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 Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

1982 ◽  
Vol 94 (3) ◽  
pp. 613-623 ◽  
Author(s):  
J Aggeler ◽  
Z Werb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
High Resolution Electron Microscopy ◽  
Peritoneal Macrophages ◽  
Coated Pits ◽  
Thin Sections ◽  
Particle Uptake ◽  
Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

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.

scholarly journals Collagen Formation by Fibroblasts of the Chick Embryo Dermis

1959 ◽  
Vol 5 (1) ◽  
pp. 153-165 ◽  
Author(s):  
Keith R. Porter ◽  
George D. Pappas
Keyword(s):  
Cell Surface ◽  
Collagen Fiber ◽  
Close Association ◽  
Supplementary Information ◽  
Cell Cortex ◽  
Intercellular Spaces ◽  
Thin Sections ◽  
Collagen Formation

This investigation has sought to determine the relation between collagen fiber and fibroblast during fibrogenesis. Toward this end the surfaces of chick fibroblasts grown under in vitro conditions have been examined with the electron microscope after fixation in OsO4. Supplementary information has been obtained from thin sections of fibroblasts fixed in situ during phases of fiber production. The evidence provided by these studies and by various conditions of the experiments indicates that the unit fibrils of collagen form in close association with the cell surface. They were never observed within the cell. When these unit fibrils form in bundles it appears as though templates of some nature, possibly coinciding with stress fibers within the cell cortex, influence the polymerization of the fibrils out of material available at the cell surface. From here the fibrils and bundles of them are shed into the intercellular spaces and there grow to limited diameters by accretion of materials from the general milieu.

scholarly journals MICAL-L1 is required for cargo protein delivery to the cell surface

Biology Open ◽  
2021 ◽  
Vol 10 (6) ◽  
Author(s):  
R. Sikora ◽  
P. Bun ◽  
L. Danglot ◽  
M. Alqabandi ◽  
P. Bassereau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Membrane Trafficking ◽  
Protein Delivery ◽  
Close Association ◽  
Super Resolution ◽  
Small Gtpase ◽  
C Terminus ◽  
Membrane Tubulation

ABSTRACT Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

scholarly journals Insulin as a surface marker on isolated cells from rat pancreatic islets.

1983 ◽  
Vol 97 (2) ◽  
pp. 433-437 ◽  
Author(s):  
D R Kaplan ◽  
J R Colca ◽  
M L McDaniel
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Islet Cells ◽  
Secretory Granules ◽  
Immunoreactive Insulin ◽  
Isolated Cells ◽  
Intact Cells ◽  
Specific Receptors ◽  
Specific Membrane ◽  
Rat Pancreatic Islet

Immunoreactive insulin was shown to exist as a surface molecule in the plasma membrane of dispersed rat pancreatic islet cells. The intact cells were stained by immunofluorescence with a guinea pig antisera specific for insulin. The hormone on the cell surface could not be accounted for by insulin bound to specific receptors or nonspecifically absorbed to cells. Thus, surface insulin was demonstrated to be a specific membrane antigen for islet cells. Furthermore, the proportion of islet cells with insulin on the cell surface was directly correlated with insulin secretion in several different settings. This correspondence was demonstrated by varying the glucose concentration in the medium, by withholding Ca2+, which inhibits secretion, and by adding theophylline, which potentiates secretion. Consequently, these results suggested that insulin as a membrane protein was a marker for cells that actively secreted the hormone and may have been derived in the fusion process of secretory granules with the plasma membrane.

scholarly journals Plasma Membrane Sterol Distribution Resembles the Surface Topography of Living Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 211-228 ◽  
Author(s):  
Daniel Wüstner
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Topography ◽  
Cell Attachment ◽  
Living Cells ◽  
Surface Structures ◽  
Surface Distribution ◽  
Correlation Lengths ◽  
Fluid Membrane ◽  
Dividing Cells

Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8–2.5 μm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1—a putative raft marker—did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane.

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.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

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.

High-voltage electron microscopic studies of inflammatory cell attachment during blood-brain barrier inflammation

1990 ◽  
Vol 48 (3) ◽  
pp. 276-277
Author(s):  
A.S. Lossinsky ◽  
M.J. Song
Keyword(s):  
High Voltage ◽  
Inflammatory Cell ◽  
Cell Attachment ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Tissue Samples ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron

Previous studies have suggested the usefulness of high-voltage electron microscopy (HVEM) for investigating blood-bram barrier (BBB) injury and the mechanism of inflammatory-cell (IC) attachment. These studies indicated that, in evaluating standard conventional thin sections, one might miss cellular attachment sites of ICs in their process of attaching to the luminal endothelial cell (EC) surface of cerebral blood vessels. Our current studies in animals subjected to autoimmune disease suggest that HVEM may be useful in localizing precise receptor sites involved in early IC attachment.Experimental autoimmune encephalomyelitis (EAE) was induced in mice and rats according to standard procedures. Tissue samples from cerebellum, thalamus or spinal cords were embedded in plastic following vascular perfusion with buffered aldehyde. Thick (0.5-0.7 μm) sections were cut on glass knives and collected on Formvar-coated slot grids stained with uranylacetate and lead citrate and examined with the AEI EM7 1.2 MV HVEM in Albany, NY at 1000 kV.

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