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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

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.
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f2: 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.

Mentions: In order to address these questions, we first explored the origin of the three Survivin pools and the mechanism by which they reach these subcellular locations. For instance, cleavage furrow-associated Survivin could originate from the metaphase centromere/kinetochore-bound fraction. Alternatively, Survivin could be recruited from the cytoplasm to specific subcellular sites during anaphase. To distinguish between these two scenarios, we tagged Survivin with the photoconvertable fluorescent protein mDendra2 and performed in vivo pulse-chase labelling experiments (see Methods). Photoconverting Survivin at the metaphase midplane with a short pulse of ultraviolet light resulted in an immediate and robust change in emission that could be traced from metaphase onwards until telophase. We found that photoconverted Survivin was localized on separating kinetochores, the central spindle population and in the furrow region (Fig. 2a,b), indicating that the Survivin molecules accumulating at the midbody by telophase, originated from the kinetochores in metaphase (100%, n=20). However, the possibility remained that newly synthesized or cytosolic Survivin relocalized to the cleavage furrow. We tested this using FRAP and bleached Survivin::GFP at the metaphase plate. In most cases, no signal was detectable at the midbody after complete quenching of Survivin::GFP (75%, n=24; Fig. 2c,d). Similarly, photoconversion experiments targeting the cytoplasm did not result in any detectable signal at the metaphase plate nor at the midbody in telophase (100%; n=6; Supplementary Fig. 2a,b). Thus, we can conclude the following: the three Survivin populations all originate from metaphase kinetochore-bound Survivin and that from anaphase onset onwards no new Survivin molecules are recruited to kinetochores, the contractile ring and the central spindle. Furthermore, these data suggest that Survivin turn-over rates are very low from metaphase onwards until telophase.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4544045&req=5

f2: 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.
Mentions: In order to address these questions, we first explored the origin of the three Survivin pools and the mechanism by which they reach these subcellular locations. For instance, cleavage furrow-associated Survivin could originate from the metaphase centromere/kinetochore-bound fraction. Alternatively, Survivin could be recruited from the cytoplasm to specific subcellular sites during anaphase. To distinguish between these two scenarios, we tagged Survivin with the photoconvertable fluorescent protein mDendra2 and performed in vivo pulse-chase labelling experiments (see Methods). Photoconverting Survivin at the metaphase midplane with a short pulse of ultraviolet light resulted in an immediate and robust change in emission that could be traced from metaphase onwards until telophase. We found that photoconverted Survivin was localized on separating kinetochores, the central spindle population and in the furrow region (Fig. 2a,b), indicating that the Survivin molecules accumulating at the midbody by telophase, originated from the kinetochores in metaphase (100%, n=20). However, the possibility remained that newly synthesized or cytosolic Survivin relocalized to the cleavage furrow. We tested this using FRAP and bleached Survivin::GFP at the metaphase plate. In most cases, no signal was detectable at the midbody after complete quenching of Survivin::GFP (75%, n=24; Fig. 2c,d). Similarly, photoconversion experiments targeting the cytoplasm did not result in any detectable signal at the metaphase plate nor at the midbody in telophase (100%; n=6; Supplementary Fig. 2a,b). Thus, we can conclude the following: the three Survivin populations all originate from metaphase kinetochore-bound Survivin and that from anaphase onset onwards no new Survivin molecules are recruited to kinetochores, the contractile ring and the central spindle. Furthermore, these data suggest that Survivin turn-over rates are very low from metaphase onwards until telophase.

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