Immunocytochemical study of the formation of striated rootlets during ciliogenesis in quail oviduct

1990 ◽  
Vol 95 (3) ◽  
pp. 423-432 ◽  
Author(s):  
M. Lemullois ◽  
M.C. Marty
Keyword(s):  
Monoclonal Antibody ◽  
Polyclonal Antibody ◽  
Ciliated Cell ◽  
Apical Part ◽  
Basal Bodies ◽  
Immunocytochemical Study ◽  
Ciliated Cells ◽  
Immunogold Staining ◽  
Dense Granules ◽  
Basal Pole

In quail oviduct, a 175K (K = 10(3) Mr) protein associated with striated rootlets was previously identified by Klotz and co-workers using monoclonal antibody CC310. As this monoclonal antibody recognizes several proteins on immunoblots of ciliated cells, we prepared a polyclonal antibody monospecific to the 175K protein by intrasplenic immunization of mice. Immunofluorescence study confirmed the distribution of the 175K protein at the apical part of the ciliated cell and its absence in other epithelial cells. Immunogold staining showed that this protein was strongly associated with the fibrillar axis of striated rootlets. The absence of labeling on striation suggested that rootlets were composed of several proteins, with one group forming the fibrillar axis and the second forming the striation. The formation of striated rootlets during ciliogenesis was studied using this polyclonal antibody. The 175K protein appeared at the beginning of centriologenesis in fibrillar material located around dense granules, and then around the generative complex. The formation of rootlets began at the basal pole of migrating basal bodies. The elongation of the rootlet axes took place when basal bodies were anchored to the plasma membrane.

Polyglutamylation and polyglycylation of alpha- and beta-tubulins during in vitro ciliated cell differentiation of human respiratory epithelial cells

1999 ◽  
Vol 112 (23) ◽  
pp. 4357-4366 ◽  
Author(s):  
K. Million ◽  
J. Larcher ◽  
J. Laoukili ◽  
D. Bourguignon ◽  
F. Marano ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Differentiation ◽  
Epithelial Cells ◽  
Beat Frequency ◽  
Ciliated Cell ◽  
Early Event ◽  
Functional Assay ◽  
Ciliated Cells ◽  
Human Nasal Epithelial Cells ◽  
Centriole Assembly

Tubulins are the major proteins within centriolar and axonemal structures. In all cell types studied so far, numerous alpha- and beta-tubulin isoforms are generated both by expression of a multigenic family and various post-translational modifications. We have developed a primary culture of human nasal epithelial cells where the ciliated cell differentiation process has been observed and quantified. We have used this system to study several properties concerning polyglutamylation and polyglycylation of tubulin. GT335, a monoclonal antibody directed against glutamylated tubulins, stained the centriole/basal bodies and the axonemes of ciliated cells, and the centrioles of non-ciliated cells. By contrast, axonemal but not centriolar tubulins were polyglycylated. Several polyglutamylated and polyglycylated tubulin isotypes were detected by two-dimensional electrophoresis, using GT335 and a specific monoclonal antibody (TAP952) directed against short polyglycyl chains. Immunoelectron microscopy experiments revealed that polyglycylation only affected axonemal tubulin. Using the same technical approach, polyglutamylation was shown to be an early event in the centriole assembly process, as gold particles were detected in fibrogranular material corresponding to the first cytoplasmic structures involved in centriologenesis. In a functional assay, GT335 and TAP952 had a dose-dependent inhibitory effect on ciliary beat frequency. TAP952 had only a weak effect while GT335 treatment led to a total arrest of beating. These results strongly suggest that in human ciliated epithelial cells, tubulin polyglycylation has only a structural role in cilia axonemes, while polyglutamylation may have a function both in centriole assembly and in cilia activity.

Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells

2004 ◽  
Vol 286 (4) ◽  
pp. L650-L657 ◽  
Author(s):  
Yingjian You ◽  
Tao Huang ◽  
Edward J. Richer ◽  
Jens-Erik Harboe Schmidt ◽  
Joseph Zabner ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basal Body ◽  
Ciliated Cell ◽  
Airway Epithelial Cells ◽  
Cell Phenotype ◽  
Basal Bodies ◽  
Airway Epithelial ◽  
Ciliated Cells ◽  
Wild Type

Factors required for commitment of an undifferentiated airway epithelial cell to a ciliated cell are unknown. Cell ultrastructure analysis indicates ciliated cell commitment activates a multistage program involving synthesis of cilia precursor proteins and assembly of macromolecular complexes. Foxj1 is an f-box transcription factor expressed in ciliated cells and shown to be required for cilia formation by gene deletion in a mouse model. To identify a specific role for foxj1 in directing the ciliated cell phenotype, we evaluated the capacity of foxj1 to induce ciliogenesis and direct cilia assembly. In a primary culture model of wild-type mouse airway epithelial cells, foxj1 expression preceded the appearance of cilia and in cultured foxj1 null cells cilia did not develop. Delivery of foxj1 to polarized epithelial cell lines and primary cultured alveolar epithelial cells failed to promote ciliogenesis. Similarly, delivery of foxj1 to wild-type airway epithelial cells did not enhance the total number of ciliated cells. In contrast, delivery of foxj1 to null cells resulted in the appearance of cilia. Analysis revealed that, in the absence of foxj1, null cells contained cilia precursor basal bodies, indicating prior commitment to ciliogenesis. However, the basal bodies were disorganized within the apical compartment and failed to dock with the apical membrane. Reconstitution of foxj1 in null cells restored normal basal body organization, resulting in axoneme growth. Thus foxj1 functions in late-stage ciliogenesis to regulate programs promoting basal body docking and axoneme formation in cells previously committed to the ciliated cell phenotype.

Growth and differentiation of tracheal epithelial progenitor cells

1994 ◽  
Vol 266 (3) ◽  
pp. L296-L307 ◽  
Author(s):  
J. Y. Liu ◽  
P. Nettesheim ◽  
S. H. Randell
Keyword(s):  
Monoclonal Antibody ◽  
Periodic Acid ◽  
Ciliated Cell ◽  
Secretory Cells ◽  
Parent Cell ◽  
Ciliated Cells ◽  
Periodic Acid Schiff ◽  
Epithelial Regeneration ◽  
Poorly Differentiated ◽  
Tracheal Epithelial

The purpose of these studies was to determine whether both basal and secretory rat tracheal epithelial (RTE) cells served as multipotent epithelial progenitors and whether both cell types gave rise to a similar "poorly differentiated" cell during the early phase of epithelial regeneration in denuded tracheal grafts. Griffonia simplicifolia I (GSI) lectin and flow cytometry were used for cell sorting. More than 98% of GSI-positive cells expressed plasma membrane alpha 1-3 terminal galactose (Gal), and 95% contained keratin 14 (K14), phenotypic markers for basal cells; < 1% were secretory or ciliated cells. Less than 2% of the GSI-negative cells expressed Gal or K14, but this fraction contained 16% ciliated cells and 54-79% secretory cells, dependent on whether periodic acid-Schiff staining or binding of an anti-secretory cell monoclonal antibody (RTE 12) was used as the criterion. Equal numbers of viable cells from either fraction were inoculated into denuded tracheal grafts, which were studied on days 1-14. At 24 h, greater numbers of GSI-negative than -positive cells were found attached to the graft wall; the keratin staining pattern of the attached cells was similar to that of the parent cell populations, but monoclonal antibody-detectable secretory and ciliated cell epitopes, originally present in the GSI-negative fraction, were lost. 5-Bromo-2'-deoxyuridine uptake was not seen at 24 h, but by 48 h all epithelial cells from both fractions entered the cell cycle. From 48 to 96 h, cells derived from either fraction were ultrastructurally indistinguishable; they were poorly differentiated and highly proliferative, and all expressed Gal and K14. A mature epithelium evolved from the poorly differentiated cells in both sets of grafts, but secretory and ciliated cells appeared earlier in grafts inoculated with GSI-negative cells. The results strongly suggest that in this model of tracheal epithelial regeneration both basal and secretory cells "dedifferentiated" into a similar highly proliferative phenotype from which a mucociliary epithelium "redifferentiated."

scholarly journals Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody.

1986 ◽  
Vol 103 (2) ◽  
pp. 613-619 ◽  
Author(s):  
C Klotz ◽  
N Bordes ◽  
M C Laine ◽  
D Sandoz ◽  
M Bornens
Keyword(s):  
Monoclonal Antibody ◽  
Striated Muscle ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Myosin Heavy Chains ◽  
Ciliated Cells ◽  
Western Blots ◽  
Apical Pole ◽  
Heavy Chains ◽  
Filamentous Material

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.

scholarly journals Immunological detection of spectrin during differentiation and in mature ciliated cells from quail oviduct

1989 ◽  
Vol 93 (4) ◽  
pp. 683-690
Author(s):  
B. Chailley ◽  
T. Frappier ◽  
F. Regnouf ◽  
M.C. Laine
Keyword(s):  
Plasma Membrane ◽  
Close Contact ◽  
Apical Part ◽  
Immunogold Labeling ◽  
Apical Plasma Membrane ◽  
Basal Bodies ◽  
Ciliated Cells ◽  
Electron Immunocytochemistry ◽  
Basal Foot ◽  
The Brain

A protein that was immunologically related to the erythrocyte and brain alpha-240-subunit and to the brain beta-235-subunit of spectrin was characterized by immunoblotting and was detected by immunofluorescence in the apical part of ciliated cells from quail oviduct. After immunogold-labeling electron immunocytochemistry, spectrin was detected mainly in a fibrillar meshwork located between the proximal parts of the basal bodies. It was also observed to be in contact with the basal foot of basal bodies, but was not found to be associated with the apical plasma membrane. Cilia and microvilli were unlabeled. In contrast, spectrin was detected in close contact with the lateral plasma membrane of mature ciliated cells as well as in stem epithelial cells in unstimulated oviduct. During ciliogenesis induced by estrogen, spectrin gradually appeared at the apex of the cells as the apical cytoskeleton differentiated.

Ultrastructure of bronchopulmonary lavage cells from bovines administered 3-methylindole: II. 24 hours post-treatment

1984 ◽  
Vol 42 ◽  
pp. 202-203
Author(s):  
Amreek Singh ◽  
Judith M. McLaren ◽  
Onkar S. Atwal ◽  
Peter Eyre
Keyword(s):  
Plasma Cells ◽  
Ciliated Cell ◽  
Lavage Fluid ◽  
Treated Group ◽  
Post Treatment ◽  
Lung Edema ◽  
Ciliated Cells ◽  
Thin Sections ◽  
Variable Diameter ◽  
Cell Pellet

Introduction3-methylindole (MI), a rumen metabolite of the amino acid L-tryptophan, has been shown to produce bovine pulmonary edema and emphysema. The airways contain free and exfoliated cells. A morphologic analysis of these cells may complement the understanding of the mechanism of lung edema. Ultrastructure of the bronchopulmonary lavage (BL) cells 24 h following MI oral administration to calves is described in this experiment. The 12 hours post-treatment results were described earlier.Materials and MethodsTwo Holstein-Friesian calves were each administered an oral dose of 0.2 g MI/Kg body weight and another two calves served as controls. The animals were euthanized with sodium pentabarbitol 24 h after receiving the compound. The lungs and trachea were removed and 0.1 M sodium phosphate buffered saline was infused into the lungs through the trachea. Glutaraldehyde fixative was added to the recovered BL fluid so as to form a 1% solution. The fluid was centrifuged and the resulting cell pellet was suspended in the buffer. The procedures were repeated on the suspension; the pellet was post-fixed in osmium tetroxide and was processed by conventional methods of section preparations for TEM examination. Lung samples from caudal lobes were fixed in 1.5% glutaraldehyde to obtain tissue sections for TEM.Results and DiscussionPulmonary alveolar macrophages (AM), neutrophils, ciliated epithelial cells, globule leukocytes and plasma cells were recovered from the BL fluid of the control and Mi-administered calves. Ciliated cells and globule leukocytes could not be harvested from the controls. The AM obtained from the treated calves (Fig. 1) in comparison with similar cells from the controls were larger, and contained large membrane-limited inclusions (phagolysosomes). There was a remarkable similarity between the lavaged AM and the AM studied in thin sections of lung (cf. Fig. 1 and Fig. 2). The neutrophil was the second most abundant cell type retrieved from the lavage fluid from the calves of control or treated group. Except for scanty pseudopodia in the neutrophils obtained from the Mi-receiving calves, the cells appeared unaltered (Fig. 3). Ciliated cells were abundant in the BL fluid of Mi-ingesting calves. A heterogeneous collection of vesicles filled the ciliated cell cytoplasm (Fig. 3). Globule leukocytes were commonly observed among BL cells of treated calves. The globule leukocytes were ca. 15 μm in diameter and contained round or elliptical nuclei with conspicuous nucleoli. The cytoplasmic granules, which are a prominent feature of globule leukocytes, were electron-opaque and had a variable diameter (0.5-3.0 μm). A one-line account of globule leukocytes in the bronchi of steers administered MI has appeared. Plasma cells were rare. Ultrastructure of BL cells is compatible with their response to chemical insult by MI.

Ciliary coordination in newt lung cells requires microtubule-based linkages between basal bodies: A correlative light and EM study

1992 ◽  
Vol 50 (1) ◽  
pp. 570-571
Author(s):  
Robert Hard ◽  
Gerald Rupp ◽  
Matthew L. Withiam-Leitch ◽  
Lisa Cardamone
Keyword(s):  
Basal Body ◽  
Untreated Control ◽  
Beat Frequency ◽  
Primary Cultures ◽  
Living Cells ◽  
Power Stroke ◽  
Ultrastructural Changes ◽  
Lung Cells ◽  
Basal Bodies ◽  
Ciliated Cells

In a coordinated field of beating cilia, the direction of the power stroke is correlated with the orientation of basal body appendages, called basal feet. In newt lung ciliated cells, adjacent basal feet are interconnected by cold-stable microtubules (basal MTs). In the present study, we investigate the hypothesis that these basal MTs stabilize ciliary distribution and alignment. To accomplish this, newt lung primary cultures were treated with the microtubule disrupting agent, Colcemid. In newt lung cultures, cilia normally disperse in a characteristic fashion as the mucociliary epithelium migrates from the tissue explant. Four arbitrary, but progressive stages of dispersion were defined and used to monitor this redistribution process. Ciliaiy beat frequency, coordination, and dispersion were assessed for 91 hrs in untreated (control) and treated cultures. When compared to controls, cilia dispersed more rapidly and ciliary coordination decreased markedly in cultures treated with Colcemid (2 mM). Correlative LM/EM was used to assess whether these effects of Colcemid were coupled to ultrastructural changes. Living cells were defined as having coordinated or uncoordinated cilia and then were processed for transmission EM.

Immunofluorescence Assay Using Monoclonal and Polyclonal Antibodies for Detection of Staphylococcal Enterotoxins A in Milk

2019 ◽  
Vol 13 (1) ◽  
pp. 137-145 ◽  
Author(s):  
Tzonka Godjevargova ◽  
Zlatina Becheva ◽  
Yavor Ivanov ◽  
Andrey Tchorbanov
Keyword(s):  
Monoclonal Antibody ◽  
Magnetic Nanoparticles ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Food Poisoning ◽  
Staphylococcal Enterotoxin ◽  
Staphylococcal Enterotoxin A ◽  
Fluorescence Immunoassay ◽  
Magnetic Separator ◽  
Milk Samples

Objectives: Staphylococcus aureus is a Gram-positive microorganism. S. aureus can grow in various foods and cause food poisoning by secreting enterotoxins. The most common enterotoxins involved in food poisoning are staphylococcal enterotoxin A and staphylococcal enterotoxin B, but Staphylococcal Enterotoxin A (SEA) is predominant. The main types of food contaminated with SEs are meat and meat products, poultry and eggs, milk and dairy products. The aim of this study was to develop a rapid and sensitive fluorescence immunoassay for detection of staphylococcal enterotoxin A in milk. Methods: Monoclonal and polyclonal antibodies for SEA were produced and characterized. Competitive fluorescence immunoassay based on Magnetic Nanoparticles (MNPs) was performed and optimized. MNPs were used as a solid carrier of the antibodies. The first step of the assay was immunoreaction between the immobilized antibody onto MNPs and SEA in milk sample. Then the fluorescein-SEA conjugate was added to the sample. Thus, competitive immunoreaction between MNP-mAb/MNP-pAb with SEA and SEA-FITC was performed. These immuno-complexes were separated by a magnetic separator and the obtained supernatants were analyzed. The fluorescent signal from the excess of conjugated SEA was proportional to the SEA contained in the milk. The assay duration was only 30 min. Results: The fluorescence immunoassays performed with polyclonal antibody had linear ranges from 5 pg/mL to 100 ng/mL SEA in a buffer, and from 50 pg/mL to 50 ng/mL SEA in spiked milk samples. While the same assays performed with monoclonal antibody had linear ranges from 1 pg/mL to 20 ng/mL SEA in buffer, and from 10 pg/mL to 10 ng/mL SEA in spiked milk samples. The detection limits of the developed immunoassays performed in milk were: 48 pg/mL with polyclonal antibody and 9 pg/mL with monoclonal antibody. Conclusion: A rapid and sensitive fluorescence immunoassay based on magnetic nanoparticles with a polyclonal and monoclonal antibody for determination of staphylococcal enterotoxin A in milk was developed.

scholarly journals Natural Herbal Estrogen-Mimetics (Phytoestrogens) Promote the Differentiation of Fallopian Tube Epithelium into Multi-Ciliated Cells via Estrogen Receptor Beta

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 722
Author(s):  
Maobi Zhu ◽  
Sen Takeda ◽  
Tomohiko Iwano
Keyword(s):  
Estrogen Receptor ◽  
Cell Differentiation ◽  
Epithelial Cells ◽  
Fallopian Tube ◽  
Ciliated Cell ◽  
Estrogen Receptor Beta ◽  
Notch Pathway ◽  
Secretory Cells ◽  
Ciliated Cells ◽  
Receptor Beta

Phytoestrogens are herbal polyphenolic compounds that exert various estrogen-like effects in animals and can be taken in easily from a foodstuff in daily life. The fallopian tube lumen, where transportation of the oocyte occurs, is lined with secretory cells and multi-ciliated epithelial cells. Recently, we showed that estrogen induces multi-ciliogenesis in the porcine fallopian tube epithelial cells (FTECs) through the activation of the estrogen receptor beta (ERβ) pathway and simultaneous inhibition of the Notch pathway. Thus, ingested phytoestrogens may induce FTEC ciliogenesis and thereby affect the fecundity. To address this issue, we added isoflavones (genistein, daidzein, or glycitin) and coumestan (coumestrol) to primary culture FTECs under air–liquid interface conditions and assessed the effects of each compound. All phytoestrogens except glycitin induced multi-ciliated cell differentiation, which followed Notch signal downregulation. On the contrary, the differentiation of secretory cells decreased slightly. Furthermore, genistein and daidzein had a slight effect on the proportion of proliferating cells exhibited by Ki67 expression. Ciliated-cell differentiation is inhibited by the ERβ antagonist, PHTPP. Thus, this study suggests that phytoestrogens can improve the fallopian tube epithelial sheet homeostasis by facilitating the genesis of multi-ciliated cells and this effect depends on the ERβ-mediated pathway.

G-proteins in mammalian gametes: an immunocytochemical study

1988 ◽  
Vol 91 (1) ◽  
pp. 21-31
Author(s):  
N.B. Garty ◽  
D. Galiani ◽  
A. Aharonheim ◽  
Y.K. Ho ◽  
D.M. Phillips ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Cumulus Cells ◽  
Bovine Brain ◽  
Regulatory Proteins ◽  
Immunocytochemical Study ◽  
Cell Cytoplasm ◽  
Sperm Tail ◽  
Immunoreactive Material

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod beta gamma-transducin (RA beta gamma T), bovine rod holotransducin (AS-1), bovine rod alpha-transducin (RA alpha T), synthetic bovine rod alpha-transducin C-terminal decapeptide (AS-6), bovine brain alpha 39Go (RA alpha 39), and two mouse monoclonal antibodies raised against frog retinal transducin (4A), and rat brain beta-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G-proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA beta gamma T, AS-1 and RA alpha 39 all stained the entire acrosome, AS-6 and RA alpha T stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat betaq-tubulin (a structural G-protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G-proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.

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