Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion

Files in this Data Supplement:

• Supplemental Material (PDF)
• Video 4 -
  Pole fragmentation of a bipolar spindle in a LIC1 and 2 morphant. Live confocal imaging of a mitotic spindle in a Xenopus gastrula stage embryo injected with both LIC1 and LIC2 MOs. GFP–α-tubulin (green) was used to label microtubules and chromosomes were visualized using mCherry-H2B (magenta). A bipolar spindle assembles but then undergoes spindle pole fragmentation: the first pole begins to fragment at ∼24 min and 20 s, and the second fragments at 36 min. Single focal plane images were collected every 10 s using a confocal microscope (FluoView FV1000; Olympus) with a 60×/1.35 NA U Plan S Apochromat objective. Time-lapse videos were constructed in ImageJ and play back at a speed of 12 frames per second. The time stamp indicates time in minutes and seconds.
• Video 3 -
  Multipolar spindle assembly in a LIC1 and 2 morphant. Live confocal imaging of a mitotic spindle in a Xenopus gastrula stage embryo injected with both LIC1 and LIC2 MOs. GFP–α-tubulin (green) was used to label microtubules, and chromosomes were visualized using mCherry-Histone2B (magenta). The spindle assembles with three poles. Single focal plane images were collected every 10 s using a confocal microscope (FluoView FV1000; Olympus) with a 60×, 1.35 NA U Plan S Apochromat objective. Time-lapse videos were constructed in ImageJ and play back at a speed of 12 frames per second. The time stamp indicates time in minutes and seconds.
• Video 6 -
  3View EM of a control spindle. A 3D reconstruction of a mitotic spindle in the epithelium of a control Xenopus morphant gastrula stage embryo. Images were collected using a microtome (3View; Gatan) within a scanning electron microscope (Quanta 250 FEG; FEI) with sections taken 100 nm apart. The imaging conditions were indicated quadrant magnification of 1,600×, accelerating voltage of 3.8 kV, pressure at 0.33 Torr, image at 4,000 × 5,000 pixels, and dwell time of 10 µs. Images were reconstructed using Imaris image analysis software (Bitplane). Chromosomes (blue) and centrioles (red) were reconstructed in Imaris using the Surfaces tool. Note that each pole of the bipolar spindle contains a pair of centrioles.
• Video 8 -
  3View EM of a LIC KD bipolar spindle. A 3D reconstruction of a mitotic spindle in the epithelium of a Xenopus morphant injected with both LIC1 and 2 MOs. Images were collected using a microtome (3View; Gatan) within a scanning electron microscope (Quanta 250 FEG; FEI) with sections taken 100 nm apart. The imaging conditions were indicated quadrant magnification of 1,600×, accelerating voltage of 3.8 kV, pressure at 0.33 Torr, image at 4,000 × 5,000 pixels, and dwell time of 10 µs. Images were reconstructed using Imaris image analysis software (Bitplane). Chromosomes (blue) and centrioles (red) were reconstructed in Imaris using the Surfaces tool. Note that although each pole contains two centrioles, these centrioles are further apart than is seen in the control bipolar spindle (Video 6).
• Video 1 -
  Loss of LIC does not affect dynein motor function in vitro. Microtubule gliding driven by dynein purified from control (left) or LIC KnD HeLaM cells (right). Imaging was performed by video-enhanced differential interference contrast microscopy at RT using a microscope (BX60; Olympus) at 25 frames per second using a camera (Newvicon) and image processor (Argus-10) and is played back at 4× real time. Each panel is 13.5-µm across. Videos are viewed best using QuickTime 7.
• Video 7 -
  3View EM of a LIC KD multipolar spindle. A 3D reconstruction of a mitotic spindle in the epithelium of a Xenopus morphant injected with both LIC1 and 2 MOs. Images were collected using a microtome (3View; Gatan) within a scanning electron microscope (Quanta 250 FEG; FEI) with sections taken 100 nm apart. The imaging conditions were indicated quadrant magnification of 1,600×, accelerating voltage of 3.8 kV, pressure at 0.33 Torr, image at 4,000 × 5,000 pixels, and dwell time of 10 µs. Images were reconstructed using Imaris image analysis software (Bitplane). Chromosomes (blue) and centrioles (red) were reconstructed in Imaris using the Surfaces tool. Note that all poles of the multipolar spindle contain only a single centriole.
• Video 5 -
  A multipolar spindle assembles in a LIC1 and 2 morphant and then undergoes pole fragmentation. Live confocal imaging of a mitotic spindle in a Xenopus gastrula stage embryo injected with both LIC1 and LIC2 MOs. GFP–α-tubulin (green) was used to label microtubules and chromosomes were visualized using mCherry-H2B (magenta). The spindle assembles with three poles, but at ∼20 min and 20 s, one of these poles fragments. Single focal plane images were collected every 10 s using a confocal microscope (FluoView FV1000; Olympus) with a 60×, 1.35 NA U Plan S Apochromat objective. Time-lapse videos were constructed in ImageJ and playback at a speed of 12 frames per second. The time stamp indicates time in minutes and seconds. To recenter the spindle in the field of view, the video position was adjusted at 17 min and 30 s.
• Video 2 -
  Mitotic spindle in a control morphant. Live confocal imaging of a mitotic spindle in a Xenopus gastrula stage embryo injected with a standard control MO. GFP–α-tubulin (green) was used to label microtubules and chromosomes were visualized using mCherry-Histone2B (magenta). Single focal plane images were collected every 6 s using a confocal microscope (FluoView FV1000; Olympus) with a 60×, 1.35 NA U Plan S Apochromat objective. Time-lapse videos were constructed in ImageJ and play back at a speed of 7 frames per second. The time stamp indicates time in minutes and seconds.