scholarly journals Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts.

1983 ◽  
Vol 96 (1) ◽  
pp. 277-281 ◽  
Author(s):  
O W Petersen ◽  
B van Deurs
Keyword(s):  
Coated Vesicles ◽  
Skin Fibroblasts ◽  
Cationized Ferritin ◽  
Serial Sections ◽  
Human Skin Fibroblasts ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Thin Sections ◽  
20 Nm ◽  
Section Analysis

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).

scholarly journals alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

1979 ◽  
Vol 82 (3) ◽  
pp. 614-625 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Coated Vesicles ◽  
The Other ◽  
Con A ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Complexes ◽  
Transmission Electron ◽  
Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

Coated pits with pinocytosis in Tetrahymena

1983 ◽  
Vol 63 (1) ◽  
pp. 209-222 ◽  
Author(s):  
J.R. Nilsson ◽  
B. van Deurs
Keyword(s):  
Cell Surface ◽  
Growth Temperature ◽  
Mammalian Cells ◽  
Inorganic Salt ◽  
Normal Growth ◽  
Cationized Ferritin ◽  
Salt Medium ◽  
Coated Pits ◽  
Food Vacuoles ◽  
Section Analysis

The possible role in pinocytosis of coated pits at the parasomal sacs of Tetrahymena has been studied using cationized ferritin (CF; pI = 8.5) as a marker of membrane and content. It is shown that CF binds evenly to the surface, including the coated pits, of Tetrahymena in an inorganic salt medium (to avoid formation of food vacuoles) at the normal growth temperature. Moreover, CF is internalized by coated vesicles (shown to be truly free by thin serial-section analysis) and delivered initially (1-5 min of incubation) to cisterna near the cell surface. Later (5-10 min) CF occurs also in autophagic vacuoles, formed as a result of starvation, and eventually (15-90 min) it is present in preformed (old) food vacuoles. These observations indicate that the coated pits at the parasomal sacs of Tetrahymena function in adsorptive pinocytosis in much the same manner as coated pits at the surface of mammalian cells.

scholarly journals Proteome Analysis of Cultured Fibroblasts from Type 1 Diabetic Patients and Normal Subjects

2006 ◽  
Vol 91 (9) ◽  
pp. 3507-3514 ◽  
Author(s):  
Lucia Puricelli ◽  
Elisabetta Iori ◽  
Renato Millioni ◽  
Giorgio Arrigoni ◽  
Peter James ◽  
...  
Keyword(s):  
Human Skin ◽  
Proteome Analysis ◽  
Normal Subjects ◽  
Skin Fibroblasts ◽  
Structural Proteins ◽  
Diabetic Patients ◽  
Human Skin Fibroblasts ◽  
Cultured Fibroblasts ◽  
Type 1 Diabetic Patients

Abstract Context: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. Objective: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. Design: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. Results: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and α-actinin 4 and a decrease of tubulin-β2 and splice isoform 2 of tropomyosin β-chain were detected. Conclusions: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.

scholarly journals Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

1977 ◽  
Vol 168 (1) ◽  
pp. 91-103 ◽  
Author(s):  
Christopher H. J. Sear ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Human Skin ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Cultured Fibroblasts ◽  
Cscl Density Gradient ◽  
Before And After

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-3H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated3H was present as [3H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [3H]fucose-labelled material before and after trypsin digestion. 3. The [3H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [3H]fucose and14C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [35S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.

scholarly journals Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes.

1981 ◽  
Vol 29 (9) ◽  
pp. 1003-1013 ◽  
Author(s):  
M C Willingham ◽  
A V Rutherford ◽  
M G Gallo ◽  
J Wehland ◽  
R B Dickson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Concanavalin A ◽  
Ruthenium Red ◽  
Coated Vesicles ◽  
Surface Markers ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Mediated Endocytosis

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

The ultrastructure of the double membrane-bound bodies and endoplasmic reticulum in serial sections of wheat spot mosaic-affected wheat plants

1990 ◽  
Vol 48 (3) ◽  
pp. 694-695
Author(s):  
M. H. Chen ◽  
C. Hiruki
Keyword(s):  
Endoplasmic Reticulum ◽  
High Resolution ◽  
Membrane Structure ◽  
Mosaic Disease ◽  
Serial Sections ◽  
Thin Sections ◽  
Double Membrane ◽  
Southern Alberta ◽  
Membrane Bound ◽  
Scanning Em

Wheat spot mosaic disease was first discovered in southern Alberta, Canada, in 1956. A hitherto unidentified disease-causing agent, transmitted by the eriophyid mite, caused chlorosis, stunting and finally severe necrosis resulting in the death of the affected plants. Double membrane-bound bodies (DMBB), 0.1-0.2 μm in diameter were found to be associated with the disease.Young tissues of leaf and root from 4-wk-old infected wheat plants were fixed, dehydrated, and embedded in Spurr’s resin. Serial sections were collected on slot copper grids and stained. The thin sections were then examined with a Hitachi H-7000 TEM at 75 kV. The membrane structure of the DMBBs was studied by numbering them individually and tracing along the sections to see any physical connection with endoplasmic reticulum (ER) membranes. For high resolution scanning EM, a modification of Tanaka’s method was used. The specimens were examined with a Hitachi Model S-570 SEM in its high resolution mode at 20 kV.

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