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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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In vivo analysis of cortical myosin light chain phosphorylation. (A–D) L. pictus eggs were incubated in the presence of 32PO4 in phosphate-free artificial sea water. Cells were monitored with interference contrast optics, and at time points when the cells were in (A) prophase, (B) metaphase, (C) anaphase, and (D) telophase (69, 78, 85, and 95 min after fertilization, respectively). Zygotes were washed and cortical cytoskeletons prepared from each time point. Cortical protein was resolved by SDS-PAGE, and light chains were excised, and digested with TPCK-trypsin. Digests were then washed and subjected to two-dimensional peptide analysis on cellulose TLC plates. The designations of the phosphopeptides are based on Satterwhite (1992). (E and F) Purified myosin II was phosphorylated in vitro using purified PKC (E) or MLCK (F). Peptide digests of cortical LC20 labeled in vivo were analyzed either alone (G) or mixed with equal counts of LC20 phosphorylated in vitro with MLCK (H).
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Figure 2: In vivo analysis of cortical myosin light chain phosphorylation. (A–D) L. pictus eggs were incubated in the presence of 32PO4 in phosphate-free artificial sea water. Cells were monitored with interference contrast optics, and at time points when the cells were in (A) prophase, (B) metaphase, (C) anaphase, and (D) telophase (69, 78, 85, and 95 min after fertilization, respectively). Zygotes were washed and cortical cytoskeletons prepared from each time point. Cortical protein was resolved by SDS-PAGE, and light chains were excised, and digested with TPCK-trypsin. Digests were then washed and subjected to two-dimensional peptide analysis on cellulose TLC plates. The designations of the phosphopeptides are based on Satterwhite (1992). (E and F) Purified myosin II was phosphorylated in vitro using purified PKC (E) or MLCK (F). Peptide digests of cortical LC20 labeled in vivo were analyzed either alone (G) or mixed with equal counts of LC20 phosphorylated in vitro with MLCK (H).

Mentions: Results of H1 kinase assays indicated that cytoskeletal-associated p34cdc2 activity cycled with kinetics delayed with respect to whole cell levels, and that this delayed activity extended into anaphase and cleavage. In light of data suggesting that p34cdc2 may regulate the timing of cytokinesis through the modulation of myosin II activity (Satterwhite et al. 1992; Yamakita et al. 1994), we asked whether the extended p34cdc2 activity is reflected in vivo by myosin regulatory light chain (LC20) phosphorylation in the cortical cytoskeleton of sea urchin embryos. Smooth muscle and cytoplasmic myosin light chains may be phosphorylated on five residues: serines 1 and 2, threonine 9, threonine 18, and serine 19 (for reviews see Sellers 1991; Satterwhite and Pollard 1992; Bresnick 1999). Whereas PKC phosphorylates LC20 on serines 1 and 2 and threonine 9 (Nishikawa et al., 1984; Bengur et al. 1987; Ikebe and Reardon 1990), p34cdc2 phosphorylates serines 1 and 2 only (Satterwhite et al. 1992). In contrast, MLCK phosphorylates both threonine 18 and serine 19 (Sellers 1991). These differential phosphorylation sites may be resolved by phosphopeptide mapping as illustrated in Fig. 2, where purified myosin II was phosphorylated in vitro by PKC or MLCK. Two phosphopeptides were detected in PKC phosphorylated light chains, corresponding to serines 1 and 2 and threonine 9 (Fig. 2 E), whereas a single phosphopeptide corresponding to serine 19 was visible in light chains phosphorylated by MLCK (Fig. 2 F) (Satterwhite et al. 1992). Peptide digests from in vitro–phosphorylated brush border or sea urchin egg myosin II were superimposable when phosphorylated by the same kinases (data not shown), indicating that the sea urchin homologue of regulatory light chain also contained these positive and negative regulatory sites.


Parameters that specify the timing of cytokinesis.

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

In vivo analysis of cortical myosin light chain phosphorylation. (A–D) L. pictus eggs were incubated in the presence of 32PO4 in phosphate-free artificial sea water. Cells were monitored with interference contrast optics, and at time points when the cells were in (A) prophase, (B) metaphase, (C) anaphase, and (D) telophase (69, 78, 85, and 95 min after fertilization, respectively). Zygotes were washed and cortical cytoskeletons prepared from each time point. Cortical protein was resolved by SDS-PAGE, and light chains were excised, and digested with TPCK-trypsin. Digests were then washed and subjected to two-dimensional peptide analysis on cellulose TLC plates. The designations of the phosphopeptides are based on Satterwhite (1992). (E and F) Purified myosin II was phosphorylated in vitro using purified PKC (E) or MLCK (F). Peptide digests of cortical LC20 labeled in vivo were analyzed either alone (G) or mixed with equal counts of LC20 phosphorylated in vitro with MLCK (H).
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Related In: Results  -  Collection

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Figure 2: In vivo analysis of cortical myosin light chain phosphorylation. (A–D) L. pictus eggs were incubated in the presence of 32PO4 in phosphate-free artificial sea water. Cells were monitored with interference contrast optics, and at time points when the cells were in (A) prophase, (B) metaphase, (C) anaphase, and (D) telophase (69, 78, 85, and 95 min after fertilization, respectively). Zygotes were washed and cortical cytoskeletons prepared from each time point. Cortical protein was resolved by SDS-PAGE, and light chains were excised, and digested with TPCK-trypsin. Digests were then washed and subjected to two-dimensional peptide analysis on cellulose TLC plates. The designations of the phosphopeptides are based on Satterwhite (1992). (E and F) Purified myosin II was phosphorylated in vitro using purified PKC (E) or MLCK (F). Peptide digests of cortical LC20 labeled in vivo were analyzed either alone (G) or mixed with equal counts of LC20 phosphorylated in vitro with MLCK (H).
Mentions: Results of H1 kinase assays indicated that cytoskeletal-associated p34cdc2 activity cycled with kinetics delayed with respect to whole cell levels, and that this delayed activity extended into anaphase and cleavage. In light of data suggesting that p34cdc2 may regulate the timing of cytokinesis through the modulation of myosin II activity (Satterwhite et al. 1992; Yamakita et al. 1994), we asked whether the extended p34cdc2 activity is reflected in vivo by myosin regulatory light chain (LC20) phosphorylation in the cortical cytoskeleton of sea urchin embryos. Smooth muscle and cytoplasmic myosin light chains may be phosphorylated on five residues: serines 1 and 2, threonine 9, threonine 18, and serine 19 (for reviews see Sellers 1991; Satterwhite and Pollard 1992; Bresnick 1999). Whereas PKC phosphorylates LC20 on serines 1 and 2 and threonine 9 (Nishikawa et al., 1984; Bengur et al. 1987; Ikebe and Reardon 1990), p34cdc2 phosphorylates serines 1 and 2 only (Satterwhite et al. 1992). In contrast, MLCK phosphorylates both threonine 18 and serine 19 (Sellers 1991). These differential phosphorylation sites may be resolved by phosphopeptide mapping as illustrated in Fig. 2, where purified myosin II was phosphorylated in vitro by PKC or MLCK. Two phosphopeptides were detected in PKC phosphorylated light chains, corresponding to serines 1 and 2 and threonine 9 (Fig. 2 E), whereas a single phosphopeptide corresponding to serine 19 was visible in light chains phosphorylated by MLCK (Fig. 2 F) (Satterwhite et al. 1992). Peptide digests from in vitro–phosphorylated brush border or sea urchin egg myosin II were superimposable when phosphorylated by the same kinases (data not shown), indicating that the sea urchin homologue of regulatory light chain also contained these positive and negative regulatory sites.

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