scholarly journals Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control.

1990 ◽  
Vol 110 (6) ◽  
pp. 2025-2032 ◽  
Author(s):  
G Sluder ◽  
F J Miller ◽  
R Cole ◽  
C L Rieder
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Translational Control ◽  
High Voltage Electron Microscopy ◽  
Sea Urchin Eggs ◽  
Voltage Electron ◽  
Sds Page ◽  
Nuclear Events ◽  
Specific Proteins ◽  
Sperm Aster

The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis. The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosome-specific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.

scholarly journals Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes.

1985 ◽  
Vol 100 (3) ◽  
pp. 897-903 ◽  
Author(s):  
G Sluder ◽  
C L Rieder
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Experimental Proof ◽  
High Voltage Electron Microscopy ◽  
Bipolar Spindle ◽  
Sea Urchin Eggs ◽  
Voltage Electron

We tested the ability of chromosomes in a mitotic cytoplasm to organize a bipolar spindle in the absence of centrosomes. Sea urchin eggs were treated with 5 X 10(-6) colcemid for 7-9 min before fertilization to block future microtubule assembly. Fertilization events were normal except that a sperm aster was not formed and the pronuclei remained up to 70 microns apart. After nuclear envelope breakdown, individual eggs were irradiated with 366-nm light to inactivate photochemically the colcemid. A functional haploid bipolar spindle was immediately assembled in association with the male chromosomes. In contrast to the male pronucleus, the female pronucleus in most of these eggs remained as a small nonbirefringent hyaline area throughout mitosis. High-voltage electron microscopy of serial semithick sections from individual eggs, previously followed in vivo, revealed that the female chromosomes were randomly distributed within the remnants of the nuclear envelope. No microtubules were found in these pronuclear areas even though the chromosomes were well-condensed and had prominent kinetochores with well-developed coronas. In the remaining eggs, a weakly birefringent monaster was assembled in the female pronuclear area. These observations demonstrate that chromosomes in a mitotic cytoplasm cannot organize a bipolar spindle in the absence of a spindle pole or even in the presence of a monaster. In fact, chromosomes do not even assemble kinetochore microtubules in the absence of a spindle pole, and kinetochore microtubules form only on kinetochores facing the pole when a monaster is present. This study also provides direct experimental proof for the longstanding paradigm that the sperm provides the centrosomes used in the development of the sea urchin zygote.

Quantitative ultrastructural reconstruction of small regions within large volumes by correlative light and high voltage electron microscopy of serial 0.25-0.50 μm sections

1987 ◽  
Vol 45 ◽  
pp. 578-581
Author(s):  
Conly L. Rieder ◽  
Frederick J. Miller ◽  
Edwin Davison ◽  
Samuel S. Bowser ◽  
Kirsten Lewis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Mitotic Spindle ◽  
Meiotic Spindle ◽  
High Voltage Electron Microscopy ◽  
Male And Female ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Differential Stability ◽  
Sperm Aster

In this abstract we Illustrate how same-section correlative light and high voltage electron microscopy (HVEM) of serial 0.25-0.50-μm sections can answer questions which are difficult to approach by EM of 60-100 nm sections.Starfish (Pisaster and Asterlas) eggs are fertilized at meiosis I when the oocyte contains two maternal centrosomes (e.g., asters) which form the poles of the first meiotic spindle. Immediately after fertilization a sperm aster is assembled in the vicinity of the male pronucleus and persists throughout meiosis. At syngamy the sperm aster splits to form the poles of the first mitotic spindle. During this time the functional and replicative properties of the maternal centrosome, inherited from the last meiotic division, are lost. The basis for this differential stability, of male and female centrosomes in the same cytoplasm, is a mystery.

scholarly journals The reproduction of centrosomes: nuclear versus cytoplasmic controls.

1986 ◽  
Vol 103 (5) ◽  
pp. 1873-1881 ◽  
Author(s):  
G Sluder ◽  
F J Miller ◽  
C L Rieder
Keyword(s):  
Cell Cycle ◽  
Electron Microscope ◽  
High Voltage ◽  
Nuclear Dna ◽  
Lytechinus Variegatus ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
Nuclear Events ◽  
Subsequent Behavior

The tight coordination normally found between nuclear events and the doubling of centrosomes at each cell cycle suggests that nuclear activities may be part of the mechanism that controls the reproduction of centrosomes. To determine if this is the case, we used a micropipette to completely remove the nucleus from eggs of the sea urchin Lytechinus variegatus at prophase of the first mitosis, leaving only one centrosome in the cell. The subsequent behavior of this centrosome was then followed in vivo with the polarization microscope. In all cases the centrosome reproduced in a precise 1:2:4:8 fashion with a periodicity that was slightly slower than the centrosome cycle of control eggs. The cell cycle-related changes in centrosome morphology were identical to those of control eggs in that: (a) the astral birefringence varied cyclically to a normal extent, (b) the astral focus enlarged and then flattened during the telophase equivalent, (c) cleavage furrows were initiated as the astral birefringence faded, and (d) daughter centrosomes separated before the increase in astral birefringence at the onset of each mitosis. To determine if centrioles also reproduced normally, enucleate eggs were followed in vivo until they contained eight centrosomes. They were then individually removed from the preparations, fixed, and embedded. Each egg was serially 0.25-micron sectioned for observation with the high voltage electron microscope. We completely reconstructed 23 centrosomes in four eggs; all centrosomes contained two centrioles apiece. These results demonstrate that the subunits for complete centrosome assembly can be stockpiled ahead of time and that the properly controlled use of these subunits for centrosome reproduction does not require nuclear transcription or nuclear DNA synthesis at each cell cycle.

scholarly journals Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase

1996 ◽  
Vol 109 (12) ◽  
pp. 2885-2893 ◽  
Author(s):  
E. Brisch ◽  
M.A. Daggett ◽  
K.A. Suprenant
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Time Course ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Spindle Poles ◽  
A Cell ◽  
Cycle Dependent ◽  
Phosphopeptide Mapping

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

scholarly journals The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote

1998 ◽  
Vol 140 (6) ◽  
pp. 1417-1426 ◽  
Author(s):  
Edward H. Hinchcliffe ◽  
Grizzel O. Cassels ◽  
Conly L. Rieder ◽  
Greenfield Sluder
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
S Phase ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Phase Arrest ◽  
Stage Specificity ◽  
S Phase Arrest ◽  
Nuclear Events ◽  
Block Protein Synthesis

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.

Cell cycle changes in water properties in sea urchin eggs

1987 ◽  
Vol 133 (1) ◽  
pp. 14-24 ◽  
Author(s):  
I. L. Cameron ◽  
K. R. Cook ◽  
D. Edwards ◽  
G. D. Fullerton ◽  
G. Schatten ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Water Properties ◽  
Sea Urchin Eggs
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