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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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Spindle folding generated a concentrated source of actin for building a new contractile ring.(A) The central spindle can be folded using a microneedle, before furrow formation. Color scheme, as in Fig. 1A. (B) Polarization microscopy sequence (Video S2). After folding (0 min), the mechanically generated monopolar spindle reorganized into a bipolar spindle. Microtubules from the pair of spindle poles radiated toward the cell cortex (11 min, arrows) where a furrow initiated (23 min, arrows). The furrow ultimately ingressed at the equator of the spindle (37–52 min), after realignment of the spindle midzone. (C) Distribution of microtubules (green), actin filaments (red), and chromosomes (blue) in cells fixed at stages similar to those in (B). Upon folding of the spindle by micromanipulation, the deformed midzone (a, arrow) and remnants of the contractile ring (e and i) remained relatively organized. As central spindle microtubules reorganized, the original midzone disappeared (b). Concurrently, actin filaments dispersed from the disintegrating ring, oriented in apparent alignment with central spindle microtubules (f and j). Meanwhile, a few very short microtubule bundles (b, arrowheads) emerged at the spindle tip and initiated formation of a new midzone, transverse to the new spindle axis (b, arrow). Around the time of furrow initiation, reorganization of the elongated bundles created a new “pole” (c), thereby establishing a bipolar spindle with a broad midzone (c, arrows). Actin filaments appeared to disassociate from spindle microtubules as they entered the new contractile ring (g and k). The furrow ingressed as microtubules at the distal pole elongated (c–d, green), shifting the midzone (c–d, arrows) and the contractile ring (g–h and k–l) toward the spindle equator. Bars, 10 µm.
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pone-0004892-g002: Spindle folding generated a concentrated source of actin for building a new contractile ring.(A) The central spindle can be folded using a microneedle, before furrow formation. Color scheme, as in Fig. 1A. (B) Polarization microscopy sequence (Video S2). After folding (0 min), the mechanically generated monopolar spindle reorganized into a bipolar spindle. Microtubules from the pair of spindle poles radiated toward the cell cortex (11 min, arrows) where a furrow initiated (23 min, arrows). The furrow ultimately ingressed at the equator of the spindle (37–52 min), after realignment of the spindle midzone. (C) Distribution of microtubules (green), actin filaments (red), and chromosomes (blue) in cells fixed at stages similar to those in (B). Upon folding of the spindle by micromanipulation, the deformed midzone (a, arrow) and remnants of the contractile ring (e and i) remained relatively organized. As central spindle microtubules reorganized, the original midzone disappeared (b). Concurrently, actin filaments dispersed from the disintegrating ring, oriented in apparent alignment with central spindle microtubules (f and j). Meanwhile, a few very short microtubule bundles (b, arrowheads) emerged at the spindle tip and initiated formation of a new midzone, transverse to the new spindle axis (b, arrow). Around the time of furrow initiation, reorganization of the elongated bundles created a new “pole” (c), thereby establishing a bipolar spindle with a broad midzone (c, arrows). Actin filaments appeared to disassociate from spindle microtubules as they entered the new contractile ring (g and k). The furrow ingressed as microtubules at the distal pole elongated (c–d, green), shifting the midzone (c–d, arrows) and the contractile ring (g–h and k–l) toward the spindle equator. Bars, 10 µm.

Mentions: In a variation on our spindle-collapsing experiment (Fig. 1), we folded a telophase spindle to induce actin redistribution into a new ring, around a reassembled spindle (Fig. 2). Although both operations brought the two spindle poles into proximity, collapsing did so by pushing segregating anaphase chromosomes back together, whereas folding bent the spindle at its equator to swing the segregated telophase chromosomes toward each other (compare Figs. 1A and 2A). Note that part of the assembling contractile ring was destroyed as the spindle equator was pushed away from the cortex by a microneedle (Fig. 2A, b). We reasoned that folding the spindle would enhance visualization of actin redistribution, because the remnants of the original ring would serve as a more concentrated source of actin, available for building the new ring. By scaling up redistribution of actin, we hoped to gain insight as to how microtubules move actin into the contractile ring.


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)

Spindle folding generated a concentrated source of actin for building a new contractile ring.(A) The central spindle can be folded using a microneedle, before furrow formation. Color scheme, as in Fig. 1A. (B) Polarization microscopy sequence (Video S2). After folding (0 min), the mechanically generated monopolar spindle reorganized into a bipolar spindle. Microtubules from the pair of spindle poles radiated toward the cell cortex (11 min, arrows) where a furrow initiated (23 min, arrows). The furrow ultimately ingressed at the equator of the spindle (37–52 min), after realignment of the spindle midzone. (C) Distribution of microtubules (green), actin filaments (red), and chromosomes (blue) in cells fixed at stages similar to those in (B). Upon folding of the spindle by micromanipulation, the deformed midzone (a, arrow) and remnants of the contractile ring (e and i) remained relatively organized. As central spindle microtubules reorganized, the original midzone disappeared (b). Concurrently, actin filaments dispersed from the disintegrating ring, oriented in apparent alignment with central spindle microtubules (f and j). Meanwhile, a few very short microtubule bundles (b, arrowheads) emerged at the spindle tip and initiated formation of a new midzone, transverse to the new spindle axis (b, arrow). Around the time of furrow initiation, reorganization of the elongated bundles created a new “pole” (c), thereby establishing a bipolar spindle with a broad midzone (c, arrows). Actin filaments appeared to disassociate from spindle microtubules as they entered the new contractile ring (g and k). The furrow ingressed as microtubules at the distal pole elongated (c–d, green), shifting the midzone (c–d, arrows) and the contractile ring (g–h and k–l) toward the spindle equator. Bars, 10 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654139&req=5

pone-0004892-g002: Spindle folding generated a concentrated source of actin for building a new contractile ring.(A) The central spindle can be folded using a microneedle, before furrow formation. Color scheme, as in Fig. 1A. (B) Polarization microscopy sequence (Video S2). After folding (0 min), the mechanically generated monopolar spindle reorganized into a bipolar spindle. Microtubules from the pair of spindle poles radiated toward the cell cortex (11 min, arrows) where a furrow initiated (23 min, arrows). The furrow ultimately ingressed at the equator of the spindle (37–52 min), after realignment of the spindle midzone. (C) Distribution of microtubules (green), actin filaments (red), and chromosomes (blue) in cells fixed at stages similar to those in (B). Upon folding of the spindle by micromanipulation, the deformed midzone (a, arrow) and remnants of the contractile ring (e and i) remained relatively organized. As central spindle microtubules reorganized, the original midzone disappeared (b). Concurrently, actin filaments dispersed from the disintegrating ring, oriented in apparent alignment with central spindle microtubules (f and j). Meanwhile, a few very short microtubule bundles (b, arrowheads) emerged at the spindle tip and initiated formation of a new midzone, transverse to the new spindle axis (b, arrow). Around the time of furrow initiation, reorganization of the elongated bundles created a new “pole” (c), thereby establishing a bipolar spindle with a broad midzone (c, arrows). Actin filaments appeared to disassociate from spindle microtubules as they entered the new contractile ring (g and k). The furrow ingressed as microtubules at the distal pole elongated (c–d, green), shifting the midzone (c–d, arrows) and the contractile ring (g–h and k–l) toward the spindle equator. Bars, 10 µm.
Mentions: In a variation on our spindle-collapsing experiment (Fig. 1), we folded a telophase spindle to induce actin redistribution into a new ring, around a reassembled spindle (Fig. 2). Although both operations brought the two spindle poles into proximity, collapsing did so by pushing segregating anaphase chromosomes back together, whereas folding bent the spindle at its equator to swing the segregated telophase chromosomes toward each other (compare Figs. 1A and 2A). Note that part of the assembling contractile ring was destroyed as the spindle equator was pushed away from the cortex by a microneedle (Fig. 2A, b). We reasoned that folding the spindle would enhance visualization of actin redistribution, because the remnants of the original ring would serve as a more concentrated source of actin, available for building the new ring. By scaling up redistribution of actin, we hoped to gain insight as to how microtubules move actin into the contractile ring.

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