Properties of microtubule-free cortical residues isolated from Paramecium tetraurelia

1989 ◽  
Vol 92 (3) ◽  
pp. 427-432 ◽  
Author(s):  
N.E. Williams ◽  
J.E. Honts ◽  
K.R. Stuart
Keyword(s):  
Immunofluorescence Microscopy ◽  
High Ionic Strength ◽  
Evolutionary Relationships ◽  
Antigenic Relationship ◽  
Paramecium Tetraurelia ◽  
Structure Development ◽  
Shape Preserving ◽  
Alpha Spectrin ◽  
Triton X ◽  
Cortical Architecture

We have found that shape-preserving residues devoid of microtubules can be prepared from Paramecium using Triton X-100 at high ionic strength. These residues contain many proteins, including one showing antigenic relationship to chicken alpha-spectrin, and three showing antigenic relationship to Tetrahymena cortical proteins. These antigens have been localized by immunofluorescence microscopy, and the isolated cortical residues have been characterized ultrastructurally. These preparations should be useful in detailed studies of the structure, development and evolutionary relationships of cortical architecture in ciliated protozoa.

Identification and localization of major cortical proteins in the ciliated protozoan, Euplotes eurystomus

1989 ◽  
Vol 92 (3) ◽  
pp. 433-439 ◽  
Author(s):  
N.E. Williams ◽  
J.E. Honts ◽  
Q. Lu ◽  
C.L. Olson ◽  
K.C. Moore
Keyword(s):  
Ionic Strength ◽  
High Ionic Strength ◽  
Basal Bodies ◽  
Cell Type ◽  
Ciliated Protozoan ◽  
Shape Preserving ◽  
Sds Page ◽  
Triton X ◽  
Development And Evolution ◽  
Integrated Structures

Shape-preserving cortical residues have been isolated from Euplotes eurystomus cells by the application of Triton X-100 at high ionic strength. These integrated structures consist of articulated plates and widely interspersed cages that formerly contained basal bodies associated with the clusters of cilia characteristic of this cell type. Using SDS-PAGE and immunolocalization procedures, we have identified major subunit proteins of both the plates (116, 110 (X10(3] Mr, and the basal body cages (86 X 10(3) Mr). The potential for studies of these proteins in contributing to our understanding of cortical development and evolution in Euplotes is discussed.

scholarly journals Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

1978 ◽  
Vol 77 (3) ◽  
pp. R27 ◽  
Author(s):  
M Osborn ◽  
RE Webster ◽  
K Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscope ◽  
Immunofluorescence Microscopy ◽  
Uranyl Acetate ◽  
Tubulin Antibodies ◽  
Ptk2 Cell ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Microtubular Structures

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

scholarly journals Change of cytokeratin filament organization during the cell cycle: selective masking of an immunologic determinant in interphase PtK2 cells.

1983 ◽  
Vol 97 (4) ◽  
pp. 1255-1260 ◽  
Author(s):  
W W Franke ◽  
E Schmid ◽  
J Wellsteed ◽  
C Grund ◽  
O Gigi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Monoclonal Antibody ◽  
Cell Cultures ◽  
Immunofluorescence Microscopy ◽  
Specific Reaction ◽  
Low Concentrations ◽  
Interphase Cells ◽  
Triton X ◽  
Cytokeratin Filaments ◽  
Striking Contrast

The organization of intermediate-sized filaments (IF) of the cytokeratin type was studied in cultures of PtK2 cells in which typical IF structures are maintained during mitosis, using a monoclonal antibody (KG 8.13). This antibody reacts, in immunoblotting experiments, with the larger of the two major cytokeratin polypeptides present in these cells but, using standard immunofluorescence microscopy procedures, does not react with the cytokeratin filaments abundant in interphase cells, in striking contrast to various antisera and other monoclonal cytokeratin antibodies. In the same cell cultures, however, the antibody does react with cytokeratin filaments of mitotic and early postmitotic cells. The specific reaction with cytokeratin filaments of mitotic cells only is due to the exposure of the specific immunologic determinant in mitosis and its masking in interphase cells. Treatment of interphase cells with both Triton X-100 as well as with methanol and acetone alters the cytokeratin filaments and allows them to react with this monoclonal antibody. A similar unmasking was noted after treatment with buffer containing 2 M urea or low concentrations of trypsin. We conclude that the organization of cytokeratin, albeit still arranged in typical IF, is altered during mitosis of PtK2 cells.

scholarly journals A polypeptide of 59 kDa is associated with bundles of cytoplasmic filaments in Neurospora crassa

1990 ◽  
Vol 268 (3) ◽  
pp. 649-655 ◽  
Author(s):  
A L Rosa ◽  
M E Alvarez ◽  
D Lawson ◽  
H J F Maccioni
Keyword(s):  
Monoclonal Antibody ◽  
Ionic Strength ◽  
Neurospora Crassa ◽  
High Ionic Strength ◽  
Differential Centrifugation ◽  
Main Constituent ◽  
Total N ◽  
Cytoplasmic Filaments ◽  
Triton X ◽  
Isolation Buffer

Complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus Neurospora crassa. They were enriched by differential centrifugation and purified by permeation chromatography. The filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. The main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kDa (P59Nc), which represents 4-5% of the total N. crassa proteins. The filamentous structures are cold-stable and are not affected by high-ionic-strength solutions or by the presence of 10 mM-EDTA or 1% (w/v) Triton X-100; they were disassembled by raising the pH of the solution or by using Tris-based buffers. The disassembled form assembled into structures sedimentable at 105,000 g after dialysis against the isolation buffer. The sedimentable structures were organized in the form of regular aggregates of 42-45 nm polypeptides and reacted weakly with anti-IFA, a monoclonal antibody which recognizes an epitope common to many of the higher-eukaryote intermediate-filament polypeptides. Immunofluorescence examination of wall-digested hyphae of N. crassa using affinity-purified antibodies prepared against P59Nc showed immunostaining of abundant filamentous and dot-shaped structures distributed in the cytoplasm.

scholarly journals Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle.

1995 ◽  
Vol 129 (2) ◽  
pp. 459-471 ◽  
Author(s):  
N Benlimame ◽  
D Simard ◽  
I R Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Mdck Cells ◽  
Electron Microscopic ◽  
Autocrine Motility Factor ◽  
Lysosomal Fraction ◽  
Motility Factor ◽  
Variable Diameter ◽  
Triton X

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule-associated tubular organelles.

scholarly journals Trypanosoma cruzi GP63 Proteins Undergo Stage-Specific Differential Posttranslational Modification and Are Important for Host Cell Infection

2009 ◽  
Vol 77 (5) ◽  
pp. 2193-2200 ◽  
Author(s):  
Manjusha M. Kulkarni ◽  
Cheryl L. Olson ◽  
David M. Engman ◽  
Bradford S. McGwire
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Host Cell ◽  
Posttranslational Modification ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antiserum ◽  
Cell Infection ◽  
Specific Expression ◽  
Metacyclic Trypomastigotes ◽  
Triton X

ABSTRACT The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

GLYCOPHOSPHATIDYLINOSITOL-ANCHORED PROTEINS INPARAMECIUM TETRAURELIA

2001 ◽  
Vol 204 (16) ◽  
pp. 2899-2910
Author(s):  
CARRIE A. PAQUETTE ◽  
VILLA RAKOCHY ◽  
ALISON BUSH ◽  
JUDITH L. VAN HOUTEN
Keyword(s):  
Cell Body ◽  
Binding Sites ◽  
Integral Membrane Protein ◽  
Paramecium Tetraurelia ◽  
Folate Binding Protein ◽  
Intact Cells ◽  
Phase Partitioning ◽  
Protein Receptor ◽  
Ethanol Washing ◽  
Triton X

SUMMARYWe have begun to characterize the glycophosphatidylinositol (GPI)-anchored proteins of the Paramecium tetraurelia cell body surface where receptors and binding sites for attractant stimuli are found. We demonstrate here (i) that inositol-specific exogenous phospholipase C (PLC) treatment of the cell body membranes (pellicles) removes proteins with GPI anchors, (ii)that, as in P. primaurelia, there is an endogenous lipase that responds differently to PLC inhibitors compared with its response to an exogenous PLC, (iii) that salt and ethanol treatment of cells removes GPI-anchored proteins from whole, intact cells, (iv) that Triton X-114 phase partitioning shows that many GPI-anchored proteins are cleaved from pellicles by the endogenous lipase and enter the aqueous phase, and (v) that integral membrane proteins are not among those cleaved with PLC or in the salt/ethanol wash.Antisera against the proteins removed by the salt/ethanol washing procedure include antibodies against large surface antigens, which we confirm in this species to be GPI-anchored, and against an array of proteins of smaller molecular mass. These antisera specifically block the chemoresponse to some stimuli, such as folate, which we suggest are signaled through GPI-anchored receptors. Responses to cyclic AMP, which we believe involve an integral membrane protein receptor, and to NH4Cl, which requires no receptor, are not affected by the antisera. Antiserum against a mammalian GPI-anchored folate-binding protein recognizes a single band among the GPI-anchored salt and ethanol wash proteins. The same antiserum specifically blocks the chemoresponse to folate.

Electroimmunochemical Characterization of Endothelial Cell Proteins: Antigenic Relationship with Platelet and Erythrocyte Membrane Proteins

1987 ◽  
Vol 58 (02) ◽  
pp. 686-693 ◽  
Author(s):  
Tone Børsum ◽  
Inger Hagen ◽  
Ole J Bjerrum
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Primary Cultures ◽  
Rabbit Antiserum ◽  
Human Platelets ◽  
Erythrocyte Membranes ◽  
Antigenic Relationship ◽  
Platelet Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryHuman endothelial cells isolated from umbilical cords and cultured in primary cultures were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogeneous labelling of the endothelial cell proteins with 35S-methionine or 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 30 or 8 immunoprecipitates, respectively. Antigenic relationship between endothelial cell proteins and proteins in human platelets or erythrocyte membranes was demonstrated by use of the corresponding antisera and by antigen addition experiments. One of the endothelial cell proteins cross-reacted with antiserum against erythrocyte membranes and showed a partial antigenic identity reaction with the band 3 protein complex of erythrocyte membranes. The same protein showed antigenic relationship also with a platelet protein. In addition, endothelial cells contain at least 7 proteins antigenically related to platelet proteins, of which at least 5 were labelled with 14C-mannose and thus were glycoproteins. Three of these glycoproteins were antigenically related to proteins from isolated platelet membranes and three were related to the release products obtained after thrombin treatment of platelets. The present study demonstrated numerous platelet and endothelial cell proteins that were antigenically related, more than previously anticipated.

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.

scholarly journals ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.

1994 ◽  
Vol 126 (2) ◽  
pp. 391-401 ◽  
Author(s):  
S Tsukita ◽  
K Oishi ◽  
N Sato ◽  
J Sagara ◽  
A Kawai ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Family Members ◽  
Integral Membrane Protein ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Bhk Cells ◽  
Triton X

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.

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