Kinetochore behavior during generative cell division inTradescantia virginiana

Summary Previous observations indicate that division of the generative cell inTradescantia virginiana is characterized by several unusual features, including persistence of surrounding microtubule (Mt) bundles during karyokinesis, lack of a distinct metaphase plate and direct contribution by mitotic Mts to the cytoskeleton of young sperm. We have further probed karyokinesis in these cells using additional antitubulin and chromosome staining, as well as kinetochore visualizations with CREST serum. The CREST antibodies reveal kinetochores as paired and single fluorescent dots similar to those seen in other species stained with this preparation. Double localizations show that the dots are located at the ends of Mt bundles previously identified as kinetochore fibers (Palevitz and Cresti 1989). Before anaphase, paired kinetochores are distributed along the length of the cell. They also tend to be located at the cell periphery or are directly connected to peripheral Mt bundles by their kinetochore (K)-fibers. Twelve pairs of dots can be counted per cell, equal to the expected number of chromosomes. During anaphase, kinetochore separation starts at various positions along the length of the cell, producing single, relatively uniformly distributed kinetochores in the crotches of forks formed by K-fiber trunks and elongating Mt branches attached to the base of the trunks. Eventually, K-fibers with attached kinetochores aggregate in stepwise fashion on thick Mt bundles at both ends of the cell. This pattern is reflected in the cytoskeleton of young sperm. These results further document the unusual distribution of chromosomes and kinetochores inTradescantia generative cells and the origin of the Mt cytoskeleton in sperm cells.Abbreviations CREST Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia - K-fiber kinetochore fiber - Mt microtubule Dedicated to the memory of Professor Oswald Kiermayer     

Organization of the mitotic apparatus during generative cell division inNicotiana tabacum

Summary In order to gain a more complete understanding of the organization of the mitotic apparatus (MA) in the generative cells (GCs) of flowering plants, pollen tubes ofNicotiana tabacum were examined using tubulin immunocytochemistry and Hoechst fluorescence. The observations were then compared with previously published information onTradescantia GCs and the MA of somatic cells. At the onset of division, the prominent microtubule (Mt) bundles characteristic of GCs are reorganized into a more random Mt network. At late prophase/prometaphase, kinetochores appear to interact with this network, resulting in the formation of K-fibers that frequently link in tree-like aggregates. The GC MA takes the form of a distinct spindle and often has pointed, focused poles; the metaphase plate is usually oblique. Karyokinesis involves both anaphase A and B; lengthening of interzonal Mts is accompanied by elongation of the spindle. In late anaphase/early telophase, phragmoplast Mts are formed in association with the proximal face of the sperm nuclei. The phragmoplast remains prominent for some time, so that its Mts as well as another population generated from the distal face of the sperm nuclei constitute the initial sperm cytoskeleton. Comparisons indicate that the spindle in tobacco GCs falls on a continuum of organization between that of somatic cells and the MA ofTradescantia GCs.Abbreviations GC generative cell - MA mitotic apparatus - Mt microtubule     

Patterns of microtubule reappearance in root cells ofVigna sinensis recovering from a colchicine treatment

Summary The reticulum of paracrystalline tubulin strands, which is assembled in meristematic root cells ofVigna sinensis treated with a 0.08% colchicine solution, disaggregates and microtubules (Mts) reappear after a 10–14 h recovery of the seedlings from the drug. In recovering interphase cells, Mts reappear in the cortical cytoplasm. Initially, they are short and aligned in different directions but finally they elongate and usually become oriented transversely to the long cell axis.A single or a pair of preprophase Mt bands (PMBs) is organized in cells enclosing one or more nuclei. Simultaneously, Mts traverse the perinuclear cytoplasm. In recovering C-mitotic cells, Mt bundles emerge from the kinetochores. Initially, they exhibit diverse orientations. Afterwards, the C-chromosomes are aligned on ametaphase plate via kinetochore Mt bundles, which become parallel to one another. As time passes non-kinetochore Mts appear among the chromosomes and anaphase proceeds. In recovering cytokinetic cells, normal, abnormally curved or branched phragmoplasts are organized. The latter arise between the nuclei of multinucleate telophase cells or between the lobes of forming polyploid nuclei. In cells which were blocked at an advanced cytokinetic stage by colchicine, phragmoplasts return to the margins of the incomplete cell walls.The observations presented here suggest that in recovering colchicine-treated root cells the Mts and the tubulin reticulum are interchangeable. Although Mts appear in cytoplasmic sites where they are expected to be nucleated, the pattern of Mt reformation differs from that operating in normal and to a smaller extent from that functioning in cells recovering from other anti-Mt drugs.     

Associations between membranes and microtubules during mitosis and cytokinesis in caulonema tip cells of the mossFunaria hygrometrica

Summary The interphase nucleus in theFunaria caulonema tip cells is associated with many non-cortical microtubules (Mts). In prophase, the cortical Mts disappear in the nuclear region; in contrast to moss leaflets, a preprophase band of Mts is not formed in the caulonema. The Mts of the early spindle are associated with the fragments of the nuclear envelope. Remnants of the nucleolus remain in the form of granular bodies till interphase. The metaphase chromosomes have distinct kinetochores; the kinetochore Mts are intermingled with non-kinetochore Mts running closely along the chromatin. Each kinetochore is associated with an ER cisterna. ER cisternae also accompany the spindle fibers in metaphase and anaphase. In telophase, Golgi vesicles accumulate in the periphery of the developing cell plate where no Mts are found. The reorientation of the cell plate into an oblique position can be inhibited by colchicine. It is concluded that the ER participates in controlling the Mt system, perhaps via calcium ions (membrane-bound calcium ions have been visualized by staining with chlorotetracycline) but that, on the other hand, the Mt system also influences the distribution of the ER. The occurrence and function of the preprophase band of Mts is discussed.     

Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes

We use liquid crystal polarized light imaging to record the life histories of single kinetochore (K-) fibers in living crane-fly spermatocytes, from their origins as nascent K-fibers in early prometaphase to their fully matured form at metaphase, just before anaphase onset. Increased image brightness due to increased retardance reveals where microtubules are added during K-fiber formation. Analysis of experimentally generated bipolar spindles with only one centrosome, as well as of regular, bicentrosomal spindles, reveals that microtubule addition occurs at the kinetochore-proximal ends of K-fibers, and added polymer expands poleward, giving rise to the robust K-fibers of metaphase cells. These results are not compatible with a model for K-fiber formation in which microtubules are added to nascent fibers solely by repetitive “search and capture” of centrosomal microtubule plus ends. Our interpretation is that capture of centrosomal microtubules—when deployed—is limited to early stages in establishment of nascent K-fibers, which then mature through kinetochore-driven outgrowth. When kinetochore capture of centrosomal microtubules is not used, the polar ends of K-fibers grow outward from their kinetochores and usually converge to make a centrosome-free pole.     

Microtubular fir-trees in mitotic spindles of onion roots

Summary The organization of kinetochore fibers was examined inAllium root cells processed for tubulin immunocytochemistry. Metaphase fibers consist of a core or trunk of Mts to which are attached numerous branches, yielding a bottle-brush of fir-tree pattern similar to that reported inHaemanthus endosperm cells. Many of the branches cross the midzone and extend into the opposite half-spindle. In addition, branch Mts associate with more than one kinetochore fiber. During anaphase, branch Mts elongate while the trunks shorten and fuse into polar caps. Our results are discussed in terms of spindle fiber organization and Mt polarity.Abbreviations K Kinetochore - Mt microtubule     

Pac-man motility of kinetochores unleashed by laser microsurgery

We report on experiments directly in living cells that reveal the regulation of kinetochore function by tension. X and Y sex chromosomes in crane fly (Nephrotoma suturalis) spermatocytes exhibit an atypical segregation mechanism in which each univalent maintains K-fibers to both poles. During anaphase, each maintains a leading fiber (which shortens) to one pole and a trailing fiber (which elongates) to the other. We used this intriguing behavior to study the motile states that X-Y kinetochores are able to support during anaphase. We used a laser microbeam to either sever a univalent along the plane of sister chromatid cohesion or knock out one of a univalent's two kinetochores to release one or both from the resistive influence of its sister's K-fiber. Released kinetochores with attached chromosome arms moved poleward at rates at least two times faster than normal. Furthermore, fluorescent speckle microscopy revealed that detached kinetochores converted their functional state from reverse pac-man to pac-man motility as a consequence of their release from mechanical tension. We conclude that kinetochores can exhibit pac-man motility, even though their normal behavior is dominated by traction fiber mechanics. Unleashing of kinetochore motility through loss of resistive force is further evidence for the emerging model that kinetochores are subject to tension-sensitive regulation.     

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.     

Density and distribution of α-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle

The polarity of kinetochore microtubules (MTs) has been studied in lysed PtK1 cells by polymerizing hook-shaped sheets of neurotubulin onto walls of preexisting cellular MTs in a fashion that reveals their structural polarity. Three different approaches are presented here: (a) we have screened the polarity of all MTs in a given spindle cross section taken from the region between the kinetochores and the poles, (b) we have determined the polarity of kinetochore MTs are more stable to cold-treated spindles; this approach takes advantage of the fact that kinetochore MTs are more stable to cold treatment than other spindle MTs; and (c) we have tracked bundles of kinetochore MTs from the vicinity of the pole to the outer layer of the kinetochore in cold- treated cells. In an anaphase cell, 90-95% of all MTs in an area between the kinetochores and the poles are of uniform polarity with their plus ends (i.e., fast growing ends) distal to the pole. In cold- treated cells, all bundles of kinetochore MTs show the same polarity; the plus ends of the MTs are located at the kinetochores. We therefore conclude that kinetochore MTs in both metaphase and anaphase cells have the same polarity as the aster MTs in each half-spindle. These results can be interpreted in two ways: (a) virtually all MTs are initiated at the spindle poles and some of the are "captured" by matured kinetochores using an as yet unknown mechanism to bind the plus ends of existing MTs; (b) the growth of kinetochore MTs is initiated at the kinetochore in such a way that the fast growing MT end is proximal to the kinetochore. Our data are inconsistent with previous kinetochore MT polarity determinations based on growth rate measurements in vitro. These studies used drug-treated cells from which chromosomes were isolated to serve as seeds for initiation of neurotubule polymerization. It is possible that under these conditions kinetochores will initiate MTs with a polarity opposite to the one described here.     

Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle

To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.     

Merotelic kinetochores in mammalian tissue cells

Merotelic kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian kinetochores typically have binding sites for about 20-25 kinetochore microtubules. In prometaphase, kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic kinetochores based on the ratio of kinetochore microtubules to the correct versus incorrect pole.     

Asynchronous entry into anaphase induced by okadaic acid: spindle microtubule organization and microtubule/kinetochore attachments

Summary We have found that a brief treatment of either PtK₂ cells or stamen hair cells ofTradescantia virginiana during metaphase with okadaic acid, a potent protein phosphatase inhibitor, results in asynchronous entry into anaphase. After this treatment, the interval for the separation of sister chromatids can be expanded from a few seconds to approximately 5 min. We have performed a series of immunolocalizations of cells with anti-tubulin antibodies and CREST serum, asking whether okadaic acid induces asynchronous entry into anaphase through changes in the organization of the spindle microtubules or through a loss in the attachment of spindle microtubules to the kinetochores. Our experiments clearly indicate that asynchronous entry into anaphase after phosphatase inhibitor treatment is not the result of either altered spindle microtubule organization or the long-term loss of microtubule attachment to kinetochores. The kinetochore fiber bundles for all of the separating chromosomes are normally of uniform length throughout anaphase, but after asynchronous entry into anaphase, different groups of kinetochore fiber bundles have distinctly different lengths. The reason for this difference in length is that once split apart, the daughter chromosomes begin their movement toward the spindle poles, with normal shortening of the kinetochore fiber bundle microtubules. Thus, okadaic acid treatment during metaphase does not affect anaphase chromosome movement once it has begun. Our results suggest that one or more protein phosphatases appear to play an important role during metaphase in the regulatory cascade that culminates in synchronous sister chromatid separation.     

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.     

Differential expression of a phosphoepitope at the kinetochores of moving chromosomes

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.     

Ultrastructure of mitosis in the aphid-pathogenic fungusErynia neoaphidis

Summary An account of mitosis in the aphid-pathogenic, entomophthoraceous fungusErynia neoaphidis is presented. The mitotic apparatus is characterized by a closed, intranuclear, polarized spindle. Chromosomes are permanently attached by kinetochore microtubules (kcMTs) to the poles during mitosis. The spindle develops as the spindle pole bodies migrate and separate. At metaphase the eccentric spindle contains only kcMTs and is located in a relatively chromatinfree zone. Paired sister kinetochores are arranged in a broad metaphase plate. During anaphase kcMTs shorten, astral and nonchromosomal microtubules develop and elongate and the interpolar distance increases.     

Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.     

Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms of Chromosome Congression and Anaphase Onset

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK₁ cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late metaphase 24.3 ± 4.9 (n = 62), and at early anaphase 27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 μM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.     

Unusual pattern of mitosis in the free-living flagellateDimastigella mimosa (Kinetoplastida)

Summary The three-dimensional ultrastructural organization of the mitotic apparatus ofDimastigella mimosa was studied by computer-aided, serial-section reconstruction. The nuclear envelope remains intact during nuclear division. During mitosis, chromosomes do not condense, whereas intranuclear microtubules are found in close association with six pairs of kinetochores. No discrete microtubule-organizing centers, except kinetochore pairs, could be found within the nucleus. The intranuclear microtubules form six separate bundles oriented at different angles to each other. Each bundle contains up to 8 tightly packed microtubules which push the daughter kinetochores apart. At late anaphase only, midzones of these bundles align along an extended interzonal spindle within the narrow isthmus between segregating progeny nuclei. The nuclear division inD. mimosa can be described as closed intranuclear mitosis with acentric and separate microtubular bundles and weakly condensed chromosomes.Abbreviation MTOC microtubule-organizing center     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin