|
Figure 1. Myo10 localizes to mitotic spindle poles in X. laevis embryos. (a) Confocal micrographs of interphase and mitotic cells in the epithelium of X. laevis embryos double stained for α-tubulin (red) and Myo10 (green). During interphase and prophase, Myo10 localizes to the nucleus. From metaphase, Myo10 can be seen localized as a band close to the pole, a position it maintains through anaphase and telophase. Throughout the cell cycle, Myo10 is also found at the cell cortex (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1) but is less obvious in these images, as fluorescent levels were reduced due to the high intensity of the nuclear staining. (b) Confocal images of an anaphase spindle double stained for γ-tubulin (red) and Myo10 (green) showing that Myo10 localizes to a region just inside the spindle pole marker γ-tubulin. (c) Confocal micrographs of spindles immunostained for α-tubulin, Myo10 (red), and TPX2 (green) showing that Myo10 and TPX2 display significant colocalization.
|
|
Figure 2. Knockdown of Myo10 leads to mitotic spindle defects. (a) Western blot showing Myo10 protein levels in uninjected (U), Myo10 MO (MO), and 5-mispair control MO (Ctrl MO) embryos. Lysates were prepared from embryos 24 h after microinjection. (b) Confocal micrographs of α-tubulin staining in embryos microinjected with nuclease-free water or Myo10 MO (MO) fixed at 12, 16, or 24 h after microinjection. (c) Quantification of mitotic spindles in water (H2O)-, Myo10 MO (MO)-, and 5-mispair control MO (Ctrl MO)-injected embryos 16 and 24 h after microinjection. At 16 and 24 h, Myo10 morphants have significantly more multipolar spindles than the mispair control (red). An increased number of bipolar spindles is seen in the morphant at 24 h, which is suggestive of a delay in mitosis (n = 15, 11, 14, 17, 18, and 18 embryos for water 16 h, MO 16 h, Ctrl MO 16 h, water 24 h, MO 24 h, and Ctrl MO 24 h, respectively). Error bars represent the standard error of the mean. (d) Box and whisker plots displaying metaphase spindle length in water (n = 92 spindles)- and Myo10 MO (n = 50 spindles)-injected embryos 24 h after injection. Spindle length measurements are shown as a percentage of total cell length to control for differences in cell size. Metaphase spindles in the Myo10 morphant are significantly longer than in water-injected controls. (e) Propidium iodide (red) and α-tubulin (green) staining of control and morphant spindles showing that chromosomes localize to the metaphase plate relatively normally in morphant spindles. (f) Confocal micrographs of α-tubulinâstained spindles assembled in vitro in the presence of either a control antibody or an anti-Myo10 antibody. In control conditions, normal bipolar spindles assemble, whereas the inhibition of Myo10 by antibody addition leads to multipolar spindles. For significance testing, unpaired Student's t tests were performed: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
|
|
Figure 3. Mitotic spindles in Myo10 morphants undergo spindle pole fragmentation, and Myo10 interacts with the spindle assembly factor TPX2. (a) Stills taken from a confocal video (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1) of mitosis in a control embryo using GFPâα-tubulin to visualize the spindle; time stamps indicate time in minutes and seconds. (b) Stills taken from a video of a Myo10 morphant spindle (Video 4) showing that it assembles in a bipolar fashion, but, subsequently, a pole fragments to form a supernumerary pole (arrows). (c) Confocal micrographs of mitotic spindles in Myo10 morphants immunostained for α-tubulin (red) and γ-tubulin (green). All supernumerary poles in the multipolar spindles possess γ-tubulin, independent of whether the poles have just arisen (top, arrows) or are more established (bottom, arrows). (d) Mitotic spindles in water-injected and Myo10 morphant embryos immunostained for α-tubulin (red) and TPX2 (green). TPX2 localization is diffuse in the Myo10 morphant compared with the more focused pole localization in controls. (e) Western blot of GST pull-down assay showing that full-length TPX2 pulls down with GST-Myo10-MyTH4/FERM (GST-Myo10-M/F) but not GST alone.
|
|
Figure 4. Spindle movement is attenuated in Myo10 morphants. (a) Stills taken from a short section of a confocal movie of GFPâα-tubulin in a control embryo (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1). Time stamps indicate time in minutes and seconds. The metaphase spindle undergoes a sudden movement between t = 1:00 and t = 2:00; this âjerkyâ rotational movement is characteristic of metaphase spindles in control embryos at embryonic stages 9 and 10. (b) Stills taken from confocal movie of GFPâα-tubulin in a Myo10 morphant (Video 2) at the same embryonic stage as the control embryo in panel a. The morphant spindle, even while still bipolar, displays a much more gradual rotational movement than the control spindle. (c) A graph displaying the rotational movement of a control spindle compared with a Myo10 morphant spindle. The time at which spindles enter metaphase and anaphase or undergo pole fragmentation are indicated by arrows and accompanying labels.
|
|
Figure 5. Rescue experiments reveal that the head and tail of Myo10 mediate different aspects of Myo10 function in mitosis. (a) Schematic diagram of the constructs used in Myo10 morphant rescue experiments. (b) Low-magnification images of α-tubulin staining in rescue experiment embryos. For these experiments, embryos were microinjected with water or Myo10 MO along with RNA encoding each of the Myo10 constructs shown in panel a. (c) Quantification of bipolar and multipolar spindles in rescue experiment embryos. n = 18, 47, 22, 16, and 12 embryos for water, MO, MO + GFP-Myo10, MO + GFP-Myo10-HMM, and MO + GFP-Myo10-IQT, respectively. Error bars represent the standard error of the mean. (d) Box and whisker plots of spindle length measurements in rescue experiment samples. Spindle length was calculated as a percentage of total cell length to allow for variation in cell size. n = 109, 141, 136, 115, and 87 spindles for water, MO, MO + GFP-Myo10, MO + GFP-Myo10-HMM, and MO + GFP-Myo10-IQT, respectively. For significance testing, unpaired Student's t tests were performed: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
|
|
Figure 6. Depolymerizing F-actin rescues spindle length but not multipolarity in Myo10 morphants. (a) Low-magnification confocal micrographs of mitotic spindles in untreated embryos (control and MO) or embryos incubated in 2.5 μM LatB (MO + LatB). In LatB-treated embryos, some spindles were improperly oriented perpendicular to the plane of the epithelium (arrows). Insets show z projection cross sections of a control spindle, oriented parallel to the plane of the epithelium, and a LatB spindle in the improper perpendicular orientation (the outermost cell cortex is indicated by a broken line in each inset). (b) High-magnification confocal micrographs of α-tubulinâstained spindles in Myo10 morphants either untreated (MO) or incubated in 2.5 μM LatB (MO + LatB). (c) Quantification of spindles in LatB experiment embryos shows that treatment with LatB does not significantly affect the number of bipolar or multipolar spindles (n = 11, 11, 17, and 15 embryos for control, LatB, MO, and MO + LatB, respectively). Error bars represent standard error of the mean. (d) Box and whisker plots of spindle length measurements in the LatB experiment; spindle length was calculated as a percentage of total cell length to allow for changes in cell size. Treatment with LatB significantly rescues the increased spindle length seen in Myo10 morphants (n = 82, 104, 86, and 59 spindles for control, LatB, MO, and MO + LatB, respectively). For significance testing, unpaired Student's t tests were performed: ****, P < 0.0001.
|
|
Figure 7. Live imaging in X. laevis embryos reveals actin cables around the mitotic spindle, which are unaffected in Myo10 morphants. (a) Images taken from a video showing F-actin organization in a control mitotic spindle (see Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1) using mCherryâα-tubulin (red) to visualize the spindle and the GFP-Utr-CH probe (green) to visualize F-actin. Highly dynamic F-actin cables surround the spindle as it assembles (t = 0:00â2:00, arrows) and are concentrated around the poles later in mitosis, especially during anaphase (t = 11:00 and 13:00, arrows). (b) An enlarged view of the uppermost spindle pole from panel a showing that F-actin (GFP-Utr-CH, green) is concentrated around the pole, with some F-actin cables appearing to emanate from the pole (t = 0:00 and 0:06, arrows) and others from the cortex (t = 0:12, arrows). (c) Stills taken from a video of a second control spindle (see Video 8) demonstrating that the assembly of F-actin cables between the spindle and the cell cortex coincide with spindle movement (t = 0:00â0:18, arrows) and concentrate as a pole is drawn toward the cortex (t = 0:24 and 0:30, arrows). (d) Images taken from a video showing F-actin (GFP-Utr-CH, green) organization during Myo10 morphant spindle assembly (see Video 9). F-actin cables associate with the morphant spindle as it assembles (arrow), just as occurs in controls. (e) Stills of a multipolar spindle in a Myo10 morphant (see Video 9) demonstrating that F-actin associates with each of the poles and follows the motion of the spindle (arrows). In each panel, time stamps indicate time in minutes and seconds.
|
|
Figure 8. Model for spindle length regulation by F-actin and Myo10. F-actin and Myo10 function antagonistically to contribute to spindle length control. F-actin promotes spindle lengthening, perhaps through interactions with astral microtubules at the cell cortex, whereas Myo10 provides a counteracting spindle shortening function. The F-actin spindle lengthening is independent of Myo10, as knockdown of Myo10 results in longer spindles. In contrast, the shortening function of Myo10 requires F-actin because disrupting both F-actin and Myo10 gives normal length spindles.
|
