Central Spindle Microtubules Research Articles

Physiological and ultrastructural analysis of elongating mitotic spindles reactivated in vitro.

We have developed a simple procedure for isolating mitotic spindles from the diatom Stephanopyxis turris and have shown that they undergo anaphase spindle elongation in vitro upon addition of ATP. The isolated central spindle is a barrel-shaped structure with a prominent zone of microtubule overlap. After ATP addition greater than 75% of the spindle population undergoes distinct structural rearrangements: the spindles on average are longer and the two half-spindles are separated by a distinct gap traversed by only a small number of microtubules, the phase-dense material in the overlap zone is gone, and the peripheral microtubule arrays have depolymerized. At the ultrastructural level, we examined serial cross-sections of spindles after 1-, 5-, and 10-min incubations in reactivation medium. Microtubule depolymerization distal to the poles is confirmed by the increased number of incomplete, i.e., c-microtubule profiles specifically located in the region of overlap. After 10 min we see areas of reduced microtubule number which correspond to the gaps seen in the light microscope and an overall reduction in the number of half-spindle microtubules to about one-third the original number. The changes in spindle structure are highly specific for ATP, are dose-dependent, and do not occur with nonhydrolyzable nucleotide analogues. Spindle elongation and gap formation are blocked by 10 microM vanadate, equimolar mixtures of ATP and AMPPNP, and by sulfhydryl reagents. This process is not affected by nocodazole, erythro-9-[3-(2-hydroxynonyl)]adenine, cytochalasin D, and phalloidin. In the presence of taxol, the extent of spindle elongation is increased; however, distinct gaps still form between the two half-spindles. These results show that the response of isolated spindles to ATP is a complex process consisting of several discrete steps including initiation events, spindle elongation mechanochemistry, controlled central spindle microtubule plus-end depolymerization, and loss of peripheral microtubules. They also show that the microtubule overlap zone is an important site of ATP action and suggest that spindle elongation in vitro is best explained by a mechanism of microtubule-microtubule sliding. Spindle elongation in vitro cannot be accounted for by cytoplasmic forces pulling on the poles or by microtubule polymerization.

Comparison Of Spindle Elongation In Vivo And In Vitro In Stephanopyxis Turris

The spindle in dividing cells of the diatom Stephanopyxis turris contains three distinct classes of microtubules: central spindle microtubules, which slide over each other and grow during anaphase spindle elongation; kinetochore-attached microtubules, which are located on the outer surface of the central spindle; and peripheral microtubules, which fan out from the spindle poles in astral-like arrays. The poles are multilayered structures, which remain attached to the spindle after isolation. In vitro, after addition of ATP, central spindles elongate and the two half-spindles slide completely apart with a concurrent decrease in the extent and magnitude of the zone of microtubule overlap. Spindle elongation takes place in spindles whose chromatin has been removed by enzymic digestion and the extent of elongation in vitro is increased by the addition of neurotubulin. After ATP addition the arrays of interdigitating microtubules in the zone of overlap become disordered and selectively depolymerize from the overlap zone polewards. In some reactivated spindles an unusual structure, a striated fibre, can be seen running from the pole plates part of the way towards the spindle midzone. The fibre has no precedent in mitotic ultrastructure and its function is unclear. These results demonstrate that we can duplicate the essential elements of anaphase B in vitro and that this system will be useful for further studies of the molecular basis of spindle elongation.

Spindle microtubule dynamics following ultraviolet-microbeam irradiations of mitotic diatoms

Lesions (“ARBs”) generated in metaphase and anaphase central spindles of Hantzschia by an ultraviolet microbeam are devoid of microtubules previously present. In vivo, the poleward transverse edge of the lesion invariably loses birefringence poleward, until this segment has vanished; the loss is slow during metaphase and faster at anaphase. The other transverse edge, proximal to the overlap, remains stable until disassembly of the whole spindle. We conclude that the central spindle microtubules are not in flux during metaphase to telophase, and that depolymerization of these microtubules takes place only from the end distal to the pole, as during normal spindle disassembly. Microtubule polarity and the creation of free ends may determine which microtubules are disassembled during later mitosis and how disassembly proceeds.

Mitosis in the cellular slime mold Polysphondylium violaceum.

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re-establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.

Meiosis in Coprinus lagopus: A comparative study with light and electron microscopy

ABSTRACT Meiosis within fruiting bodies of Coprinus lagopus Fr. is closely synchronized. This conveniently facilitates joint light- and electron-microscope observations. Before nuclear fusion the chromatin appears diffuse in the light microscope; after nuclear fusion individual chromosomes can be recognized. In the electron micrographs the chromatin of pre-fusion and early fusion nuclei cannot be recognized as denned structures with the fixation and staining procedures employed. At the time of synapsis the lateral components of the synaptinemal complexes can be seen in the micrographs. The pairing process of the two chromosomes of the homologous pairs is believed to involve two steps: (1) two homologous chromosomes become aligned in parallel, and (2) pairing occurs by formation of the synaptinemal complex including the central synaptic component. The term synaptic centre is coined for the central component, which is believed to be the zone where crossing-over occurs. The formation of this structure in relation to homologous pairing, and the structural organization of the synaptinemal complexes are discussed. At meiotic metaphase, the chromosomes congregate around the central spindle microtubules. They are contracted and contain densely packed chromatin fibrils. Two types of spindle microtubules are demonstrated: (1) the chromosomal microtubules directly connecting the chromosomes to the centrosomes, and (2) the central spindle microtubules connecting the two centrosomes. The centrosomes are round, fibril-containing bodies approximately 0-3 fi in diameter. They have been observed outside the nuclear envelope at pachytene, but do not show the characteristic structure normally found in animal cells.