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R Nuydens

Publications and source records attributed to R Nuydens.

At least 37 records · Page 2Linked to original sources
PubMed1990

Automatic quantification of fast axonal transport in neuronal cell cultures.

A method is presented which allows the automatic quantification of the fast axonal transport of endogenous organelles in neurites of cultured neuronal cells. Stretches of videotape recordings from Allen video enhanced contrast (AVEC) microscopy are digitized by currently available image processor hardware and analysed off-line on a MicroVAX II. Movements along the axon are calculated in great detail, allowing statistically significant changes to be detected. Interaction from the operator is minimised, thereby bypassing tedious manual analysis. This paper further reports the application of this system to the effect of vanadate treatment on axonal transport in cultures of rat embryonic hippocampal neurons.

Animals↗
PubMed1989

The effect of flunarizine on intracellular calcium in isolated rat cardiomyocytes. A digital image processing study.

The molecular mechanisms associated with the effects of various pathological stimuli on myocardial tissue, as well as the mechanisms by which Ca2+ antagonists exert their protective effect, are poorly understood. With the use of digital image processing of Fura-2 fluorescence, we have shown that the mean intracellular free Ca2+ concentration of single isolated rat cardiomyocytes is increased upon exposure to various pathological stimuli (high extracellular Ca2+, veratrine). This increased Ca2+ content coincided with an increased number of hypercontracted cells. Pretreatment with flunarizine under these experimental conditions lowered the free intracellular Ca2+ concentration, thereby reducing the number of hypercontracted cells. Verapamil had no effect. The kinetics of changes in intracellular Ca2+ in electrically paced cardiomyocytes were not affected by flunarizine, but were significantly altered by the beta agonist isoprenaline. In addition, isoprenaline increased the mean diastolic intracellular free Ca2+ concentration of paced cardiomyocytes, whereas it remained unchanged in flunarizine treated cells. We conclude that flunarizine reduces intracellular free Ca2+ levels in isolated cardiomyocytes under pathological conditions, but does not affect physiological processes mediated by Ca2+. The report also illustrates the possibilities of digital imaging microscopy in the study of ion distributions in living cells.

Animals↗
PubMed1988

Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy.

Transferrin receptors labeled with the B3/25 monoclonal antibody-gold complexes were followed in living A431 cells by using video-enhanced contrast microscopy. Initially, the antibody-gold complexes bind to receptors which are freely mobile on the upper cell surface; they then become trapped at the inner margins of the peripheral lamellae and internalize. During endocytosis discrete gold-loaded vesicular elements first appear, and then, as they fuse, a heterogenous peripheral endosomal compartment forms. The endosomes from this compartment then begin to migrate centripetally through the cytoplasm in a saltatory way so that within 15 min gold label accumulates in a juxtanuclear endosome compartment. This compartment, which consists mainly of multivesicular bodies, is thus formed by the influx and retention of peripheral endosomal elements and their continued fusion in the juxtanuclear area. Although their overall migration is inward, saltating endosomes frequently reverse their direction of movement. As label builds up in the juxtanuclear area, small vesicles containing gold label continuously pinch off from the larger elements and migrate toward the cell periphery. Experiments with nocodazole and sodium azide show that the saltatory movements, the accumulation and retention of endosomes in the juxtanuclear area, and the separation of vesicles from endosomes are driven by a microtubule-associated, ATP-dependent, motility-generating mechanism. Analysis of the movements shows that although each individual vesicle saltation can occur unpredictably toward the centre or the periphery of the cell, a net centripetal flux is observed. Moreover, it is evident that the probability of migration toward and maintenance in the juxtanuclear area is related to the diameter of the vesicles. We propose a mechanism by which bidirectional saltation along microtubules forming a radial network may be instrumental in the selective concentration of large endosomes in the juxtanuclear area while small vesicles are left free to return to the periphery. This process may be responsible for the sorting of receptors and ligands destined either for intracellular degradation in juxtanuclear lysosomes or, alternatively, for recycling to the plasma membrane.

Alkaloids↗
PubMed1987

Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy.

We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection.

Animals↗
PubMed1986

The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell.

We describe a new approach to probe the molecular biology of the living cell that uses small colloidal gold particles coupled to specific ligands. They are visualized in cells by bright-field, video enhanced contrast microscopy. We describe the basic aspects of the technique and provide examples of applications to intracellular motility, cell membrane dynamics, receptor translocation, internalization, and intracellular routing. We also provide examples of the use of this approach in immunospecific labelling of cells and tissue sections.

Animals↗
PubMed1986

Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells.

Immunofluorescence with specific peptide antibodies has previously established that tyrosinated (Tyr) and detyrosinated (Glu) tubulin, the two species generated by posttranslational modification of the COOH-terminus of alpha-tubulin, are present in distinct, but overlapping, subsets of microtubules in cultured cells (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789). Similar results were observed by light microscopic immunogold staining in the two cell types used in this study, CV1 and PtK2 cells: most microtubules were stained with the Tyr antibody, whereas only a few were stained with the Glu antibody. We have examined immunogold-stained preparations by electron microscopy to extend these results. In general, electron microscopic localization confirmed results obtained at the light microscopic level: the majority of the microtubules in CV1 and PtK2 cells were nearly continuously labeled with the Tyr antibody, whereas only a few were heavily labeled with the Glu antibody. However, in contrast to the light microscopic staining, we found that all microtubules of interphase and mitotic CV1 and PtK2 cells contained detectable Tyr and Glu immunoreactivity at the electron microscopic level. No specific localization of either species was observed in microtubules near particular organelles (e.g., mitochondria or intermediate filaments). Quantification of the relative levels of Glu and Tyr immunoreactivity in individual interphase and metaphase microtubules showed that all classes of spindle microtubules (i.e., kinetochore, polar, and astral) contained nearly the same level of Glu immunoreactivity; this level of Glu immunoreactivity was lower than that found in all interphase microtubules. Most interphase microtubules had low levels of Glu immunoreactivity, whereas a few had relatively high levels; the latter corresponded to morphologically sinuous microtubules. Quantification of the relative levels of Tyr and Glu immunoreactivity in segments along individual microtubules suggested that the level of Tyr (or Glu) tubulin in a given microtubule was uniform along its length. Understanding how microtubules with different levels of Tyr and Glu tubulin arise will be important for understanding the role of tyrosination/detyrosination in microtubule function. Additionally, the coexistence of microtubules with different levels of the two species may have important implications for microtubule dynamics in vivo.

Animals↗
PubMed1985

Probing microtubule-dependent intracellular motility with nanometre particle video ultramicroscopy (nanovid ultramicroscopy).

Colloidal gold particles of 20 to 40 nm diameter stabilized with polyethylene glycol (PEG) were microinjected in PTK2 cells. Aggregates and individual particles, which are smaller than the theoretical limit of resolution of the optical microscope and invisible to the eye are discernible from organelles by reflection of polarized light. They are optimally visualized using transmitted light and electronic subtraction of diffuse background light. The gold particles show saltatory motion. The direction, speed, median distance travelled and frequency of saltations are indiscernible from measurements made on cell organelles in the same preparations. Because microtubule treadmilling has been implicated as a potential motor for organelle motility, gold particles coupled to monoclonal antibodies, recognizing the alpha-subunit of tubulin (Kilmartin et al., 1982), were injected. These particles, often forming linear arrays, assumed entirely fixed positions in the cell. The results suggest that there is a transport system associated with microtubules which can carry synthetic particles through the cell without the need for them being covered with specific proteins. Microtubule treadmilling does not seem to be involved. The possibility of following 20-40 nm particles and probably even smaller ones, that can be coupled to most proteins, within living cells provides a tool of wide applicability to study the fate and behaviour of such proteins. It is suggested that this new method be called nanoparticle video ultramicroscopy or nanovid ultramicroscopy.

Animals↗
PubMed1983

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↗
PubMed1981

Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores.

Taxol, a potent promoter of microtubule polymerization in vitro, induces massive assembly of free microtubules in cultured cells as visualized by immunocytochemistry and electron microscopy. The centrosomes and kinetochores largely lost their capacity to organize microtubule assembly, as became evident by the disappearance of the cytoplasmic microtubule complex and the mitotic spindle. The taxol-induced microtubules were partially resistant to nocodazole, an inhibitor of tubulin polymerization. Moreover, taxol induced microtubule assembly in cells pretreated with nocodazole. Increasing the ratio of nocodazole to taxol restored the ability of the centrosomes and kinetochores to specifically induce microtubule assembly in their immediate vicinity. The data suggest that taxol lowers the critical tubulin concentration in vivo as well as in vitro and that the organizing capacity of the microtubule-organizing centers depends on the cytoplasmic polymerization threshold.

Alkaloids↗
PubMedDate not supplied

Detection of gold probes with video-enhanced contrast microscopy: nanovid microscopy.

This contribution describes a new microscopic technique aimed at visualizing colloidal gold particles of 20-40 nm diameter as dynamic markers at the light microscopic level. The technique, called nanovid microscopy, is based on the use of contrast enhancement by video techniques and digital image processing. Two applications on living cells are discussed. The first is the receptor-mediated endocytosis of the transferrin receptor, which for the first time can be followed in real time. A second application documents the motion of cell-surface glycoproteins on PTK2-cells. In addition, nanovid microscopy allows the number of gold particles in immunogold staining to be quantified easily. With this tool, the translocation of specific molecules in living cells becomes one of the most enjoyable activities to observe.

Biological Transport↗