scholarly journals Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.

1982 ◽  
Vol 92 (2) ◽  
pp. 378-386 ◽  
Author(s):  
U W Goodenough ◽  
P A Detmers ◽  
C Hwang
Keyword(s):  
Cell Fusion ◽  
Mating Type ◽  
Central Zone ◽  
Uranyl Acetate ◽  
Surface Coat ◽  
Wild Type ◽  
En Bloc ◽  
Mating Structure ◽  
Fusion Images ◽  
Coat Material

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.

scholarly journals The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

1997 ◽  
Vol 137 (7) ◽  
pp. 1537-1553 ◽  
Author(s):  
Nedra F. Wilson ◽  
Mary J. Foglesong ◽  
William J. Snell
Keyword(s):  
Cell Fusion ◽  
Mating Type ◽  
Plasma Membranes ◽  
Surface Proteins ◽  
Filamentous Actin ◽  
Mating Structure ◽  
Cell Protrusion ◽  
Isolation And Characterization ◽  
In Vitro Adhesion

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ∼3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 106 fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes.

scholarly journals ISOLATION AND GENETIC ANALYSIS OF MUTANT STRAINS OF CHLAMYDOMONAS REINHARDI DEFECTIVE IN GAMETIC DIFFERENTIATION

Genetics ◽  
1976 ◽  
Vol 82 (2) ◽  
pp. 169-186
Author(s):  
Ursula W Goodenough ◽  
Carol Hwang ◽  
Howard Martin
Keyword(s):  
Genetic Analysis ◽  
Cell Fusion ◽  
Mating Type ◽  
Normal Growth ◽  
The Other ◽  
Genetic Dissection ◽  
Structural Studies ◽  
Tetrad Analysis ◽  
Mating Structure ◽  
Mutant Strains

ABSTRACT Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-6, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt  - or mt  + gametes; these strains have been induced to form rare zygotes with mt  - gametes and the mutations are shown to be unlinked to the mt locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-1 strain agglutinate well with mt  - gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt  + locus, and fine-structural studies reveal that imp-1gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.

Distribution of concanavalin a binding carbohydrates during mating in Chlamydomonas

1984 ◽  
Vol 66 (1) ◽  
pp. 223-239
Author(s):  
B.E. Millikin ◽  
R.L. Weiss
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Concanavalin A ◽  
Sodium Dodecyl ◽  
Fluorescein Isothiocyanate ◽  
Affinity Column ◽  
Wild Type ◽  
Mating Structure ◽  
Autolytic Activity ◽  
Surface Carbohydrates

Cell surface carbohydrates, detected by fluorescein isothiocyanate/concanavalin A (FITC-ConA), were identified at four locations on gametes of Chlamydomonas reinhardtii. (1) The cell wall: uniform labelling with FITC-ConA was observed; a substantial number of sites were localized in the sodium dodecyl sulphate-insoluble inner wall, which contains the flagellar collars. (2) The periplasm: a crescent-shaped area was visualized with FITC-ConA and localized by ferritin-ConA. We were able to recover autolytic activity on a ConA affinity column from the mating medium of wild-type cells after the release of these periplasmic sites. The cell-wall-less mutant CW15 displays no periplasmic sites and demonstrates a corresponding inability to release autolytic activity after mating for 60 min. A model for wall lysis is presented, which considers the involvement of these sites in the lytic process. (3) The mating structure: during mating a small fluorescent plaque-like site was observed on cells at a location corresponding to the carbohydrate-like zone of the mating type minus mating structure and may indicate the involvement of ConA binding material in gametic cell fusion. (4) Secreted products: following cell fusion zygotes begin to secrete ConA positive material at about 1 1/2 h. After 24 h a ConA positive zygote wall and pellicle appear.

scholarly journals Transient increase in calcium efflux accompanies fertilization in Chlamydomonas.

1983 ◽  
Vol 97 (2) ◽  
pp. 397-404 ◽  
Author(s):  
R A Bloodgood ◽  
E N Levin
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Mating Type ◽  
Transient Increase ◽  
Calcium Efflux ◽  
Efflux Rate ◽  
Mating Structure ◽  
Flagellar Membrane ◽  
Intracellular Storage ◽  
Cell Cell

Mating in Chlamydomonas is a complex process initiated by contact of gametic flagellar surfaces, resulting in transmission of a signal from the flagella to the cell bodies. This signal triggers later events of cell wall loss, mating structure activation, and cell-cell fusion. Little is known about the nature of the signal or the role of Ca in these events. It was found that extracellular Ca is not necessary for successful mating in Chlamydomonas. However, cells will take up Ca from the medium in a linear manner for many hours and will accumulate micromolar concentrations, presumably by sequestering Ca within intracellular storage sites. If gametic cells of one mating type (preloaded with 45Ca) are mated with gametes of the opposite mating type (preloaded with unlabeled calcium), there is a rapid, transient increase in calcium efflux rate (20 times that of the control) that lasts approximately 6 min. This effect is not associated with cell-cell fusion, since the same observation is made if (+) gametes preloaded with 45-Ca are agglutinated by isolated flagella from (-) gametes preloaded with unlabeled Ca. Other experiments have shown that the increased efflux rate is not a simple consequence of cell wall release. Ca efflux in unmated gametes is greatly reduced in deflagellated cells, suggesting that much of the Ca movement is associated with the flagellar membrane. Although signaling itself may involve Ca fluxes across the flagellar membrane, it is also possible that a consequence of signaling is release of Ca from intracellular storage sites (perhaps functional equivalents of the sarcoplasmic reticulum). The observed transient increase in Ca efflux rate may reflect a transient increase in the cytoplasmic free-Ca concentration. This increase in cytoplasmic Ca may regulate the later events in mating (such as cell wall release and mating structure activation).

scholarly journals Gametic differentiation in Chlamydomonas reinhardtii. III. Cell wall lysis and microfilament-associated mating structure activation in wild-type and mutant strains.

1975 ◽  
Vol 67 (3) ◽  
pp. 623-637 ◽  
Author(s):  
U W Goodenough ◽  
R L Weiss
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Mating Type ◽  
Experimental Manipulation ◽  
Wild Type ◽  
Opposite Mating Type ◽  
Mating Structure ◽  
Cell Wall Lysis ◽  
Gamete Formation ◽  
Mutant Strains

Cell fusion between mating type plus (mt+) and minus (mt-) gametes of Chlamydomonas reinhardtii is analyzed structurally and subjected to experimental manipulation. Cell wall lysis, a necessary prelude to fusion, is shown to require flagellar agglutination between competent gametes; glutaraldehyde-fixed gametes ("corpses") of one mating type will elicit both agglutination and cell wall lysis in the opposite mating type, whereas nonagglutinating impotent (imp) mutant strains are without effect. The fusion process is mediated by a narrow fertilization tubule which extends from the mt+ gamete and establishes contact with the mt- gamete. Formation of the tubule requires the "activation" of a specialized mating structure associated with the ml+ cell membrane; activation causes microfilaments to polymerize from the mating structure into the growing fertilization tubule. Mating structure activation is shown to depend on gametic flagellar agglutination; isoagglutination mediated by the lectin concanavalin A has no effect. Gametes carrying the imp-l mt+ mutation are able to agglutinate but not fuse with mt- cells; the imp-l gametes are shown to have structurally defective mating structures that do not generate microfilaments in response to gametic agglutination.

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Cellular Contacts Within the Interhemal Membrane of the Rat Placenta at Term

1974 ◽  
Vol 32 ◽  
pp. 146-147
Author(s):  
William P. Jollie
Keyword(s):  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Capillary Endothelium ◽  
En Bloc ◽  
Aldehyde Fixation ◽  
Chorioallantoic Placenta

By routine EM preparative techniques, the tissues which, collectively, separate maternal and fetal bloods in the fully formed chorioallantoic placenta of the rat have been shown to consist of three chorionic layers, or trophoblast, and a layer of allantoic capillary endothelium [Fig. 1]. Relationships between these layers are best demonstrated by special techniques, viz., cacodylate-buffered aldehyde fixation, collidine-buffered osmium tetroxide postfixation, and en bloc staining with uranyl acetate. By using this method on placentas at term, the cells of the outermost chorionic layer (Trophoblast 1) appear to be attached to each other by means of maculae adherentes which sometimes occur in clusters [Fig. 2].

Ultrastructural comparison of prolactin adenomas of pituitary in men and women

1988 ◽  
Vol 46 ◽  
pp. 346-347
Author(s):  
R.C. Caughey ◽  
U.P. Kalyan-Raman
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Phosphate Buffer ◽  
Pituitary Adenomas ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Men And Women ◽  
Transmission Electron

Prolactin producing pituitary adenomas are ultrastructurally characterized by secretory granules varying in size (150-300nm), abundance of endoplasmic reticulum, and misplaced exocytosis. They are also subclassified as sparsely or densely granulated according to the amount of granules present. The hormone levels in men and women vary, being higher in men; so also the symptoms vary between both sexes. In order to understand this variation, we studied 21 prolactin producing pituitary adenomas by transmission electron microscope. This was out of a total of 80 pituitary adenomas. There were 6 men and 15 women in this group of 21 prolactinomas.All of the pituitary adenomas were fixed in 2.5% glutaraldehyde, rinsed in Millonig's phosphate buffer, and post fixed with 1% osmium tetroxide. They were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole's non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin.

Microprobe analysis of inclusion bodies related to two benzofuran derivatives

1987 ◽  
Vol 45 ◽  
pp. 672-673
Author(s):  
T. L. Benning ◽  
P. Ingram ◽  
J. D. Shelburne
Keyword(s):  
Inclusion Bodies ◽  
Uranyl Acetate ◽  
Multiple Organ ◽  
High Dose ◽  
En Bloc ◽  
Tissue Samples ◽  
Transmission Electron ◽  
Organ Systems ◽  
Dense Inclusion ◽  
Melanin Complex

Two benzofuran derivatives, chlorpromazine and amiodarone, are known to produce inclusion bodies in human tissues. Prolonged high dose chlorpromazine therapy causes hyperpigmentation of the skin with electron-dense inclusion bodies present in dermal histiocytes and endothelial cells ultrastructurally. The nature of the deposits is not known although a drug-melanin complex has been hypothesized. Amiodarone may also cause cutaneous hyperpigmentation and lamellar lysosomal inclusion bodies have been demonstrated within the cells of multiple organ systems. These lamellar bodies are believed to be the product of an amiodarone-induced phospholipid storage disorder. We performed transmission electron microscopy (TEM) and energy dispersive x-ray microanalysis (EDXA) on tissue samples from patients treated with these drugs, attempting to detect the sulfur atom of chlorpromazine and the iodine atom of amiodarone within their respective inclusion bodies.A skin biopsy from a patient with hyperpigmentation due to prolonged chlorpromazine therapy was fixed in 4% glutaraldehyde and processed without osmium tetroxide or en bloc uranyl acetate for Epon embedding.

An ultrastructural study of sertoli cells in two geographically isolated populations of Aphanius dispar

1992 ◽  
Vol 50 (1) ◽  
pp. 548-549
Author(s):  
Taber A. Ba-Omar ◽  
Philip F. Prentis
Keyword(s):  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Freshwater Teleost ◽  
Isolated Populations ◽  
En Bloc ◽  
The North ◽  
Group A ◽  
Ultrathin Sections ◽  
Group B ◽  
Geographically Isolated

We have recently carried out a study of spermiogenic differentiation in two geographically isolated populations of Aphanius dispar (freshwater teleost), with a view to ascertaining variation at the ultrastructural level. The sampling areas were the Jebel Al Akhdar in the north (Group A) and the Dhofar region (Group B) in the south. Specimens from each group were collected, the testes removed, fixed in Karnovsky solution, post fixed in OsO, en bloc stained with uranyl acetate and then routinely processed to Agar 100 resin, semi and ultrathin sections were prepared for study.

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