Direct visualization of the 10-nm (100-A)-filament network in whole and enucleated cultured cells

1978 ◽  
Vol 31 (1) ◽  
pp. 393-409
Author(s):  
J.V. Small ◽  
J.E. Celis
Keyword(s):  
Cytochalasin B ◽  
Cultured Cells ◽  
Marked Effect ◽  
High Electron ◽  
Perinuclear Region ◽  
Structural Elements ◽  
3T3 Cells ◽  
10 Nm Filaments ◽  
Triton X ◽  
Filament Network

Following extraction of actomyosin and tubulin from cultured cells treated with Triton X-100, a cytoskeleton remains which is composed predominantly of the cell nucleus encompassed by a network of 10-nm filaments. After negative staining the dense perinuclear region appears as a densely woven filament net punctuated by patches of high electron density. Enucleation of 3T3 cells with cytochalasin B gives rise to karyoplasts surronunded by 10-nm filaments and cytoplasts in which 10-nm filaments remain situated in the central region of the cytoplasm. While the 10-nm filaments occurred mainly as single filaments in human skin fibroblasts and 3T3 cells, in epithelioid PtK1 and PtK2 cells they were commonly associated in prominent meandering bundles. In addition, in these latter cells after Triton extraction the remaining ribosomes were bound specifically to the 10-nm-filament net. After exposure of 3T3 cells to cytochalasin B the 10-nm filaments formed branches that radiated from the perinuclear region into the immobile cell extensions. Concavalin A had no marked effect on the distribution of the 10-nm-filament net. The results suggest that the 10-nm filaments act primarily as structural elements, serving, in particular, to support and constrain the nucleus in its position in the cell.

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

Characterization of the intermediate (10 nm) filaments of cultured cells using an autoimmune rabbit antiserum

Cell ◽  
1978 ◽  
Vol 13 (2) ◽  
pp. 249-261 ◽  
Author(s):  
William E. Gordon ◽  
Anne Bushnell ◽  
Keith Burridge
Keyword(s):  
Cultured Cells ◽  
Rabbit Antiserum ◽  
10 Nm Filaments

Leukosialin (CD43, sialophorin) redistribution in uropods of polarized neutrophils is induced by CD43 cross-linking by antibodies, by colchicine or by chemotactic peptides

1997 ◽  
Vol 110 (13) ◽  
pp. 1465-1475
Author(s):  
S. Seveau ◽  
S. Lopez ◽  
P. Lesavre ◽  
J. Guichard ◽  
E.M. Cramer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Motility ◽  
Small Proportion ◽  
Cytochalasin B ◽  
Cell Polarization ◽  
Cross Linking ◽  
Chemotactic Peptides ◽  
Butanedione Monoxime ◽  
Major Surface ◽  
Triton X

We investigated a possible association of leukosialin (CD43), the major surface sialoglycoprotein of leukocytes, with neutrophil cytoskeleton. We first analysed the solubility of CD43 in Triton X-100 and observed that CD43 of resting neutrophils was mostly soluble. The small proportion of CD43 molecules, which ‘spontaneously’ precipitated in Triton, appeared associated with F-actin, as demonstrated by the fact that this insolubility did not occur when cells were incubated with cytochalasin B or when F-actin was depolymerized with DNase I in the Triton precipitate. Cell stimulation with anti-CD43 mAb (MEM59) enhanced this CD43-cytoskeleton association. By immunofluorescence as well as by electron microscopy, we observed a redistribution of CD43 on the neutrophil membrane, initially in patches followed by caps, during anti-CD43 cross-linking at 37 degrees C. This capping did not occur at 4 degrees C and was inhibited by cytochalasin B and by a myosin disrupting drug butanedione monoxime, thus providing evidence that the actomyosin contracile sytem is involved in the capping and further suggesting an association of CD43 with the cytoskeleton. Some of the capped cells exhibited a front-tail polarization with CD43 caps located in the uropod at the rear of the cell. Surprisingly, colchicine and the chemotactic factor fNLPNTL which induce neutrophil polarization associated with cell motility, also resulted in a clustering of CD43 in the uropod, independently of a cross-linking of the molecule by mAbs. An intracellular redistribution of F-actin, mainly at the leading front and of myosin in the tail, was observed during CD43 clustering induced by colchicine and in cells polarized by anti-CD43 mAbs cross-linking. We conclude that neutrophil CD43 interacts with the cytoskeleton, either directly or indirectly, to redistribute in the cell uropod under antibodies stimulation or during cell polarization by colchicine, thus highly suggesting that CD43 may be involved in cell polarization.

Transport of phagosomes in mouse peritoneal macrophages

1989 ◽  
Vol 94 (1) ◽  
pp. 143-153
Author(s):  
A. Toyohara ◽  
K. Inaba
Keyword(s):  
Cytochalasin B ◽  
Large Diameter ◽  
Sulfate Solution ◽  
Peritoneal Macrophages ◽  
Transport Systems ◽  
Perinuclear Region ◽  
Microtubule Network ◽  
Mouse Macrophages ◽  
Hank’S Solution ◽  
Mouse Peritoneal Macrophages

Mouse macrophages were elicited by the peritoneal injection of chondroitin sulfate solution, harvested and purified, and used as experimental materials. Small and large (diameter: 0.9 microns and 3.0 microns, respectively) polystyrene beads (PB) were used as ingested particles. When the macrophages were incubated with Hank's solution containing small or large PB for 30 min, the phagosomes containing small or large PB were usually randomly distributed. When the macrophages were further incubated for 45 min in PB-free medium, both small and large phagosomes containing PB accumulated at the perinuclear region. The transport of large phagosomes containing 3.0 microns PB was inhibited by cytochalasin B, but not by vinblastine or podophyllotoxin. Conversely, the transport of small phagosomes containing 0.9 microns PB was not inhibited by cytochalasin B but was inhibited by vinblastine or podophyllotoxin. Immunofluorescence microscopy showed that the small phagosomes appeared to accumulate at the central region of the microtubule network. The large phagosomes, on the other hand, appeared to be surrounded by actin-rich cytoplasm, and in some cells actin filament-like structures could be seen around large phagosomes. These results suggest that there are two different transport systems of phagosomes in macrophages. Phagosomes smaller than 0.9 microns in diameter are, probably, mainly transported to the perinuclear region by a microtubule-based motility system and those larger than 3.0 microns in diameter by an actin-based mechanism. It was observed electron-microscopically that accumulated phagosomes containing PB could fuse with each other and form larger phagosomes.

Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits

1999 ◽  
Vol 77 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Jean-Martin Beaulieu ◽  
Janice Robertson ◽  
Jean-Pierre Julien
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Intermediate Filament Protein ◽  
Physiological Conditions ◽  
Filament Protein ◽  
Filament Network ◽  
Filament Type

Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of neurofilaments can have an impact on peripherin assembly in vivo.Key words: peripherin, neurofilament, SW13 cells, intermediate filament.

Effect of vinblastine and cytochalasin B on the cytoskeletal domains in 3T3 cells

1981 ◽  
Vol 48 (1) ◽  
pp. 55-73
Author(s):  
J.H. Temmink ◽  
H. Spiele
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Cytochalasin B ◽  
Morphological Changes ◽  
Ultrastructural Changes ◽  
3T3 Cells ◽  
Cytoplasmic Material ◽  
Transmission Electron ◽  
Cytoskeletal Elements ◽  
Scanning Electron

Normal 3T3 cells were exposed to vinblastine and cytochalasin B in an attempt to correlate the morphological changes of the cell surface as seen in the scanning electron microscope with ultrastructural changes of the cytoskeletal elements as seen in critical-point-dried cells in the transmission electron microscope. Special attention was given to the changes in the cytoplasmic domains distinguished in a previous paper. Cytochalasin B primarily affects the ultrastructure of the cytocortical domain by inducing the formation of condensation foci on the cytoplasmic material. Vinblastine not only induces the depolymerization of microtubules and the perinuclear concentration of intermediate filaments, but it also causes the disappearance of stress fibres from the cortical cytoplasm and the widening of the cytocortex at the expense of the endoplasmic domain. These results support the hypothesis that the differentiation in ultrastructural domains is dependent on the spreading of the cells and their adhesion to substrate.

Interaction of plasma membrane-associated filaments and H-2 histocompatibility antigens before and after induced patching and capping

1987 ◽  
Vol 88 (3) ◽  
pp. 313-325
Author(s):  
C.A. Feltkamp ◽  
H. Spiele ◽  
E. Roos
Keyword(s):  
Plasma Membrane ◽  
Short Distance ◽  
Coated Pits ◽  
Apparent Absence ◽  
Histocompatibility Antigens ◽  
Before And After ◽  
Prevailing Direction ◽  
Sequential Addition ◽  
Triton X ◽  
Filament Network

The interaction of H-2 antigens and plasma membrane-associated filaments was studied on dry-cleaved preparations of immunogold-labelled lymphoma cells. In prefixed cells, the plasma membrane-associated network was isotropic without any prevailing direction of the filaments, and the gold-labelled H-2 antigens were preferentially localized over or at a very short distance from membrane-associated filaments. Incubation of unfixed cells with anti-H-2 antibodies followed by fixation and incubation with anti-Ig, did not induce detectable redistribution of H-2 antigens or of the filament network. Notwithstanding this apparent absence of rearrangement of H-2 antigens and filaments, a detergent-resistant linkage to the cytoskeleton was induced. Before immune incubations, virtually all H-2 antigens were solubilized by extraction with Triton X-100, while after incubation with anti-H-2 antibodies about 50% of the H-2 antigens were linked to the Triton X-100-insoluble cytoskeleton. Sequential addition of anti-H-2 and anti-Ig antibodies to unfixed cells induced formation of patches and caps of H-2 antigens. Under these conditions, the majority of the H-2 antigens became linked to the detergent-resistant cytoskeleton. Redistribution into patches and caps was often accompanied by a local rearrangement of the isotropic network into bundles of parallel filaments immediately adjacent to the plasma membrane. Patches were seen to overly both isotropic networks and these parallel filaments. Large sheets of plasma membrane overlying parallel filaments were frequently devoid of gold-labelled H-2 antigens and coated pits, and thus most probably represented areas away from caps. This observation suggests that capping is accompanied by a rearrangement of filaments close to the membrane.

Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells

2000 ◽  
Vol 113 (21) ◽  
pp. 3805-3814 ◽  
Author(s):  
J. Xu ◽  
D. Ziemnicka ◽  
G.S. Merz ◽  
L. Kotula
Keyword(s):  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Binding Protein ◽  
Sh3 Domain ◽  
Water Soluble ◽  
Protein Markers ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.

Plasminogen Activator (PA) Activity In Metastatic Cells: An Amidolytic Approach

1981 ◽  
Author(s):  
D Coen ◽  
A Bini ◽  
G Balconi ◽  
F Delaini ◽  
L Mussoni ◽  
...  
Keyword(s):  
Primary Tumor ◽  
Fibrinolytic Activity ◽  
Tumor Tissue ◽  
Cultured Cells ◽  
Cell Extracts ◽  
Mechanical Disruption ◽  
Metastatic Cells ◽  
Triton X ◽  
Selection Of

It has been proposed that fibrinolytic activity can play an important role in the process of metastasis formation. Nevertheless, it is not yet clear in which phase of the tumor growth and dissemination this activity is involved. We measured the fibrinolytic activity of cells from primary tumor and metastatic nodules of 3LL, an i.m. implanted murine tumor which selectively metastasizes to the lungs. Tumor cells have been studied both immediately after mechanical disruption of tumor tissue and after in vitro culturing to confluence. Their P.A. activity was tested by an amidolytic assay in which cells were incubated with purified plasminogen (3CU/ml) and 4mM S-2251 (Kabi Diagnostica, Stockholm, Sweden), a plasmin specific chromogenic substrate. After 3 hour incubation at 37°C, the reaction was stopped with acetic acid and absorbance read at 405 nm.Cells from the primary tumor and metastatic nodules showed a similar fibrinolytic activity, which was in both cases in- increased 3 to 4 fold in cell extracts obtained after preincubation with TRITON X-100. A dose-response curve plotted with increasing urokinase concentrations showed a parallel course. This data suggests that, in the 3LL model, PA activity is not one of the properties characterizing the selection of metastatic cells.On the other hand,cultured cells presented consistently higher levels of PA than their native counterparts, suggesting that adhesion of cells in culture may stimulate PA production or, alternatively, that cultured cells are a selected population in comparison to the overall number of native cells.

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