Shuster CB and Burgess DR (1999), Parameters that specify the timing of cytokinesis.

ECB-ART-37279

J Cell Biol 1999 Sep 06;1465:981-92. doi: 10.1083/jcb.146.5.981.

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Parameters that specify the timing of cytokinesis.

Shuster CB , Burgess DR .

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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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Genes referenced: cep152 LOC100887844 LOC115919910 LOC576786 LOC581963 LOC594261 LOC752081 LOC756768 pole

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	Figure 1. Characterization of cortical histone H1 kinase activity. (A) At the times either before (Uf) or following fertilization (20â€“140 min after fertilization), zygotes were washed and both whole cell lysates and detergent-extracted cytoskeletons were prepared, snap frozen, then assayed for H1 kinase activity. The first and second cleavages occurred at 90 and 140 min after fertilization, and are denoted with arrows. (B) Detergent-extracted cortical cytoskeletons prepared from dividing blastomeres were assayed for H1 kinase activity in the presence of 10 Î¼M H-7, 5 Î¼M roscovitine, 10 Î¼M olomoucine, or 10 Î¼M isoolomoucine. Note that while the PKC inhibitor H-7 has no effect on H1 kinase activities, the CDK inhibitors roscovitine and olomoucine (but not the inactive isomer, isoolomoucine) strongly inhibit H1 kinase activity.
	Figure 2. In vivo analysis of cortical myosin light chain phosphorylation. (Aâ€“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).
	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 [Î³-32P]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.
	Figure 4. Mitotic arrest and needle displacement in Î”90 cyclin-injected sand dollar embryos. Zygotes were cultured at 15Â°C through the first division. Shortly before prophase of the second cell division, one blastomere was injected with Î”90 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 Î”90-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 Î¼m.
	Figure 5. Cleavage formation in a cylindrical cell. Zygotes were cultured through the first division at 15Â°C. Upon appearance of a mitotic spindle, one blastomere was drawn into a fire-polished capillary pipette. At 150 min after fertilization, the anaphase asters become visible, and a bilateral furrow appears shortly afterward. Note that by the time the control blastomere begins to cleave (D), the cylindrical blastomere has completed cytokinesis. (Aâ€“D) 145, 150, 153, and 159 min after fertilization, respectively. Dots indicate the positions of the aster centers. Bar, 80 Î¼m.
	Figure 6. Cleavage furrow formation in a cylindrical, Î”90-arrested cell. One blastomere of a two cell embryo was injected with Î”90 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 âˆ¼3 min later, a furrow becomes visible that progresses along the long axis of the cell. (Aâ€“D) 190, 193, 197, 201 min after fertilization, respectively. Bar, 80 Î¼m.
	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 âˆ¼7 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 Î”90 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.

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