Limits...
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.

Show MeSH

Related in: MedlinePlus

Spatial relationships between the spindle poles and the cell surface specify the timing of cytokinesis in echinoderm eggs. (A) In spherical control blastomeres, the onset of anaphase is accompanied by an elaboration of astral microtubules that contact the surface. After a short latent period, a cleavage furrow forms that ingresses to completion in ∼7 min. (B) If blastomeres are manipulated into a cylindrical form, the distance between the spindle poles and the surface is reduced, and a furrow forms precociously in comparison with spherical controls (this report). (C) Treatment of mitotic sand dollar eggs with 60 mM urethane results in an inhibition of astral microtubule elongation (Rappaport 1971; Rappaport and Rappaport 1984) and a failure to cleave. The inhibition of cytokinesis may be overcome by physically shifting the position of the mitotic apparatus towards the surface. (D) Blastomeres arrested in mitosis by injection of Δ90 cyclin B undergo normal chromosome separation and anaphase B movements, but the spindle fails to induce a furrow (Wheatley et al. 1997; Hinchcliffe et al. 1998; this report). However, reduction of the distance between the spindle poles and the surface may overcome the inhibition of cleavage by physical displacement of the asters towards the surface.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169486&req=5

Figure 7: Spatial relationships between the spindle poles and the cell surface specify the timing of cytokinesis in echinoderm eggs. (A) In spherical control blastomeres, the onset of anaphase is accompanied by an elaboration of astral microtubules that contact the surface. After a short latent period, a cleavage furrow forms that ingresses to completion in ∼7 min. (B) If blastomeres are manipulated into a cylindrical form, the distance between the spindle poles and the surface is reduced, and a furrow forms precociously in comparison with spherical controls (this report). (C) Treatment of mitotic sand dollar eggs with 60 mM urethane results in an inhibition of astral microtubule elongation (Rappaport 1971; Rappaport and Rappaport 1984) and a failure to cleave. The inhibition of cytokinesis may be overcome by physically shifting the position of the mitotic apparatus towards the surface. (D) Blastomeres arrested in mitosis by injection of Δ90 cyclin B undergo normal chromosome separation and anaphase B movements, but the spindle fails to induce a furrow (Wheatley et al. 1997; Hinchcliffe et al. 1998; this report). However, reduction of the distance between the spindle poles and the surface may overcome the inhibition of cleavage by physical displacement of the asters towards the surface.

Mentions: The necessity of bringing the asters of Δ90-injected cells into close proximity with the cortex suggests that at least one determinant of the timing of cytokinesis is the geometrical relationship between the spindle poles and the surface. The spatial relationship between the spindle poles and the surface in normal and geometrically or chemically altered cells, and the respective effects on the timing of cleavage furrow formation is illustrated in Fig. 7. Under normal conditions, the cleavage plane is specified shortly after the metaphase–anaphase transition (Rappaport 1996). With the onset of anaphase and the decline of p34cdc2 activity, there is an extensive elaboration and elongation of astral microtubules along with an accompanying loss of spindle birefringence (Fig. 7 A) (Salmon and Wolniak 1990). Contact between astral microtubules and the surface requires as little as 1 min to specify the position of the furrow, and after a brief latent period the contractile ring is induced (Rappaport and Ebstein 1965). Under conditions where the distance between the spindle poles and the surface is reduced, as in the case of a cylindrical cell (Fig. 7 B), contractile rings are induced and progress to completion before spherical controls (Fig. 5). Thus, while both spherical and cylindrical cells enter anaphase at the same time, the timing of furrow formation is a function of the distance that the putative cleavage stimulus has to travel to reach the surface, and not an abrupt cell cycle transition.


Parameters that specify the timing of cytokinesis.

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

Spatial relationships between the spindle poles and the cell surface specify the timing of cytokinesis in echinoderm eggs. (A) In spherical control blastomeres, the onset of anaphase is accompanied by an elaboration of astral microtubules that contact the surface. After a short latent period, a cleavage furrow forms that ingresses to completion in ∼7 min. (B) If blastomeres are manipulated into a cylindrical form, the distance between the spindle poles and the surface is reduced, and a furrow forms precociously in comparison with spherical controls (this report). (C) Treatment of mitotic sand dollar eggs with 60 mM urethane results in an inhibition of astral microtubule elongation (Rappaport 1971; Rappaport and Rappaport 1984) and a failure to cleave. The inhibition of cytokinesis may be overcome by physically shifting the position of the mitotic apparatus towards the surface. (D) Blastomeres arrested in mitosis by injection of Δ90 cyclin B undergo normal chromosome separation and anaphase B movements, but the spindle fails to induce a furrow (Wheatley et al. 1997; Hinchcliffe et al. 1998; this report). However, reduction of the distance between the spindle poles and the surface may overcome the inhibition of cleavage by physical displacement of the asters towards the surface.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169486&req=5

Figure 7: Spatial relationships between the spindle poles and the cell surface specify the timing of cytokinesis in echinoderm eggs. (A) In spherical control blastomeres, the onset of anaphase is accompanied by an elaboration of astral microtubules that contact the surface. After a short latent period, a cleavage furrow forms that ingresses to completion in ∼7 min. (B) If blastomeres are manipulated into a cylindrical form, the distance between the spindle poles and the surface is reduced, and a furrow forms precociously in comparison with spherical controls (this report). (C) Treatment of mitotic sand dollar eggs with 60 mM urethane results in an inhibition of astral microtubule elongation (Rappaport 1971; Rappaport and Rappaport 1984) and a failure to cleave. The inhibition of cytokinesis may be overcome by physically shifting the position of the mitotic apparatus towards the surface. (D) Blastomeres arrested in mitosis by injection of Δ90 cyclin B undergo normal chromosome separation and anaphase B movements, but the spindle fails to induce a furrow (Wheatley et al. 1997; Hinchcliffe et al. 1998; this report). However, reduction of the distance between the spindle poles and the surface may overcome the inhibition of cleavage by physical displacement of the asters towards the surface.
Mentions: The necessity of bringing the asters of Δ90-injected cells into close proximity with the cortex suggests that at least one determinant of the timing of cytokinesis is the geometrical relationship between the spindle poles and the surface. The spatial relationship between the spindle poles and the surface in normal and geometrically or chemically altered cells, and the respective effects on the timing of cleavage furrow formation is illustrated in Fig. 7. Under normal conditions, the cleavage plane is specified shortly after the metaphase–anaphase transition (Rappaport 1996). With the onset of anaphase and the decline of p34cdc2 activity, there is an extensive elaboration and elongation of astral microtubules along with an accompanying loss of spindle birefringence (Fig. 7 A) (Salmon and Wolniak 1990). Contact between astral microtubules and the surface requires as little as 1 min to specify the position of the furrow, and after a brief latent period the contractile ring is induced (Rappaport and Ebstein 1965). Under conditions where the distance between the spindle poles and the surface is reduced, as in the case of a cylindrical cell (Fig. 7 B), contractile rings are induced and progress to completion before spherical controls (Fig. 5). Thus, while both spherical and cylindrical cells enter anaphase at the same time, the timing of furrow formation is a function of the distance that the putative cleavage stimulus has to travel to reach the surface, and not an abrupt cell cycle transition.

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