Intracellular relationship between actin and alpha-actinin in a whole corneal epithelial tissue

1993 ◽  
Vol 106 (3) ◽  
pp. 703-717
Author(s):  
W. Khoory ◽  
E. Wu ◽  
K.K. Svoboda
Keyword(s):  
Cell Surface ◽  
Basal Cell ◽  
Laser Scanning ◽  
Cytochalasin D ◽  
Confocal Laser ◽  
Epithelial Tissue ◽  
Control Medium ◽  
Cell Nuclei ◽  
Double Labeling ◽  
Corneal Epithelial

Alpha-actinin is an actin crosslinking protein that may be one of the proteins involved in the attachment of the actin cytoskeletal framework to the plasma membrane. We investigated the distribution of alpha-actinin in whole-mount embryonic chick corneal epithelia using confocal laser scanning analysis. The intracellular alpha-actinin distribution was compared with F-actin using phalloidin, or total actin using an anti-actin antibody. Corneal epithelial tissues were isolated with or without the basal lamina (+ or -BL), and fixed immediately. In addition, epithelia isolated -BL were cultured for 2 hours with either control medium, laminin-supplemented medium or laminin and cytochalasin D (CD)-containing medium. The single- and double-labeled epithelia showed that alpha-actinin delineated the cell borders and microvilli of the periderm cells in the most apical optical sections of control and laminin-treated epithelia. At the optical plane through the basal cell nuclei, the alpha-actinin was distributed diffusely throughout the cytoplasm, whereas the actin was sparse, only associated with the lateral cell membranes. Epithelia (-BL) cultured in control medium had cytoplasmic protrusions or blebs on the basal cell surface. The blebs contained both actin and alpha-actinin. In epithelial cultured with laminin, the basal cell surface was flat. The actin cortical mat became reorganized within two hours. Actin and alpha-actinin were colocalized in the re-formed basal cytoskeletal network. In cells cultured with cytochalasin D (CD) and laminin the actin cortical mat was not reorganized. Actin networks from both cell layers were eliminated and replaced by aggregates scattered throughout the cytoplasm. The alpha-actinin remained diffusely distributed in the cytoplasm and failed to colocalize with the actin aggregates. The alpha-actinin appeared closer to the basal cell membrane than the actin in cross-sectional views of the tissue. Results from these double-labeling experiments confirmed the intimate association of alpha-actinin and actin in the laminin-stimulated actin cortical mat reorganization. This study is the first to demonstrate that CD-aggregated F-actin does not capture the alpha-actinin. The alpha-actinin appeared to remain diffuse throughout the cytoplasm and separate from F-actinin; however, there was some overlap with G-actin.

scholarly journals New approaches to the estimation of endometrial dysfunction

2017 ◽  
Vol 66 (3) ◽  
pp. 8-15 ◽  
Author(s):  
Edvard K. Aylamazyan ◽  
Gulrukhsor Kh. Tolibova ◽  
Tatyana G. Tral ◽  
Igor U. Kogan ◽  
Mariya I. Yarmolinskaya ◽  
...  
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Uterine Cavity ◽  
Immunohistochemical Analysis ◽  
Endometrial Biopsy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Nuclei

Introduction. The application of modern methods for assessing the morphofunctional state of the endometrium to verify and study the expression of sex steroid hormones, proinflammatory markers and markers of angiogenesis using confocal laser scanning microscopy will allow an objective study of the role of studied markers in the pathogenesis of the endometrial dysfunction. The aim of the study was to evaluate the expression of ER and PR receptors in endometrium in patients with endometrial dysfunction. Material and methods. Endometrial biopsy specimens obtained with the aid of a pile-biopsy or a scraping from the uterine cavity are used to conduct the method of immunofluorescent confocal laser scanning microscopy. It is possible to use both cryostat material and paraffin blocks to provide the immunohistochemical analysis. Monoclonal antibodies to ER (1 : 60, Dako, Denmark) and PR (1 : 50, Dako, Denmark) are used as primary antibodies, antibodies conjugated with fluorochrome Alexa Fluor 647 (1 : 1000, Abcam, England) are used as secondary antibodies). Hoechst 33258 (Sigma, USA) is used for staining of cell nuclei. Results. A method of confocal laser scanning microscopy makes it possible to conduct qualitative and quantitative evaluation of the studied markers in different structures of the endometrium.

Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia

1991 ◽  
Vol 100 (1) ◽  
pp. 23-33 ◽  
Author(s):  
K.K. Svoboda
Keyword(s):  
Endoplasmic Reticulum ◽  
In Situ Hybridization ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Confocal Laser ◽  
Type I ◽  
Basal Cells ◽  
Double Labeling ◽  
Collagen Mrna

The intracellular distribution of endoplasmic reticulum (ER) and types I and II collagen mRNA was analyzed in whole-mount preparations of freshly isolated corneal epithelia using in situ hybridization combined with confocal laser scanning analysis. The ER stained with DiOC6 (3) was prominent in both the periderm and basal cells. The basal cell ER distribution was perinuclear in the center of the cells, but below the nucleus the ER occupied nearly all of the cytoplasm in a reticular pattern similar to that seen with TEM cross-sections. Initial single label in situ hybridization studies showed that both the periderm and basal cells were positive for both types I and II collagen mRNA. The collagen cDNA probes appeared perinuclear in the center of the basal cells, similar to the DiOC6(3) staining pattern. In double-labeling experiments, the two mRNAs that translate chains of type I collagen, alpha 1 and alpha 2, colocalized within the same cell. However, the hybridization of probes specific for type I and II collagen mRNAs had separate, but overlapping, distributions within the same cell.

A Facile Synthesis of Acid-Activatable Nanoparticles for Efficient Intracellular Doxorubicin Release

2017 ◽  
Vol 46 ◽  
pp. 20-30 ◽  
Author(s):  
Cao Ming ◽  
Xiao Wan Song ◽  
Yu Jiao Zhang ◽  
Chang Zhi Xu ◽  
Peng Chen ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Hela Cells ◽  
Laser Scanning ◽  
Human Cancer ◽  
Polymeric Nanoparticles ◽  
Confocal Laser Scanning Microscope ◽  
Ph Sensitive ◽  
Confocal Laser ◽  
Cell Nuclei

pH responsive polymeric nanoparticles have emerged as a promising technology platform for targeted and controlled drug delivery in recent years. In this paper, endosomal pH-activatable doxorubicin (DOX) and core-crosslinked polymeric nanoparticles (DCNPs) were prepared and investigated for potent growth inhibition of human cancer cells in vitro. In vitro drug release studies, DOX conjugated nanoparticles with hydrazone bond showed a pH sensitive release phenomenon, that is, the releasing is significantly faster at mildly acidic condition with pH of 5.5 than that at physiological condition. Confocal laser scanning microscope (CLSM) observations revealed that DOX conjugated nanoparticles delivered and released DOX into the cytosols as well as cell nuclei of Hela cells following 6 h incubation. MTT assays demonstrated that these pH-sensitive DOX nanoparticles exhibited high antitumor effect to HeLa cells. The conjugated DOX polymeric nanoparticles may be a promising candidate as a nanoscale and pH-sensitive drug delivery vehicle for cancer therapy.

scholarly journals Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937.

1991 ◽  
Vol 39 (7) ◽  
pp. 981-985 ◽  
Author(s):  
S B Por ◽  
M A Cooley ◽  
S N Breit ◽  
R Penny ◽  
P W French
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Cell Line ◽  
Laser Scanning ◽  
Confocal Laser ◽  
Staining Procedure ◽  
Intermediate Filament Protein ◽  
U937 Cells ◽  
Monocytic Cell ◽  
Monocytic Cell Line

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.

scholarly journals A Straightforward DOPE (Double Labeling of Oligonucleotide Probes)-FISH (FluorescenceIn SituHybridization) Method for Simultaneous Multicolor Detection of Six Microbial Populations

2012 ◽  
Vol 78 (15) ◽  
pp. 5138-5142 ◽  
Author(s):  
Faris Behnam ◽  
Andreas Vilcinskas ◽  
Michael Wagner ◽  
Kilian Stoecker
Keyword(s):  
Laser Scanning ◽  
Simultaneous Detection ◽  
Laser Scanning Microscopy ◽  
Clinical Samples ◽  
Confocal Laser ◽  
Oligonucleotide Probes ◽  
Double Labeling ◽  
Attractive Option ◽  
Higher Sensitivity

ABSTRACTFluorescencein situhybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.

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