The role of microtubules in contractile ring function.

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.     

Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells.

Myosin II is a major component of a contractile ring. To examine if myosin II turns over in contractile rings, fluorescence of GFP-myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 +/- 2.62 s. This recovery was not caused by lateral movement of myosin II from the nonbleached area, but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP-3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.     

The action of salt-solutions on the unfertilized and fertilized eggs of Arbacia,and of other animals

Summary The last two experiments show that after 4 1/2 hours in the solutions employed the unfertilized eggs divided excellently when returned to sea-water. The last experiment shows that potassium chloride, 1.5%, gives the same results as the other salts.     

Eosinophi-mediated destruction of Schistosoma mansoni eggs in vitro. II. The role of cytophilic antibody

Peritoneal exudative eosinophils obtained from Schistosoma mansoni-infected CBA/J mice cause morphological damage to isolated S. mansoni eggs in a 24 hr co-cultivation system in vitro. This egg-destructive activity was complement-independent and was abolished by trypsinization of the cells prior to co-cultivation. Trypsinized cells could be passively sensitized to renewed egg-destructive capacity by preincubation or co-gcultivation with immune sera, containing antibodies against a soluble egg antigenic preparation (SEA). Solid phase absorption of immune sera with SEA coupled to Sepharose 4B lowered the anti-egg antibody titers of these sera and eliminated their ability to sensitize trypsinized eosinophils. Sera from uninfected mice or from mice infected with Trichinella spiralis did not sensitize trypsinized cells. Addition of immune sera to eosinophil-rich cell populations obtained from uninfected mice also enhanced the egg-destructive capacity of these otherwise non-reactive cells. Therefore, eosinophil-mediated destruction of S. mansoni eggs may be directed by cytophilic antigen-specific factors in sera from S. mansoni infected hosts.     

The onset of DNA synthesis and its relation to morphogenetic events of the pronuclei in activated eggs of the sea urchin, Arbacia punctulata

Experiments were carried out in order to investigate the onset of DNA synthesis and its possible relation to pronuclear morphogenesis prior to, during, and following pronuclear fusion in the sea urchin, Arbacia punctulata. Analysis of the acid-soluble radioactivity of zygotes continuously incubated in tritiated thymidine (³H-TdR) at 22°C demonstrated the production of thymidine triphosphate prior to and during pronuclear migration (5–10 min post-insemination) which continued to increase until prophase (about 45 min post-insemination). DNA synthesis was initiated subsequent to pronuclear fusion, i.e., 15–20 min post-insemination. Little or no cytoplasmic label was detected during fertilization. Silver grains were first detected over the zygote nucleus in sections prepared for light and electron microscopic radioautography from zygotes fixed at 18 min post-insemination. Incubation of Arbacia zygotes at 12°C dramatically slowed development but did not alter the temporal relation between pronuclear fusion and DNA synthesis. Radioautographs of polyspermic zygotes demonstrated that unfused male pronuclei were able to synthesize DNA and substantiate the claim that pronuclear fusion is not required for the initiation of DNA synthesis. Analysis of autoradiographs of artificially activated eggs also lead to the same conclusion. Uptake of ³H-TdR or ³H-bromodeoxyuridine was not found in spermatozoa incorporated into polyspermic oocytes regardless of whether or not they had initiated or developed into male pronuclei. It is concluded that the relation between DNA synthesis and pronuclear fusion is not close or a direct one, i.e., pronuclear fusion does not appear to be a prerequisite for the onset of DNA synthesis in Arbacia.     

The eggs and half-eggs of Arbacia punctulata and the plutei,as photographed by ultraviolet,visible and infrared light

Normal and centrifuged eggs, the halves obtained by strong centrifugal force and the plutei of Arbacia have been photographed with visible, ultraviolet (1,537Å) and infrared (8,000–10,000Å) light. Differences in appearance with these different light sources are described and some of the photographs reproduced.     

Phosphorylation of myosin II regulatory light chain controls its accumulation, not that of actin, at the contractile ring in HeLa cells

During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells.     

A contractile ring and cortical changes found in the dividing Tetrahymena pyriformis

A contractile ring consisting mainly of microfilaments was found in the fission zone of dividing Tetrahymena pyriformis. Diameters of the microfilaments were widely distributed from 2.5 to 15 nm. Ring-associated structures such as lateral stripes, linkers and beads with siender tails were recognized. Lateral stripes arranged at regular intervals of about 84 nm on some parts of microfilament bundles were found in both tangential and transverse sections, suggesting that they correspond to bands fastening the contractile ring microfilaments. Linkers that connect individual lateral stripes to the epiplasmic layer were present. Beads or beads with slender tails were found to be arranged on some microfilaments.The results of the present paper also indicate that drastic morphological changes occur in the cortex of the fission zone, especially in the epiplasmic layer, accompanying contraction of the division furrow. The epiplasmic layer which was proved to be a compact filamentous network in this study has been known to exist at the periphery of cytoplasm in immediate contact with one of the cell surface membranes, the inner alveolar membrane; however, in the fission zone of the dividing ceil, it was frequently separated from the membrane and subsided into the cytoplasm. The subsided epiplasmic layer was then loosened and dispersed. The subsidence of the epipiasmic layer appears to be caused by the force generated by the contraction of the contractile ring and transmitted with the linkers to the epiplasmic layer. The changes observed in the epiplasmic layer are presumably indispensable for the rigid cortical layer contraction involved in cytokinesis of Tetrahymena.