Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm

Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time- dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly.     

Dynamic changes in autosomal spindle fibers during prometaphase in crane fly spermatocytes

Different prometaphase stages of Pales ferruginea spermatocytes were serially sectioned and the regions between kinetochores and poles analysed by counting and measuring spindle microtubules. These regions are characterized by an intermingling of kinetochoric (kMTs) and non-kinetochoric microtubules (nkMTs). A considerable proportion of nkMTs is skewed with respect to kMTs, thus being responsible for microtubule disorder in these spindle areas. The degree of disorder expressed by the percentage of skew microtubules was found to decrease from early prometaphase to metaphase, parallel with an increase in kMT number. A possible causal relation between pulling forces and morphological changes in the spindle is discussed.     

The arrangement of microtubules and the attachment of chromosomes to the spindle during anaphase in tipulid spermatocytes

Analysis of serial sections oriented parallel to the interpolar spindle axis revealed the following results. Autosomes in anaphase of the 1. meiotic division of Pales ferruginea spermatocytes are attached to the spindle in two ways: 1. The short kinetochoric microtubules (kMTs) diverge and interdigitate with the axial mass of non-kinetochoric microtubules (nkMTs). 2. The chromosome surface shows projections which protrude between the mass of nkMTs. — At the level of anaphase plates the concentration of nkMTs is higher than in the interzone. — The lagging sex chromosomes at the equator become stretched by anaphase forces during autosomal movement. — The mean length of nkMTs in metaphase is 3.0±0.1 μm, in anaphase 2.6±0.1 μm, possibly indicating an overall MT shortening in anaphase. Spindle architecture and aspects of anaphase forces are discussed.     

Kinetochore microtubules in crane-fly spermatocytes: untreated, 2 degrees C-treated, and 6 degrees C-grown spindles

We investigated the involvement of kinetochore microtubules (kMTs) in mediating chromosome-to-pole connections in crane-fly (Nephrotoma suturalis and Nephrotoma ferruginea) spermatocytes. Two experimental treatments were used to yield spindles with reduced numbers of nonkinetochore microtubules (nkMTs). Short-term (10-15 min) exposure of spermatocytes to 2 degrees C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long-term (24h) exposure to 2 degrees C followed by recovery at 6 degrees C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2 degrees C-treated spindle but fewer than an untreated spindle, in which the kMT:(kMT + nkMT) ratio was 0.27. The numbers and lengths of kMTs in 6 degrees C-grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6 degrees C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (greater than 3 microns), and many extended to the polar regions of the spindle. Thus, the crane-fly spindle appears not to be as atypical as it was previously suggested to be.     

The three-dimensional architecture of chromosome fibres in the crane fly

Microtubules of two chromosome fibres in metaphase and two in anaphase of the first meiotic division of Pales ferruginea spermatocytes were traced in serial sections. The number and lengths of microtubules constituting the fibres were determined and the fibres reconstructed. The chromosome fibres were found to contain only about 20% (in metaphase) and 9% (in anaphase) kinetochore microtubules (kMT), taking the sum of all individual microtubule lengths as the measure. The remainder consisted of free microtubules (fMT), a certain percentage of which were oriented obliquely with respect to the kMTs (skew fMTs). Between metaphase and anaphase most kMTs and fMTs shortened, causing a decrease in mean length of about onehalf in the case of kMTs. The proportion of skew fMTs increased considerably in anaphase, leading to more disorder in the fibre. The absolute mass of microtubules in the fibre seems to be irrelevant for spindle function. This can be deduced from the observed variability of absolute mass. A statistical correlation was found between the total mass of microtubules (kMTs+fMTs) and the number of kMTs, suggesting that some functional interrelation exists between these parameters.Dedicated to Prof. Hans Bauer on the occasion of his 80th birthday     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.     

The spatial distribution of spindle microtubules in anaphase 3Y1 cells

Summary— The spatial distribution of the microtubules (MT) in the rat 3Y1 cells in mitosis was investigated by immunoelectron microscopy and computer-graphic reconstruction of serial thin sections. In anaphase the interzone-MT increased in number gradually with advancing phase, while the kinetochore-MT in half-spindles decreased. The interzone-MT overlapped with each other at the equatorial region of the cell, and they formed a specific structure called the ‘stem bodies’. The ends of the interzone-MT opposite to the stem bodies often attached to chromosomes but not to the poles. The stem bodies were not labeled with immunogold particles of anti-α tubulin. Some of the stem bodies or MT which originate from stem bodies were found just beneath the plasma membrane in the equatorial region where abundant actin filaments appear showing the formation of the contractile ring and subsequently the cleavage furrow begins. On the basis of these observations it is assumed that the interzone-MT is involved both in the separation of chromosomes in anaphase and in the formation of the cleavage furrow in telophase.     

Selective reduction of anaphase B in quinacrine-treated PtK1 cells

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non-kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10-19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 microM to determine energy requirements for chromosome motion. The rate and extent of chromosome-to-pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore-kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore-kinetochore separation by 20%, and addition of 12 microM quinacrine reduced the kinetochore-kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongation, quinacrine-treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373-379, 1985). Metaphase cells treated with 2-10 microM concentrations of quinacrine for 2-5 min reduced spindle lengths by 10-50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose-induced elongation was concomitantly diminished. These data suggest that quinacrine-sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose-induced metaphase spindle elongation.     

The kinetochore fiber structure in the acentric spindles of the green algaOedogonium

Summary The microtubule (MT) arrangement in three kinetochore fibers in the acentric spindles of the green algaOedogonium cardiacum were reconstructed from serial sections of prometaphase and metaphase cells. The majority of the MTs attached to the kinetochore (kMTs) are relatively short, extending less than a third of the distance to the putative spindle pole region, and none extended the full distance. Fine filaments and a matrix described earlier (Schibler andPickett-Heaps 1980) were associated with the MTs all along the fibers. Live cells ofOedogonium were also studied by time lapse cinematography for correlation with the ultrastructural observations. Late prometaphase and metaphase kinetochore fibers appear to move independently as if unattached at their poleward ends. These observations suggest that kinetochore fibers inOedogonium are not attached to a specific pole structure from late prometaphase until the inception of anaphase. The results are discussed with reference to spindle structure and function in general.     

Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation

Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.     

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.     

Anaphase B precedes anaphase A in the mouse egg

Segregation of chromosomes at the time of cell division is achieved by the microtubules and associated molecules of the spindle. Chromosomes attach to kinetochore microtubules (kMTs), which extend from the spindle pole region to kinetochores assembled upon centromeric DNA. In most animal cells studied, chromosome segregation occurs as a result of kMT shortening, which causes chromosomes to move toward the spindle poles (anaphase A). Anaphase A is typically followed by a spindle elongation that further separates the chromosomes (anaphase B). The experiments presented here provide the first detailed analysis of anaphase in a live vertebrate oocyte and show that chromosome segregation is initially driven by a significant spindle elongation (anaphase B), which is followed by a shortening of kMTs to fully segregate the chromosomes (anaphase A). Loss of tension across kMTs at anaphase onset produces a force imbalance, allowing the bipolar motor kinesin-5 to drive early anaphase B spindle elongation and chromosome segregation. Early anaphase B spindle elongation determines the extent of chromosome segregation and the size of the resulting cells. The vertebrate egg therefore employs a novel mode of anaphase wherein spindle elongation caused by loss of k-fiber tension is harnessed to kick-start chromosome segregation prior to anaphase A.     

The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work.

During metaphase and anaphase in newt lung cells, tubulin subunits within the kinetochore microtubule (kMT) lattice flux slowly poleward as kMTs depolymerize at their minus-ends within in the pole. Very little is known about how and where the force that moves the tubulin subunits poleward is generated and what function it serves during mitosis. We found that treatment with the drug taxol (10 microM) caused separated centrosomes in metaphase newt lung cells to move toward one another with an average velocity of 0.89 microns/min, until the interpolar distance was reduced by 22-62%. This taxol-induced spindle shortening occurred as kMTs between the chromosomes and the poles shortened. Photoactivation of fluorescent marks on kMTs revealed that taxol inhibited kinetochore microtubule assembly/disassembly at kinetochores, whereas minus-end MT disassembly continued at a rate typical of poleward flux in untreated metaphase cells. This poleward flux was strong enough to stretch the centromeric chromatin between sister kinetochores as much as it is stretched in control metaphase cells. In anaphase, taxol blocked kMT disassembly/assembly at the kinetochore whereas minus-end disassembly continued at a rate similar to flux in control cells (approximately 0.2 microns/min). These results reveal that the mechanism for kMT poleward flux 1) is not dependent on kMT plus-end dynamics and 2) produces pulling forces capable of generating tension across the centromeres of bioriented chromosomes.     

Interpolar spindle microtubules in PTK cells

Spindle microtubules (MTs) in PtK1 cells, fixed at stages from metaphase to telophase, have been reconstructed using serial sections, electron microscopy, and computer image processing. We have studied the class of MTs that form an interdigitating system connecting the two spindle poles (interpolar MTs or ipMTs) and their relationship to the spindle MTs that attach to kinetochores (kMTs). Viewed in cross section, the ipMTs cluster with antiparallel near neighbors throughout mitosis; this bundling becomes much more pronounced as anaphase proceeds. While the minus ends of most kMTs are near the poles, those of the ipMTs are spread over half of the spindle length, with at least 50% lying > 1.5 microns from the poles. Longitudinal views of the ipMT bundles demonstrate a major rearrangement of their plus ends between mid- and late anaphase B. However, the minus ends of these MTs do not move appreciably farther from the spindle midplane, suggesting that sliding of these MTs contributes little to anaphase B. The minus ends of ipMTs are markedly clustered in the bundles of kMTs throughout anaphase A. These ends lie close to kMTs much more frequently than would be expected by chance, suggesting a specific interaction. As sister kinetochores separate and kMTs shorten, the minus ends of the kMTs remain associated with the spindle poles, but the minus ends of many ipMTs are released from the kMT bundles, allowing the spindle pole and the kMTs to move away from the ipMTs as the spindle elongates.     

Malorientation in half-bivalents at anaphase: analysis of autosomal laggards in untreated, cold-treated, and cold-recovering crane fly spermatocytes

Exposing crane fly larvae to 6 degrees C or returning them to 22 degrees C after exposure to 6, 2, or 0.2 degrees C can induce any number of autosomes in their primary spermatocytes to lag near the spindle equator at anaphase. Autosomal laggards in cold-recovering cells are contained in bivalents until anaphase (Janicke, M. A., and J. R. LaFountain, 1982, Chromosoma, 85:619-631). We report here documentation that lagging autosomes in cold-treated and cold- recovering cells are maloriented. During meiosis I, half-bivalents usually associate with only one pole via kinetochore fibers, with sister chromatids being oriented to the same pole. In contrast, laggards had kinetochore microtubules (kMTs) extending from them toward both poles: one sister was oriented to one pole and the other had some or all of its kMTs extending toward the opposite pole. Bipolar malorientation of autosomal laggards also was observed in one untreated cell. The number of kMTs per half-bivalent was similar in lagging and non-lagging autosomes, and those kMTs were contained in long birefringent kinetochore fibers. The overall spindle structure in cold- recovering cells was similar to that observed in untreated anaphase cells. Giemsa-stained centromeric dots of sister chromatids were contiguous in non-laggards and separated in laggards at anaphase. We conclude that bipolar malorientations can exist at anaphase in chromosomes that remain paired until anaphase, that cold recovery increases the frequency of that anomaly, and that such malorientations may be one cause of anaphase lag.     

Chromosome movement and spindle birefringence in locally heated cells: Interaction versus local control

A microheater was used to produce a temperature gradient within the mitotic spindle of living cells. The slope of the temperature gradient was estimated from thermal conductivity calculations and confirmed by measurements of spindle birefringence and by experiments on striated muscle. When the microheater was placed at one spindle pole or at one group of kinetochores, the gradient was steep enough to cause a large difference in birefringence between the two half-spindles, but the velocity of chromosome movement in anaphase was nearly the same in the warmer and cooler half-spindles. When the heater was shifted from the pole toward the interzone, the average velocity of chromosome movement increased approximately two-fold but was, again, nearly uniform in the two half-spindles. The rate of spindle elongation was especially sensitive to the site of heating, increasing ten-fold when the heater was shifted from the pole to the interzone. Regardless of heater position, the rate of chromosome movement was determined largely by the temperature of the coolest spindle region —chromosomes in the warmer half-spindle moved more slowly than expected from estimates of the temperature in that region. Since the microheater produces a substantial temperature gradient within the spindle, the near uniformity of chromosome velocity in both half-spindles must be due to some biological property of the spindle. Two very different explanations for the results are considered the most likely. According to one explanation, the near uniformity of velocity in both half-spindles is determined by the structure of the interpolar spindle, while changes in velocity involve force producers located both in the half-spindles and in the interzone. On the other explanation, the velocity is nearly the same in both half-spindles because the force producers are located exclusively in the interzone (Margolis et al., 1978).This paper is dedicated to Professor Sally Hughes-Schrader with admiration and affection     

Model of chromosome motility in Drosophila embryos: adaptation of a general mechanism for rapid mitosis

During mitosis, ensembles of dynamic MTs and motors exert forces that coordinate chromosome segregation. Typically, chromosomes align at the metaphase spindle equator where they oscillate along the pole-pole axis before disjoining and moving poleward during anaphase A, but spindles in different cell types display differences in MT dynamicity, in the amplitude of chromosome oscillations and in rates of chromatid-to-pole motion. Drosophila embryonic mitotic spindles, for example, display remarkably dynamic MTs, barely detectable metaphase chromosome oscillations, and a rapid rate of "flux-pacman-dependent" anaphase chromatid-to-pole motility. Here we develop a force-balance model that describes Drosophila embryo chromosome motility in terms of a balance of forces acting on kinetochores and kMTs that is generated by multiple polymer ratchets and mitotic motors coupled to tension-dependent kMT dynamics. The model shows that i), multiple MTs displaying high dynamic instability can drive steady and rapid chromosome motion; ii), chromosome motility during metaphase and anaphase A can be described by a single mechanism; iii), high kinetochore dynein activity is deployed to dampen metaphase oscillations, to augment the basic flux-pacman mechanism, and to drive rapid anaphase A; iv), modulation of the MT rescue frequency by the kinetochore-associated kinesin-13 depolymerase promotes metaphase chromosome oscillations; and v), this basic mechanism can be adapted to a broad range of spindles.     

In vivo effects of ortho-vanadate on spindle structure and dynamics of locust spermatocytes I

After external application of vanadate, a potent inhibitor of several ATPases including dynein, the following effects on living spermatocytes I are detectable: spherical metaphase cells change to a lemon shape due to a concentration dependent elongation of the spindle, apparently achieved by pulling the plasma membrane-inserted poles apart, presumably through the assistance of cytoskeletal filaments. The observed dismembering of the spindle seems to be due to the separation of the half-spindle fibres, composed of usually interdigitating kinetochore microtubules (kMTs), free MTs (fMTs) and polar MTs (pMTs). As revealed by microcinematographic recordings, the lengthening of the half-spindles is accompanied by counter-clockwise twisting movements of the polar regions which, after prolonged vanadate treatment, lead to the formation of filiform appendices. Bundles of 5 nm microfilaments, which could be identified by indirect immunofluorescence microscopy (IIF) as actin, are concentrated within these appendices. In spite of a certain derangement of spindle architecture, half of the metaphases in 1 mM vanadate are capable of entering anaphase, but the rates of chromosome-to-pole movement have changed depending on the incubation time and the cell shape developed, respectively. Thus, chromosomes move with the highest speed in lemon-shaped cells but lag in cells with filiform appendices. However, it remains an open question whether the acceleration of chromosome migration is the result of spindle dismemberment or whether the slowing of anaphase motion is the consequence of a far-reaching displacement of the filamentous component from the spindle framework.     

Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells

Summary Treatment of metaphase PtK₁ cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK₁ metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 m in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 m in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.Abbreviations DIC differential interference contrast - kMT(s) kinetochore microtubule(s) - MT(s) microtubule(s) - nkMT(s) non-kinetochore microtubule(s)     

Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells

Summary Quinacrine, an acridine derivative, has previously been shown to disrupt lateral associations between non-kinetochore microtubules (nkMTs) of opposite polarity in PtK₁ metaphase spindles such that the balance of spindle forces is significantly altered. We extended the analysis of the spatial relationship of spindle microtubules (MTs) in this study by using quinacrine to compare ATP-dependent requirements for early prometaphase centrosome separation and spindle formation. The route used for centrosome migration can take a variety of pathways in PtK₁ cells, depending on the location of the centrosomes at the time of nuclear envelope breakdown. Following quinacrine treatment centrosome separation decresased by 1.9 to 14.0 m depending on the pathway utilized. However, birefringence of the centrosomal region increased approximately 50% after quinacrine treatment. Quinacrine-treated mid-prometaphase cells, where chromosome attachment to MTs had occurred, showed a decrease in spindle length of approximately 6.0 m with only a slight increase in astral birefringence. Computer-generated reconstructions of quinacrine-treated prometaphase cells were used to confirm changes in MT reorganization. Early-prometaphase cells showed more astral MTs (aMTs) of varied length while mid-prometaphase cells showed only a few short aMTs. Late prometaphase cells again showed a large number of aMTs. Our results suggest that: (1) quinacrine treatment affects centrosome separation, (2) recruitment of nkMTs by kinetochores is quinacrine-sensitive, and (3) development of the prometaphase spindle is dependent on quinacrine-sensitive lateral interactions between nkMTs of opposite polarity. These data also suggest that lateral interactions between MTs formed during prometaphase are necessary for centrosome separation and normal spindle formation but not necessarily chromosome motion.Abbreviations aMT(s) astral microtubule(s) - DIC differential interference contrast - MT(s) microtubule(s) - kMT(s) kinetochore microtubule(s) - NEB nuclear envelope breakdown - nkMT(s) non-kinetochore microtubule(s)