scholarly journals Filopodelike projections induced with dimethyl sulfoxide and their relevance to cellular polarity in Dictyostelium.

1983 ◽  
Vol 96 (3) ◽  
pp. 857-865 ◽  
Author(s):  
S Yumura ◽  
Y Fukui
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Dimethyl Sulfoxide ◽  
Cell Polarity ◽  
Cytoplasmic Streaming ◽  
Anterior Part ◽  
Cellular Polarity ◽  
Microtubule Organizing Center ◽  
Cell Organelles ◽  
Thin Sections

When 5% dimethyl sulfoxide (DMSO) was applied to Dictyostelium cells, the cells rounded up in shape and cytoplasmic streaming ceased. The cells resumed both cytoplasmic streaming and locomotion in 20 min. SDS PAGE of isolated plasma membrane fractions showed that actin and myosin apparently became dissociated from the plasma membrane by the action of DMSO. Scanning electron microscopy revealed that many filopodelike projections formed on the surface of cells treated with 5% DMSO for 5 min. Interestingly, the projections were formed on a restricted portion of the cell surface. The phagokinetic track technique of Albrecht-Buehler (1977, Cell, 11: 395-404) showed that the projection region corresponded to the anterior part of a migrating cell. The possible relationship between the DMSO-induced projection region on the cell surface and intracellular organization of cell organelles was investigated using serial thin sections. The DMSO-induced projections contained arrays of microfilaments; and the microtubule organizing center (MTOC), nucleus, and vesicular structure were usually located in this order from the anterior end of the cell. The indirect immunofluorescent study using monoclonal anti-alpha-tubulin antibody was performed with a new fixation technique, which greatly improved the phase as well as immunofluorescent microscopy. It was verified that the intracellular positioning of the MTOC and nucleus had significant correlation with the cell polarity. The results show that DMSO is a powerful tool with which to manipulate the cellular microfilaments and to make visible the differentiation in the cortex layer, which apparently is relevant to the intracellular positioning of cell organelles and cell polarity.

Effect of different colchicine concentrations on microtubules and membranes in tomato root tip cells

1988 ◽  
Vol 46 ◽  
pp. 306-307
Author(s):  
T.E. Jensen
Keyword(s):  
Plasma Membrane ◽  
Phosphate Buffer ◽  
Cell Types ◽  
Root Tip ◽  
Distilled Water ◽  
Root Tips ◽  
Cell Organelles ◽  
Tomato Root ◽  
Thin Sections ◽  
Numerous Cell

The effect of colchicine on microtubules has been investigated in numerous cell types. In this present study we have used different concentrations of colchicine to determine if the two major groups of microtubules in plant cells, plasma membrane associated and mitotic, are differentially sensitive to this drug.Tomato seeds “Michigan forcing” were germinated on filter paper saturated with distilled water. Radicles 1.5 to 2.0cm were selected and placed into either distilled water or colchicine, 0.001, 0.01, 0.03, 0.04, 0.05, 0.1%, for 20 hrs. During this time they were kept at 20°C in dim light. Root tips were fixed in 3% glutaraldehyde in phosphate buffer at pH7.2 for lh at 4°C. After 5 rinses in buffer they were placed in 1% OsO4 in phosphate buffer at pH7.2 for 1h at 4°C. Root tips were then dehydrated in ethanol, treated with propylene and embedded in Epon.No growth occurred in any of the colchicine treated radicles. Observation of thin sections of control cells revealed many plasma membrane associated microtubules, many cells in division stages and the usual arrangement of cell organelles.

scholarly journals SUBPLASMALEMMAL MICROFILAMENTS AND MICROTUBULES IN RESTING AND PHAGOCYTIZING CULTIVATED MACROPHAGES

1973 ◽  
Vol 59 (1) ◽  
pp. 12-27 ◽  
Author(s):  
Eve P. Reaven ◽  
Stanton G. Axline
Keyword(s):  
Plasma Membrane ◽  
Free Surface ◽  
Cell Surface ◽  
Cell Attachment ◽  
Close Association ◽  
Microtubule Organization ◽  
Thin Sections ◽  
Attached Cell ◽  
Specific Membrane

The subplasmalemmal organization of the free and glass-attached surfaces of resting and phagocytizing cultivated macrophages were examined in an attempt to define specific membrane-associated structures related to phagocytosis. From analysis of serial thin sections of oriented cells it was found that the subplasmalemmal region of the attached cell surface has a complex microfilament and microtubule organization relative to the subplasmalemmal area of the free surface. A filamentous network composed of 40–50-Å microfilaments extended for a depth of 400–600 Å from the attached plasma membrane. Immediately subjacent to the filamentous network was a zone of oriented bundles of 40–50-Å microfilaments and a zone of microtubules. Additional microtubules were found to extend from the plasma membrane to the interior of the cell in close association with electron-dense, channellike structures. In contrast, the free aspect of the cultivated macrophage contained only the subplasmalemmal filamentous network. However, after a phagocytic pulse with polystyrene particles (14 µm diam) microtubules and oriented filaments similar to those found on the attached surface were observed surrounding the ingested particles. The observations reported in this paper provide support for the hypothesis that microfilaments and/or microtubules play a role in the translocation of plasma membrane required for the functionally similar processes of phagocytosis and cell attachment to glass.

scholarly journals Regulated internalization of caveolae.

1994 ◽  
Vol 127 (5) ◽  
pp. 1199-1215 ◽  
Author(s):  
R G Parton ◽  
B Joggerst ◽  
K Simons
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Alkaline Phosphatase Activity ◽  
Kinase Inhibitor ◽  
Cytochalasin D ◽  
Microtubule Organizing Center ◽  
Hypertonic Medium

Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network.

scholarly journals Intracellular Redirection of Plasma Membrane Trafficking after Loss of Epithelial Cell Polarity

2000 ◽  
Vol 11 (9) ◽  
pp. 3045-3060 ◽  
Author(s):  
Seng Hui Low ◽  
Masumi Miura ◽  
Paul A. Roche ◽  
Anita C. Valdez ◽  
Keith E. Mostov ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Polarity ◽  
Membrane Traffic ◽  
Cellular Polarity ◽  
Madin Darby Canine Kidney ◽  
Apical Compartment ◽  
Basolateral Plasma Membrane ◽  
Darby Canine Kidney ◽  
Canine Kidney

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.

scholarly journals A CRISPR Screen Identifies the Cell Polarity Determinant Crumbs 3 as an Adeno-associated Virus Restriction Factor in Hepatocytes

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Victoria J. Madigan ◽  
Tyne O. Tyson ◽  
Julianne A. Yuziuk ◽  
Minakshi Pillai ◽  
Sven Moller-Tank ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Surface ◽  
Cell Polarity ◽  
Cell Types ◽  
Host Factors ◽  
Cellular Polarity ◽  
A Genome ◽  
Organ Systems ◽  
Gene Therapy Vectors ◽  
Crispr Screen

ABSTRACTAdeno-associated viruses (AAV) are helper-dependent parvoviruses that have been developed into promising gene therapy vectors. Many studies, including a recent unbiased genomic screen, have identified host factors essential for AAV cell entry, but no genome-wide screens that address inhibitory host factors have been reported. Here, we utilize a novel CRISPR screen to identify AAV restriction factors in a human hepatocyte cell line. The major hit from our gain-of-function screen is the apical polarity determinant Crumbs 3 (Crb3). Knockout (KO) of Crb3 enhances AAV transduction, while overexpression exerts the opposite effect. Further, Crb3 appears to restrict AAV transduction in a serotype- and cell type-specific manner. Particularly, for AAV serotype 9 and a rationally engineered AAV variant, we demonstrate that increased availability of galactosylated glycans on the surfaces of Crb3 KO cells, but not the universal AAV receptor, leads to increased capsid attachment and enhanced transduction. We postulate that Crb3 could serve as a key molecular determinant that restricts the availability of AAV glycan attachment factors on the cell surface by maintaining apical-basal polarity and tight junction integrity.IMPORTANCEAdeno-associated viruses (AAVs) have recently emerged at the forefront as gene therapy vectors; however, our understanding of host factors that influence AAV transduction in different cell types is still evolving. In the present study, we perform a genome-scale CRISPR knockout screen to identify cellular host factors that restrict AAV infection in hepatocyte cultures. We discover that Crumbs 3, which determines cellular polarity, also influences the distribution of certain carbohydrate attachment factors on the cell surface. This in turn affects the ability of virions to bind and enter the cells. This study underscores the importance of cell polarity in AAV transduction and provides a potential molecular basis for the differential infectious mechanism(s) in cell culture versus organ systems.

scholarly journals Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

1958 ◽  
Vol 4 (6) ◽  
pp. 711-716 ◽  
Author(s):  
Edward Essner ◽  
Alex B. Novikoff ◽  
Bertha Masek
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Function ◽  
Molecular Transport ◽  
Enzyme Reaction ◽  
Bile Canaliculus ◽  
Nucleotidase Activity ◽  
Reaction Products ◽  
Thin Sections

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.

Cell Fine Structure as Revealed in Dessicator-Dried Resinless Sections

1981 ◽  
Vol 39 ◽  
pp. 622-623
Author(s):  
R. P. Becker ◽  
J. J. Wolosewick ◽  
J. Ross-Stanton
Keyword(s):  
Fine Structure ◽  
Tannic Acid ◽  
Three Dimensional ◽  
Albino Rats ◽  
Cell Organelles ◽  
Vascular Perfusion ◽  
Thin Sections ◽  
Carbon Coated ◽  
Embedding Medium ◽  
Polyethylene Glycol 6000

Methodology has been introduced recently which allows transmission and scanning electron microscopy of cell fine structure in semi-thin sections unencumbered by an embedding medium. Images obtained from these “resinless” sections show a three-dimensional lattice of microtrabeculfee contiguous with cytoskeletal structures and membrane-bounded cell organelles. Visualization of these structures, especially of the matiiDra-nous components, can be facilitated by employing tannic acid in the fixation step and dessicator drying, as reported here.Albino rats were fixed by vascular perfusion with 2% glutaraldehyde or 1.5% depolymerized paraformaldehyde plus 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4). Tissues were removed and minced in the fixative and stored overnight in fixative containing 4% tannic acid. The tissues were rinsed in buffer (0.2M cacodylate), exposed to 1% buffered osmium tetroxide, dehydrated in ethyl alcohol, and embedded in pure polyethylene glycol-6000 (PEG). Sections were cut on glass knives with a Sorvall MT-1 microtome and mounted onto poly-L-lysine, formvar-carbon coated grids while submerged in a solution of 95% ethanol containing 5% PEG.

Quantification of cell-surface expressed antigenic sites with 5-nm gold markers: Getting close to biologically relevant data

1989 ◽  
Vol 47 ◽  
pp. 1050-1051
Author(s):  
Etienne de Harven ◽  
Hilary Christensen ◽  
Richard Leung ◽  
Cameron Ackerley
Keyword(s):  
Cell Surface ◽  
Human Peripheral Blood ◽  
Contour Length ◽  
Thin Sections ◽  
Gold Particles ◽  
Biologically Relevant ◽  
Transmission Electron ◽  
Entire Cell ◽  
Electron Imaging ◽  
Cell Contour

The T-derived subset of human peripheral blood normal lymphocytes has been selected as a model system to study the usefulness of 5 nm gold markers for quantification of single epitopes expressed on cell surfaces. The chosen epitopes are parts of the CD3 and CD5 molecules and can be specifically identified by hybridoma produced monoclonal antibodies (MoAbs; LEU-4 and LEU-1; Becton-Dick- inson, Mountain view, CA) . An indirect immunolabeling procedure, with goat anti-murine IgG adsorbed on the surface of 5 nm colloidal gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) has been used. Backscattered Electron Imaging (BEI) in a field emission scanning electronmicroscope (SEM) and transmission electron microscopy of thin sections of lymphocytes labeled before plastic embedding, were both used to identify and quantitate gold labeled cell surface sites, Estimating that the thickness of “silver” sections is approximately 60 nm and counting the number of gold particles on the entire cell perimeter, we calculated that, for LEU-4, the number of markers per um2 of cell surface is in the 140-160 range (Fig.l). Cell contour length measurements indicated that the surface of one lymphocyte is approximately 130-160 um2 that of a smooth sphere of identical diameter, reflecting the role of microvilli in expanding the surface area. The total number of gold labeled sites on the surface of one lymphocyte averages, therefore between 20,000 and 24,000 per cell.

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

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