Regional differentiation of the membrane skeleton in Tetrahymena

1987 ◽  
Vol 87 (3) ◽  
pp. 457-463
Author(s):  
N.E. Williams ◽  
J.E. Honts ◽  
R.F. Jaeckel-Williams
Keyword(s):  
Cell Surface ◽  
Cell Shape ◽  
Surface Membrane ◽  
Tetrahymena Pyriformis ◽  
Membrane Skeleton ◽  
Regional Differentiation ◽  
Basal Bodies ◽  
Structural Elements ◽  
Molecular Weights ◽  
Triton X

Antisera have been raised in rabbits against three high molecular weight proteins that are present in Triton X-100-insoluble residues of Tetrahymena pyriformis GL cells. These proteins, called A, B and C, have apparent molecular weights of 235, 135 and 125 (X 10(3)), respectively, in SDS-polyacrylamide gels. The antisera obtained are specific for these proteins, as shown by immunoblotting. Immunolocalization studies are reported that suggest that these proteins are present throughout the epiplasmic layer beneath the cell surface (membrane skeleton). Images obtained with the fluorescence microscope, however, suggest that the membrane skeleton is modified in discrete zones: (1) around somatic basal bodies, (2) within the oral apparatus, (3) in the cytoproct, (4) in contractile vacuole pores, (5) in the fission zone in late division, and (6) at the mating junction in conjugating cells. These regions may represent areas of increased rigidity at the cell surface. The transition from pliable to rigid epiplasm in spatially delimited areas is apparently a recurring theme in cortical morphogenesis in Tetrahymena. Together, the two types of epiplasm probably allow for extensive changes in cell shape while preserving essential relationships between structural elements within the cortex.

The Membrane Skeleton of Pseudomicrothorax

1991 ◽  
Vol 100 (4) ◽  
pp. 707-715 ◽  
Author(s):  
IRM HUTTENLAUCH ◽  
ROBERT K. PECK
Keyword(s):  
Spatial Organization ◽  
Membrane Skeleton ◽  
Basal Bodies ◽  
Structural Elements ◽  
Or Groups ◽  
Positive Immunoreactivity ◽  
Cortical Membranes ◽  
Cytoskeletal Elements

The membrane skeleton, or epiplasm, is part of the structurally complex ciliate cortex. It is thought to have skeletal functions concerning the spatial organization of cortical elements such as the basal bodies. Here we report the biochemical and immunological characterization of some components of the purified epiplasm of Pseudomicrothorax dubius. The epiplasm proteins consist of two quantitatively major groups of proteins, one of 76–80x103Mr, the other of 11–13x103Mr, which appear to be the principal structural elements of the epiplasm, and a series of minor components of 62–18x103Mr. Based upon lectin labeling and glycosidase treatment, some of the latter have been identified as glycoproteins. Using affinity-purified antibodies specific for individual glycoproteins or groups of glycoproteins, we were able to localize them in situ by immunoelectron microscopical methods. This in situ localization demonstrates that the glycosylated epitopes, unlike the glycoresidues of membrane proteins, are distributed throughout the entire epiplasmic layer rather than being restricted to regions adjacent to the cortical membranes. Thus, these proteins represent glycosylated, cytoskeletal elements. At least one of these glycoproteins (Mr 62x103) shows positive immunoreactivity with a monoclonal antibody (Pruss anti-IFA) recognizing most intermediate filament (IF) proteins, indicating that IF proteins might be present in protozoan cytoskeletons.

LABELLING OF THE THYROID CELL SURFACE WITH 125I

1978 ◽  
Vol 77 (3) ◽  
pp. 353-NP ◽  
Author(s):  
PAMELA M. POVEY ◽  
B. REES SMITH ◽  
R. HALL
Keyword(s):  
Cell Surface ◽  
Species Differences ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Molecular Weights ◽  
Electrophoretic Patterns ◽  
High Viability ◽  
Method Evidence

The surface membrane proteins of cultured porcine thyroid cells have been labelled with 125I by the lactoperoxidase method. Evidence that the labelling was restricted to the cell surface was supported by the high viability of the cells in suspension, the high proportion of labelled material in the particulate fraction after homogenization and electronmicroscopic autoradiographic studies. The labelled proteins were analysed by electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate and this indicated the presence of ten major labelled protein bands with approximate molecular weights of 175 000, 155 000, 135 000, 88 000, 80 000, 52 300, 39 000, 30 000, 21 000 and 14 300. Comparison of the electrophoretic patterns obtained with cultured human and porcine thyroid cells suggested that there were species differences in the proportions of lower-molecular-weight proteins.

Isolation of pure pellicles containing intact basal bodies of Tetrahymena pyriformis

1985 ◽  
Vol 77 (1) ◽  
pp. 155-165
Author(s):  
A. Tiedtke
Keyword(s):  
Pore Size ◽  
Tetrahymena Pyriformis ◽  
Basal Bodies ◽  
Electron Microscopic ◽  
Encapsulated Cells ◽  
Low Concentrations ◽  
Granular Matrix ◽  
Mass Isolation ◽  
Cytoskeletal Elements ◽  
Triton X

A new procedure for mass isolation of pure pellicles containing intact basal bodies of Tetrahymena pyriformis is reported. The success of the procedure depends on the elimination of the sticky mucocyst contents before fractionation of the cells, which is induced by Alcian Blue 8GS. Under appropriate ionic conditions greater than 95% of the cells are able to form a capsule by simultaneous extrusion of all mature mucocysts. About 50% of these cells are able to escape from their capsules, which are now devoid of mature mucocysts. These cells are separated from the empty capsules and encapsulated cells by passage through layers of gauze of 10 microns pore size. The fractionation of mucocyst-free cells in homogenization buffer yields pure pellicles, which are retained when the homogenate is sieved through steel sieves of 5 microns pore size. Electron-microscopic controls show that the isolated pellicles are not contaminated with subcellular particles. Cells homogenized in the presence of low concentrations of Triton X-100 yield pellicles consisting of the known cell-surface-associated cytoskeletal elements, together with basal bodies. The cilia are detached just above the kinetosomal plate. The basal bodies of isolated pellicles are obviously undamaged, since all the known structures of native basal bodies are preserved. Even the granular matrix, a labile structure in the lumen of the basal body that probably contains RNA, is preserved.

A monoclonal antibody study of protein distribution in the membrane skeleton of the ciliate Pseudomicrothorax

1992 ◽  
Vol 103 (4) ◽  
pp. 1117-1125 ◽  
Author(s):  
S. Curtenaz ◽  
R.K. Peck
Keyword(s):  
Monoclonal Antibody ◽  
Cell Shape ◽  
Membrane Skeleton ◽  
The Other ◽  
Regional Differentiation ◽  
Protein Distribution ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Western Blots

The membrane skeleton, or epiplasm, of the ciliated protozoon Pseudomicrothorax dubius is a chemically and structurally complex layer. It is responsible for the cell shape and the positioning of some cortical organelles. One may expect that its possible morphogenetic role can be achieved only via a regional differentiation of the protein distribution in the epiplasm. We have tried to demonstrate such differentiation by preparing an epiplasm extract, which consists predominantly of concanavalin A (ConA)-positive glycoproteins. This fraction, either untreated or deglycosylated, was used to raise monoclonal antibodies (mAbs), whose specificity was tested on western blots of either untreated or deglycosylated epiplasm. The recognized polypeptides were then localized in situ by fluorescence and electron microscopic immunocytochemistry. Six mAbs are presented here. Four of them are directed against ConA-positive glycoproteins and show a localization of the latter on the outer surface of the epiplasm. The two others are directed against other epiplasmic polypeptides: one is specific for a common epitope shared by most of the epiplasmic proteins, but not by the glycoproteins, and labels the entire membrane skeleton, whereas the other recognizes three minor polypeptides, which seem localized to the inner part of the epiplasm.

scholarly journals The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets.

1975 ◽  
Vol 64 (2) ◽  
pp. 408-420 ◽  
Author(s):  
R E Stephens
Keyword(s):  
Sodium Dodecylsulfate ◽  
Potassium Thiocyanate ◽  
Basal Bodies ◽  
Molecular Weights ◽  
Unique Protein ◽  
Structural Details ◽  
Basal Apparatus ◽  
Mass Isolation ◽  
Triton X

The basal apparatus, consisting of an array of interconnected basal bodies bearing bifurcating striated rootlets encompassing a nucleus, has been isolated from hypertonically deciliated columnar gill epithelial cells of the bay scallop Aequipecten irradians through gentle lysis with Triton X-100. The rootlets, 8-10 mum in length, were not easily preserved with conventional electron microscope fixatives, suggesting that the extent of their contribution to cellular architecture has been somewhat underestimated, even though Englemann described many of the structural details of the basal apparatus in 1880. The striated rootlets were soluble at high but not at low pH, in 2 M solutions of sodium azide and potassium thiocyanate but not sodium or potassium chloride, in 1% deoxycholate but not digitonin, and in the denaturing solvents 6 M guanidine-HC1, 8 M urea, and 1% sodium dodecylsulfate at 100 degrees C. The protein found consistently when rootlets were solubilized migrated on SDS-polyacrylamide gels as a closely spaced doublet with apparent molecular weights of 230,000 and 250,000 daltons. This unique protein, distinct from tropocollagen or various muscle components, has been named ankyrin because of the rootlet's anchor-like function in the cell.

Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes

2000 ◽  
Vol 113 (2) ◽  
pp. 279-290 ◽  
Author(s):  
Z.A. Khayat ◽  
P. Tong ◽  
K. Yaworsky ◽  
R.J. Bloch ◽  
A. Klip
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Surface Membrane ◽  
Dorsal Surface ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
L6 Myotubes ◽  
Triton X ◽  
Phosphatidylinositol 3

We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucose transporters in L6 myotubes expressing myc-tagged glucose transporter 4 (GLUT4myc). Scanning electron microscopy revealed a dynamic distortion of the dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimulated cells, phalloidin-labeled actin filaments ran parallel to the longitudinal axis of the cell. Immunostaining of the p85 regulatory subunit of phosphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuclear. After 3 minutes of insulin treatment, actin reorganized to form structures; these structures protruded from the dorsal surface of the myotubes by 10 minutes and condensed in the myoplasm into less prominent foci at 30 minutes. The p85 polypeptide colocalized with these structures at all time points. Actin remodeling and p85 relocalization to actin structures were prevented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the actin-rich projections was also observed, but only after 10 minutes of insulin treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble material that was also enriched with actin. In contrast, vp165, a transmembrane aminopeptidase that morphologically colocalized with GLUT4 vesicles, was fully soluble in Triton X-100 extracts of both insulin-treated and control myotubes. Transient transfection of dominant inhibitory Rac1 (N17) into L6 myotubes prevented formation of dorsal actin structures and blocked insulin-induced GLUT4myc translocation to the cell surface. We propose that insulin-dependent formation of actin structures facilitates the association of PI3-K (p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.

scholarly journals PARTIAL PURIFICATION AND PHOSPHOTUNGSTATE SOLUBILIZATION OF BASAL BODIES AND KINETODESMAL FIBERS FROM TETRAHYMENA PYRIFORMIS

1973 ◽  
Vol 57 (3) ◽  
pp. 601-612 ◽  
Author(s):  
Robert W. Rubin ◽  
William P. Cunningham
Keyword(s):  
Basal Body ◽  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Tetrahymena Pyriformis ◽  
Partial Purification ◽  
Basal Bodies ◽  
Thin Sectioning ◽  
Triton X

Previously devised methods for the isolation of basal bodies from ciliate protozoans were found to be inadequate for chemical analysis. We have modified and expanded these procedures and developed a method which gives preparations containing mainly basal bodies and kinetodesmal fibers. This procedure involved fixation of cells in 30% ETOH followed by digitonin or Triton X-100 solubilization and homogenization with a Brinkmann Polytron. This is followed by sucrose gradient centrifugation. Negative staining and thin sectioning revealed these preparations to be substantially more pure than those of previous workers. It was also found that neutralized phosphotungstate (PTA) solubilized many of the components present in fixed Tetrahymena. Neutralized 1.0% PTA solubilized axonemes, cortical, axonemal, and basal body microtubules as well as kinetodesmal fibers. These results have been confirmed by both electron microscope observations and gel electrophoresis of 100,000 g supernatants of the PTA extracts. A solution of 0.1% PTA did not affect the fibers but did solubilize basal bodies. Running 1.0% PTA extracts from our basal body fractions on sodium dodecyl sulfate (SDS) polyacrylamide gels allowed us to tentatively identify the peptides of basal bodies and kinetodesmal fibers. The latter structures appear to consist of a single 21,000 mol wt peptide. These results also suggest that great caution should be taken in interpreting PTA images, especially of microtubules and axonemes.

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

Oncornavirus assembly at the cell surface revealed in replicas of freeze-dried cells

1978 ◽  
Vol 36 (2) ◽  
pp. 354-355
Author(s):  
Anthony Demsey ◽  
Christopher W. Stackpole
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Viral Origin ◽  
Intact Cells ◽  
Structural Formation ◽  
Virus Core ◽  
Freeze Dried ◽  
Cacodylate Buffer ◽  
Surface Replica ◽  
Leukemia Viruses

The murine leukemia viruses are type-C oncornaviruses, and their release from the host cell involves a “budding” process in which the newly-forming, RNA-containing virus core becomes enveloped by modified cell surface membrane. Previous studies revealed that the released virions possess a dense array of 10 nm globular projections (“knobs”) on this envelope surface, and that these knobs contain a 70, 000 MW glycoprotein (gp70) of viral origin. Taking advantage of this distinctive structural formation, we have developed a procedure for freeze-drying and replication of intact cells which reveals surface detail superior to other surface replica techniques, and sufficient to detect even early stages of virus budding by localized aggregation of these knobs on the cell surface.Briefly, cells growing in monolayer are seeded onto round glass coverslips 10-12 mm in diameter. After a period of growth, cells are fixed in situ for one hour, usually with 1% OsO4 in 0. 1 M cacodylate buffer, and rinsed in distilled water.

The organization of cell surface membrane domains

1989 ◽  
Vol 47 ◽  
pp. 804-805
Author(s):  
Michael Edidin
Keyword(s):  
Cell Surface ◽  
Membrane Lipids ◽  
Surface Membrane ◽  
Lateral Diffusion ◽  
Cell Types ◽  
Membrane Domains ◽  
Membrane Biology ◽  
Morphological Polarity ◽  
Discrete Domains ◽  
Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

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