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Parameters that specify the timing of cytokinesis.

Shuster CB, Burgess DR - J. Cell Biol. (1999)

Bottom Line: To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division.We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin.These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.

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Mitotic arrest and needle displacement in Δ90 cyclin-injected sand dollar embryos. Zygotes were cultured at 15°C through the first division. Shortly before prophase of the second cell division, one blastomere was injected with Δ90 cyclin B and marked by a small oil droplet. 35 min after the second division of the control blastomere (at 150 min after fertilization), small asters remain visible in the Δ90-injected embryo, and a third aster is visible, most likely the result of spindle pole splitting (Hinchcliffe, 1998) (A). Needles were then placed across the surface, isolating two aster centers and forcing them into a confined region of cytoplasm (B). 5 min after the initial displacement, a unilateral furrow becomes visible, which continues to ingress after one needle is removed (C and D). The control blastomeres went on to divide 10 min later. Dots indicate the position of the aster centers in the injected blastomere. Bar, 30 μm.
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Figure 4: Mitotic arrest and needle displacement in Δ90 cyclin-injected sand dollar embryos. Zygotes were cultured at 15°C through the first division. Shortly before prophase of the second cell division, one blastomere was injected with Δ90 cyclin B and marked by a small oil droplet. 35 min after the second division of the control blastomere (at 150 min after fertilization), small asters remain visible in the Δ90-injected embryo, and a third aster is visible, most likely the result of spindle pole splitting (Hinchcliffe, 1998) (A). Needles were then placed across the surface, isolating two aster centers and forcing them into a confined region of cytoplasm (B). 5 min after the initial displacement, a unilateral furrow becomes visible, which continues to ingress after one needle is removed (C and D). The control blastomeres went on to divide 10 min later. Dots indicate the position of the aster centers in the injected blastomere. Bar, 30 μm.

Mentions: Results of in vivo labeling and phosphopeptide mapping suggest that despite the enriched and extended levels of p34cdc2 activity associated with the cortex, cytoskeletal myosin II was not subject to a light chain-based negative regulation during mitosis. In an effort to directly assess the light chain–based model for the timing of cytokinesis in vivo, we asked whether the cortex could be induced to form a cleavage furrow in the presence of chronically extended p34cdc2 activity. Towards these ends, a truncated, nondegradable form of cyclin B was produced in Escherichia coli (Δ90 cyclin) (Murray et al. 1989; Glotzer et al. 1991). Nondegradable forms of cyclin B have been introduced in cultured cells as well as Xenopus and sea urchin eggs (Murray et al. 1989; Wheatley et al. 1997; Hinchcliffe, 1998). In each case, chromatin remains condensed, and cleavage is arrested. However, because there are dramatic effects on microtubule dynamics and spindle behavior in Δ90 cyclin–arrested cells, it is difficult to attribute the inhibition of cytokinesis to either a suppression of myosin II–based contractility or a failure to deliver the cleavage stimulus to the cortex. To differentiate between these possibilities, recombinant Δ90 cyclin was produced in bacteria, purified to homogeneity, and tested in Xenopus cell–free extracts for its ability to arrest cycling extracts in a mitotic state (data not shown). Δ90 cyclin was then concentrated and injected into blastomeres of two cell E. parma embryos. Injection of Δ90 into blastomeres shortly before nuclear envelope breakdown resulted in mitotic arrest in 81% of the cells injected (n = 42). In six cases where Δ90 cyclin was injected during metaphase, cells divided but arrested in the following cell cycle. In contrast, 93% of cells injected with buffer alone (n = 31) went on to develop past the mesenchymal blastula stage. Examination of injected cells revealed that although injected blastomeres failed to divide, the mitotic apparatus was still visible and spindle poles underwent an anaphase B–like separation as reported previously (Holloway et al. 1993; Wheatley et al. 1997; Hinchcliffe, 1998), and in some cases, the spindle poles split to form three or four individual aster centers (Fig. 4 A). Arrested blastomeres remained viable for up to 3 h, at which time the cells underwent membrane blebbing, and died soon after (Hinchcliffe et al. 1998). Vital staining with Hoescht No. 33342 revealed that throughout this period, chromatin remained condensed.


Parameters that specify the timing of cytokinesis.

Shuster CB, Burgess DR - J. Cell Biol. (1999)

Mitotic arrest and needle displacement in Δ90 cyclin-injected sand dollar embryos. Zygotes were cultured at 15°C through the first division. Shortly before prophase of the second cell division, one blastomere was injected with Δ90 cyclin B and marked by a small oil droplet. 35 min after the second division of the control blastomere (at 150 min after fertilization), small asters remain visible in the Δ90-injected embryo, and a third aster is visible, most likely the result of spindle pole splitting (Hinchcliffe, 1998) (A). Needles were then placed across the surface, isolating two aster centers and forcing them into a confined region of cytoplasm (B). 5 min after the initial displacement, a unilateral furrow becomes visible, which continues to ingress after one needle is removed (C and D). The control blastomeres went on to divide 10 min later. Dots indicate the position of the aster centers in the injected blastomere. Bar, 30 μm.
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Related In: Results  -  Collection

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Figure 4: Mitotic arrest and needle displacement in Δ90 cyclin-injected sand dollar embryos. Zygotes were cultured at 15°C through the first division. Shortly before prophase of the second cell division, one blastomere was injected with Δ90 cyclin B and marked by a small oil droplet. 35 min after the second division of the control blastomere (at 150 min after fertilization), small asters remain visible in the Δ90-injected embryo, and a third aster is visible, most likely the result of spindle pole splitting (Hinchcliffe, 1998) (A). Needles were then placed across the surface, isolating two aster centers and forcing them into a confined region of cytoplasm (B). 5 min after the initial displacement, a unilateral furrow becomes visible, which continues to ingress after one needle is removed (C and D). The control blastomeres went on to divide 10 min later. Dots indicate the position of the aster centers in the injected blastomere. Bar, 30 μm.
Mentions: Results of in vivo labeling and phosphopeptide mapping suggest that despite the enriched and extended levels of p34cdc2 activity associated with the cortex, cytoskeletal myosin II was not subject to a light chain-based negative regulation during mitosis. In an effort to directly assess the light chain–based model for the timing of cytokinesis in vivo, we asked whether the cortex could be induced to form a cleavage furrow in the presence of chronically extended p34cdc2 activity. Towards these ends, a truncated, nondegradable form of cyclin B was produced in Escherichia coli (Δ90 cyclin) (Murray et al. 1989; Glotzer et al. 1991). Nondegradable forms of cyclin B have been introduced in cultured cells as well as Xenopus and sea urchin eggs (Murray et al. 1989; Wheatley et al. 1997; Hinchcliffe, 1998). In each case, chromatin remains condensed, and cleavage is arrested. However, because there are dramatic effects on microtubule dynamics and spindle behavior in Δ90 cyclin–arrested cells, it is difficult to attribute the inhibition of cytokinesis to either a suppression of myosin II–based contractility or a failure to deliver the cleavage stimulus to the cortex. To differentiate between these possibilities, recombinant Δ90 cyclin was produced in bacteria, purified to homogeneity, and tested in Xenopus cell–free extracts for its ability to arrest cycling extracts in a mitotic state (data not shown). Δ90 cyclin was then concentrated and injected into blastomeres of two cell E. parma embryos. Injection of Δ90 into blastomeres shortly before nuclear envelope breakdown resulted in mitotic arrest in 81% of the cells injected (n = 42). In six cases where Δ90 cyclin was injected during metaphase, cells divided but arrested in the following cell cycle. In contrast, 93% of cells injected with buffer alone (n = 31) went on to develop past the mesenchymal blastula stage. Examination of injected cells revealed that although injected blastomeres failed to divide, the mitotic apparatus was still visible and spindle poles underwent an anaphase B–like separation as reported previously (Holloway et al. 1993; Wheatley et al. 1997; Hinchcliffe, 1998), and in some cases, the spindle poles split to form three or four individual aster centers (Fig. 4 A). Arrested blastomeres remained viable for up to 3 h, at which time the cells underwent membrane blebbing, and died soon after (Hinchcliffe et al. 1998). Vital staining with Hoescht No. 33342 revealed that throughout this period, chromatin remained condensed.

Bottom Line: To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division.We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin.These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.

Show MeSH