scholarly journals Unveiling a novel function of CD9 in surface compartmentalization of oocytes

Development ◽  
2020 ◽  
Vol 147 (15) ◽  
pp. dev189985
Author(s):  
Naokazu Inoue ◽  
Takako Saito ◽  
Ikuo Wada
Keyword(s):  
Crucial Role ◽  
Molecular Organization ◽  
Cortical Actin ◽  
Gamete Fusion ◽  
Fusion Site ◽  
Molecular Profiles ◽  
Fusion Ability ◽  
Unique Surface

ABSTRACTGamete fusion is an indispensable process for bearing offspring. In mammals, sperm IZUMO1–oocyte JUNO recognition essentially carries out the primary step of this process. In oocytes, CD9 is also known to play a crucial role in gamete fusion. In particular, microvilli biogenesis through CD9 involvement appears to be a key event for successful gamete fusion, because CD9-disrupted oocytes produce short and sparse microvillous structures, resulting in almost no fusion ability with spermatozoa. In order to determine how CD9 and JUNO cooperate in gamete fusion, we analyzed the molecular profiles of each molecule in CD9- and JUNO-disrupted oocytes. Consequently, we found that CD9 is crucial for the exclusion of GPI-anchored proteins, such as JUNO and CD55, from the cortical actin cap region, suggesting strict molecular organization of the unique surface of this region. Through distinct surface compartmentalization due to CD9 governing, GPI-anchored proteins are confined to the appropriate fusion site of the oocyte.

scholarly journals Positional relationship between the gamete fusion site and the first division plane in the rice zygote

2010 ◽  
Vol 61 (11) ◽  
pp. 3101-3105 ◽  
Author(s):  
Keisuke Nakajima ◽  
Takao Uchiumi ◽  
Takashi Okamoto
Keyword(s):  
Gamete Fusion ◽  
Division Plane ◽  
Fusion Site

Molecular organization of the desmoglein-plakoglobin complex

1998 ◽  
Vol 111 (14) ◽  
pp. 1941-1949 ◽  
Author(s):  
N.A. Chitaev ◽  
A.Z. Averbakh ◽  
R.B. Troyanovsky ◽  
S.M. Troyanovsky
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gradient Centrifugation ◽  
Intercellular Junctions ◽  
Molecular Organization ◽  
Cortical Actin ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Desmosomal Cadherins ◽  
E Cadherin

Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and alpha-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with alpha-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent co-immunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1–3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with alpha-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as alpha-catenin and desmosomal cadherins.

scholarly journals Correlative Light-Electron Microscopy Reveals the Tubular-Saccular Ultrastructure of Carriers Operating between Golgi Apparatus and Plasma Membrane

2000 ◽  
Vol 148 (1) ◽  
pp. 45-58 ◽  
Author(s):  
Roman S. Polishchuk ◽  
Elena V. Polishchuk ◽  
Pierfrancesco Marra ◽  
Saverio Alberti ◽  
Roberto Buccione ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Molecular Organization ◽  
Maximum Diameter ◽  
Intracellular Traffic ◽  
Fusion Site ◽  
Green Fluorescent

Transport intermediates (TIs) have a central role in intracellular traffic, and much effort has been directed towards defining their molecular organization. Unfortunately, major uncertainties remain regarding their true structure in living cells. To address this question, we have developed an approach based on the combination of the green fluorescent protein technology and correlative light-electron microscopy, by which it is possible to monitor an individual carrier in vivo and then take a picture of its ultrastructure at any moment of its lifecycle. We have applied this technique to define the structure of TIs operating from the Golgi apparatus to the plasma membrane, whose in vivo dynamics have been characterized recently by light microscopy. We find that these carriers are large (ranging from 0.3–1.7 μm in maximum diameter, nearly half the size of a Golgi cisterna), comprise almost exclusively tubular-saccular structures, and fuse directly with the plasma membrane, sometimes minutes after docking to the fusion site.

Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1995-2002 ◽  
Author(s):  
Guo-Zhang Zhu ◽  
Brent J. Miller ◽  
Claude Boucheix ◽  
Eric Rubinstein ◽  
Christopher C. Liu ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Point Mutation ◽  
Active Site ◽  
Gene Knockout ◽  
Extracellular Loop ◽  
Gamete Fusion ◽  
Extracellular Loops ◽  
Egg Membrane ◽  
Large Extracellular Loop ◽  
Fusion Ability

Gamete fusion is the fundamental first step initiating development of a new organism. Female mice with a gene knockout for the tetraspanin CD9 (CD9 KO mice) produce mature eggs that cannot fuse with sperm. However, nothing is known about how egg surface CD9 functions in the membrane fusion process. We found that constructs including CD9’s large extracellular loop significantly inhibited gamete fusion when incubated with eggs but not when incubated with sperm, suggesting that CD9 acts by interaction with other proteins in the egg membrane. We also found that injecting developing CD9 KO oocytes with CD9 mRNA restored fusion competence to the resulting CD9 KO eggs. Injecting mRNA for either mouse CD9 or human CD9, whose large extracellular loops differ in 18 residues, rescued fusion ability of the injected CD9 KO eggs. However, when the injected mouse CD9 mRNA contained a point mutation (F174 to A) the gamete fusion level was reduced fourfold, and a change of three residues (173-175, SFQ to AAA) abolished CD9’s activity in gamete fusion. These results suggest that SFQ in the CD9 large extracellular loop may be an active site which associates with and regulates the egg fusion machinery.

Bimolecular and Monomolecular Lipid Films: Selected Area Electron Diffraction

1969 ◽  
Vol 27 ◽  
pp. 338-339
Author(s):  
Robert M. Glaeser ◽  
David W. Deamer
Keyword(s):  
Electron Diffraction ◽  
Membrane Structure ◽  
Molecular Organization ◽  
Membrane Material ◽  
X Ray Diffraction ◽  
Membrane Systems ◽  
X Ray ◽  
Selected Area Electron Diffraction ◽  
Lipid Films ◽  
Ultimate Objective

In the investigation of the molecular organization of cell membranes it is often supposed that lipid molecules are arranged in a bimolecular film. X-ray diffraction data obtained in a direction perpendicular to the plane of suitably layered membrane systems have generally been interpreted in accord with such a model of the membrane structure. The present studies were begun in order to determine whether selected area electron diffraction would provide a tool of sufficient sensitivity to permit investigation of the degree of intermolecular order within lipid films. The ultimate objective would then be to apply the method to single fragments of cell membrane material in order to obtain data complementary to the transverse data obtainable by x-ray diffraction.

Evaluation of Freezing Methods by Low Temperature X-Ray Diffraction

1977 ◽  
Vol 35 ◽  
pp. 330-333
Author(s):  
T. Gulik-Krzywicki ◽  
M.J. Costello
Keyword(s):  
Biological Materials ◽  
Molecular Organization ◽  
X Ray Diffraction ◽  
Glass Capillary ◽  
X Ray ◽  
Rate Dependent ◽  
Before And After ◽  
Freeze Etching ◽  
Diffraction Patterns ◽  
Set Up

Freeze-etching electron microscopy is currently one of the best methods for studying molecular organization of biological materials. Its application, however, is still limited by our imprecise knowledge about the perturbations of the original organization which may occur during quenching and fracturing of the samples and during the replication of fractured surfaces. Although it is well known that the preservation of the molecular organization of biological materials is critically dependent on the rate of freezing of the samples, little information is presently available concerning the nature and the extent of freezing-rate dependent perturbations of the original organizations. In order to obtain this information, we have developed a method based on the comparison of x-ray diffraction patterns of samples before and after freezing, prior to fracturing and replication.Our experimental set-up is shown in Fig. 1. The sample to be quenched is placed on its holder which is then mounted on a small metal holder (O) fixed on a glass capillary (p), whose position is controlled by a micromanipulator.

On the structural organization of biological pumps and channels

1984 ◽  
Vol 42 ◽  
pp. 138-141
Author(s):  
G. Zampighi ◽  
M. Kreman
Keyword(s):  
Active Transport ◽  
Transport System ◽  
Plasma Membranes ◽  
Transport Proteins ◽  
Molecular Organization ◽  
Passive Transport ◽  
Concentration Gradients ◽  
Cell Functions ◽  
Natural Concentration ◽  
Active Transport System

The plasma membranes of most animal cells contain transport proteins which function to provide passageways for the transported species across essentially impermeable lipid bilayers. The channel is a passive transport system which allows the movement of ions and low molecular weight molecules along their concentration gradients. The pump is an active transport system and can translocate cations against their natural concentration gradients. The actions and interplay of these two kinds of transport proteins control crucial cell functions such as active transport, excitability and cell communication. In this paper, we will describe and compare several features of the molecular organization of pumps and channels. As an example of an active transport system, we will discuss the structure of the sodium and potassium ion-activated triphosphatase [(Na+ +K+)-ATPase] and as an example of a passive transport system, the communicating channel of gap junctions and lens junctions.

High-resolution measurement of birefringent fine structure in living cells using a new polarized light microscope

1995 ◽  
Vol 53 ◽  
pp. 54-55
Author(s):  
Rudolf Oldenbourg
Keyword(s):  
Light Microscope ◽  
Fluorescent Dyes ◽  
Polarized Light ◽  
Processing System ◽  
Video Camera ◽  
Molecular Organization ◽  
Computer Assisted ◽  
Image Recording ◽  
Birefringent Crystal ◽  
Technological Advances

The recent renaissance of the light microsope is fueled in part by technological advances in components on the periphery of the microscope, such as the laser as illumination source, electronic image recording (video), computer assisted image analysis and the biochemistry of fluorescent dyes for labeling specimens. After great progress in these peripheral parts, it seems timely to examine the optics itself and ask how progress in the periphery facilitates the use of new optical components and of new optical designs inside the microscope. Some results of this fruitful reflection are presented in this symposium.We have considered the polarized light microscope, and developed a design that replaces the traditional compensator, typically a birefringent crystal plate, with a precision universal compensator made of two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at ALL POINTS of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen.

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