The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10(-8) M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10(-7) M oryzalin stopped migration abruptly. Oryzalin (1.0·10(-7) M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Paul's scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (K i ) of 2.59·10(6) M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [(14)C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (K app) of 1.19·10(5) M(-1). Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.

The oscillations of chromosomes associated with a single spindle pole in monocentric and bipolar spindles were analysed by time-lapse cinematography in mitosis of primary cultures of lung epithelium from the newt Taricha granulosa. Chromosomes oscillate toward and away from the pole in all stages of mitosis including anaphase . The duration, velocity, and amplitude of such oscillations are the same in all stages of mitosis. The movement away from the pole in monocentric spindle is rapid enough to suggest the existence of a previously unrecognized active component in chromosome movement, presumably resulting from a pushing action of the kinetochore fiber. During prometaphase oscillations, chromosomes may approach the pole even more closely than at the end of anaphase . Together, these observations demonstrate that a monopolar spindle is sufficient to generate the forces for chromosome transport, both toward and away from the pole .The coordination of the aster/centrosome migration in prophase with the development of the kinetochore fibers determines the course of mitosis . After the breaking of the nuclear envelope in normal mitosis, aster/centrosome separation is normally followed by the rapid formation of bipolar chromosomal fibers . There are two aberrant extremes that may result from a failure in coordination between these processes: (a) A monocentric spindle will arise when aster separation does not occur, and (b) an anaphaselike prometaphase will result if the aster/ centrosomal complexes are already well-separated and bipolar chromosomal fibers do not form . In the latter case, the two monopolar prometaphase half-spindles migrate apart, each containing a random number of two chromatid (metaphase) monopolar-oriented chromosomes. This random segregation of prometaphase chromosomes displays many features of a standard anaphase and may be followed by a false cleavage . The process of polar separation during prometaphase occurs without any visible interzonal structures . Aster/centrosomes and monopolar spindles migrate autonomously by an unknown mechanism. There are, however, firm but transitory connections between the aster center and the kinetochores as demonstrated by the occasional synchrony of centrosome-kinetochore movement . The data suggest that aster motility is important in the progress of both prometaphase and anaphase in normal mitosis.The bipolar mitotic apparatus normally provides the structural basis for equal chromosome segregation . The observations reported here demonstrate, however, that a bipolar spindle is not necessary either for chromosome-to-pole movement or for polar migration. Thus, a monopolar spindle or "half-spindle" appears to be the basic functional unit of mitosis .In a standard bipolar mitotic spindle, two essential steps assure precise segregation of the genetic material . In the first, double-chromatid chromosomes are bipolar oriented; in the second, they are aligned in an equatorial, metaphase position . Thus, the sister kinetochores come to point toward two opposite

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.

Abstract. The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizing organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self-reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubulerelated processes. BSERVATIONS of microtubule reorganization in a newexperimental system, anucleate cell fragments in Haemanthus endosperm (7), have provided evidence for a previously unknown property of higher plant microtubules (MTs) t in vivo: the tendency of an initially irregular MT meshwork to reorganize itself until a very stable structure is formed. Such reorganization may be an intrinsic property of higher plant MTs, as it has not been observed in animal tissue culture (28). This is a sequential reorganization that follows always the same steps and is triggered merely by removing endosperm from the ovule. The final result may be the formation of chromosome-free spindles/phragmoplasts, or spirals that may close and form tings. Further observations of MT reorganization have revealed the presence of unexpected MT configurations, appearing under specific conditions, whose morphological structure often resembles that of fir trees (35). These MT converging centers, with the shape of J Abbreviations used in this paper. DIC, differen...

Abstract. Metaphase and anaphase spindles in cultured newt and PtK~ cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation. THE UV microbeam offers a means by which structures containing microtubules (MTs) 1 can be experimentally manipulated by local disruption. This possibility exists because MTs are sensitive to irradiation of between 260-300 nM (20,52). The technique has been used mostly for studying spindle structure and function (for example, see references 2, 12, 18-23, 26, 27), particularly by Forer and his colleagues (for example, see references 4, 7-9, 40, 41); on occasion, it has been used to probe other MT-based motility systems (for example, see references 25, 28; see also Using our first UV microbeam apparatus and working with diatoms, our previous observations on spindle MT dynamics (26,27) were significantly different from those reported by Forer in crane fly spermatocytes (7,8; see Discussion). Specifically, Forer describes areas of reduced birefringence (ARBs) created by the irradiation, as moving polewards at metaphase and anaphase at about the rate of anaphase chromosome movement. This observation was widely interpreted as indicating the existence of a polewards flux of MT subunits in spindle fibers with the likelihood that the MTs were being assembled at the kinetochores during metaphase and disassembled at the poles. In our experiments on diatom central spindles, the two cut ends of MTs in the ARB behaved quite differently, with the polewards end of severed MTs disassembling polewards (increasing the size of...