The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line - PubMed (original) (raw)
The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line
Gohta Goshima et al. J Cell Biol. 2003.
Abstract
Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.
Figures
Figure 1.
Mitosis of untreated S2. (A) Untreated S2 cells expressing GFP-tubulin (green) were fixed and stained with γ-tubulin antibody (red) and Hoechst 33342 (blue). Bar, 5 μm. (B) Time-lapse observation of GFP-tubulin in untreated cells, which have variable numbers of prophase MTOCs (Table II). Bipolar spindle was formed directly from two MTOCs (top) or in an extreme case, from eight MTOCs joined through fusion process (middle). The bottom cells failed to complete MTOC fusion, and a tripolar spindle was formed. Images were taken every 10 s (top; single optical section) or 20 s (middle and bottom; Z-projection, 0.66 μm × 10 and 0.58 μm × 10, respectively) using a spinning-disk confocal microscopy. See also Videos 1–3 (available at
http://www.jcb.org/cgi/content/full/jcb.200303022/DC1
). Bar, 5 μm. (C) Time-lapse phase-contrast images of a GFP-tubulin cell from anaphase to cytokinesis. Phase images were taken every 30 s using wide-field microscopy. The image of GFP-tubulin was obtained at the last frame (1980 s). See also Video 4. Bar, 10 μm.
Figure 2.
Reduction of kinesins and dynein after RNAi. (A) Reduction of seven microtubule motor proteins after dsRNA addition. Immunoblotting of KHC, Ncd, Klp3A, Pav, Cos2, Klp61F, and Dhc64C for cultures subjected to RNAi for 3 (Pav and Klp61F), 4 (others), or 7 (Dhc64C) days. All lanes contained an equal load of total proteins (not depicted). Quantitative analyses (not depicted) indicated >90% of reduction of each protein. (B) Specificity of RNAi. dsRNA against KHC or Ncd did not affect the levels of the other five kinesins. (C) Triple RNAi (KHC/Ncd/Klp3A, right lane) yielded efficient protein reduction of all three kinesins.
Figure 3.
Bipolar spindle formation defects caused by Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] RNAi. (A) Abnormal spindle formation after RNAi of indicated four kinesins. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (DNA; green) at d 3 (Klp61F) or d 4 (Ncd, Klp10A and Klp67A). The majority of the mitotic cells had monopolar spindles after Klp61, Klp10A, or Klp67A RNAi, whereas reduction of Ncd induced multipolar spindle formation. In the case of Klp10A or Klp67A RNAi, long bipolar spindles were also observed. (B) γ-Tubulin staining (green) after RNAi. Spindle (red) was visualized by GFP-tubulin expression. Bipolar spindle formed in Klp10A and Klp67A RNAi cells often had only one of the two poles stained for γ-tubulin. Quantitative data and additional cell images are presented in Table II, Table III, and Figs. S2–S8. Prometa, prometaphase; Meta, metaphase; Ana, anaphase. Bars, 10 μm.
Figure 4.
Real-time imaging of GFP-tubulin during the abnormal spindle formation in RNAi cells. Images were taken every 10 s at single optical section using spinning-disk confocal microscopy. (A) A Klp61F [BimC/Eg5] RNAi cell. A monopolar spindle is formed through MTOC fusion. (B) A Ncd [Kin C] RNAi cell. Multiple spindles are formed during prometaphase. (C) Klp10A [Kin I] RNAi cells. Monopolar spindle is initially formed, but is converted to bipolar spindle through acentrosomal pole fusion. (D) Klp67A [Kip3] RNAi cells. A monopolar spindle is maintained for 34 min (top panels), or is eventually converted to a monastral bipolar spindle (bottom panels). See also Videos 5–12 (available at
http://www.jcb.org/cgi/content/full/jcb.200303022/DC1
. Bars, 5 μm.
Figure 5.
Chromosome congression defects after CENP-meta [CENP-E], Klp3A [chromokinesin], and Nod [Kid] RNAi. Cells were treated with dsRNA targeting for indicated genes and were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 4. Bar in the left column represents 10 μm. Magnified images of the chromosomes are shown in the right column (Bar, 5 μm). Control nontreated cell is shown in A. Misaligned chromosomes were frequently detected after CENP-meta (B) and Klp3A (C) RNAi, whereas stretched chromatin was observed after Nod RNAi (D). More severe misalignment phenotypes appeared when RNAi of a kinetochore kinesin and two chromokinesins were combined in E (see also Table IV). Additional images are presented in Fig. S9 and Fig. S10.
Figure 6.
Accumulation of metaphase by cytoplasmic dynein knockdown. (A) Mitotic index of Dhc64C [DHC] RNAi at d 4 was 1.9-fold higher than controls. (B) Percentage of metaphase and anaphase in mitotic cells. Metaphase cells were accumulated, whereas anaphase was less frequently observed in RNAi samples. (C) Metaphase cells with congressed chromosomes. Cells treated with dsRNA for Dhc64C [DHC] were fixed and stained by γ-tubulin (red) and Hoechst 33342 (green) at d 4. Spindle-like structure was visualized by the overexposure of anti- γ-tubulin signals. Majority of the metaphase cells (64%; n = 22) had two punctate signals of γ-tubulin like untreated cells, and no apparent morphological defects in the spindle were detected (not depicted). Bar, 10 μm.
Figure 7.
Pav [MKLP1] functions in the formation and maintenance of the central spindle bundling during cytokinesis. (A) Cells with binuclei are abundant after Pav RNAi. Cells were fixed and stained by anti-tubulin antibodies (red) and Hoechst 33342 (green) at d 3. Bar, 10 μm. (B and C) Real-time imaging of GFP-tubulin during cytokinesis in an untreated cell (B) and Pav RNAi cells (C) on Con A–coated dish. Arrow indicates the microtubules splayed apart from the bundle. See also Videos 13–15 (available at
http://www.jcb.org/cgi/content/full/jcb.200303022/DC1
). Bars, 5 μm.
Figure 8.
Roles of microtubule-based motors in the sequential steps of mitosis in S2 cells. Proper bipolar spindle assembly requires Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] (left pathway). Once a bipolar spindle is formed, chromosomes congress to the metaphase plate by the redundant actions of four chromosomal kinesins, CENP-meta [CENP-E], Klp3A [chromokinesin], Nod [Kid], and Klp67A [Kip3]. If a cell fails to assemble a bipolar spindle initially and a monopolar spindle is formed, it can be eventually converted to monastral bipolar spindle by chromatin-directed, acentrosomal pole-focusing mechanism that requires Klp61F [BimC/Eg5] and possibly Ncd [Kin C] functions (right pathway). After establishment of metaphase, Dhc64C [DHC] (cytoplasmic dynein) controls the timing of anaphase onset. Anaphase B spindle elongation is caused by sliding of antiparallel microtubules, which may require the functions of certain motor proteins that were not identified in our screening. Finally, central spindle formation and cytokinesis requires Pav [MKLP1].
References
- Antonio, C., I. Ferby, H. Wilhelm, M. Jones, E. Karsenti, A.R. Nebreda, and I. Vernos. 2000. Xkid, a chromokinesin required for chromosome alignment on the metaphase plate. Cell. 102:425–435. - PubMed
- Bloom, K. 2001. Nuclear migration: cortical anchors for cytoplasmic dynein. Curr. Biol. 11:R326–R329. - PubMed
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