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. 2015 Mar 20:6:6551.
doi: 10.1038/ncomms7551.

Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways

Michaela Roth  1 , Chantal Roubinet  1 , Niklas Iffländer  1 , Alexia Ferrand  2 , Clemens Cabernard  1
Affiliations

Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways

Michaela Roth et al. Nat Commun. .

Abstract

Precise cleavage furrow positioning is required for faithful chromosome segregation and cell fate determinant distribution. In most metazoan cells, contractile ring placement is regulated by the mitotic spindle through the centralspindlin complex, and potentially also the chromosomal passenger complex (CPC). Drosophila neuroblasts, asymmetrically dividing neural stem cells, but also other cells utilize both spindle-dependent and spindle-independent cleavage furrow positioning pathways. However, the relative contribution of each pathway towards cytokinesis is currently unclear. Here we report that in Drosophila neuroblasts, the mitotic spindle, but not polarity cues, controls the localization of the CPC component Survivin. We also show that Survivin and the mitotic spindle are required to stabilize the position of the cleavage furrow in late anaphase and to complete furrow constriction. These results support the model that two spatially and temporally separate pathways control different key aspects during asymmetric cell division, ensuring correct cell fate determinant segregation and neuroblast self-renewal.

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Figures

Figure 1
Figure 1. Myosin is localized to the cleavage furrow prior to Survivin.
(a) Third instar larval neuroblasts were imaged along the xy axis (horizontal) or (c) en-face (vertical); intensity measurements were performed along a line through the midplane or at the level of the ingressing cleavage furrow, respectively. (b) and (d) Image sequence of a wild-type neuroblast expressing Survivin::GFP (green in overlay) and Sqh::mCherry (Myo; magenta in overlay). Intensity plots (Survivin, green; Myosin, magenta) are shown underneath the corresponding images. Intensity curves exceeding the scale were cut off (horizontal grey line). The vertical dashed lines refer to the position of the cortex. For all subsequent figures, coloured squares refer to the mitotic stage indicated below. Yellow and red arrowheads highlight the level of Survivin on the contractile ring and the central spindle, respectively. (e) Fluorescence intensity was measured for Survivin::GFP and Sqh::mCherry to determine the time when Survivin (green) reaches the cortical position of the future cleavage furrow in relation to Myosin (magenta; n=7). (f) Kymograph along the division axis. Note that towards the end of mitosis, Survivin shifts basally. The light green line and measurement point refer to Survivin appearing at the central spindle (CS). The dark green line and measurement point indicate Survivin shifting basally to the midbody (MB). Time in min:s; scale bar, 5 μm.
Figure 2
Figure 2. Cortical Survivin originates from the kinetochore.
(a) Image sequence of a representative wild-type neuroblast expressing Survivin::mDendra2. Top row shows unconverted (green), middle row photoconverted (white) Survivin::mDendra, respectively. Photoconversion (PC) was performed on the metaphase plate (yellow square). Red and yellow arrowheads refer to photoconverted Survivin at the central spindle and contractile ring, respectively. Schematic representation shown below. (b) Quantification of the photoconversion experiment. Number of scored neuroblasts is highlighted in bars. (c) FRAP experiments performed at the metaphase plate (yellow square) on Survivin::GFP (top row). Sqh::mCherry (Myo) was imaged to visualize the cortex and cleavage furrow. Dashed lines outline the neuroblast. (d) Quantification of the FRAP experiment. Number of scored neuroblasts is highlighted in bars. Coloured squares refer to the cell cycle stage as defined in Fig. 1. Time in min:s; scale bar, 5 μm.
Figure 3
Figure 3. Survivin’s relocalization depends on the spindle and anaphase entry.
(a) 3D-SIM images of third instar larval brain wild-type neuroblasts, expressing Survivin::GFP (green) and stained with α-Tubulin (magenta). High-magnification pictures of areas of interest (blue and orange arrows) are shown below the overview pictures. Coloured arrowheads highlight Survivin clusters in association with microtubules. (b) Image sequence of a representative rodH4.8 mutant neuroblast treated with colcemid, imaged with Survivin::GFP (top row) and the spindle marker mCherry::Jupiter (middle row). Schematic below; green dots represent Survivin molecules fading away. (c) Intensity measurements of Survivin::GFP in rodH4.8 and (d) wild-type neuroblasts treated with colcemid. The graph shows average intensity. Error bars correspond to s.d. (wt; n=10, rod and colcemid; n=8). (e) Image sequence of a representative dlgm52;;pinsP89 mutant neuroblast expressing Survivin::GFP (top row) and Sqh::mCherry (Myo; second row), dividing symmetrically. (f) Measured distance ratios between the indicated Survivin pools in wild-type, dlgm52;;pinsP89 and mud4 mutant neuroblasts. Number of scored cells are indicated in the grey box. Asterisk (*) denotes statistical significance. P=0.000023 (two-sample unequal variance t-test). NS, not significant; P>0.01 (based on two-sample equal or unequal variance t-test). Time in min:s; scale bar, 2 μm in panel (a) and 5 μm in all subsequent panels. wt, wild type.
Figure 4
Figure 4. Survivin’s localization is independent of the polarity pathway.
(a) Schematic representation of the genetic spindle rotation experiment to uncouple the orientation of the mitotic spindle in relation to the neuroblast intrinsic polarity axis (apical, blue; basal, red). (b) Image sequence of a representative mud4 mutant neuroblast expressing Survivin::GFP (top row) and Sqh::mCherry (Myo; second row), dividing symmetrically and forming a polar lobe (06:30; orange arrowhead). Blue arrowheads highlight Survivin localization at the spindle-induced furrow. (c) Quantification of Survivin appearance at polarity-dependent cleavage furrow (polar lobe). Number of cells scored is highlighted in bars. (d) Survivin was ectopically localized at the apical neuroblast cortex (green); intensity measurements were performed along the dashed yellow line from apical to basal. (e) Third instar larval neuroblast expressing ALD-Survivin::EGFP (top row; green) and Sqh::mCherry (bottom row; white). Intensity measurements (green, ALD-Survivin::EGFP; grey, sqh::mCherry) are shown below. (f) Quantification of ectopic furrowing and Myosin distribution. Number of cells scored is highlighted in bars. Ap, apical; Ba, basal. Time in min:s; scale bar, 5 μm.
Figure 5
Figure 5. Survivin is required for cleavage furrow constriction.
(a) Image sequence of a representative wild-type neuroblast expressing Sqh::GFP (green in overlay) and mCherry::Jupiter (white in overlay). (b) Quantification of wild-type neuroblast diameter measurements. (c) Constriction rates of wild-type neuroblasts. Averages and s.d. are shown (n=11). (d) Image sequence of a representative scpoz2775/Df(3R)5780 mutant neuroblast expressing Sqh::GFP (green in overlay) and mCherry::Jupiter. (e) Diameter measurements. The grey dashed line indicates the minimal diameter. (f) Corresponding constriction rates of scpoz2775/Df(3R)5780 mutant neuroblasts (n=8). Time in min:s; scale bar, 5 μm.
Figure 6
Figure 6. The mitotic spindle regulates furrow constriction through the CPC.
(a) Image sequence of a representative rodH4.8 mutant neuroblast treated with colcemid, expressing Sqh::GFP (green in overlay) and mCherry::Jupiter (white in overlay). The mitotic spindle is completely depolymerized. (b) Diameter measurements of rodH4.8 and colcemid neuroblasts. The grey dashed line indicates the minimal diameter. (c) Corresponding constriction rates of rodH4.8 and colcemid neuroblasts. Averages and s.d. are shown (n=15). (d) Image sequence of a sas4 mutant neuroblast, expressing Sqh::GFP (green in overlay) and mCherry::Jupiter (white in overlay). (e) Diameter measurements and (f) constriction rates of sas4 mutant neuroblasts (n=10). (g) Image sequence of a representative neuroblasts, expressing RNAi against aurB, Sqh::GFP (green in overlay) and mCherry::Jupiter (white in overlay). (h) Diameter measurements and (i) constriction rates (n=4). Time in min:s; scale bar, 5 μm.
Figure 7
Figure 7. The spindle-dependent pathway stabilizes furrow positioning.
Myosin intensity was measured along the neuroblast cortex (green gradient line) and averaged for wild-type, scpoz2775/Df(3R)5780 and dlgm52;;pinsP89 mutant neuroblasts at (a) metaphase, (b) anaphase and (c) telophase. Vertical dashed lines represent the forming cleavage furrow. Horizontal dashed lines indicate the difference between the lowest and the highest intensity values. These intensity differences are plotted in (d) for the indicated genotypes. Average values were derived from at least five neuroblasts. Error bars indicate s.d. (e) Cleavage furrow positioning was independently measured at the onset of furrowing for wild type (blue ball), dlg;;pins (green ball), scpo (orange ball) and rod and colcemid (brown ball). The A/B ratio (A, distance from the furrow to the apical cortex; B, distance from the furrow to the basal cortex) was plotted as a ratio in (f). Asterisk (*) denotes statistical significance. P=3.4 × 10−9 (two-sample equal variance t-test; wt vs dlg;;pins), P=0.00054 (two-sample equal variance t-test; wt vs scpo), P=0.00094 (two-sample unequal variance t-test; wt vs rod and colcemid). NS, not significant; P>0.01 (based on two-sample equal or unequal variance t-test). Dashed orange line outlines the cell boundaries. Dashed white line highlights the position of the cleavage furrow. Scale bar, 5 μm. wt, wild type.
Figure 8
Figure 8. Model.
(a) Schematic representation of Survivin’s (orange) dynamic redistribution during asymmetric cell division. By metaphase, the entire Survivin pool is associated with chromosomes and lines up at the metaphase plate. After anaphase onset, a fraction of this Survivin pool is loaded onto microtubules and redistributed to the central spindle as well as the forming furrow region, which is shifted towards the basal cortex. Microtubules decorated with Survivin (black lines), contact the cortex at the cleavage furrow, forming a Survivin ring (orange ring) just inside the Myosin-containing contractile ring (green ring). This Survivin ring lines up with the Survivin pool at the central spindle during late anaphase/telophase, and both pools contribute to the forming midbody. (b) Two pathways with distinct functions control the generation of physical and molecular asymmetry in Drosophila neuroblasts. (b) (1) The polarity-dependent pathway induces asymmetric Myosin localization via Pins and Dlg shortly after anaphase onset. The molecular composition of this pathway is not known. Asymmetric Myosin localization is required for basal cleavage furrow positioning. (c) The spindle-dependent pathway not only stabilizes the basal furrow position (2) but also controls cleavage furrow ingression and completion (3). The vertical dashed line indicates the polarity/division axis. Both pathways act spatially and temporally separate to generate a large, self-renewed neuroblast and a differentiating small GMC. The mitotic spindle is responsible to relocalize the CPC, which activates the centralspindlin complex (Pav/Tum). Whether the centralspindlin complex maintains Myosin constriction through Rho1 is unclear in this system. Other CPC targets could act redundantly to complete cytokinesis. Nb, neuroblast; GMC, ganglion mother cell.
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