Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies

1989 ◽  
Vol 93 (3) ◽  
pp. 491-500 ◽  
Author(s):  
A. Woods ◽  
T. Sherwin ◽  
R. Sasse ◽  
T.H. MacRae ◽  
A.J. Baines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Complex Mixtures ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Tubulin Isotypes ◽  
Definition Of ◽  
Attachment Zone

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

scholarly journals Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle.

1995 ◽  
Vol 128 (6) ◽  
pp. 1163-1172 ◽  
Author(s):  
D R Robinson ◽  
T Sherwin ◽  
A Ploubidou ◽  
E H Byard ◽  
K Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Daughter Nucleus ◽  
Basal Bodies ◽  
Cortical Microtubules ◽  
Cell Extension ◽  
Microtubule Polarity ◽  
Check Points ◽  
High Degree ◽  
Attachment Zone

Trypanosoma brucei has a precisely ordered microtubule cytoskeleton whose morphogenesis is central to cell cycle events such as organelle positioning, segregation, mitosis, and cytokinesis. We have defined microtubule polarity and show the + ends of the cortical microtubules to be at the posterior end of the cell. Measurements of organelle positions through the cell cycle reveal a high degree of coordinate movement and a relationship with overall cell extension. Quantitative analysis of the segregation of the replicated mitochondrial genome (the kinetoplast) by the flagellar basal bodies identifies a new G2 cell cycle event marker. The subsequent mitosis then positions one "daughter" nucleus into the gap between the segregated basal bodies/kinetoplasts. The anterior daughter nucleus maintains its position relative to the anterior of the cell, suggesting an effective yet cryptic nuclear positioning mechanism. Inhibition of microtubule dynamics by rhizoxin results in a phenomenon whereby cells, which have segregated their kinetoplasts yet are compromised in mitosis, cleave into a nucleated portion and a flagellated, anucleate, cytoplast. We term these cytoplasts "zoids" and show that they contain the posterior (new) flagellum and associated basal-body/kinetoplast complex. Examination of zoids suggests a role for the flagellum attachment zone (FAZ) in defining the position for the axis of cleavage in trypanosomes. Progression through cytokinesis, (zoid formation) while mitosis is compromised, suggests that the dependency relationships leading to the classical cell cycle check points may be altered in trypanosomes, to take account of the need to segregate two unit genomes (nuclear and mitochondrial) in this cell.

scholarly journals Monoclonal Antibodies to a Subfraction of Merino Wool High-tyrosine Proteins

1986 ◽  
Vol 39 (4) ◽  
pp. 341 ◽  
Author(s):  
DR Hewish ◽  
PW French
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Immunofluorescent Staining ◽  
Immunogold Electron Microscopy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Location ◽  
Merino Wool

Monoclonal antibodies were prepared which react with members of the high-tyrosine type proteins from Merino wool. Specificity was confirmed by the use of Western transfer immunoassays and by enzyme-linked immunosorbent assay on purified fractions. Immunofluorescent staining of sections of wool follicles using the antibodies showed that the proteins were present in the developing wool shaft but that staining was asymmetric, indicating specific location of the proteins in the orthocortex of the fibres. Immunogold-electron microscopy confirmed that one of the antibodies bound to the keratin microfibril bundles.

scholarly journals The 17β-oestradiol dehydrogenase of pig endometrial cells is localized in specialized vesicles

1993 ◽  
Vol 290 (3) ◽  
pp. 777-782 ◽  
Author(s):  
J Adamski ◽  
B Husen ◽  
F Marks ◽  
P W Jungblut
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Immunogold Electron Microscopy ◽  
The Novel ◽  
Electron Dense Material ◽  
Endometrial Cells ◽  
Functional Aspects ◽  
Cytoplasmic Vesicles ◽  
Similar Appearance ◽  
High Degree

Two monoclonal antibodies against the 17 beta-oestradiol dehydrogenase of pig endometrial cells have been used in localization studies with immunogold electron microscopy. The antibodies attach both to a fraction of dehydrogenase-rich cytoplasmic vesicles isolated from homogenates and to vesicles of similar appearance in cells. The vesicles are filled with electron-dense material. Their tagging intensity indicates a high degree of specialization. Endometrial cells from mature animals contain a host of dehydrogenase vesicles, and cells from prepubertal animals only a few. Functional aspects of the novel organelle are discussed.

scholarly journals Orientation within the Exosporium and Structural Stability of the Collagen-Like Glycoprotein BclA of Bacillus anthracis

2005 ◽  
Vol 187 (15) ◽  
pp. 5310-5317 ◽  
Author(s):  
Jeremy A. Boydston ◽  
Ping Chen ◽  
Christopher T. Steichen ◽  
Charles L. Turnbough
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Triple Helix ◽  
Thermal Denaturation ◽  
Sodium Dodecyl ◽  
Basal Layer ◽  
Structural Data ◽  
Immunogold Electron Microscopy

ABSTRACT Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T m ) of 37°C, like mammalian collagen. However, rBclA trimers exhibited T m values of 84°C and 95°C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T m values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.

Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation

1997 ◽  
Vol 110 (3) ◽  
pp. 311-322 ◽  
Author(s):  
K. Fujimoto ◽  
A. Nagafuchi ◽  
S. Tsukita ◽  
A. Kuraoka ◽  
A. Ohokuma ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gap Junction ◽  
Immunofluorescence Microscopy ◽  
Immunogold Electron Microscopy ◽  
Cell Contact ◽  
Contact Sites ◽  
Junction Formation ◽  
Intramembrane Proteins ◽  
Junction Proteins ◽  
E Cadherin

We examined the dynamics of connexins, E-cadherin and alpha-catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or alpha-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

scholarly journals Identification of a 70,000-D protein in lens membrane junctional domains.

1985 ◽  
Vol 101 (1) ◽  
pp. 28-35 ◽  
Author(s):  
J Kistler ◽  
B Kirkland ◽  
S Bullivant
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
High Frequency ◽  
Immunofluorescence Microscopy ◽  
Eye Lens ◽  
Vertebrate Species ◽  
Outer Cortex ◽  
Lens Fibers ◽  
Sds Page

A 70,000-D membrane protein (MP70), which is restricted to the eye lens fibers and is present in immunologically homologous form in many vertebrate species, has been identified. By use of anti-MP70 monoclonal antibodies for immunofluorescence microscopy and electron microscopy, this polypeptide was localized in lens membrane junctional domains. Both immunofluorescence microscopy and SDS PAGE reveal an abundance of MP70 in the lens outer cortex that coincides with a high frequency of fiber gap junctions in the same region.

scholarly journals An analogue-sensitive approach identifies basal body rotation and flagellum attachment zone elongation as key functions of PLK in Trypanosoma brucei

2013 ◽  
Vol 24 (9) ◽  
pp. 1321-1333 ◽  
Author(s):  
Ana Lozano-Núñez ◽  
Kyojiro N. Ikeda ◽  
Thomas Sauer ◽  
Christopher L. de Graffenried
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Rna Interference ◽  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Basal Bodies ◽  
Body Rotation ◽  
The Many ◽  
Attachment Zone

Polo-like kinases are important regulators of cell division, playing diverse roles in mitosis and cytoskeletal inheritance. In the parasite Trypanosoma brucei, the single PLK homologue TbPLK is necessary for the assembly of a series of essential organelles that position and adhere the flagellum to the cell surface. Previous work relied on RNA interference or inhibitors of undefined specificity to inhibit TbPLK, both of which have significant experimental limitations. Here we use an analogue-sensitive approach to selectively and acutely inhibit TbPLK. T. brucei cells expressing only analogue-sensitive TbPLK (TbPLKas) grow normally, but upon treatment with inhibitor develop defects in flagellar attachment and cytokinesis. TbPLK cannot migrate effectively when inhibited and remains trapped in the posterior of the cell throughout the cell cycle. Using synchronized cells, we show that active TbPLK is a direct requirement for the assembly and extension of the flagellum attachment zone, which adheres the flagellum to the cell surface, and for the rotation of the duplicated basal bodies, which positions the new flagellum so that it can extend without impinging on the old flagellum. This approach should be applicable to the many kinases found in the T. brucei genome that lack an ascribed function.

scholarly journals Polo-like kinase phosphorylation of bilobe-resident TbCentrin2 facilitates flagellar inheritance in Trypanosoma brucei

2013 ◽  
Vol 24 (12) ◽  
pp. 1947-1963 ◽  
Author(s):  
Christopher L. de Graffenried ◽  
Dorothea Anrather ◽  
Freia Von Raußendorf ◽  
Graham Warren
Keyword(s):  
Trypanosoma Brucei ◽  
Slow Growth ◽  
In Vivo Studies ◽  
Basal Bodies ◽  
Domain Duplication ◽  
Kinase Phosphorylation ◽  
Attachment Zone ◽  
Single Flagellum

In the protist parasite Trypanosoma brucei, the single Polo-like kinase (TbPLK) controls the inheritance of a suite of organelles that help position the parasite's single flagellum. These include the basal bodies, the bilobe, and the flagellar attachment zone (FAZ). TbCentrin2 was previously shown to be a target for TbPLK in vitro, and this is extended in this study to in vivo studies, highlighting a crucial role for serine 54 in the N-terminal domain. Duplication of the bilobe correlates with the presence of TbPLK and phospho-TbCentrin2, identified using phosphospecific antiserum. Mutation of S54 leads to slow growth (S54A) or no growth (S54D), the latter suggesting that dephosphorylation is needed to complete bilobe duplication and subsequent downstream events necessary for flagellum inheritance.

Isoforms of caveolin-1 and caveolar structure

2000 ◽  
Vol 113 (19) ◽  
pp. 3509-3517 ◽  
Author(s):  
T. Fujimoto ◽  
H. Kogo ◽  
R. Nomura ◽  
T. Une
Keyword(s):  
Electron Microscopy ◽  
Hepg2 Cells ◽  
Immunofluorescence Microscopy ◽  
Human Fibroblasts ◽  
Freeze Fracture ◽  
Molecular Composition ◽  
Immunogold Electron Microscopy ◽  
Caveolin 1 ◽  
Different Depths ◽  
The Relationship

The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.

scholarly journals Differential utilization of beta-tubulin isotypes in differentiating neurites.

1989 ◽  
Vol 109 (2) ◽  
pp. 663-673 ◽  
Author(s):  
H C Joshi ◽  
D W Cleveland
Keyword(s):  
Electron Microscopy ◽  
Immunogold Electron Microscopy ◽  
Functional Genes ◽  
Specific Class ◽  
Class Iii ◽  
Beta Tubulin ◽  
Specific Antibodies ◽  
Tubulin Isotypes ◽  
Functional Differences

beta-Tubulin is encoded in vertebrate genomes by a family of six to seven functional genes that produce six different polypeptide isotypes. We now document that although rat PC-12 cells express five of these isotypes, only two (classes II and III) accumulate significantly as a consequence of nerve growth factor-stimulated neurite outgrowth. In contrast to previous efforts that have failed to detect in vivo distinctions among different beta-tubulin isotypes, we demonstrate using immunoblotting with isotype-specific antibodies that three beta-tubulin polypeptides (classes I, II, and IV) are used preferentially for assembly of neurite microtubules (with approximately 70% of types I and II assembled but only approximately 50% of type III in polymer). Immunofluorescence localization shows that an additional isotype (V) is partially excluded from neurites. Distinctions in in vivo localization of the neuron-specific, class III isotype have also been directly observed using immunofluorescence and immunogold electron microscopy. The sum of these efforts documents that some in vivo functional differences between tubulin isotypes do exist.

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