Activation-dependent and activation-independent localisation of calmodulin to the mitotic apparatus during the first cell cycle of the Lytechinus piçtus embryo.

Zygote ◽  
1995 ◽  
Vol 3 (3) ◽  
pp. 219-224 ◽  
Author(s):  
Martin Wilding ◽  
Katalin Török ◽  
Michael Whitaker
Keyword(s):  
Cell Cycle ◽  
Quantitative Analysis ◽  
Confocal Microscopy ◽  
Sea Urchin ◽  
Competitive Inhibitor ◽  
Mitotic Apparatus ◽  
Lytechinus Pictus ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Astral Microtubule

SummaryWe have used confocal microscopy and a fluorescent calmodulin probe to examine the mechanism of localisation of calmodulin during the first cell cycle of the sea urchin zygote. Using fluoresceincalmodulin, calmodulin can be observed within the nucleus and interphase astral microtubule arrays as cells approach mitosis. During mitosis, calmodulin redistributes to the mitotic apparatus and to condensed chromosomes. Quantitative analysis with reference to a control dye (fluorescein-dextran) shows that the distribution of calmodulin is specific. We used a competitive inhibitor of calcium-dependent calmodulin binding (Trp-peptide; Török & Trentham (1994) Biochemistry 33, 12807–20) to test whether the cell cycle localisation of calmodulin was due to its binding to targets on activation. The Trp-peptide eliminates localisation of calmodulin within the nucleus. However, microtubule localisation persists in the presence of the Trp-peptide. These data show that calmodulin can localise by calcium (and hence activation)-dependent as well as calcium-independent mechanisms. This suggests that distinct mechanisms of localisation may be involved in the regulation of the differential functions of calmodulin, at least during the 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.

Involvement of l(–)-rhamnose in sea urchin gastrulation. Part II: α-l-Rhamnosidase

Zygote ◽  
2015 ◽  
Vol 24 (3) ◽  
pp. 371-377 ◽  
Author(s):  
Jing Liang ◽  
Heghush Aleksanyan ◽  
Stan Metzenberg ◽  
Steven B. Oppenheimer
Keyword(s):  
Sea Urchin ◽  
Sea Urchins ◽  
Competitive Inhibitor ◽  
Innate Immune ◽  
Lytechinus Pictus ◽  
Sea Urchin Embryo ◽  
Low Concentrations ◽  
Health And Disease ◽  
Dose Dependent ◽  
Dose Dependent Effect

SummaryThe sea urchin embryo is recognized as a model system to reveal developmental mechanisms involved in human health and disease. In Part I of this series, six carbohydrates were tested for their effects on gastrulation in embryos of the sea urchin Lytechinus pictus. Only l-rhamnose caused dramatic increases in the numbers of unattached archenterons and exogastrulated archenterons in living, swimming embryos. It was found that at 30 h post-fertilization the l-rhamnose had an unusual inverse dose-dependent effect, with low concentrations (1–3 mM) interfering with development and higher concentrations (30 mM) having little to no effect on normal development. In this study, embryos were examined for inhibition of archenteron development after treatment with α-l-rhamnosidase, an endoglycosidase that removes terminal l-rhamnose sugars from glycans. It was observed that the enzyme had profound effects on gastrulation, an effect that could be suppressed by addition of l-rhamnose as a competitive inhibitor. The involvement of l-rhamnose-containing glycans in sea urchin gastrulation was unexpected, since there are no characterized biosynthetic pathways for rhamnose utilization in animals. It is possible there exists a novel l-rhamnose-containing glycan in sea urchins, or that the enzyme and sugar interfere with the function of rhamnose-binding lectins, which are components of the innate immune system in many vertebrate and invertebrate species.

scholarly journals Water ordering during the cell cycle: nuclear magnetic resonance studies of the sea-urchin egg

1985 ◽  
Vol 79 (1) ◽  
pp. 247-257
Author(s):  
S. Zimmerman ◽  
A.M. Zimmerman ◽  
G.D. Fullerton ◽  
R.F. Luduena ◽  
I.L. Cameron
Keyword(s):  
Cell Cycle ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Low Temperature ◽  
Sea Urchin ◽  
Water Proton ◽  
Proton Relaxation ◽  
Mitotic Apparatus ◽  
Microtubule Protein ◽  
Nuclear Magnetic

Nuclear magnetic resonance was used to measure spin-lattice water proton relaxation times (T1) during the first cell cycle in sea-urchin zygotes of packed Strongylocentrotus purpuratus. Following insemination there was a 90% increase in the T1 value. The increase in T1 at fertilization could be accounted for by the accumulation of extracellular fluid between the egg surface and the fertilization envelope. The T1 value then remained without change during the first cell cycle, except at metaphase when there was a significant 13% decrease. The lowered T1 values measured at metaphase were not related to a change in the water content of the packed cells, which remained fairly constant throughout the cell cycle. High hydrostatic pressure, low temperature and colchicine (agents that depolymerize mitotic apparatus microtubules) did not affect the T1 values in fertilized eggs. Treatment in vitro of a microtubule protein preparation with low temperature and colchicine resulted in an increased T1, which accompanied the depolymerization of microtubule protein. Since depolymerization of the microtubules associated with the mitotic apparatus by high pressure, colchicine or low temperature does not alter the T1 of water protons in the cell, it is proposed that the increased state of ordered water molecules at metaphase is maintained by nonmicrotubular factor(s) of the metaphase egg.

scholarly journals Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm.

1988 ◽  
Vol 106 (5) ◽  
pp. 1625-1633 ◽  
Author(s):  
J S Tash ◽  
M Krinks ◽  
J Patel ◽  
R L Means ◽  
C B Klee ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Sea Urchin ◽  
Protein Phosphatase ◽  
Flagellar Motility ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Functional Correlation ◽  
B Subunit ◽  
Dependent Protein ◽  
A Subunit

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.

The Effects of Spaceflight Conditions on Calcium-Dependent Secretion and Cytoskeletal Organization During Fertilization and Cell Divisions In the Sea Urchin Lytechinus Pictus

1999 ◽  
Vol 5 (S2) ◽  
pp. 1070-1071
Author(s):  
Heide Schatten ◽  
Amitabha Chakrabarti ◽  
Howard Levine ◽  
Mario Runco ◽  
Ken Anderson ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Research Facility ◽  
Cytoskeletal Organization ◽  
Lytechinus Pictus ◽  
Sea Urchin Eggs ◽  
Calcium Dependent ◽  
Kennedy Space Center ◽  
Cell Divisions ◽  
Kennedy Space ◽  
Calcium Loss

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. To investigate the effects of spaceflight on calcium-dependent secretion and cytoskeletal formation in a less complex system we utilized sea urchin eggs and embryos which were fertilized and cultured under spaceflight conditions during the STS-77 shuttle mission. Sea urchin eggs were fertilized and cultured in the newly developed aquatic research facility (ARF) which allowed culture of eggs and embryos in microgravity and in a 1g centrifuge in space. This allowed analysis of the comparison of microgravity and 1g spaceflight treatments with samples cultured on ground. Eggs and embryos were maintained in Standard Container Assemblies (SCAs) with identical sets prepared for culture in microgravity, and at 1g in the middeck compartment of the shuttle Endeavor, as well as for ground observations at the Kennedy Space Center.

Centrosomes are phosphorylated in sea urchin eggs throughout the cell cycle, during artificial activation, and when microtubule growth is inhibited

1994 ◽  
Vol 52 ◽  
pp. 296-297
Author(s):  
Heide Schatten ◽  
Amitabha Chakrabarti
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Sea Urchin ◽  
Immunofluorescence Microscopy ◽  
Mitotic Apparatus ◽  
Zygote Nucleus ◽  
Transmission Electron ◽  
Microtubule Growth ◽  
Artificial Activation

In most animal systems, microtubules are nucleated and organized by centrosomes which undergo considerable modifications during the cell cycle. Typically, centrosomes are phoshorylated at the transition from interphase to mitosis as shown with MPM2, an antibody directed against phosphoproteins. Using the MPM2 antibody, we show in this paper with transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) that in the sea urchin system centrosomes are phosphorylated in the sperm before fertilization and at every stage of the first cell cycle. MPM2 exhibits identical staining patterns as Ah-6 and 5051 previously shown to reliably identify centrosomal material in sea urchin cells. After centrosomal material is brought into the egg by the sperm, it spreads around the zygote nucleus where it gets distributed and becomes bipolar and compacted to form the mitotic apparatus. Typically, at these mitotic stages, centrosomal material exhibits the brightest staining with MPM2, Ah-6 and 5051 Since in this system phoshorylated centrosomal material is contributed by the sperm, the egg's competence for centrosome phosphorylation was analyzed by activating centrosomal material in the unfertilized egg by treatment with either A23187, ammonia, D2O, or taxol.

Lithium blocks cell cycle transitions in the first cell cycles of sea urchin embryos, an effect rescued by myo-inositol

Development ◽  
1997 ◽  
Vol 124 (6) ◽  
pp. 1099-1107 ◽  
Author(s):  
A. Becchetti ◽  
M. Whitaker
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Sea Water ◽  
Cleavage Stage ◽  
Teratogenic Effects ◽  
Cell Cycles ◽  
Lytechinus Pictus ◽  
Nuclear Envelope Breakdown ◽  
Phosphoinositide Pathway

Lithium is a classical inhibitor of the phosphoinositide pathway and is teratogenic. We report the effects of lithium on the first cell cycles of sea urchin (Lytechinus pictus) embryos. Embryos cultured in 400 mM lithium chloride sea water showed marked delay to the cell cycle and a tendency to arrest prior to nuclear envelope breakdown, at metaphase and at cytokinesis. After removal of lithium, the block was reversed and embryos developed to form normal late blastulae. The lithium-induced block was also reversed by myo- but not epi-inositol, indicating that lithium was acting via the phosphoinositide pathway. Lithium microinjection before fertilization caused arrest prior to nuclear envelope breakdown at much lower concentrations (3-5 mM). Co-injection of myo-inositol prevented the block. Microinjection of 1–2 mM lithium led to block at the cleavage stage. This was also reversed by coinjection of myo-inositol. Embryos blocked by lithium microinjection proceeded rapidly into mitosis after photolysis of caged inositol 1,4,5-trisphosphate. These data demonstrate that a patent phosphoinositide signalling pathway is essential for the proper timing of cell cycle transitions and offer a possible explanation for lithium's teratogenic effects.

scholarly journals Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)

2009 ◽  
Vol 2009 ◽  
pp. 1-12 ◽  
Author(s):  
Karen J. Loechner ◽  
Wendy C. Salmon ◽  
Jie Fu ◽  
Shipra Patel ◽  
James T. McLaughlin
Keyword(s):  
Cell Cycle ◽  
Calcium Channels ◽  
Intracellular Calcium ◽  
Cell Line ◽  
Time Lapse ◽  
Mitotic Apparatus ◽  
Calcium Dependent ◽  
Voltage Dependent ◽  
Specific Localization ◽  
Cycle Dependent

Changes in intracellular calcium are necessary for the successful progression of mitosis in many cells. Both elevation and reduction in intracellular calcium can disrupt mitosis by mechanisms that remain ill defined. In this study we explore the role of transmembrane voltage-gated calcium channels (CaV channels) as regulators of mitosis in the mouse corticotroph cell line (AtT-20). We report that the nifedipine-sensitive isoform CaV1.2 is localized to the “poleward side” of kinetechores during metaphase and at the midbody during cytokinesis. A second nifedipine-sensitive isoform, CaV1.3, is present at the mid-spindle zone in telophase, but is also seen at the midbody. Nifedipine reduces the rate of cell proliferation, and, utilizing time-lapse microscopy, we show that this is due to a block at the prometaphase stage of the cell cycle. Using Fluo-4 we detect calcium fluxes at sites corresponding to the mid-spindle zone and the midbody region. Another calcium dye, Fura PE3/AM, causes an inhibition of mitosis prior to anaphase that we attribute to a chelation of intracellular calcium. Our results demonstrate a novel, isoform-specific localization of CaV1 channels during cell division and suggest a possible role for these channels in the calcium-dependent events underlying mitotic progression in pituitary corticotrophs.

scholarly journals Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos.

1996 ◽  
Vol 135 (1) ◽  
pp. 191-199 ◽  
Author(s):  
M Wilding ◽  
E M Wright ◽  
R Patel ◽  
G Ellis-Davies ◽  
M Whitaker
Keyword(s):  
Cell Cycle ◽  
Confocal Microscopy ◽  
Sea Urchin ◽  
Calcium Release ◽  
Flash Photolysis ◽  
Mitotic Cell ◽  
Calcium Transients ◽  
Perinuclear Region ◽  
Calcium Signals ◽  
Sea Urchin Embryos

Using calcium-sensitive dyes together with their dextran conjugates and confocal microscopy, we have looked for evidence of localized calcium signaling in the region of the nucleus before entry into mitosis, using the sea urchin egg first mitotic cell cycle as a model. Global calcium transients that appear to originate from the nuclear area are often observed just before nuclear envelope breakdown (NEB). In the absence of global increases in calcium, confocal microscopy using Calcium Green-1 dextran indicator dye revealed localized calcium transients in the perinuclear region. We have also used a photoinactivatable calcium chelator, nitrophenyl EGTA (NP-EGTA), to test whether the chelator-induced block of mitosis entry can be reversed after inactivation of the chelator. Cells arrested before NEB by injection of NP-EGTA resume the cell cycle after flash photolysis of the chelator. Photolysis of chelator triggers calcium release. TreatmenT with caFfeine to enhance calcium-induced calcium release increases the amplitude of NEB-associated calcium transients. These results indicate that calcium increases local to the nucleus are required to trigger entry into mitosis. Local calcium transients arise in the perinuclear region and can spread from this region into the cytoplasm. Thus, cell cycle calcium signals are generated by the perinuclear mitotic machinery in early sea urchin embryos.

scholarly journals A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo.

1989 ◽  
Vol 109 (1) ◽  
pp. 149-161 ◽  
Author(s):  
J H Henson ◽  
D A Begg ◽  
S M Beaulieu ◽  
D J Fishkind ◽  
E M Bonder ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Calcium Release ◽  
Immunofluorescent Staining ◽  
Frozen Sections ◽  
Electron Microscopic ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Antibody Staining ◽  
Intracellular Ca

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.

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