Nanovid microscopy.
By combining small colloidal gold probes with video-enhanced quantitative microscopy, the intracellular dynamics of specific proteins in living cells can now be studied.
Axonal Transport↗
Biomedical subjects
M de Brabander
Publications and source records attributed to M de Brabander.
Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy.
A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.
Adenosine Triphosphate↗
Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling.
Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.
Adenosine Triphosphate↗
Effect of tubulozole, a new synthetic microtubule inhibitor, on the induction of casein gene expression by prolactin.
Colchicine and related drugs are known to inhibit milk secretion. They are also able to prevent stimulation of casein and DNA synthesis by prolactin in the mammary gland. The present report reports data obtained with tubulozole, a new antimitotic drug. Tubulozole C added to culture medium of isolated rabbit epithelial mammary cells strongly inhibited their multiplication. Simultaneously, at a concentration of 1 microM, it prevented almost completely the induction of beta-casein mRNA. Induced cells were rapidly deinduced by addition of the drug to the medium. A similar inhibition was observed when the induction was obtained with prolactin alone or with its two stimulators insulin and glucocorticoids. Tubulozole T, an isomer of tubulozole C which is known to be ineffective in disrupting microtubules, did not alter prolactin actions. These data and those obtained with other tubulin-binding drugs strongly suggest that the integrity of microtubules is required for prolactin to deliver its message to the mammary cell.
Animals↗
The effects of tubulazole, a new synthetic microtubule inhibitor on experimental neoplasms.
Tubulazole, a new synthetic microtubule inhibitor in vitro, is tested in vivo upon three experimental neoplasms: MO4 sarcoma, L1210 leukemia and TA3 carcinoma. The compound is tested using different treatment schedules upon different inoculation routes of the cells. All trials show the compound to have distinct antineoplastic properties in vivo by prolonging the median survival time. The best treatment schedule seems to be an intermittent one, i.e. treatment every fourth day starting 1 day after tumor inoculation. Comparison with cyclophosphamide and vincristine is in favor of tubulazole for treating TA3 mammacarcinoma, while cyclophosphamide and vincristine give somewhat better results upon L1210 leukemia. The effects of tubulazole and cyclophosphamide upon MO4 fibrosarcoma are comparable, while vincristine has no effect in this system. Worthwhile noting is that all the in vivo, as well as in vitro, activity of tubulazole resides in the cis isomer, while the trans isomer has no effect at all.
Animals↗
Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method.
Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.
Actinin↗
The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole.
Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MT) and intermediate filaments (IF) in MO cells treated with various combinations of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MT bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated cells a close association between these two filamentous systems is observed. In cells pretreated with nocodazole followed by addition of taxol, to induce the bundles of free MT, the preexisting IF coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MT determine the spatial distribution of IF which most probably involves some kind of physicochemical link.
Alkaloids↗
Microtubules and microfilaments in ageing hamster embryo fibroblasts in vitro.
Microtubules and microfilaments were investigated in hamster lung fibroblasts, during their in vitro life-span. These cells show a senescence process characterized by a drastic phenotypic change, resulting in two phenotypes: the type 1 cells, characteristic of young cultures and the type 2 cells appearing progressively with culture passages. Microtubules and microfilaments were observed at the TEM and also visualized by the unlabelled peroxidase-anti-peroxidase method. Moreover, the susceptibility of microtubules to nocodazole was tested in type 1 and 2 cells. We could not provide evidence for a different susceptibility to the drug. However the depolymerization wave occurred centripetally in type 1 cells whilst centrifugally in type 2 cells. These observations are discussed in relationship with the early arrest of division growth of the type 2 differentiated cells.
Animals↗
The activity of ketoconazole in mixed cultures of leukocytes and Candida albicans.
A system is described which allows the semi-quantitative investigation of the interaction between Candida albicans and leukocytes in culture with and without the addition of chemotherapeutic agents. Both polymorphonuclear leukocytes and macrophages avidly engulfed added yeast cells. However, they did not succeed in eradicating the fungus even when only 450 yeast cells were added to 3 X 10(6) leukocytes. This is probably due to several factors, including the decline in the functiontional capacity of the leukocytes with time in culture. The major way for the fungus to escape intracellular killing, however, seems to be the switch to the mycellial form in the presence of leukocytes. Engulfed yeasts produce germ tubes, grow out of the leukocytes and form hyphae which are much more resistant to the lytic action of the leukocytes. The leukocytes become necrotic through their interaction with the mycelia. Ketoconazole, a potent, orally active systemic antifungal agent inhibited the growth of C. albicans and completely suppressed the formation of mycelia in culture at very low concentrations (0.01 microgram ml-1). It was toxic to the leukocytes themselves only at 100 microgram ml-1. Addition of ketoconazole (10 (10-1.01 microgram ml-1) to mixed cultures of leukocytes and C. albicans allowed complete elimination of the fungus, probably because the leukocytes could easily remove the remaining yeast cells. The data show the usefulness of the system in the search for systemic antifungals and provide a possible explanation for the efficacy of ketoconazole in vivo.
Antifungal Agents↗
Methyl (5-(2-thienylcarbonyl)-1H-benzimidazole-2-yl) carbamate, (R17934), a synthetic microtubule inhibitor, prevents malignant invasion in vitro.
The effect of malignant invasion of methyl (5-(2-thienylcarbonyl)-1H-benzimidazole-2-yl) carbamate, (R17934), a microtubule inhibitor, was examined in organotypical co-cultures of mouse sarcoma virus transformed cells (MO4) and embryonic chick tissues. In contrast with control cultures, 1 microgram/ml R17934 prevented invasion of MO4 cells into the host tissue. Since mitostatic doses of 5-fluorouracil did not inhibit invasion, we presumed that the anti-invasive properties of R17934 were correlated with a loss of microtubule mediated directional cell migration.
Antineoplastic Agents↗
Quantitative determination of tubulin by radioimmunoassay.
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Animals↗
Immune responses in elderly cuti-negative subjects and the effect of levamisole.
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Aged↗