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.

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.

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.

Immunofluorescence microscopy of the flagella and multilayered structure in two mosses, Sphagnum palustre L. and Polytrichum juniperinum Hedw

1983 ◽  
Vol 61 (1) ◽  
pp. 71-86
Author(s):  
C.C. Miller ◽  
J.G. Duckett ◽  
P. Sheterline ◽  
Z.B. Carothers
Keyword(s):  
Immunofluorescence Microscopy ◽  
Strong Support ◽  
Multilayered Structure ◽  
Cross Reactivity ◽  
Basal Bodies ◽  
Plant Structure ◽  
Porcine Brain ◽  
Granular Matrix ◽  
Triton X ◽  
Sphagnum Palustre

Antibody against tubulin from porcine brain was used to examine the distribution of tubulin in developing spermatids of Polytrichum and mature spermatozoids of Sphagnum. Cells were prepared for indirect immunofluorescence microscopy after fixation in buffered paraformaldehyde and brief incubation in cellulase. Pretreatment with cold methanol resulted in considerably enhanced immunofluorescence but exposure to Triton X-100, with or without sonication, had no effect. The antibody showed similar immunological cross-reactivity with the flagella (both basal bodies and axonemes) and the spline microtubules of the multilayered structure. This is the first direct evidence that this rigid array of stable cytoskeletal microtubules consists of tubulin. Particularly intense fluorescence from the lamellar strata of the MLS in developing spermatids provides strong support for the notion that the lamellae comprise a highly structured microtubule organizing centre (MTOC), responsible for the ordered assembly of the overlying spline tubules. The demonstration of immunological cross-reactivity with antitubulin from porcine brain tubulin, within a plant structure other than fully formed microtubules, suggests that immunocytochemistry may have considerable potential for the detection of other MTOCs. By contrast, no detectable fluorescence emanated from the granular matrix cementing the flagellar basal bodies to the spline or the spindle-shaped sheath of fibres present in the spermatozoids of Sphagnum. Disruption of the mature gametes by sonication and treatment with Triton X-100 reveals the presence of particularly strong links between the spline and subjacent nuclear envelope.

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.

SEM of non-coated hepatocellular cytoskeleton of perfused livers

1984 ◽  
Vol 42 ◽  
pp. 306-307
Author(s):  
S.W. French ◽  
N.C. Benson ◽  
C. Davis-Scibienski
Keyword(s):  
Ambient Temperature ◽  
Critical Point ◽  
Protease Inhibitors ◽  
Metal Deposition ◽  
Heat Damage ◽  
Detergent Extraction ◽  
Cytoskeletal Elements ◽  
Triton X ◽  
Excised Tissue

Previous SEM studies of liver cytoskeletal elements have encountered technical difficulties such as variable metal coating and heat damage which occurs during metal deposition. The majority of studies involving evaluation of the cell cytoskeleton have been limited to cells which could be isolated, maintained in culture as a monolayer and thus easily extracted. Detergent extraction of excised tissue by immersion has often been unsatisfactory beyond the depth of several cells. These disadvantages have been avoided in the present study. Whole C3H mouse livers were perfused in situ with 0.5% Triton X-100 in a modified Jahn's buffer including protease inhibitors. Perfusion was continued for 1 to 2 hours at ambient temperature. The liver was then perfused with a 2% buffered gluteraldehyde solution. Liver samples including spontaneous tumors were then maintained in buffered gluteraldehyde for 2 hours. Samples were processed for SEM and TEM using the modified thicarbohydrazide procedure of Malich and Wilson, cryofractured, and critical point dried (CPD). Some samples were mechanically fractured after CPD.

scholarly journals Effect of Inflammation on Gingival Mesenchymal Stem/Progenitor Cells’ Proliferation and Migration through Microperforated Membranes: An In Vitro Study

2020 ◽  
Vol 2020 ◽  
pp. 1-10 ◽  
Author(s):  
M. Al Bahrawy ◽  
K. Ghaffar ◽  
A. Gamal ◽  
K. El-Sayed ◽  
V. Iacono
Keyword(s):  
Pore Size ◽  
Progenitor Cells ◽  
Cellular Migration ◽  
Gingival Tissue ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Electron Microscopic ◽  
Healthy Human ◽  
Membrane Pores ◽  
Scanning Electron

Background. In the field of periodontal guided tissue regeneration, microperforated membranes have recently proved to be very promising periodontal regenerative tissue engineering tools. Regenerative periodontal approaches, employing gingival mesenchymal stem/progenitor cells in combination with these novel membranes, would occur mostly in inflamed microenvironmental conditions intraorally. This in turn entails the investigation into how inflammation would affect the proliferation as well as the migration dynamics of gingival mesenchymal stem/progenitor cells. Materials and Methods. Clones of human gingival mesenchymal stem/progenitor cells (GMSCs) from inflamed gingival tissues were characterized for stem/progenitor cells’ characteristics and compared to clones of healthy human GMSCs (n=3), to be subsequently seeded on perforated collagen-coated poly-tetra-floro-ethylene (PTFE) membranes with a pore size 0.4 and 3 microns and polycarbonic acid membranes of 8 microns pore size in Transwell systems. The population doubling time and the MTT test of both populations were determined. Fetal bovine serum (FBS) was used as a chemoattractant in the culturing systems, and both groups were compared to their negative controls without FBS. Following 24 hours of incubation period, migrating cells were determined on the undersurface of microperforated membranes and the membrane-seeded cells were examined by scanning electron microscopy. Results. GMSCs demonstrated all predefined stem/progenitor cell characteristics. GMSCs from inflamed gingival tissues showed significantly shorter population doubling times. GMSCs of inflamed and healthy tissues did not show significant differences in their migration abilities towards the chemoattractant, with no cellular migration observed in the absence of FBS. GMSCs from healthy gingival tissue migrated significantly better through larger micropores (8 microns). Scanning electron microscopic images proved the migratory activity of the cells through the membrane pores. Conclusions. Inflammation appears to boost the proliferative abilities of GMSCs. In terms of migration through membrane pores, GMSCs from healthy as well as inflamed gingival tissues do not demonstrate a difference in their migration abilities through smaller pore sizes, whereas GMSCs from healthy gingival tissues appear to migrate significantly better through larger micropores.

scholarly journals Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

1997 ◽  
Vol 8 (3) ◽  
pp. 533-545 ◽  
Author(s):  
T Harder ◽  
R Kellner ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Independent Manner ◽  
Low Concentrations ◽  
Early Endosomes ◽  
Associated Proteins ◽  
Cytoskeletal Elements

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

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.

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