scholarly journals Isolation, ultrastructure, and protein composition of the flagellar rootlet of Naegleria gruberi.

1981 ◽  
Vol 89 (3) ◽  
pp. 424-432 ◽  
Author(s):  
D E Larson ◽  
A D Dingle
Keyword(s):  
Protein Composition ◽  
Basal Bodies ◽  
Maximum Width ◽  
Subunit Molecular Weight ◽  
Striated Rootlet ◽  
Body Complex ◽  
Partial Dissociation ◽  
Triton X ◽  
Naegleria Gruberi

Attached to the basal bodies of Naegleria gruberi flagellates is a striated rootlet or rhizoplast. The rootlet-basal body complex has been isolated by Triton X-100 lysis of deflagellated cells and differential centrifugation through a 25% glycerol medium. Rootlets isolated from mature flagellates are approximately 13 micrometers long but vary from 8 to 15 micrometers in length: they taper at both ends from a maximum width of approximately 0.25 micrometers in the vicinity of the basal bodies. They are highly stable during isolation but can be solubilized by urea, high salt, low pH, or detergent (Sarkosyl). Partial dissociation of rootlets with 1 M urea reveals that they are composed of filaments, approximately 5 nm diameter, associated in a linear fashion to yield the characteristic 21-nm cross-banded appearance. Differential solubilization of rootlets and their associated contaminants allowed identification of a major rootlet protein, comprising at least 50% of any purified rootlet preparation, with an apparent subunit molecular weight of 170,000. The localization of rootlets in situ by indirect immunofluorescence using a specific antibody directed against the purified rootlet protein demonstrated unequivocally that this 170,000-dalton protein is an organelle component.

Studies of the rhizoplast from Naegleria gruberi

1981 ◽  
Vol 47 (1) ◽  
pp. 277-293
Author(s):  
P.R. Gardiner ◽  
R.H. Miller ◽  
M.C. Marsh
Keyword(s):  
Gel Electrophoresis ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chemical Properties ◽  
Biochemical Properties ◽  
Optical Diffraction ◽  
Subunit Molecular Weight ◽  
Low Ionic Strength ◽  
Triton X ◽  
Naegleria Gruberi

A procedure utilizing homogenization and centrifugation in a low ionic strength buffer containing Triton X-100, has been used to facilitate the isolation of the rhizoplast from flagellates of Naegleria gruberi. This has enabled a study to be made of the physical and biochemical properties of this organelle. The rhizoplast is shown to be a proteinaceous structure with chemical properties similar to those of the molluscan gill ciliary rootlet. Polyacrylamide gel electrophoresis gives a possible subunit molecular weight of approximately 240 000 Daltons. Studies with antisera raised against the rhizoplast fraction demonstrated the absence of rhizoplast antigens in amoeboid forms of Naegleria gruberi and is taken as evidence that the organelle is synthesized de novo during transformation of the amoeba to the flagellate form. Results of optical diffraction studies on isolated rhizoplasts are also presented.

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 Epithelial structure revealed by chemical dissection and unembedded electron microscopy.

1984 ◽  
Vol 99 (1) ◽  
pp. 203s-208s ◽  
Author(s):  
E G Fey ◽  
D G Capco ◽  
G Krochmalnic ◽  
S Penman
Keyword(s):  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Protein Composition ◽  
Intact Cells ◽  
Thin Sections ◽  
Transmission Electron ◽  
Kidney Epithelial Cell ◽  
Epithelial Structure ◽  
Triton X ◽  
Whole Mounts

Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton was further fractionated by extraction with (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei. A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrix intermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions. Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique. Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct.

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.

scholarly journals Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates.

1984 ◽  
Vol 98 (2) ◽  
pp. 449-456 ◽  
Author(s):  
C Walsh
Keyword(s):  
Monoclonal Antibody ◽  
Protein Synthesis ◽  
Indirect Immunofluorescence ◽  
Cell Protein ◽  
Basal Bodies ◽  
Immunofluorescence Method ◽  
Nonionic Detergent ◽  
Cytoplasmic Microtubules ◽  
Spherical Cells ◽  
Naegleria Gruberi

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.

In situ preparation of the nuclear matrix of Physarum polycephalum: ultrastructural and biochemical analysis of different matrix isolation procedures

1988 ◽  
Vol 90 (4) ◽  
pp. 621-628 ◽  
Author(s):  
W. Waitz ◽  
P. Loidl
Keyword(s):  
Nuclear Matrix ◽  
Physarum Polycephalum ◽  
Matrix Isolation ◽  
Protein Composition ◽  
Biochemical Properties ◽  
Agarose Beads ◽  
In Situ Preparation ◽  
The Matrix ◽  
Important Approach

A novel method for in situ preparation of nuclear matrix from whole plasmodia of Physarum polycephalum without isolation of nuclei is presented. Plasmodia are encapsulated in agarose beads and after solubilization of the cytoplasm the nuclear matrix is prepared. With this quick and easy technique nuclear matrix can be reproducibly prepared with perfect recovery. We compared the ultrastructural and biochemical properties of the matrix after three different matrix isolation procedures: preparation with high salt, ammonium sulphate and lithium diiodosalicylic acid. The results show that the ultrastructure and protein composition of the three types of matrix are very similar or even identical. We conclude that many of the conflicting results on nuclear matrix in the literature are due to perturbations of nuclear integrity during the isolation of nuclei. For this reason the new in situ method is an important approach in the standardization of nuclear matrix isolation.

Localization of actin in the Tetrahymena basal body-cage complex

1992 ◽  
Vol 103 (3) ◽  
pp. 629-641 ◽  
Author(s):  
J.G. Hoey ◽  
R.H. Gavin
Keyword(s):  
Basal Body ◽  
Specific Binding ◽  
Ultrastructural Level ◽  
Basal Bodies ◽  
Muscle Actin ◽  
Cage Complex ◽  
Contractile System ◽  
Chicken Muscle ◽  
Filament Bundles

In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify anti-chicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahymena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.

scholarly journals A possible skeletal substructure of the macronucleus of tetrahymena

1980 ◽  
Vol 84 (1) ◽  
pp. 160-171 ◽  
Author(s):  
J Wolfe
Keyword(s):  
Citric Acid ◽  
Inner Core ◽  
Protein Composition ◽  
High Salt ◽  
Skeletal Structure ◽  
Digestion Procedure ◽  
Salt Extraction ◽  
Nonhistone Protein ◽  
Fibrillar Component ◽  
Triton X

Upon removal of chromatin from isolated macronuclei of tetrahymena, residual structures are obtained, the organization of which faithfully reflects the distinctive architecture of the macronucleus. Macronuclei are isolated by a new procedure in which cells are lysed by immersion in citric acid and Triton X-100. This method is rapid and efficient and leaves the nuclear structures stripped of nuclear envelope and nucleoli. The remaining interconnected chromatin bodies are structurally differentiated into a dense outer shell and a fibrillar inner core. The fibrillar component is identified as chromatin because it is removed upon digestion with DNase and extraction with 2 M NaCl. The dense shell of the chromatin body is unaffected by the digestion procedure, which leaves a skeletal structure comprised of hollow spherical bodies. Analysis of the protein composition by SDS acrylamide gel electrophoresis before and after digestion with DNase and RNase and high-salt extraction shows that histones are diminished, whereas the nonhistone protein composition remains unchanged. It was found the DNase not only extracts chromatin but also protects the nonchromatin structure from the otherwise disruptive effects of high-salt extraction. The method used for isolating the nuclei also affects the structure remaining after the digestion procedure the citric acid/Triton X-100 method enhances the stability of the interconnected spherical bodies. The results indicate that the method for isolating nuclei and the procedure by which chromatin is extracted are both major factors contributing to the detection of a possible nonchromatin nuclear skeleton.

scholarly journals The centromere-kinetochore complex: a repeat subunit model.

1991 ◽  
Vol 113 (5) ◽  
pp. 1091-1110 ◽  
Author(s):  
R P Zinkowski ◽  
J Meyne ◽  
B R Brinkley
Keyword(s):  
Three Dimensional ◽  
Protein Composition ◽  
Cytoplasmic Dynein ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Microtubule Motor ◽  
A Subunit ◽  
Kinetochore Region ◽  
Subunit Organization

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

scholarly journals Observations of the marginal band system of nucleated erythrocytes.

1978 ◽  
Vol 78 (1) ◽  
pp. 260-273 ◽  
Author(s):  
W D Cohen
Keyword(s):  
Phase Contrast ◽  
Band System ◽  
Proteolytic Enzymes ◽  
Mirror Image ◽  
Structural Components ◽  
Microtubule Polymerization ◽  
Transmission Electron ◽  
Marginal Band ◽  
Triton X

The marginal band (MB) of nucleated erythrocytes (thos of nonmammalian vertebrates) is a continuous peripheral bundle of microtubules normally obscured by hemoglobin. Treatment of these elliptical cells with modified microtubule polymerization media containing Triton X-100 yields a semilysed system in which MB, nucleus, and trans-MB material (TBM) are visible under phase contrast. The TBM apparently interconnects structural components, passing around opposite sides of the nucleus and suspending it in native position. In uranyl acetatestained whole whole mounts (goldfish) examined by transmission electron microscopy, the TBM appears as a network. MBs of semilysed cells are relatively planar initially, but twist subsequently into a range of "figure-8" shapes with one of the two possible mirror-image configurations predominant. Nuclei and MBs can be released using proteolytic enzymes, to which the TBM seems most rapidly vulnerable. MBs thus freed are birefringent, generally untwisted, and much more circular than they are in situ. As a working hypothesis, it is prosposed that the flattened, elliptical shape of nucleated erythrocytes is a result of TBM tension applied asymmetrically across an otherwise more circular MB, and that the firure-8 configuration occurs when there is extreme TBM shrinkage or contraction.

Sign in / Sign up

Export Citation Format

Share Document