scholarly journals Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

1977 ◽  
Vol 75 (3) ◽  
pp. 899-914 ◽  
Author(s):  
NK Detering ◽  
GL Decker ◽  
ED Schmell ◽  
WJ Lennarz
Keyword(s):  
Plasma Membrane ◽  
Specific Radioactivity ◽  
Surface Glycoprotein ◽  
Soluble Form ◽  
Cortical Granule ◽  
Initial Surface ◽  
Cortical Granules ◽  
Isolation And Characterization

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.

Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome

1992 ◽  
Vol 102 (1) ◽  
pp. 91-102 ◽  
Author(s):  
M. Kallajoki ◽  
K. Weber ◽  
M. Osborn
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Nuclear Matrix ◽  
Essential Role ◽  
Soluble Form ◽  
Microtubule Nucleation ◽  
Brain Microtubules ◽  
Mitotic Cells

The SPN antigen plays an essential role in mitosis, since microinjection of antibodies causes mitotic arrest. Here we show, by examination of the relative locations of SPN antigen, the centrosomal 5051 antigen and tubulin in normal mitotic, and in taxol-treated mitotic cells, that the SPN antigen is involved in organizing the microtubules of the spindle. The 210 kDa protein defined as SPN antigen relocates from the nuclear matrix to the centrosome at prophase, remains associated with the poles at metaphase and anaphase, and dissociates from the centrosomes in telophase. In taxol-treated mitotic cells, SPN staining shows a striking redistribution while 5051 antigen remains associated with centrosomes. SPN antigen is seen at the plasma membrane end of the rearranged microtubules. SPN antigen is always at the center of the multiple microtubule asters (5 to 20 per cell) induced by taxol, whereas 5051 again remains associated with the centrosomal complex (1 to 2 foci per cell). Microtubule nucleation is associated with the SPN antigen rather than with the 5051 antigen. Microinjection of SPN-3 antibody into taxol-treated mitotic PtK2 cells causes disruption of the asters as judged by tubulin staining of the same cells. Finally, SPN antigen extracted in soluble form from synchronized mitotic HeLa cells binds to, and sediments with, pig brain microtubules stabilized by taxol. This association of SPN antigen with microtubules is partially dissociated by 0.5 M NaCl but not by 5 mM ATP. Thus SPN antigen binds to microtubules in vitro and seems to act as a microtubular minus-end organizer in mitotic cells in vivo.

Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function

1983 ◽  
Vol 218 (1213) ◽  
pp. 397-413 ◽  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granule ◽  
Regulatory Protein ◽  
Calcium Sensitivity ◽  
Magnesium Concentration ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Calcium Dependent ◽  
Cortical Granule Exocytosis

Egg cortical granules remain attached to the egg plasma membrane when the egg is ruptured. We present evidence that demonstrates that, when the cytoplasmic face of the egg plasma membrane is exposed to micromolar calcium concentrations, an exocytosis of the cortical granules occurs which corresponds to the cortical granule exocytosis seen when the egg is fertilized. The calcium sensitivity of the preparation is decreased by an increase in magnesium concentration and increased by a decrease in magnesium concentration. Exocytosis is inhibited by trifluoperazine (half inhibition at 6 μm), a drug that inhibits the action of the calciumdependent regulatory protein calmodulin. Colchicine, vinblastine, nocodazole, cytochalasin B, phalloidin N -ethylmaleimide-modified myosin subfragment 1, and antibody to actin are without effect on this in vitro exocytosis at concentrations that far exceed those required to disrupt microtubules and microfilaments. Conditions are such that penetration to the exocytotic site is optimal. It is unlikely, therefore, that either actin or tubulin participate intimately in exocytosis. Our data also exclude on quantitative grounds several other mechanisms postulated to account for the fusion of the secretory granule with the plasma membrane.

359 MORPHOLOGIC AND BIOCHEMISTRY ALTERATIONS ON BOVINE OOCYTE MATURATION IN VITRO WITH NITRIC OXIDE AND ITS IMPACT ON EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 336 ◽  
Author(s):  
K. S. Viana ◽  
M. C. C. Bussiere ◽  
C. S. Paes de Carvalho ◽  
B. L. Dias ◽  
M. R. Faes ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Plasma Membrane ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Control Group ◽  
Cortical Granule ◽  
Bovine Oocyte ◽  
Cortical Granules ◽  
Maturation In Vitro

The aim of this study was to evaluate morphologic and biochemistry alterations caused by the addition of sodium nitroprusside (SNP), a NO donor, on bovine oocyte maturation in vitro. Bovine ovaries were collected at a local abattoir. COC were cultured in TCM-199 with 10% FCS, 0.5 μg mL-1 FSH, 5.0 μg mL-1 LH, and antibiotics. Analysis of variance was conducted and the means were compared by t-test at a level of 5%. Experimental design: (1) evaluation of the oocyte plasma membrane viability and integrity using Annexin V/propidium iodide (PI) and Hoechst 33342/PI staining, respectively; (2) microtubule and microfilament organization, and migration of cortical granules by immunofluorescence; (3) oocyte glutathione content and concentration of NO3-/NO2-using the method of Griess (Ricart-Jane D et al. 2002 Nitric Oxide 6, 178-185) and (4) embryo development. In Experiment 1, the addition of 1 mM SNP caused cellular death in the majority of the oocytes [100%, AnnexinV/PI (+) and 80.7% Hoescht/IP (+)] differing from the control group and the 0.01 mM SNP (P < 0.05). In Experiment 2, the microtubule staining was observed in the cytoplasm in both control group and 0.01 mM SNP; however, the group treated with 1 mM of SNP exhibited clear defects in spindle and chromatin arrangements (P < 0.05). No alterations in microfilaments disposition was observed in the control group and 0.01 mM SNP. However, after the addition of 1 mM, the microfilaments arranged into clusters, and not below of the cortex. Oocytes treated with 1 mM SNP (68.2%) showed total cortical granule migration to the periphery of the ooplasm and were similar to the control group (72.2%) (P > 0.05). Nevertheless, in the group treated with 0.01 mM SNP the total cortical granule migration was greater (86.8%, P < 0.05). In Experiment 3, the glutathione content of oocytes cultured in the presence of 1 mM SNP was lower (4.4p mol) when compared to the control group (5.4p mol) and 0.01 mM SNP (5.5 pmol) (P > 0.05). The concentration of NO in the medium were similar to both control group (6.0 ± 3.0 μM) and treated with 0.01 mM SNP (15.8 ± 1.9 μM), however, the treatment with 1 mM SNP increased 10 times (59.9 ± 12.0 μM; P < 0.05) this concentration. In Experiment 4, cleavage rates and embryo development were similar for groups control and 0.01 mM SNP (P > 0.05). Even so, in the group treated with 0.01 mM there was a greater blastocyst cell number when compared to the control group (256.8 ± 52.5 and 196.9 ± 54.0, respectively-P < 0.05). These results indicate that: (1) the addition of 0.01 mM SNP increased the quality of the oocyte maturation, leading to a higher percentage of cortical granules migration and blastocyst cell numbers, in a different pathway from that of glutathione; (2) the addition of 1 mM of SNP caused a cytotoxic effect, leading to cellular death with loss of viability and integrity of plasma membrane, absence of nuclear maturation/organization of cytoskeleton and reduction of the glutathione content, although with no intervention in the migration of cortical granules.

scholarly journals Galectin–glycan lattices regulate cell-surface glycoprotein organization and signalling

2008 ◽  
Vol 36 (6) ◽  
pp. 1472-1477 ◽  
Author(s):  
Omai B. Garner ◽  
Linda G. Baum
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Targeted Delivery ◽  
Cell Types ◽  
Surface Glycoprotein ◽  
Membrane Domains ◽  
Cellular Functions ◽  
Cell Surface Glycoprotein

The formation of multivalent complexes of soluble galectins with glycoprotein receptors on the plasma membrane helps to organize glycoprotein assemblies on the surface of the cell. In some cell types, this formation of galectin–glycan lattices or scaffolds is critical for organizing plasma membrane domains, such as lipid rafts, or for targeted delivery of glycoproteins to the apical or basolateral surface. Galectin–glycan lattice formation is also involved in regulating the signalling threshold of some cell-surface glycoproteins, including T-cell receptors and growth factor receptors. Finally, galectin–glycan lattices can determine receptor residency time by inhibiting endocytosis of glycoprotein receptors from the cell surface, thus modulating the magnitude or duration of signalling from the cell surface. This paper reviews recent evidence in vitro and in vivo for critical physiological and cellular functions that are regulated by galectin–glycoprotein interactions.

Cortical granules of the sea urchin translocate early in oocyte maturation

Development ◽  
1997 ◽  
Vol 124 (9) ◽  
pp. 1845-1850
Author(s):  
L.K. Berg ◽  
G.M. Wessel
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown

Cortical granules are secretory vesicles poised at the cortex of an egg that, upon stimulation by sperm contact at fertilization, secrete their contents. These contents modify the extracellular environment and block additional sperm from reaching the egg. The role of cortical granules in blocking polyspermy is conserved throughout much of phylogeny. In the sea urchin, cortical granules accumulate throughout the cytoplasm during oogenesis, but in mature eggs the cortical granules are attached to the plasma membrane, having translocated to the cortex at some earlier time. To study the process of cortical granule translocation to the cell surface we have devised a procedure for maturation of sea urchin oocytes in vitro. Using this procedure, we examined the rate of oocyte maturation by observing the movement and breakdown of the germinal vesicle, the formation of polar bodies and the formation of the egg pronucleus. We find that oocyte maturation takes approximately 9 hours in the species used here (Lytechinus variegatus), from the earliest indication of maturation (germinal vesicle movement) to formation of a distinct pronucleus. We then observed the translocation of cortical granules in these cells by immunolocalization using a monoclonal antibody to hyalin, a protein packaged specifically in cortical granules. We found that the translocation of cortical granules in in vitro-matured oocytes begins with the movement of the germinal vesicle to the oocyte cell surface, and is 50% complete 1 hour after germinal vesicle breakdown. In the in vitro-matured egg, 99% of the cortical granules are at the cortex, indistinguishable from translocation in oocytes that mature in vivo. We have also found that eggs that mature in vitro are functionally identical to eggs that mature in vivo by four criteria. (1) The matured cells undergo a selective turnover of mRNA encoding cortical granule contents. (2) The newly formed pronucleus begins transcription of histone messages. (3) Cortical granules that translocate in vitro are capable of exocytosis upon activation by the calcium ionophore, A23187. (4) The mature egg is fertilizable and undergoes normal cleavage and development. In vitro oocyte maturation enables us to examine the mechanism of cortical granule translocation and other processes that had previously only been observed in static sections of fixed ovaries.

scholarly journals Isolation and characterization of sea urchin egg cortical granules.

1982 ◽  
Vol 95 (3) ◽  
pp. 924-932 ◽  
Author(s):  
G S Kopf ◽  
G W Moy ◽  
V D Vacquier
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Cortical Granules ◽  
Electrophoretic Patterns ◽  
Isolation And Characterization ◽  
Sea Urchin Egg

A method has been developed to isolate cortical granules (CG) free in suspension. It involves the mechanical disruption of the CG from CG lawns (CGL; Dev. Biol. 43:62-74, 1975) and concentration of the CG by low speed centrifugation. The isolated CG are intact and are a relatively pure population as judged by electron microscopy. Granule integrity is confirmed by the fact that isolated intact CG are radioiodinated to only 0.05% of the specific activity of hypotonically lysed CG. Purity of the CG preparation is assessed by the enrichment (four- to sevenfold) of CG marker enzymes and the absence or low activity of plasma membrane, mitochondrial, cytoplasmic, and yolk platelet marker enzyme activities. CG isolated from 125I-surface-labeled eggs have a very low specific radioactivity, demonstrating that CG contamination by the plasma membrane-vitelline layer (PM-VL) is minimal. CG yield is approximately 1% of the starting egg protein. The CG isolation method is simple and rapid, 4 mg of CG protein being obtained in 1 h. Isolated CG and PM-VL display distinct electrophoretic patterns on SDS gels. Actin is localized to the PM-VL, and all bands present in the CGL are accounted for in the CG and PM-VL. Calmodulin is associated with the CGL, CG, and PM-VL fractions, but is not specifically enriched in these fractions as compared with whole egg homogenates. This method of isolating intact CG from unfertilized sea urchin eggs may be useful for exploring the mechanism of Ca2+-mediated CG exocytosis.

scholarly journals Extraction of active RhoGTPases by RhoGDI regulates spatiotemporal patterning of RhoGTPases

2019 ◽  
Author(s):  
Adriana Golding ◽  
Ilaria Visco ◽  
Peter Bieling ◽  
William Bement
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Bound States ◽  
Molecular Switches ◽  
Current Understanding ◽  
Soluble Form ◽  
Direct Visualization ◽  
Spatial Regulation

AbstractThe RhoGTPases are characterized as membrane-associated molecular switches cycling between active, GTP-bound and inactive, GDP-bound states. However, 90-95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these tools, we identified pools of active and inactive RhoGTPase associated with the membrane, showed that RhoGDI can actively extract both inactive and active RhoGTPases, and that the extraction of active RhoGTPase contributes to their spatial regulation around wounds. In contrast to the textbook model of the RhoGTPase cycle, these results indicate that RhoGDI actively contributes to spatiotemporal patterning by removing active RhoGTPases from the plasma membrane.

scholarly journals Extraction of active RhoGTPases by RhoGDI regulates spatiotemporal patterning of RhoGTPases

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Adriana E Golding ◽  
Ilaria Visco ◽  
Peter Bieling ◽  
William M Bement
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Bound States ◽  
Molecular Switches ◽  
Current Understanding ◽  
Soluble Form ◽  
Direct Visualization ◽  
Spatial Regulation

The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active, GTP-bound and inactive, GDP-bound states. However, 90–95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these methods, we identified pools of active and inactive RhoGTPase associated with the membrane, found that RhoGDI can extract both inactive and active RhoGTPases, and found that extraction of active RhoGTPase contributes to their spatial regulation around cell wounds. These results indicate that RhoGDI directly contributes to the spatiotemporal patterning of RhoGTPases by removing active RhoGTPases from the plasma membrane.

Radiolabeling of Fibrinogen Using the Iodogen Technique

1981 ◽  
Vol 46 (03) ◽  
pp. 593-596 ◽  
Author(s):  
Linda C Knight ◽  
Andrei Z Budzynski ◽  
Stephanie A Olexa
Keyword(s):  
Specific Radioactivity ◽  
Oxidizing Agent ◽  
Iodine Monochloride ◽  
Human Fibrinogen

SummaryThe properties of human fibrinogen labeled with 125-Iodine using Iodogen (1, 3, 4, 6-tetrachloro-3α, 6α-diphenylglycoluril) as an oxidizing agent were compared with those of an iodine monochloride labeled counterpart. It was found that thrombin clottability, binding to staphylococci, the relative specific radioactivity of the Aα, Bβ, and γ chains and in vivo clearance from plasma in rabbits were the same in these two labeled fibrinogen preparations. Labeling efficiency was higher when iodogen was used. It is concluded that human fibrinogen labeled with radioiodine using the Iodogen technique is suitable for studies in vitro and in vivo.

ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

2020 ◽  
Author(s):  
Avik Sotira Scientific
Keyword(s):  
Social Life ◽  
Plaque Assay ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Crystallographic Structure ◽  
Therapeutic Implications ◽  
Biochemical Assays ◽  
Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

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