scholarly journals Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs.

1976 ◽  
Vol 70 (1) ◽  
pp. 75-85 ◽  
Author(s):  
G Sluder
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Maximum Rate ◽  
Spindle Assembly ◽  
Experimental Manipulation ◽  
Lytechinus Variegatus ◽  
Tubulin Polymerization ◽  
Treated Cell ◽  
Sea Urchin Eggs ◽  
Nuclear Envelope Breakdown

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.

scholarly journals Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown.

1990 ◽  
Vol 111 (5) ◽  
pp. 1763-1773 ◽  
Author(s):  
C Baitinger ◽  
J Alderton ◽  
M Poenie ◽  
H Schulman ◽  
R A Steinhardt
Keyword(s):  
Protein Kinase ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Mitotic Division ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Sea Urchin Eggs ◽  
Nuclear Envelope Breakdown ◽  
Dependent Protein

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.

On the Calcium Pulse during Nuclear Envelope Breakdown (NEB) in Sea Urchin Eggs

1992 ◽  
Vol 183 (2) ◽  
pp. 370-371 ◽  
Author(s):  
C. L. Browne ◽  
A. L. Miller ◽  
R. E. Palazzo ◽  
L. F. Jaffe
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Sea Urchin Eggs ◽  
Nuclear Envelope Breakdown

scholarly journals Role of spindle microtubules in the control of cell cycle timing.

1979 ◽  
Vol 80 (3) ◽  
pp. 674-691 ◽  
Author(s):  
G Sluder
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Control Cell ◽  
Microtubule Assembly ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Cells ◽  
Spindle Microtubules

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

scholarly journals Activation of sea urchin eggs by inositol phosphates is independent of external calcium

1988 ◽  
Vol 252 (1) ◽  
pp. 257-262 ◽  
Author(s):  
I Crossley ◽  
K Swann ◽  
E Chambers ◽  
M Whitaker
Keyword(s):  
Sea Urchin ◽  
Calcium Ions ◽  
Sea Water ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Egg Activation ◽  
Lytechinus Variegatus ◽  
Lytechinus Pictus ◽  
Sea Urchin Eggs ◽  
External Calcium

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.

scholarly journals Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

2000 ◽  
Vol 11 (3) ◽  
pp. 897-914 ◽  
Author(s):  
Mark Terasaki
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Permeability Barrier ◽  
Cell Cycles ◽  
Nuclear Envelope Breakdown ◽  
Quantitative Measurements ◽  
Green Fluorescent

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

scholarly journals Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote

1999 ◽  
Vol 112 (8) ◽  
pp. 1139-1148 ◽  
Author(s):  
E.H. Hinchcliffe ◽  
E.A. Thompson ◽  
F.J. Miller ◽  
J. Yang ◽  
G. Sluder
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Dna Breaks ◽  
Male Pronucleus ◽  
Nuclear Envelope Breakdown ◽  
Female Pronucleus

In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.

The effects of mitotic inhibitors on the structure of vinblastine-induced tubulin paracrystals from sea-urchin eggs

1976 ◽  
Vol 20 (1) ◽  
pp. 91-100
Author(s):  
D. Starling
Keyword(s):  
Sea Urchin ◽  
Ultrastructural Level ◽  
Tubulin Polymerization ◽  
Sea Urchin Eggs ◽  
Mitotic Inhibitors ◽  
Vinblastine Sulphate ◽  
Tubulin Paracrystals ◽  
Careful Standardization

Vinblastine sulphate (VLB) is known to induce in vivo formation of tubulin paracrystals in sea-urchin eggs. Corresponding paracrystals have been prepared in the presence of both vinblastine sulphate and other mitoclasic agents. Careful standardization of conditions was required to restrict the formation of alternative forms of the paracrystals induced by vinblastine alone. Comparisons were made between preparations in terms of paracrystal shape, size, proportion of eggs containing paracrystals, number per egg and their relative times of first appearance. A correlation between such properties were established. Comparison of paracrystals at the ultrastructural level showed them all to be similar regardless of the drugs present during their formation. The implications of tubulin polymerization in the presence of mitoclasic agents are discussed and mechanisms for paracrystal enhancement by combinations of such drugs are suggested. Some similarities of paracrystal and microtubule seeding are discussed together with the activation of tubulin in the pool.

Effect of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, on the first mitotic divisions of the fertilized sea urchin egg

1998 ◽  
Vol 111 (17) ◽  
pp. 2507-2518 ◽  
Author(s):  
C. De Nadai ◽  
P. Huitorel ◽  
S. Chiri ◽  
B. Ciapa
Keyword(s):  
Sea Urchin ◽  
Mitotic Spindle ◽  
Second Messengers ◽  
Cellular Systems ◽  
Phosphatidylinositol 3 Kinase ◽  
Sea Urchin Eggs ◽  
Cellular Processes ◽  
Nuclear Envelope Breakdown ◽  
Sea Urchin Egg ◽  
Messenger System

We have reported earlier that the polyphosphoinositide messenger system may control mitosis in sea urchin eggs. Besides phospholipase C activation and its second messengers, phosphatidylinositol (PI) 3-kinase has been proposed to affect a wide variety of cellular processes in other cellular systems. Therefore, we have investigated whether PI 3-kinase could play a role in regulating the sea urchin early embryonic development. Our data presented here suggest that PI 3-kinase is present in sea urchin eggs. We found that wortmannin, an inhibitor of PI 3-kinase, led to arrest of the cell cycle. Chromosome condensation, nuclear envelope breakdown, microtubular aster polymerization, protein and DNA synthesis were not affected when fertilization was performed in the presence of the drug. However, maturation-promoting factor (MPF) activation was inhibited and centrosome duplication was perturbed preventing the formation of a bipolar mitotic spindle in wortmannin treated eggs. We discuss how PI 3-kinase might be involved in the cascade of events leading to the first mitotic divisions of the fertilized sea urchin egg.

scholarly journals Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate and GTP-binding protein.

1986 ◽  
Vol 102 (1) ◽  
pp. 70-76 ◽  
Author(s):  
P R Turner ◽  
L A Jaffe ◽  
A Fein
Keyword(s):  
Sea Urchin ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Lytechinus Variegatus ◽  
Nucleotide Analogs ◽  
Sea Urchin Eggs ◽  
Sequence Of Events ◽  
Inositol 1,4,5 Trisphosphate ◽  
Vesicle Exocytosis ◽  
Nucleotide Binding Proteins

To investigate the roles of inositol 1,4,5-trisphosphate (InsP3) and guanyl nucleotide binding proteins (G-proteins) in the transduction mechanism coupling fertilization and exocytosis of cortical vesicles in sea urchin eggs, we microinjected InsP3 and guanyl nucleotide analogs into eggs of Lytechinus variegatus. Injection of 28 nM InsP3 caused exocytosis. However, if the egg was first injected with EGTA ([Cai] less than or equal to 0.1 microM; EGTA = 1.6 mM), InsP3 injection did not cause exocytosis, supporting the hypothesis that InsP3 acts by causing a rise in intracellular free calcium. Injection of 28 microM guanosine-5'-0-(3-thiotriphosphate) (GTP-gamma-S), a hydrolysis-resistant analog of GTP, caused exocytosis, but exocytosis did not occur if the egg was pre-injected with EGTA. Injection of 3 mM guanosine-5'-0-(2-thiodiphosphate) (GDP-beta-S), a metabolically stable analog of GDP, prevented sperm from stimulating exocytosis. However, injection of GDP-beta-S did not prevent the stimulation of exocytosis by InsP3. These results suggested the following sequence of events. The sperm activates a G-protein, which stimulates production of InsP3. InsP3 elevates intracellular free calcium, which causes exocytosis.

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