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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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Compartmentalization of serine 1,2 phosphorylation. (A) L. pictus eggs were incubated in the presence of inorganic 32PO4 in phosphate-free artificial sea water. Zygotes were cultured up to metaphase of the first cell division, washed, and lysed in a buffer containing 0.5% NP-40 and protease and phosphatase inhibitors. Cortical cytoskeletons were removed by centrifugation, and myosin II was isolated from the supernatant by immunoprecipitation. Light chains were subjected to proteolysis and two dimensional phosphopeptide analysis. (A, panel A) LC20 immunoprecipitated from metaphase zygotes (77 min after fertilization). (A, panel B) LC20 phosphorylated in vitro with both PKC and MLCK to identify both activating and inhibitory phosphorylation sites. (B) Cortices were prepared from interphase zygotes (30 min after fertilization) and incubated with [γ-32P]ATP in the absence (lanes 1 and 2) or presence of purified p34cdc2 (lanes 3 and 4) or PKC (lanes 5 and 6) for 30 min. The suspension was then clarified by centrifugation, and the supernatant (lanes 1, 3, and 5) and pellet (cytoskeletal) fractions (lanes 2, 4, and 6) were analyzed by Western blotting and autoradiography.
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Figure 3: Compartmentalization of serine 1,2 phosphorylation. (A) L. pictus eggs were incubated in the presence of inorganic 32PO4 in phosphate-free artificial sea water. Zygotes were cultured up to metaphase of the first cell division, washed, and lysed in a buffer containing 0.5% NP-40 and protease and phosphatase inhibitors. Cortical cytoskeletons were removed by centrifugation, and myosin II was isolated from the supernatant by immunoprecipitation. Light chains were subjected to proteolysis and two dimensional phosphopeptide analysis. (A, panel A) LC20 immunoprecipitated from metaphase zygotes (77 min after fertilization). (A, panel B) LC20 phosphorylated in vitro with both PKC and MLCK to identify both activating and inhibitory phosphorylation sites. (B) Cortices were prepared from interphase zygotes (30 min after fertilization) and incubated with [γ-32P]ATP in the absence (lanes 1 and 2) or presence of purified p34cdc2 (lanes 3 and 4) or PKC (lanes 5 and 6) for 30 min. The suspension was then clarified by centrifugation, and the supernatant (lanes 1, 3, and 5) and pellet (cytoskeletal) fractions (lanes 2, 4, and 6) were analyzed by Western blotting and autoradiography.

Mentions: Although the increase in LC20 phosphorylation on activating residues is consistent with data from cultured cells where there is an increase in serine 19 phosphorylation upon anaphase onset (DeBiasio et al. 1996; Matsumura et al. 1998; Murata-Hori et al. 1998), we were surprised to find no evidence of serine 1,2 phosphorylation on light chains associated with cytoskeletal myosin heavy chain at any time during mitosis (Fig. 2), especially in light of our data regarding p34cdc2 activity associated with cytoskeleton (Fig. 1 A). Light chain phosphorylation on serine 1,2 has been detected in vivo in tissue culture cells arrested in mitosis using microtubule-destabilizing drugs (Yamakita, 1994), as well as Xenopus (Satterwhite, 1992) and sea urchin (Mishima and Mabuchi 1996; Totsukawa et al. 1996) extracts. This apparent discrepency might be explained by either: (a) the soluble and cytoskeletal myosin populations were subject to differential regulation; (b) the presence of a phosphatase activity that prevented our detection of cdc2 phosphorylation of light chain; or (c) a selective destabilization or solubilization of myosin filaments from the cortical cytoskeleton following serine 1,2 phosphorylation (Nishikawa et al., 1984; Bengur et al. 1987; Ikebe and Reardon 1990). To control for the first two possibilities, sea urchin eggs were labeled in vivo, samples collected from cultures during metaphase, and LC20 was immunoprecipitated from detergent-soluble supernatants using anti–myosin heavy chain antibodies and subjected to tryptic digestion and phosphopeptide analysis. As shown in Fig. 3 A, a major phosphopeptide could be detected in samples prepared from metaphase zygotes (Fig. 3 A, panel A) that comigrated with phosphopeptides derived from in vitro–phosphorylated LC20 (Fig. 3 A, panel B). A second, minor phosphopeptide could be detected comigrating with threonine 9–containing phosphopeptides. These results suggested that our experimental conditions did allow for the detection of serine 1,2 phosphorylation in vivo, and suggested that serine 1,2 modulation of myosin II activity may be dependent on the cytoplasmic compartment in which the myosin molecules reside.


Parameters that specify the timing of cytokinesis.

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

Compartmentalization of serine 1,2 phosphorylation. (A) L. pictus eggs were incubated in the presence of inorganic 32PO4 in phosphate-free artificial sea water. Zygotes were cultured up to metaphase of the first cell division, washed, and lysed in a buffer containing 0.5% NP-40 and protease and phosphatase inhibitors. Cortical cytoskeletons were removed by centrifugation, and myosin II was isolated from the supernatant by immunoprecipitation. Light chains were subjected to proteolysis and two dimensional phosphopeptide analysis. (A, panel A) LC20 immunoprecipitated from metaphase zygotes (77 min after fertilization). (A, panel B) LC20 phosphorylated in vitro with both PKC and MLCK to identify both activating and inhibitory phosphorylation sites. (B) Cortices were prepared from interphase zygotes (30 min after fertilization) and incubated with [γ-32P]ATP in the absence (lanes 1 and 2) or presence of purified p34cdc2 (lanes 3 and 4) or PKC (lanes 5 and 6) for 30 min. The suspension was then clarified by centrifugation, and the supernatant (lanes 1, 3, and 5) and pellet (cytoskeletal) fractions (lanes 2, 4, and 6) were analyzed by Western blotting and autoradiography.
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Figure 3: Compartmentalization of serine 1,2 phosphorylation. (A) L. pictus eggs were incubated in the presence of inorganic 32PO4 in phosphate-free artificial sea water. Zygotes were cultured up to metaphase of the first cell division, washed, and lysed in a buffer containing 0.5% NP-40 and protease and phosphatase inhibitors. Cortical cytoskeletons were removed by centrifugation, and myosin II was isolated from the supernatant by immunoprecipitation. Light chains were subjected to proteolysis and two dimensional phosphopeptide analysis. (A, panel A) LC20 immunoprecipitated from metaphase zygotes (77 min after fertilization). (A, panel B) LC20 phosphorylated in vitro with both PKC and MLCK to identify both activating and inhibitory phosphorylation sites. (B) Cortices were prepared from interphase zygotes (30 min after fertilization) and incubated with [γ-32P]ATP in the absence (lanes 1 and 2) or presence of purified p34cdc2 (lanes 3 and 4) or PKC (lanes 5 and 6) for 30 min. The suspension was then clarified by centrifugation, and the supernatant (lanes 1, 3, and 5) and pellet (cytoskeletal) fractions (lanes 2, 4, and 6) were analyzed by Western blotting and autoradiography.
Mentions: Although the increase in LC20 phosphorylation on activating residues is consistent with data from cultured cells where there is an increase in serine 19 phosphorylation upon anaphase onset (DeBiasio et al. 1996; Matsumura et al. 1998; Murata-Hori et al. 1998), we were surprised to find no evidence of serine 1,2 phosphorylation on light chains associated with cytoskeletal myosin heavy chain at any time during mitosis (Fig. 2), especially in light of our data regarding p34cdc2 activity associated with cytoskeleton (Fig. 1 A). Light chain phosphorylation on serine 1,2 has been detected in vivo in tissue culture cells arrested in mitosis using microtubule-destabilizing drugs (Yamakita, 1994), as well as Xenopus (Satterwhite, 1992) and sea urchin (Mishima and Mabuchi 1996; Totsukawa et al. 1996) extracts. This apparent discrepency might be explained by either: (a) the soluble and cytoskeletal myosin populations were subject to differential regulation; (b) the presence of a phosphatase activity that prevented our detection of cdc2 phosphorylation of light chain; or (c) a selective destabilization or solubilization of myosin filaments from the cortical cytoskeleton following serine 1,2 phosphorylation (Nishikawa et al., 1984; Bengur et al. 1987; Ikebe and Reardon 1990). To control for the first two possibilities, sea urchin eggs were labeled in vivo, samples collected from cultures during metaphase, and LC20 was immunoprecipitated from detergent-soluble supernatants using anti–myosin heavy chain antibodies and subjected to tryptic digestion and phosphopeptide analysis. As shown in Fig. 3 A, a major phosphopeptide could be detected in samples prepared from metaphase zygotes (Fig. 3 A, panel A) that comigrated with phosphopeptides derived from in vitro–phosphorylated LC20 (Fig. 3 A, panel B). A second, minor phosphopeptide could be detected comigrating with threonine 9–containing phosphopeptides. These results suggested that our experimental conditions did allow for the detection of serine 1,2 phosphorylation in vivo, and suggested that serine 1,2 modulation of myosin II activity may be dependent on the cytoplasmic compartment in which the myosin molecules reside.

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
Related in: MedlinePlus