scholarly journals Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts.

1976 ◽  
Vol 71 (3) ◽  
pp. 704-714 ◽  
Author(s):  
R E Kane
Keyword(s):  
Sea Urchin ◽  
Low Temperatures ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Complex Structure ◽  
The Other ◽  
Cytoplasmic Proteins ◽  
Sea Urchin Egg ◽  
Low Concentrations ◽  
Egg Extracts

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 22,000 mol wt. When combined in 0.1 M KCl, even at 0 degrees C, these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm. This association of F-actin with the other proteins to form a gel is very likely the last step fo the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous F-actin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.

scholarly journals Interconversion of structural and contractile actin gels by insertion of myosin during assembly.

1983 ◽  
Vol 97 (6) ◽  
pp. 1745-1752 ◽  
Author(s):  
R E Kane
Keyword(s):  
Sea Urchin ◽  
Actin Polymerization ◽  
Small Volume ◽  
Final Concentration ◽  
Cytoplasmic Structure ◽  
Cytoplasmic Proteins ◽  
Sea Urchin Egg ◽  
Low Salt ◽  
Sequential Combination ◽  
Characteristic Banding

Extracts of the soluble cytoplasmic proteins of the sea urchin egg form gels of different composition and properties depending on the temperature used to induce actin polymerization. At temperatures that inactivate myosin, a gel composed of actin, fascin, and a 220,000-mol-wt protein is formed. Fascin binds actin into highly organized units with a characteristic banding pattern, and these actin-fascin units are the structural core of the sea urchin microvilli formed after fertilization and of the urchin coelomocyte filopods. Under milder conditions a more complex myosin-containing gel is formed, which contracts to a small fraction of its original volume within an hour after formation. What has been called "structural" gel can be assembled by combining actin, fascin, and the 220,000-mol-wt protein in 50-100 mM KCl; the aim of the experiments reported here was to determine whether myosin could be included during assembly, thereby interconverting structural and contractile gel. This approach is limited by the aggregation of sea urchin myosin at the low salt concentrations utilized in gel assembly. A method has been devised for the sequential combination of these components under controlled KCl and ATP concentrations that allows the formation of a gel containing dispersed myosin at a final concentration of 60-100 mM KCl. These gels are stable at low (approximately 10 micron) ATP concentrations, but contract to a small volume in the presence of higher (approximately 100 micron) ATP. Contraction can be controlled by forming a stable gel at low ATP and then overlaying it with a solution containing sufficient ATP to induce contraction. This system may provide a useful model for the study of the interrelations between cytoplasmic structure and motility.

scholarly journals Preparation and purification of polymerized actin from sea urchin egg extracts.

1975 ◽  
Vol 66 (2) ◽  
pp. 305-315 ◽  
Author(s):  
R E Kane
Keyword(s):  
Sea Urchin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mammalian Brain ◽  
Simple Method ◽  
Cytoplasmic Proteins ◽  
Sea Urchin Egg ◽  
Egg Extracts ◽  
Low Levels ◽  
Birefringent Fibrils

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

scholarly journals pH regulates the polymerization of actin in the sea urchin egg cortex.

1979 ◽  
Vol 83 (1) ◽  
pp. 241-248 ◽  
Author(s):  
D A Begg ◽  
L I Rebhun
Keyword(s):  
Sea Urchin ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Filamentous Actin ◽  
Cortical Granules ◽  
Isolation Medium ◽  
Large Numbers ◽  
Sea Urchin Egg ◽  
Isolated Cortex ◽  
Egg Cortex

The state of actin in the isolated cortex of the unfertilized sea urchin egg can be controlled by experimentally manipulating the pH of the isolation medium. Cortices isolated at the pH of the unfertilized egg (6.5--6.7) do not contain filamentous actin, while those isolated at the pH of the fertilized egg (7.3--7.5) develop large numbers of microvilli which contain bundles of actin filaments. Cortices that are isolated at pH 6.5 and then transferred to isolation medium buffered at pH 7.5 also develop actin filaments. However, the filaments are not arranged in bundles and microvilli do not form. Although the cortical granules in cortices isolated at pH 6.5 discharge at a free Ca++ concentration of approximately 10 micrometer, actin polymerization is not induced by increasing the Ca++ concentration of the isolation medium. These results suggest that the increase in cytoplasmic pH which occurs following fertilization induces the polymerization of actin in the egg cortex.

scholarly journals A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

1984 ◽  
Vol 99 (3) ◽  
pp. 994-1001 ◽  
Author(s):  
H Hosoya ◽  
I Mabuchi
Keyword(s):  
Sea Urchin ◽  
Actin Filament ◽  
Actin Polymerization ◽  
Initial Rate ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Capping Protein ◽  
Sea Urchin Egg ◽  
Rate Of Polymerization ◽  
Hemicentrotus Pulcherrimus

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.

Wave of cortical actin polymerization in the sea urchin egg

1987 ◽  
Vol 7 (1) ◽  
pp. 46-53 ◽  
Author(s):  
Shigenobu Yonemura ◽  
Issei Mabuchi
Keyword(s):  
Sea Urchin ◽  
Actin Polymerization ◽  
Cortical Actin ◽  
Sea Urchin Egg

scholarly journals Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3573
Author(s):  
Nunzia Limatola ◽  
Jong Tai Chun ◽  
Sawsen Cherraben ◽  
Jean-Louis Schmitt ◽  
Jean-Marie Lehn ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Early Development ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Cortical Granule ◽  
Cortical Actin ◽  
Phenotypic Changes ◽  
Sea Urchin Egg ◽  
Cortical Granule Exocytosis

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg’s fertilization response.

Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose

2000 ◽  
Vol 113 (24) ◽  
pp. 4413-4420 ◽  
Author(s):  
H.C. Lee ◽  
R. Aarhus
Keyword(s):  
Endoplasmic Reticulum ◽  
Sea Urchin ◽  
The Other ◽  
Calcium Stores ◽  
Cellular Functions ◽  
Large Size ◽  
Opposite Pole ◽  
Visual Evidence ◽  
Sea Urchin Egg ◽  
Cyclic Adp Ribose

Cells possess multiple Ca(2+) stores and their selective mobilization provides the spatial-temporal Ca(2+) signals crucial in regulating diverse cellular functions. Except for the inositol trisphosphate (IP(3))-sensitive Ca(2+) stores, the identities and the mechanisms of how these internal stores are mobilized are largely unknown. In this study, we describe two Ca(2+) stores, one of which is regulated by cyclic ADP-ribose (cADPR) and the other by nicotinic acid adenine dinucleotide phosphate (NAADP). We took advantage of the large size of the sea urchin egg and stratified its organelles by centrifugation. Using photolysis to produce either uniform or localized increases of cADPR and NAADP from their respective caged analogs, the two separate stores could be visually identified by Ca(2+) imaging and shown to be segregated to the opposite poles of the eggs. The cADPR-pole also contained the IP(3)-sensitive Ca(2+) stores, the egg nucleus and the endoplasmic reticulum (ER); the latter was visualized using Bodipy-thapsigargin. On the other hand, the mitochondria, as visualized by rhodamine 123, were segregated to the opposite pole together with the NAADP-sensitive calcium stores. Fertilization of the stratified eggs elicited a Ca(2+) wave starting at the cADPR-pole and propagating toward the NAADP-pole. These results provide the first direct and visual evidence that the NAADP-sensitive Ca(2+) stores are novel and distinct from the ER. During fertilization, communicating signals appear to be transmitted from the ER to NAADP-sensitive Ca(2+) stores, leading to their activation.

scholarly journals Actin, microvilli, and the fertilization cone of sea urchin eggs.

1980 ◽  
Vol 87 (3) ◽  
pp. 771-782 ◽  
Author(s):  
L G Tilney ◽  
L A Jaffe
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Germinal Vesicle ◽  
Rapid Phase ◽  
Thin Sections ◽  
Step Process ◽  
Sea Urchin Eggs ◽  
Early Stages

Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges.

scholarly journals Studies on Sulfhydryl Groups during Cell Division of Sea Urchin Egg

1962 ◽  
Vol 45 (3) ◽  
pp. 427-438 ◽  
Author(s):  
Hikoichi Sakai
Keyword(s):  
Cell Division ◽  
Sea Urchin ◽  
Soluble Fraction ◽  
Water Soluble ◽  
The Other ◽  
Original Sample ◽  
Water Soluble Fraction ◽  
Sea Urchin Egg ◽  
Soluble Fractions

The contractility of the thread model prepared from the KCl-soluble proteins of the egg and in vivo factors for the contraction are investigated in Hemicentrotus, Anthocidaris, and Pseudocentrotus eggs. The contractility of the thread model induced by metal ions or cystine changes during development in the characteristic pattern of high at the metaphase and low at the monaster and the interkinetic stages. The change in contractility is paralleled by the change in the —SH content of the protein. The water-soluble fraction of the eggs has activity in causing contraction of the thread model. This activity changes during development in the same way as the contractility itself. The contraction of the thread induced by the water-soluble fractions is accompanied by a decrease in the —SH content of the thread. The activity of the water-soluble fraction in inducing the contraction is proportional to its ability to decrease the number of —SH groups. On boiling, the activity is largely destroyed. The activity is due to two components, one being non-dialyzable and the other dialyzable. Separately each component has little effect, but when mixed, the activity of the original sample is completely restored.

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