spindle, which, in turn, promotes MT polymerization. FAM29A preferentially associates with kinetochore MTs and knockdown of FAM29A reduces the number of MTs in a kinetochore fiber, activates the spindle checkpoint, and delays the mitotic progression. Our study provides a biochemical mechanism for MT-dependent MT amplification and for the maturation of kinetochore fibers in mammalian cells.

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Figures

Figure 1.

FAM29A is a spindle protein preferentially associated with kinetochore MTs. (A) HeLa S3 cells were synchronized at the G1/S boundary by a double-thymidine arrest and harvested at the indicated time after release. Cell cycle profile was analyzed by FACS with anti-MPM2 antibody staining and propidium iodide staining. Protein levels were determined by Western blotting with Hsp70 as a loading control. (B–E) Maximum projections from deconvolved z stacks of representative HeLa cells (B, D, and E) or a HeLa cells transiently expressing GFP-FAM29A (C). Cells were stained for FAM29A (B, D, and E)/GFP (C) (green), β-tubulin (B–D)/centrin (B)/Hec1 (E) (red), and DNA (blue). In D, the cell was incubated with Monastrol (4 μM) for 4 h and then stained. The inset in E shows a single focal plane of the boxed region. (F) HeLa cells were cultured at 37°C and then incubated at 4°C for the indicated time. Cells were stained for FAM29A, β-tubulin, and DNA. MT and FAM29A fluorescence intensity in the mitotic spindle of metaphase cells (n = 10 cells for each time point) were quantified and normalized to their respective intensity at the 0-min time point. Error bars, SEM. Bars, 5 μm.

Figure 2.

FAM29A controls mitotic progression. (A) HeLa cells and HeLa/GFP-FAM29A cells were transfected with either control (siControl) or FAM29A-specific siRNAs (siFAM29A-A and B) and analyzed by Western blotting. (B and C) HeLa/GFP-H2B cells were transfected with siRNAs and imaged for GFP by time lapse, starting from 50 h after transfection. Cell images were captured every 3 min to monitor mitotic progression. Representative still images are shown in C and the time stamp is in h:min:s (Video 1 for siControl, and Video 2 for siFAM29A-A; Videos available at

http://www.jcb.org/cgi/content/full/jcb.200807046/DC1

). The time when a cell enters metaphase is set to 0 min. Mitotic cells were divided into different categories based on the duration of metaphase (from the initial metaphase plate formation to anaphase onset) (30 cells quantified for each transfection) (B). (D–G) A single focal plane from deconvolved z stacks of representative HeLa cells transfected with siRNAs and stained for CREST (green), Hec1 (D)/BubR1 (F) (red), and DNA (blue). Insets show the details of the boxed regions. Inter-kinetochore (Inter-KT) distances (E; n > 100 kinetochore pairs from five cells for each quantification) and kinetochore BubR1 signals (G; n = 100 kinetochores) in prometaphase and metaphase cells were quantified and plotted. In E: *, P < 4.7 × 10−24; in G: *, P < 5.0 × 10−4 (two-tailed t test relative to siControl metaphase cells). Error bars, SEM. Bars: 10 μm (C); 5 μm (D and F).

Figure 3.

FAM29A controls spindle structure. (A–C) Maximum projections from deconvolved z stacks of representative HeLa cells transfected with siRNAs and stained for FAM29A (green), β-tubulin (red), and DNA (blue). Long exposures of the β-tubulin images are presented here to show the increased amounts of astral MTs. Arrowheads point to astral MTs outside spindle poles. The percentage of metaphase cells with detectable astral MTs among total metaphase cells (B; n = 100 cells for each quantification) as well as the MT fluorescence intensity in metaphase cells (C; n = 10 cells) were quantified and plotted. In C: *, P < 0.01 (two-tailed t test relative to siControl metaphase cells). (D) Lysates of prometaphase HeLa S3 cells were incubated with increasing amounts of exogenously added, taxol-stabilized MTs, followed by sedimentation of MTs. Pellets and supernatants were analyzed by Western blotting. (E) Lysates of prometaphase HeLa S3 cells were incubated with taxol-stabilized MTs, fixed, centrifugated onto coverslips, and stained for FAM29A (green) and β-tubulin (red). Error bars, SEM. Bars, 5 μm.

Figure 4.

FAM29A recruits NEDD1 onto spindle MTs. (A and B) Maximum projections from deconvolved z stacks of representative HeLa or HeLa/GFP-FAM29A cells transfected with siRNAs and stained for NEDD1 (green), β-tubulin (red), and DNA (blue) (A). NEDD1 fluorescence intensity on spindle (excluding spindle pole signals) was quantified (B; n = 10 metaphase cells). *, P < 0.01 (two-tailed t test relative to siControl metaphase cells). (C and D) Maximum projections from deconvolved z stacks of representative HeLa cells transfected with siRNAs and stained for FAM29A (green), NEDD1 (C)/γ-tubulin (D) (red), and DNA (blue). Error bars, SEM. Bars, 5 μm.

Figure 5.

FAM29A interacts with NEDD1. (A) Myc-NEDD1 was cotransfected with GFP-FAM29A or GFP into HeLa cells. Lysates of transfected HeLa cells were immunoprecipitated (IP) with anti-GFP antibodies or with nonspecific IgG followed by Western blotting. (B) HeLa S3 cells were synchronized at prometaphase by a thymidine-nocodazole treatment (TN0). Lysates from asynchronous cells (AS) or prometaphase cells were immunoprecipitated with anti-FAM29A antibodies or with nonspecific IgG. The immunoprecipitates were treated with or without λ-phosphatase followed by Western blotting. (C and D) HeLa S3 cells were synchronized at prometaphase by a thymidine-nocodazole treatment (TN) (C) or at the G1/S boundary by a double-thymidine treatment (TT) (D). Cells were released and harvested at the indicated times (hours). Cell cycle profile was determined by FACS. Lysates and the anti-FAM29A immunoprecipitates were analyzed by Western blotting. In B–D, arrowheads point to nonspecific cross-reacting proteins on Western blot. “ap” (in C), apoptotic cells.

Figure 6.

FAM29A controls MT polymerization in the spindle. (A) Still frames from confocal time-lapse microscopy of HeLa/GFP–α-tubulin cells transfected with siRNAs. Cells were treated with nocodazole for 15 min, washed with PBS, and released into fresh media (t = 0 min) (Video 9 for siControl and Video 10 for siFAM29A-A; Videos available at

http://www.jcb.org/cgi/content/full/jcb.200807046/DC1

). Time, min:sec. (B–E) HeLa cells transfected with siRNAs were treated with 1 μg/ml nocodazole for 15 min, washed, and released into fresh media for the indicated time. Shown are maximum projections from deconvolved z stacks of representative cells stained for FAM29A (green), β-tubulin (B)/NEDD1 (D) (red), and DNA (blue). Fluorescence intensities of β-tubulin (C) and NEDD1 (E) were quantified and plotted (n = 10 mitotic cells). *, P < 0.003 (two-tailed t test relative to siControl cells). (F–H) Maximum projections from deconvolved z stacks of representative HeLa cells transfected with siRNAs and stained for FAM29A (green), EB1 (red), and DNA (blue) (F). The EB1 fluorescence density inside and outside the spindle (G), as well as the ratio of inside vs. outside spindle (H) were quantified and plotted (n = 10 mitotic cells for each quantification). The EB1 fluorescence density is defined as the EB1 fluorescence intensity in a fixed circular area. *, P < 0.005; **, P < 0.008; ***, P < 0.003 (two-tailed t test relative to siControl metaphase cells). Error bars, SEM. Bars: 10 μm (A); 5 μm (B, D, and F).

Figure 7.

FAM29A controls the maturation of kinetochore MT fibers. (A) HeLa cells transfected with siRNAs were incubated at 4°C for 10 min, fixed, and stained for CREST (green), β-tubulin (red), and DNA (blue). Shown are maximum projections from deconvolved z stacks of representative cells. Insets show single focal planes of the boxed regions. Total intensity of spindle MTs as well as the intensity of k-MTs (the MT signal from a defined area immediately next to the CREST signal) were quantified and plotted (n = 10 metaphase cells for each quantification). *, P < 0.05; **, P < 5.6 × 10−5 (two-tailed t test relative to siControl metaphase cells). AU, arbitrary units. (B–D) HeLa cells transfected with siRNAs were incubated at 37°C or at 4°C for 10 min, fixed, and stained for CREST (B)/Mad2 (C and D) (green), β-tubulin (B)/Hec1 (C and D) (red), and DNA (blue). Shown in B and C are maximum projections from deconvolved z stacks of representative cells. Insets show single focal planes of the boxed regions. Fluorescence intensities of kinetochore Mad2 in prometaphase or metaphase cells under different conditions were quantified and plotted in D (n = 120 kinetochores). For metaphase cells in D, only kinetochores on chromosomes aligned at the metaphase plate were quantified. *, P < 1.06 × 10−4; **, P < 1.14 × 10−7 (two-tailed t test relative to siControl metaphase cells). Error bars, SEM. Bars, 5 μm.

Figure 8.

A model for FAM29A-mediated MT amplification in spindle assembly and in k-fiber maturation. (A) Three major pathways for MT nucleation in mitosis: centrosome-dependent (I), chromatin-dependent (II), and FAM29A/MT-dependent (III). (B and C) Role of FAM29A-mediated MT amplification in spindle assembly and in k-fiber maturation. FAM29A recruits the NEDD1–γ-tubulin complex to MTs derived from centrosomes and chromatin to promote their amplification (IIIa and IIIb, respectively). Similarly, FAM29A also targets the NEDD1–γ-tubulin complex to a kinetochore MT, which then nucleates additional MT to promote k-fiber maturation (IIIc). MT flux then transports newly synthesized MTs to spindle poles. Drawings for various cellular structures are representative, but not to scale.

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FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle - PubMed (original) (raw)