Taxol, a potent promoter of microtubule polymerization in vitro, induces massive assembly offree 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 ofthe 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 ofnocodazole to taxol restored the ability of the centrosomes and kdnetochores 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.Taxol, an experimental antitumor drug (1) isolated from Taxus brevifolia, was recently shown by Schiff et aL (2, 3) to affect microtubule assembly in vitro and in living cells. It essentially eliminated the initial lag phase and decreased the critical tubulin concentration to less than 0.01 mg/ml. The rate and extent of the polymerization was increased and the microtubules were relatively resistant to depolymerization by cold and CaCl2. In living cells (3), taxol was shown to be a potent inhibitor of HeLa and mouse. fibroblast replication. The cells were blocked in the G2 and M phases of the cell cycle. It was inferred that this was due to the stabilization of cytoplasmic microtubules. Indirect immunofluorescence was used to show that taxoltreated cells displayed bundles of microtubules radiating from a common site in addition to their cytoplasmic microtubules. These microtubules were resistant to treatment with steganacin or incubation of the cells at low temperature, both of which disintegrated microtubules in control cells. Ultrastructural observations showed that the mitotic cells contained microtubule bundles but no normal spindle. It was concluded that the inability of the cells to form a mitotic spindle in the presence of taxol could be due to the fact that the cells were unable to depolymerize their microtubule cytoskeletons (3).We have investigated the effects of taxol on the cytoplasmic microtubule complex and the mitotic spindle in cultured cells. Our observations and conclusions differ partly from those published previously (3). Taxol apparently induces the assembly of free microtubules in the cytoplasm, not attached to the centrosomes or kinetochores. The preexisting microtubules, attached to the organizing centers, are not stabilized and disappear gradually.The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. MATERIAL AND METHODSPt K2 potoroo cells, C3H mouse 3T3 cells, and human...

No abstract

Abstract. Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment l, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction.The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to a-actinin: a-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with a-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to a-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

A procedure is presented for the immunocytochemical visualization of microtubules in interphase and mitotic cells of Haemanthus endosperm. It includes preservation of microtubules (MTs) with glutaraldehyde and uses colloidal gold, coated with secondary antibodies, in a novel indirect-light microscopic technique: the immuno-gold staining method. This immunocytochemical stain allows us to follow the changes in distribution of MTs during mitosis with greater precision and specificity than allowed by other light microscopic techniques. Many aspects of MT arrangements, as reported from ultrastructural studies, are corroborated and extended. This demonstrates the reliability of the technique. In addition, a number of significant observations were made. These concern (i) the presence of a network of MTs in interphase cells, (ii) the transformation of this network into a spindle-like cage of MTs (the clear zone) surrounding the nucleus during prophase, (iii) the drastic rearrangement of MT distribution during prometaphase, (iv) new evidence for the formation of aster-like arrays of polar MTs during anaphase, and (v) the development of the phragmoplast.

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.