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

Roth M, Roubinet C, Iffländer N, Ferrand A, Cabernard C - Nat Commun (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f3: 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.

Mentions: Next, we investigated how kinetochore-bound Survivin reaches the contractile ring. Previous reports suggest that Survivin’s dynamic redistribution depends on microtubules (reviewed in ref. 10). To test this, we first used super-resolution microscopy to image larval brains expressing Survivin::GFP and stained with anti-αTubulin (see Methods). Survivin::GFP accurately reflects Survivin’s localization but provides better signal-to-noise ratio compared with the anti-Survivin antibody18 (Supplementary Fig. 3a,b). We found that Survivin clusters generally colocalized with microtubule fibres. For instance, Survivin was associated with MTs forming the central spindle. Furthermore, we detected Survivin clusters, colocalizing with MTs extending towards the furrow region in anaphase and telophase cells (Fig. 3a).


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

Roth M, Roubinet C, Iffländer N, Ferrand A, Cabernard C - Nat Commun (2015)

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.
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f3: 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.
Mentions: Next, we investigated how kinetochore-bound Survivin reaches the contractile ring. Previous reports suggest that Survivin’s dynamic redistribution depends on microtubules (reviewed in ref. 10). To test this, we first used super-resolution microscopy to image larval brains expressing Survivin::GFP and stained with anti-αTubulin (see Methods). Survivin::GFP accurately reflects Survivin’s localization but provides better signal-to-noise ratio compared with the anti-Survivin antibody18 (Supplementary Fig. 3a,b). We found that Survivin clusters generally colocalized with microtubule fibres. For instance, Survivin was associated with MTs forming the central spindle. Furthermore, we detected Survivin clusters, colocalizing with MTs extending towards the furrow region in anaphase and telophase cells (Fig. 3a).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland.

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.

No MeSH data available.


Related in: MedlinePlus