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Redistribution of actin during assembly and reassembly of the contractile ring in grasshopper spermatocytes.

Alsop GB, Chen W, Foss M, Tseng KF, Zhang D - PLoS ONE (2009)

Bottom Line: The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin.Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force.Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oregon State University, Corvallis, OR, USA.

ABSTRACT
Cytokinesis in animal cells requires the assembly of an actomyosin contractile ring to cleave the cell. The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin. To investigate this process in grasshopper spermatocytes, we mechanically manipulated the spindle to induce actin redistribution into ectopic contractile rings, around reassembled lateral spindles. To enhance visualization of actin, we folded the spindle at its equator to convert the remnants of the partially assembled ring into a concentrated source of actin. Filaments from the disintegrating ring aligned along reorganizing spindle microtubules, suggesting that their incorporation into the new ring was mediated by microtubules. We tracked incorporation by speckling actin filaments with Qdots and/or labeling them with Alexa 488-phalloidin. The pattern of movement implied that actin was transported along spindle microtubules, before entering the ring. By double-labeling dividing cells, we imaged actin filaments moving along microtubules near the contractile ring. Together, our findings indicate that in one mechanism of actin redistribution, actin filaments are transported along spindle microtubule tracks in a plus-end-directed fashion. After reaching the spindle midzone, the filaments could be transported laterally to the ring. Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force. Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules.

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Cortical flow and de novo synthesis of actin filaments in grasshopper spermatocytes.(A) Time-lapse sequence of actin filaments undergoing cortical flow (from Video S11). Flow of actin was induced by collapsing the spindle and repositioning it near one side of the cortex. The final location of the spindle is marked by “sp”. Actin filaments were labeled by microinjection with Alexa 488 phalloidin. Note the clearing over time of the region closest to the spindle, as the actin flows to the cortex on the opposite side of the cell. Spindle-associated mitochondria were seen as a pair of large bright patches (labeled “m”) that autofluoresced in the FITC channel. Because mitochondria were localized to the cytoplasm, their displacement was not related to cortical flow, but rather was driven by the elongation of dynamic microtubules with which they were associated. Time in minutes. (B) A confocal micrograph showing actin aggregates (red, marked by arrows) localized to the tips of bundled microtubules (green) at the spindle midzone. These aggregates were strikingly similar in their location, timing of appearance and morphology to aggregates found in silkworm spermatocytes [17], which have been shown to be assembled de novo at the midzone. Actin was labeled by microinjection of rhodamine phalloidin during anaphase, and microtubules were labeled with paclitaxel green. A cell division scar (red circle, lower edge of cell) was visible. Bars, 10 µm.
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pone-0004892-g008: Cortical flow and de novo synthesis of actin filaments in grasshopper spermatocytes.(A) Time-lapse sequence of actin filaments undergoing cortical flow (from Video S11). Flow of actin was induced by collapsing the spindle and repositioning it near one side of the cortex. The final location of the spindle is marked by “sp”. Actin filaments were labeled by microinjection with Alexa 488 phalloidin. Note the clearing over time of the region closest to the spindle, as the actin flows to the cortex on the opposite side of the cell. Spindle-associated mitochondria were seen as a pair of large bright patches (labeled “m”) that autofluoresced in the FITC channel. Because mitochondria were localized to the cytoplasm, their displacement was not related to cortical flow, but rather was driven by the elongation of dynamic microtubules with which they were associated. Time in minutes. (B) A confocal micrograph showing actin aggregates (red, marked by arrows) localized to the tips of bundled microtubules (green) at the spindle midzone. These aggregates were strikingly similar in their location, timing of appearance and morphology to aggregates found in silkworm spermatocytes [17], which have been shown to be assembled de novo at the midzone. Actin was labeled by microinjection of rhodamine phalloidin during anaphase, and microtubules were labeled with paclitaxel green. A cell division scar (red circle, lower edge of cell) was visible. Bars, 10 µm.

Mentions: In addition to transport of actin along microtubules as one mechanism for redistributing actin toward the contractile ring, actin redistribution in grasshopper spermatocytes could occur by cortical flow as it does in silkworm spermatocytes [17] and cultured mammalian cells [18], [31]. A comparison of panels e–g (Fig. 2C) with panel h suggests the existence of cortical flow, as a significant clearing of actin (presumably cortical) occurred from the region outside of the cortical ring. To conclusively demonstrate the existence of cortical flow in grasshopper spermatocytes, we repeated an experiment previously performed in silkworm spermatocytes [17]. By collapsing the spindle with a microneedle and repositioning it near the cortex, we induced cortical flow of Alexa 488 phalloidin-labeled actin away from the spindle (Fig. 8A; Video S11; n = 10). The movement of actin filaments, either individual or bundled, was readily apparent in a representative cell (panel 8A). The pair of large bright patches (labeled “m”) was generated by two bundles of spindle-associated mitochondria, which autofluoresced in the FITC channel and shifted when microtubules in the reorganizing spindle elongated to drive cortical flow. Thus, even though the mitochondria also moved away from the spindle, their motion was associated only indirectly with cortical flow.


Redistribution of actin during assembly and reassembly of the contractile ring in grasshopper spermatocytes.

Alsop GB, Chen W, Foss M, Tseng KF, Zhang D - PLoS ONE (2009)

Cortical flow and de novo synthesis of actin filaments in grasshopper spermatocytes.(A) Time-lapse sequence of actin filaments undergoing cortical flow (from Video S11). Flow of actin was induced by collapsing the spindle and repositioning it near one side of the cortex. The final location of the spindle is marked by “sp”. Actin filaments were labeled by microinjection with Alexa 488 phalloidin. Note the clearing over time of the region closest to the spindle, as the actin flows to the cortex on the opposite side of the cell. Spindle-associated mitochondria were seen as a pair of large bright patches (labeled “m”) that autofluoresced in the FITC channel. Because mitochondria were localized to the cytoplasm, their displacement was not related to cortical flow, but rather was driven by the elongation of dynamic microtubules with which they were associated. Time in minutes. (B) A confocal micrograph showing actin aggregates (red, marked by arrows) localized to the tips of bundled microtubules (green) at the spindle midzone. These aggregates were strikingly similar in their location, timing of appearance and morphology to aggregates found in silkworm spermatocytes [17], which have been shown to be assembled de novo at the midzone. Actin was labeled by microinjection of rhodamine phalloidin during anaphase, and microtubules were labeled with paclitaxel green. A cell division scar (red circle, lower edge of cell) was visible. Bars, 10 µm.
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Related In: Results  -  Collection

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pone-0004892-g008: Cortical flow and de novo synthesis of actin filaments in grasshopper spermatocytes.(A) Time-lapse sequence of actin filaments undergoing cortical flow (from Video S11). Flow of actin was induced by collapsing the spindle and repositioning it near one side of the cortex. The final location of the spindle is marked by “sp”. Actin filaments were labeled by microinjection with Alexa 488 phalloidin. Note the clearing over time of the region closest to the spindle, as the actin flows to the cortex on the opposite side of the cell. Spindle-associated mitochondria were seen as a pair of large bright patches (labeled “m”) that autofluoresced in the FITC channel. Because mitochondria were localized to the cytoplasm, their displacement was not related to cortical flow, but rather was driven by the elongation of dynamic microtubules with which they were associated. Time in minutes. (B) A confocal micrograph showing actin aggregates (red, marked by arrows) localized to the tips of bundled microtubules (green) at the spindle midzone. These aggregates were strikingly similar in their location, timing of appearance and morphology to aggregates found in silkworm spermatocytes [17], which have been shown to be assembled de novo at the midzone. Actin was labeled by microinjection of rhodamine phalloidin during anaphase, and microtubules were labeled with paclitaxel green. A cell division scar (red circle, lower edge of cell) was visible. Bars, 10 µm.
Mentions: In addition to transport of actin along microtubules as one mechanism for redistributing actin toward the contractile ring, actin redistribution in grasshopper spermatocytes could occur by cortical flow as it does in silkworm spermatocytes [17] and cultured mammalian cells [18], [31]. A comparison of panels e–g (Fig. 2C) with panel h suggests the existence of cortical flow, as a significant clearing of actin (presumably cortical) occurred from the region outside of the cortical ring. To conclusively demonstrate the existence of cortical flow in grasshopper spermatocytes, we repeated an experiment previously performed in silkworm spermatocytes [17]. By collapsing the spindle with a microneedle and repositioning it near the cortex, we induced cortical flow of Alexa 488 phalloidin-labeled actin away from the spindle (Fig. 8A; Video S11; n = 10). The movement of actin filaments, either individual or bundled, was readily apparent in a representative cell (panel 8A). The pair of large bright patches (labeled “m”) was generated by two bundles of spindle-associated mitochondria, which autofluoresced in the FITC channel and shifted when microtubules in the reorganizing spindle elongated to drive cortical flow. Thus, even though the mitochondria also moved away from the spindle, their motion was associated only indirectly with cortical flow.

Bottom Line: The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin.Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force.Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oregon State University, Corvallis, OR, USA.

ABSTRACT
Cytokinesis in animal cells requires the assembly of an actomyosin contractile ring to cleave the cell. The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin. To investigate this process in grasshopper spermatocytes, we mechanically manipulated the spindle to induce actin redistribution into ectopic contractile rings, around reassembled lateral spindles. To enhance visualization of actin, we folded the spindle at its equator to convert the remnants of the partially assembled ring into a concentrated source of actin. Filaments from the disintegrating ring aligned along reorganizing spindle microtubules, suggesting that their incorporation into the new ring was mediated by microtubules. We tracked incorporation by speckling actin filaments with Qdots and/or labeling them with Alexa 488-phalloidin. The pattern of movement implied that actin was transported along spindle microtubules, before entering the ring. By double-labeling dividing cells, we imaged actin filaments moving along microtubules near the contractile ring. Together, our findings indicate that in one mechanism of actin redistribution, actin filaments are transported along spindle microtubule tracks in a plus-end-directed fashion. After reaching the spindle midzone, the filaments could be transported laterally to the ring. Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force. Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules.

Show MeSH
Related in: MedlinePlus