Microtubule dynamics in the spindle. II. A thermodynamic and kinetic description.

We have previously presented a model for the assembly and disassembly of mitotic spindle microtubules (MTs) (Pickett-Heaps et al., 1986). In this paper, we describe the thermodynamics of such spindle MT assembly and present equations to describe the polymerization kinetics of different classes of spindle MTs. These equations are used to predict, in terms of kinetics parameters, the magnitude of forces extant on spindle MTs and to define the critical force needed to halt MT assembly. We calculate several of these forces for a hypothetical model cell; our predicted value for the force generated along kinetochore fibers is in close agreement with measured values taken from living cells. The model and its implications are discussed with reference to other recent models of spindle and MT dynamics.

Microtubule dynamics in the spindle. Theoretical aspects of assembly/disassembly reactions in vivo.

The mitotic spindle contains several classes of microtubules (MTs) whose lengths change independently during mitosis. Precise control over MT polymerization and depolymerization during spindle formation, anaphase chromosome movements, and spindle breakdown is necessary for successful cell division. This model proposes the site of addition and removal of MT subunits in each of four classes of spindle MTs at different stages of mitosis, and suggests how this addition and removal is controlled. We propose that spindle poles and kinetochores significantly alter the assembly-disassembly kinetics of associated MT ends. Control of MT length is further modulated by localized forces affecting assembly and disassembly kinetics of individual sets of MTs.

The organization of microtubules during anaphase and telophase spindle elongation in the rust fungus Puccinia.

The entire framework of microtubules (MTs) in the meiotic spindle of the rust fungus Puccinia has been reconstructed during the later stages of meiosis I, by tracking MTs through transverse serial sections. This spindle is of special interest because it elongates considerably during anaphase spindle elongation, from 5 microns at metaphase to 15 microns at telophase. The spindle is composed mainly of MTs from opposite poles which interdigitate or overlap in the middle of the spindle. In the overlap region, MTs from one pole seek out as near neighbors, MTs from the opposite pole at a preferred spacing of 43 to 55 nm. During anaphase elongation three changes in spindle structure occur: 1) the region of overlap decreases, but this reduction in overlap cannot account for all the increase in spindle length; 2) interdigitated MTs (MTs from one pole that are within 80 nm of a MT from the opposite pole) dramatically increase in length by MT polymerization and; 3) kinetochore MTs, free MTs (those unattached to the poles) and non-interdigitated polar MTs shorten and disappear. The mechanism of anaphase elongation and the control over MT polymerization and depolymerization during anaphase are discussed.

Near-neighbor analysis of spindle microtubules in the alga Ochromonas.

The near-neighbor spacing of microtubules (MTs) in the spindle of the alga Ochromonas is analyzed. The technique of near-neighbor analysis of MTs (as developed by McDonald et al. [9]) in the mid-region of the Ochromonas spindle (overlap) shows that MTs from one pole preferentially associate with MTs from the opposite pole at a center-to-center distance of 35 to 43 nm. However, in the half spindle between the chromosomes and the poles, kinetochore MTs (kMTs) do not preferentially associate with other MTs in the half spindle but instead are arranged essentially at random. Individual polar MTs (MTs attached to one pole), kMTs and free MTs (MTs unattached to the poles) were selected for near-neighbor analysis over their entire lengths. The spacing of MTs in the overlap is compatible with those models for mitosis which propose that separation of the poles is accomplished by sliding between closely spaced MTs of opposite polarity. In contrast to the overlap, the arrangement of MTs in the half spindle is not compatible with MT2MT sliding theories that propose that chromosome movement is accomplished by sliding between kMTs and polar MTs.

Organization of spindle microtubules in Ochromonas danica.

The entire framework of microtubules (MTs) in the mitotic apparatus of Ochromonas danica is reconstructed (except at the spindle poles) from transverse serial sections. Eleven spindles were sectioned and used for numerical data, but only four were reconstructed: a metaphase, an early anaphase, a late anaphase, and telophase. Four major classes of MTs are observed: (a) free MTs (MTs not attached to either pole); (b) interdigitated MTs (MTs attached to one pole which laterally associate with MTs from the opposite pole); (c) polar MTs (MTs attached to one pole); (d) kinetochore MTs (kMTs). Pole-to-pole MTs are rare and may be caused by tracking errors. During anaphase, the kMTs, free MTs, and polar MTs shorten until most disappear, while interdigitated MTs lengthen. In the four reconstructed spindles, the number of MTs decreases between early anaphase and telophase from 881 to 285, while their average length increases from 1.66 to 4.98 micron. The total length of all the MTs in the spindle (placed end to end) remains at 1.42 +/- 0.04 mm between these stages. At late anaphase and telophase the spindle is comprised mainly of groups of interdigitated MTs. Such MTs from opposite poles form a region of overlap in the middle of the spindle. During spindle elongation (separation of the poles), the length of the overlap region does not decrease. These results are compatible with theories that suggest that MTs directly provide the force that elongates the spindle, either by MT polymerization alone or by MT sliding with concomitant MT polymerization.

Cell division in two large pennate diatoms Hantzschia and Nitzschia III. A new proposal for kinetochore function during prometaphase.

Prometaphase in two large species of diatoms is examined, using the following techniques: (a) time-lapse cinematography of chromosome movements in vivo; (b) electron microscopy of corresponding stages: (c) reconstruction of the microtubules (MTs) in the kinetochore fiber of chromosomes attached to the spindle. In vivo, the chromosomes independently commence oscillations back and forth to one pole. The kinetochore is usually at the leading edge of such chromosome movements; a variable time later both kinetochores undergo such oscillations but toward opposite poles and soon stretch poleward to establish stable bipolar attachment. Electron microscopy of early prometaphase shows that the kinetochores usually laterally associate with MTs that have one end attached to the spindle pole. At late prometaphase, most chromosomes are fully attached to the spindle, but the kinetochores on unattached chromosomes are bare of MTs. Reconstruction of the kinetochore fiber demonstrates that most of its MTs (96%) extend past the kinetochore and are thus apparently not nucleated there. At least one MT terminates at each kinetochore analyzed. Our interpretation is that the conventional view of kinetochore function cannot apply to diatoms. The kinetochore fiber in diatoms appears to be primarily composed of MTs from the poles, in contrast to the conventional view that many MTs of the kinetochore fiber are nucleated by the kinetochore. Similarly, chromosomes appear to initially orient their kinetochores to opposite poles by moving along MTs attached to the poles, instead of orientation effected by kinetochore MTs laterally associating with other MTs in the spindle. The function of the kinetochore in diatoms and other cell types is discussed.

Light and electron microscopic observations on cell division in two large pennate diatoms, Hantzschia and Nitzschia. I. Mitosis in vivo.

Mitosis and cytokinesis have been followed in live cells using Nomarski and birefringence optics. Prophase is protracted. The small square or rectangular spindle is brillantly birefringent and situated close to one side of the cell. It grows very slowly until suddenly it begins to elongate rapidly, doubling or trebling its length in a few minutes. This prometaphase stage is immediately accompanied by very active, oscillatory movements of the previously quiescent chromosomes, along invisible tracks directed at either pole. Independently, each pair of chromatids soon attaches to the other pole as well; immediately, its oscillations cease, and it becomes stretched across the central spindle. Some chromosomes attach almost as soon as the spindle enters the nucleus, others much later. The overlap in the central spindle becomes discernable during mid prometaphase; it stays roughly the same length while the total length of the spindle increases to a temporarily stable maximum at metaphase which is quiescent except for late chromosome attachments and which lasts around 10 mins. Then suddenly and synchronously, the chromatids split and immediately move polewards as if tension has been released in the. About a minute later, the spindle recommences elongation, but now the overlap diminishes in step with elongation. At this stage, Nitzschia and Hantzschia differ markedly in behavior. In Hantzschia, like other diatoms, the half spindles become coarsely striated near the poles, and the elongated central spindle stays intact after reaching its maximum length, until broken by the cleavage furrow, whereupon the broken halves slowly disassemble. In contrast, the half spindles in Nitzschia never display such striations, and after maximum elongation, the central spindle rapidly breaks down entirely, before cleavage is complete. The cleavage furrow grows inward very slowly during metaphase. Its ingrowth is stimulated during late anaphase, and it moves inwards at about 20 micrometer/min. Most of the cleavage is accomplished in about 4 mins. The chloroplasts are pulled inwards and finally pinched in two by the furrow. These events are discussed with emphasis on the dynamics and mechanics of spindle assembly, elongation and disassembly.

Light and electron microscopic observations on cell division in two large pennate diatoms. Hantzschia and Nitzschia. II. Ultrastructure.

Cells, preselected to cover all stages of mitosis, were sectioned accurately for investigating changes in spindle structure that accompany mitosis. During spindle formation, the interphase Microtubule Center (MC) breaks down. Numerous tiny foci, each apparently nucleating one microtubule (MT) and derived from the MC, line up along the two polar plates; lateral interaction between these two sets of (oppositely polarized--?) MTs is presumed to generate the MT packing arrangement characteristic of the diatom spindle's overlap. Later, when the elongating central spindle enters the nucleus at prometaphase, the MTs from each polar plate have either interacted thus to generate the central spindle proper, or else they radiate into the nucleus. This latter population of MTs interacts with the kinetochores and most become thereby organized into kinetochore fibres. The zone of overlap quickly develops ragged edges, suggesting that it is labile (i.e., by irregular sliding and/or growth of MTs) even at early prometaphase. Metaphase spindle structure is as expected from light microscopy. The collar material is difficult to discern, but it apparently permeates the kinetochore fibres. During anaphase, the overlap diminishes and disappears as the spindle elongates. The chromosomes always move past the ends of the spindle, a movement accomplished without any apparent involvement of MTs. In N. sigmoidea, the spindle invariably breaks down upon completion of elongation, and the scattered remnants of its MTs soon disappear. In contrast, the central spindle of H. amphioxys persists until it is broken by the cleavage furrow; the MTs in the half spindle away from the overlap always exhibit pronounced clumping. These observations are integrated with extensive observations on mitosis in vivo, with a view to understanding the mechanisms of spindle formation, function and disassembly.

Analysis of the distribution of spindle microtubules in the diatom Fragilaria.

The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.

On the mechanism of anaphase spindle elongation in Diatoma vulgare.

Central spindles from five dividing cells (one metaphase, three anaphase, and one telophase) of Diatoma vulgare were reconstructed from serial sections. Each spindle is made up of two half-spindles that are composed almost entirely of polar microtubules. A small percentage of continuous microtubules and free microtubules were present in every stage except telophase. The half-spindles interdigitate at the midregion of the central spindle, forming a zone of overlap where the microtubules from one pole intermingle with those of the other. At metaphase the overlap zone is fairly extensive, but as elongation proceeds, the spindle poles move apart and the length of the overlap decreases because fewer microtubules are sufficiently long to reach from the pole to the zone of interdigitation. At telophase, only a few tubules are long enough to overlap at the midregion. Concurrent with the decrease in the length of the overlap zone is an increase in the staining density of the intermicrotubule matrix at the same region. These changes in morphology can most easily be explained by assuming zone mechanochemical interaction between microtubules in the overlap zone which results in a sliding apart of the two half-spindles.

Apparent amitosis in the binucleate dinoflagellate Peridinium balticum.

Mitosis and cytokinesis in the free-living binucleate dinoflagellate Peridinium balticum are described, P. balticum contains 2 nuclei; one is a typical dinoflagellate nucleus and the other resembles the interphase nuclei of some eucaryotic cells and is here named the supernumerary nucleus (formerly called the eucaryotic nucleus). The dinoflagellate nucleus divides in the characteristic manner already described for certain other dinoflagellates. The supernumerary nucleus does not undergo normal mitosis; its chromatin does not condense, a spindle is not differentiated for its division, nor are any microtubules present inside the nucleus during any stage of its division. Instead the supernumerary nucleus divides by simple cleavage, which is concurrent with cytoplasmic cleavage. The nucleus cleaves first on its side facing the wall, but later it cleaves circumferentially as the cytoplasmic cleavage furrow draws closer. Invariably at late cytokinesis, a portion of the dividing nucleus passes through the only remaining uncleaved area of the cell. The final separation of the supernumerary nucleus is probably accomplished by the ingrowing furrow pinching the nucleus in two. There is no apparent precise segregation of genetic material during division, nor are there any structural changes inside the dividing nucleus which distinguish it from the interphase nucleus. Certain aspects of amitosis, and previously postulated theories concerning the endosymbiont origin of the second nucleus, are discussed.