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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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Cleavage furrow formation in a cylindrical, Δ90-arrested cell. One blastomere of a two cell embryo was injected with Δ90 cyclin, and 30 min after cleavage of the uninjected control, the arrested cell was drawn into a pipette. Although the exact position of the aster centers is not clearly visible in this specimen, the arrows indicate a cleared zone in the cytoplasm, where ∼3 min later, a furrow becomes visible that progresses along the long axis of the cell. (A–D) 190, 193, 197, 201 min after fertilization, respectively. Bar, 80 μm.
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Figure 6: Cleavage furrow formation in a cylindrical, Δ90-arrested cell. One blastomere of a two cell embryo was injected with Δ90 cyclin, and 30 min after cleavage of the uninjected control, the arrested cell was drawn into a pipette. Although the exact position of the aster centers is not clearly visible in this specimen, the arrows indicate a cleared zone in the cytoplasm, where ∼3 min later, a furrow becomes visible that progresses along the long axis of the cell. (A–D) 190, 193, 197, 201 min after fertilization, respectively. Bar, 80 μm.

Mentions: The physical manipulation of the asters in Δ90-arrested cells suggested not only that the cortical cytoskeleton retains the capacity to assemble a contractile ring in the presence of chronically elevated MPF levels, but also suggested that the timing of cytokinesis may be a function of the spindle's capacity to deliver the signal to the surface. To further explore this notion, a second method was employed to alter the geometry of normal and Δ90-arrested cells. Blastomeres were carefully drawn into a fire-polished capillary pipette, resulting in a cylindrical cell and a reduced distance between the spindle pole and the cell surface (Rappaport 1981, Rappaport 1997). As shown in Fig. 5, when an uninjected blastomere was drawn into a pipette, the mitotic apparatus is usually drawn into the distal portion of the cell. Just after the appearance of anaphase asters (Fig. 5 B), a cleavage furrow was induced and furrowing was complete before the spherical control had commenced cleavage, even though anaphase onset and astral microtubule elongation occurred simultaneously in the two cells. When Δ90-arrested cells were drawn into a pipette, cleavage furrows could also be observed. In the embryo shown in Fig. 6, the aster centers could not be clearly delineated, but a localized contraction was induced adjacent to a cleared zone (arrow), and because the spindle was aligned slightly oblique to the axis of the cylinder, the furrow attempted to progress along the long axis of the cell. Similar results have been obtained with normal cylindrical cells when the axis of the spindle is normal to the long axis of the cell (Rappaport and Ratner 1967; Rappaport and Rappaport 1987). Similar results were obtained using injected mRNA in L. pictus and Dendraster excentricus. Furrows induced in cylindrical, Δ90-injected embryos were irregular, and while none (5/5 blastomeres) progressed to completion, contractility activity was observed in all cells whose capillary diameter was not >60 μm.


Parameters that specify the timing of cytokinesis.

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

Cleavage furrow formation in a cylindrical, Δ90-arrested cell. One blastomere of a two cell embryo was injected with Δ90 cyclin, and 30 min after cleavage of the uninjected control, the arrested cell was drawn into a pipette. Although the exact position of the aster centers is not clearly visible in this specimen, the arrows indicate a cleared zone in the cytoplasm, where ∼3 min later, a furrow becomes visible that progresses along the long axis of the cell. (A–D) 190, 193, 197, 201 min after fertilization, respectively. Bar, 80 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Cleavage furrow formation in a cylindrical, Δ90-arrested cell. One blastomere of a two cell embryo was injected with Δ90 cyclin, and 30 min after cleavage of the uninjected control, the arrested cell was drawn into a pipette. Although the exact position of the aster centers is not clearly visible in this specimen, the arrows indicate a cleared zone in the cytoplasm, where ∼3 min later, a furrow becomes visible that progresses along the long axis of the cell. (A–D) 190, 193, 197, 201 min after fertilization, respectively. Bar, 80 μm.
Mentions: The physical manipulation of the asters in Δ90-arrested cells suggested not only that the cortical cytoskeleton retains the capacity to assemble a contractile ring in the presence of chronically elevated MPF levels, but also suggested that the timing of cytokinesis may be a function of the spindle's capacity to deliver the signal to the surface. To further explore this notion, a second method was employed to alter the geometry of normal and Δ90-arrested cells. Blastomeres were carefully drawn into a fire-polished capillary pipette, resulting in a cylindrical cell and a reduced distance between the spindle pole and the cell surface (Rappaport 1981, Rappaport 1997). As shown in Fig. 5, when an uninjected blastomere was drawn into a pipette, the mitotic apparatus is usually drawn into the distal portion of the cell. Just after the appearance of anaphase asters (Fig. 5 B), a cleavage furrow was induced and furrowing was complete before the spherical control had commenced cleavage, even though anaphase onset and astral microtubule elongation occurred simultaneously in the two cells. When Δ90-arrested cells were drawn into a pipette, cleavage furrows could also be observed. In the embryo shown in Fig. 6, the aster centers could not be clearly delineated, but a localized contraction was induced adjacent to a cleared zone (arrow), and because the spindle was aligned slightly oblique to the axis of the cylinder, the furrow attempted to progress along the long axis of the cell. Similar results have been obtained with normal cylindrical cells when the axis of the spindle is normal to the long axis of the cell (Rappaport and Ratner 1967; Rappaport and Rappaport 1987). Similar results were obtained using injected mRNA in L. pictus and Dendraster excentricus. Furrows induced in cylindrical, Δ90-injected embryos were irregular, and while none (5/5 blastomeres) progressed to completion, contractility activity was observed in all cells whose capillary diameter was not >60 μm.

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