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Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo.

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 organizating 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 microtubule-related processes.

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 new experimental 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, differential interference contrast; IGS, immunogold stain; KF, kinetochore fiber; KFC, kinetochore fiber complex; MT, microtubule; MTFT, microtubular fir tree; NA, numerical aperture. microtubular fir trees (MTFTs), are of special interest because they were also identified as components of the higher plant acentriolar spindle and may be directly related to the spindle function. These unexpected observations revise our understanding of anaphase, emphasizing the role of the polar region in spindle elongation. Data reported earlier (9,35) suggest that a functional MT fiber has a complex organization: it consists of MTs terminating in the kinetochore at the one end, and extends to the pole as a more difficult to define MT organization, often seen as an MTFT. Relating these observations of MT behavior in vivo to known properties of MTs in vitro suggests a role for MTFTs during anaphase and leads to a new concept of spindle elongation. Inou6 and Ritter (24) suggested that in typical standard anaphase the kinetochore-to-pole movements are accompanied by shortening of half-spindles, and polar separation is accompanied by an active elongation of the interzone (anaphase A and B). However, our observations show that in the higher plant Haemanthus the half-spindle does not shorten during the first two-thirds of anaphase, i.e., anaphase B occurs before anaphase A.
The present observations extend the data on the behavior of birefringent chromosomal fibers (21) to the whole halfspindle by including reorganization within the polar region. We conclude that during anaphase the kinetochore fiber (KF) shortens, while the entire kinetochore fiber complex (KFC) elongates due to new MT assembly in the polar region. Moreover, observation of MT reorganization in the absence of chromosomes demonstrates how some intrinsic properties of higher plant MTs might be expressed during mitosis.
The following MT reorganization in vivo from early stages was unsuccessful because these processes were completed before the preparation was mounted for the light microscopic observations . The final stage, however, formation of the cell plates within the pharagmoplasts, can be easily followed. This work is based, therefore, on precisely timed fixation after preparation and correlation of these data with observations in vivo . The minimum time after preparation we were able to fix the cells using the sandwich technique was 60 ± 10 s. The moment the ovule was opened was counted as 0, and the time in seconds was measured until the fixative covered the whole preparation .
Methods for low temperature shocks, which partly disassemble MTs, and recovery after the treatment, were described previously (7) .
The immunogold stain (IGS) method for MTs (14) was used as described (15). Monospecific affinity column purified rabbit antitubulin antibody against dog brain tubulin was used as a primary antibody . The technique, using 15and 20-nm IGS, does not stain cells that died during endosperm preparation (7,15) . The endosperm cells are free-floating and during IGS processing -30% of large cells are lost, as well as perhaps more than 90% of cell fragments, which prevents meaningful statistics . Improper handling results in 100% loss . The use of polylysine-coated slides to improve cell attachment was not successful due to decreased cell survival .
The maximum absorption of immunogold is at 520 rim after dehydration and embedding in permount (Fisher Scientific Co., Pittsburg, PA, or similar medium) . The micrographs of cells treated with IGS were taken with a narrow band interference filter (520 nm) using a Zeiss 63x Plan-Ape 1 .4 numerical aperture (NA) objective, a Zeiss condenser of 1 .4 NA (Carl Zeiss, Inc ., Thomwood, NY), and a Xenon 75-W are lamp. Measurements (Tables I-III) were made with a measuring eyepiece . Kodak negative film, type 2415, processed in Kodak HC 110 developer (dilution B), was used. The dehydration and mounting (in Permount) procedure may result in a slight (5%) shrinkage of the spindle, which we assume is uniform for all cells .
Our image enhancement system is based on rectified differential interference contrast (DIC) (21) as described elsewhere (36). This system permits us to analyze both water medium mounted and dehydrated preparations with stained chromosomes . It permits also elimination of cells not stained properly due to insufficient immunogold penetration .
Both in cytoplasts and in postmitotic phragmoplasts, the cell plate is not stained due to a dense accumulation of vesicles around MTs which prevents antibody penetration . At least some interzonal MTs are trapped within the cell plate, while most polar MTs terminate within this structure . The vesicles are The Journal of Cell Biology, Volume 102, 1986 264 not seen with standard optics in dehydrated preparations but can be visualized with an image enhancement system (Fig. 3d) . The relation of polar MTs to interzonal and their role in the cell plate formation is beyond the scope of this paper. MT arrangement after IGS in about 80 cells in different stages of mitosis was compared with the distribution of birefringence in the same cell in vivo . This is the first report where the same endosperm cells, in a living state and after glutaraldehyde fixation followed by immunoprocessing, are shown . The time between recording in vivo and fixation for IGS was I-4 min . A Leitz polarizing microscope (E. Leitz, Inc ., Rockleigh, NJ) was used in conjunction with strain-free rectified Nikon optics (Nikon Inc., Garden City, NY) (40x objective, 0 .65 NA with 16-mm condenser, 0.52 NA and 100x objective, 1 .25 NA with 8-mm condenser, 1 .15 NA ; micrographs included were taken with 40x objective) and a Leitz l/30 a compensator. The light source was an HBO 100-W mercury arc lamp. The illumination was filtered through a liquid CuSO filter, two Zeiss Calfex heat reflecting filters, and a green 550-nm multilayer high transmission interference filter (Baird Corp ., Bedford. MA, nominal half band width 7 nm) with its accessory high and low cut-off filters .
Statistical analysis of the measurements was performed using the SPSSX statistical package on an IBM 431 computer (IBM Corp ., Danbury, CT) . Unless indicated otherwise, all fixations were made at room temperature, 22°-24°C .

Terminology
MT organization seen in IGS processed cells does not correspond precisely to the organization observed either with an electron microscope or with birefringent pattern in the living cells detected with a polarizing microscope, DIC, etc . This is due to specifics of immunocolloidal gold reaction with MTs combined with its visualization in the light microscope . The problem is beyond the scope of this paper. Therefore spindle components as seen in IGS-processed cells require definitions and comparison to the existing terminology.
Chromosomal Fiber. The chromosomal fiber is the birefringent fiber connected to a chromosome (5), detected in living cells with a polarizing microscope . Images of bireffngent fibers depend on extinction of the polarizing microscope and orientation of the spindle . The fibers in flattened cells often show weak birefringence and do not correspond precisely (area and shape wise) to MT distribution in the same cells after IGS processing. Figs. 5 and 7 illustrate these differences and the measured parameters (see also Fig. 14). The birefringent chromosomal fibers in Fig. 7 are shorter than the half-spindle. The length of chromosomal fibers in vivo cannot be well defined (cf. Figs . 5 and 7) .
KF and KFC. Our use of the term KF is somewhat different in meaning from that used previously (27) . The latter (27) corresponds to kinetochore/ centromere-associated MTs (4l) and is only a few ym long . We define KF as densely stained with the IGS part of the chromosomal fiber associated with the kinetochore region. The fiber is several ium long and is longer than the chromosomal fiber detected under 45°orientation with crossed polarizers in a polarizing microscope. It should be distinguished from the broader term KFC, which with other often skewed MTs (which are not associated with the kinetochore region), form the entire KFC (9,35) . As the length of chromosomal fibers in vivo, the measurements of 576 KFs in IGS preparations could not be done as precisely as that of the half-spindle . A precise comparison ofbirefringent chromosomal fiber with MT distribution within KF is beyond the scope of the present work.
The KFC includes the portion of the fiber that penetrates the polar region of the spindle and includes a less defined structure shaped often as an MTFT . KFC extends all the way to the spindle periphery and is usually longer than the h after preparation . (ef) Medium-sized spherical cytoplasts 30 min after preparation . e shows an advanced stage of the formation in a circular structure : the elongating head of the cone has nearly entered the tail . Since there was no more space for growth, the tail curved and both ends of the structure interconnected, forming a spiral or a ring. e is an earlier stage of development than f. (g-h) Chromosome-free spindles, structures similar, both structurally and functionally, to mitotic spindles . Granules of undetermined origin have been transported toward the poles, where they form pseudo-nuclei (thin arrows) . MTs radiating from the poles probably correspond to polar MTs during mitosis. 1 1h h after preparation . g is an earlier stage ofdevelopment than h, where a cell plate has already been formed. Thick arrows show polar MTs . (i) Multiple chromosome-free spindles in the cytoplasm of an interphase cell at different stages of their development. Configurations of converging centers are complex and may indicate that phragmoplasts, which are the final stage, may form in diverse ways . 1 h after preparation . (j) A typical MT converging center formed at room temperature during recovery from low temperature shock (5°C) . (k) A growing MT converging center 25 min after preparation. (l) Multiple chromosome-free spindles before cell plate formation in a very large cytoplast, 1 h after preparation. Bar, 10 Mm .
KF (see Fig. 7 for additional explanation). It should be stressed that such differentiation is detected only in IGS-processed cells and has not been detected in vivo. The KF in flattened cells may be arranged under an angle to the spindle axis.
Half-spindle and lnterzone. The whole MT flattened cone-like structure in front of kinetochores and the surrounding cytoplasm is a half-spindle. It includes the polar region with the irregular arrangements of MTs (which can only be seen after immunoprocessing). Time-lapse films of Haemanthus endosperm taken under a polarizing microscope suggest that poorly defined ends of chromosomal fibers do not penetrate the polar region.
The length of the half-spindle in IGS preparations was measured from the kinetochores to the border of the polar region along its long axis and does not include asters. Therefore the half-spindle may be shorter than tilted KFCs located at the spindle periphery and is the measure of the KFCs located on the spindle axis. These measurements depend on the light microscope resolution and contain little error.
lnterzone. Interzone is the region between the separating chromosome groups in anaphase. Therefore the metaphase spindle is composed from two half-spindles only, while anaphase consists of two half-spindles and an interzone. The interzone transforms into a phragmoplast during the anaphasetelophase transition.
Pole and Polar Region. In unflattened cells MTs of the half spindle terminate at the relatively small pointed area called the pole, and in flattened ones in the broad, polar region that is often well defined due to the presence of birefringent granules at the spindle periphery. The broad polar region may also be interpreted as multiple poles.

Materials
Endosperm. Free cells and syncytia of young endosperm have no cellulose cell walls but have the ability to form such walls between sister and non-sister nuclei. Endosperm of Haemanthus is a triploid tissue (n = 9). All cells reproduced have 27 chromosomes. Due to the size, the spindle can be sectioned optically. Such sections can contain only few chromosomes and make a false impression of lower chromosome number (e.g., Fig. 6, a and b). The main difference between the endosperm and cellular higher plant tissues is the lack in the former of the preprophase band of MTs (45).
According to our knowledge it has never been reported that Haemanthus endosperm has two extreme populations of cells and mitotic spindles: large and very small (lengths ratios up to 1:4). The origin of the small cells is unknown, and they cannot be flattened in our hands. Additionally, there is a considerable variation in the size of the cells within each population and in different ovules. In this and previously published work on endosperm, mostly large cells partly flattened were studied (see exception in reference 17). The present work includes also medium-sized cells. Therefore some values are somewhat lower than previously reported.
Intact Cells. Endosperm cells of Haemanthus katherinae (South African globe or blood lily) have the largest known mitotic spindle (40-60-~m long in metaphase). There is considerable variation in the organization of the spindle pole, or the whole polar region. Polar regions are often not well defined, and multiple subpoles may be seen. However, they gradually fuse during anaphase, and multipolar divisions are rare (below 5 %). The preparatory technique always results in a partial flattening of large and medium-sized cells. Cells flatten due to the action of the surface tension and not mechanical compression. The spindle becomes wider, but the length does not seem to be affected. The spindle can be further dissociated by flattening, or occasionally during preparation into separate functional units containing few, or even one chromosome, which proceed normally through mitosis and cytokinesis in over 95% of cells.
Cytoplasts. Anucleate cell fragments (cytoplasts) containing MTs (7) arise both from interphase and from mitotic cells, as demonstrated by occasionally persisting partial connections. The number of cytoplasts varies from zero in old ovules to a few hundred in young ones. MTs in anucleate cell fragments retain their ability to assemble-disassemble and rearrange for several hours. The majority of cytoplasts is lost during the IGS processing, which prevents meaningful statistics.

Chromosome-free Spindles Reorganization of MTs in Cytoplasts.
In the slides fixed 1-5 min after preparation, irregular arrays of MTs are found in all cytoplasts, regardless of their size (Fig. 1). No other MT configurations were ever found in controls, i.e., in preparations not treated with drugs. A strict relationship exists be-tween the size and shape of cytoplasts and the mode of their future MT reorganization: MTs in small and large fragments behave quite differently. No reorganization is observed in very small fragments (Fig. l, c and d), even several hours after preparation. The rate of MT reorganization can be experimentally modified by such factors as temperature and other conditions influencing critical concentration of tubulin (work in progress). The time scale of rearrangement described here applies to our standard preparatory conditions at room temperature.
Irregular arrays of MTs in large size fragments are rarely seen after 10-15 min, and only exceptionally 2 h after the preparation. Regular arrays are occasionally seen as early as 5 min after preparation (Fig. I b). Intermediate configurations between such arrays and MT cones indicate that they originate from regular arrays.
Two most characteristic configurations found in small cytoplasts are cones and rings. Cones with MTs converging toward one center are seen in medium and large sized cytoplasts ( Fig. 1, j and k) on slides fixed between 8 and 45 min after endosperm preparation. The considerable loss of cytoplasts during the IGS procedure prevents statistical analysis. The rings are not found 8 min after preparation and appear only later. Several intermediate configurations demonstrate that converging centers are unstable intermediates in spherical cytoplasts. They transform into bushy spirals or rings, a first type of configuration that is very stable, and do not reorganize any further. This transformation occurs through the formation of MTFTs. In large and not spherical cytoplasts the MT cones transform into spindle/phragmoplasts. This is indicated by the low number or absence of cones in preparations fixed after 1-2 h. Several intermediate stages between an MT cone and a closed ring were observed. The first stage is the formation of MTFTs, which may be seen as soon as l0 min after preparation. The MTFT subsequently elongates (Fig. I k). A closed ring is formed when the head enters the tail (Fig. 1, e and f). A considerable asynchrony is seen in both the formation of MT cones and closed rings.
Large converging centers are very seldom observed after 2 h and transform into cytoplasmic spindles/phragmoplasts, with cell plate (Fig. l, g and h). They have been called (7) cytoplasmic chromosome-free spindles. We could not distinguish among the variety of MT configurations, which might be intermediates between the large converging centers and the spindles (Fig. l, i and l). There is also a considerable asynchrony of reorganization. In most cytoplasts the process is completed (cell plate formed) in about 20-45 min. Very large fragments always develop multiple, remarkably uniform sized spindles/phragmoplasts (Fig. 1, i and l) with cell plates. In single phragmoplasts a small incomplete aster may occasionally develop at one or two poles (cf. Fig. 1 h). We have never observed a single spindle/phragmoplast longer than a metaphase spindle (40-60 ~m) or shorter than a metaphase halfspindle. MT converging centers may also occasionally give rise to asymmetrical spindles/phragmoplasts. We do not know yet whether or in what conditions these asymmetrical phragmoplasts, after several hours, finally transform into symmetrical ones (work in progress).
The nonkinetochore transport (7,27), also called passive transport (5) or particle and/or state movements (1), within these structures is the same as in the mitotic spindle. It distributes inclusions (such as starch grains, etc.) randomly toward the two poles, where they accumulate and form pseudo-nuclei (Fig. 1, g and h). The transport appears before the cell plate is seen and continues for several hours after its appearance.
Nucleated Cells and Syneytia. As in cytoplasts, MTs form a uniform meshwork in intact interphase and early prophase cells immediately after the preparation (Fig. 2a). Only occasionally is this meshwork somewhat denser around the nu-cleus. In less than 5% of the cells, part of this meshwork transforms into cytoplasmic spindles/phragmoplasts within -3 0 rain.
The events essentially follow the same pattern in most interphase and late telophase cells (Fig. 2, a-d and Fig. 3, a and f). The uniform distribution of MTs found in interphase and early telophase gradually differentiates. The region around the nucleus is gradually depleted of MTs, and a dense band of MTs arranged perpendicularly to the cell periphery is formed (Fig. 2, b and c and Fig. 3f). This accumulation forms either a continuous band or separate patches. A continuous band is usually observed in cells with a regular shape (Figs. 2b and 3f), while patches are observed in cells with an irregular shape (Fig. 2c) and longer time after preparation. Thus, during the progress of telophase MTs grow outward from the nuclear region toward the cell periphery and eventually, the nuclear region becomes depleted of MTs (see also figures in reference 7.) Similar processes are seen in interphase cells (Fig. 2 b).
MT patches at the cell periphery often undergo further transformations. When two MT bundles curve and their ends become opposed, a phragmoplast forms. It usually arises within 30-45 min at room temperature (Fig. 2, c and d). The early stages of such cytoplasmic spindle/phragmoplast formation are difficult to recognize after IGS procedure. This precludes in vivo predetermination of the position in the cell at which the cytoplasmic spindles/phragmoplasts will form. Consequently, attempts to follow the reorganization of the MT bundle from very early stages in vivo have been unsuccessful, although fully developed cytoplasmic phragmoplasts are easily detected in the living cells (see figures in reference 7).

Mitotic Spindle/Phragmoplast
The Formation of KFC in Late Prometaphase-Metaphase.
The formation of KFs at the onset of prometaphase was not followed. Our observations begin in mid-prometaphase. The fir tree organization of individual KFCs is gradually established in late prometaphase. MTs attached to the kinetochore represent the trunk, and wispy non-kinetochore MTs are the branches (polar MTs in later stages of anaphase). The bending of the branches toward the cell equator, at angles up to 45", is a pronounced feature of metaphase and some late prometaphase spindles, especially in flattened cells (Fig. 4, a and  b). The branches, which are often S-shaped with the distal ends pointing toward the equator, fuse with adjacent KFC. Individual KFC also may be twisted. However, this has been seen only in 12 out of hundreds of observed cases. A righthanded twist (looking from the kinetochore to the pole) was observed in 9 of these fibers.
The length of the KF after IGS corresponds approximately to the length of the chromosomal fiber as seen in the polarizing microscope in the living cell (Fig. 5, a and b). The region between the end of the chromosomal fiber seen in vivo with the polarizing microscope (22, 25) and the outline of the spindle occupied by MTs, i.e., the polar termination of the KFC as seen after IGS, is not resolved by the polarizing microscope. The difference is especially striking in spindles with a low number of MTs (Fig. 5).
Anaphase. The analysis of KFCs during anaphase without an image enhancement system is possible only in very fiat cells. In the majority of cells only the branches, or polar MTs, are seen at the periphery of half-spindles as a hairy outline (Fig. 6a).
Such organization of whole half-spindles becomes increasingly pronounced during the progress of anaphase (Fig. 6 d). The polar MTs (branches of MTFTs), which form abundantly in anaphase (15), penetrate the interzonal region after midanaphase in an asynchronous manner and associate laterally with the interzonal MTs often present around the trailing chromosome arms.
The number and spatial organization of the interzone MTs vary markedly in different cells. Some cells contain large numbers of MTs while others are practically devoid of them by the end of anaphase (compare Fig. 8 c, and Fig. 6, a and  d). As a rule the number is low in unflattened cells. In cells fixed less than 5 min after preparation, the number of interzonal MTs is often lower than in any figure reproduced here, and the number of polar MTs is very high, implying that such relations exist in the ovules in situ. In both extreme cases anaphase progresses in a normal fashion; we have, however, never seen a control anaphase without abundant polar MTs. The arching and bending of adjacent polar and interzonal MTs is often variable (Figs. 6 b and 7 c), demonstrating that such bends do not result from the shrinkage during the IGS procedure unless the shrinkage is non-uniform for different MTs, which we feel is unlikely.
Anaphase Elongation. The direct measurements of separation of the diffuse polar regions of the acentriolar spindle in Haemanthus endosperm in vivo cannot be done precisely because the border between the MT-occupied region and the cytoplasm in most cells is not sharp. The granules around the spindle allow such measurements in some cells (Fig. 9) and the data seem to be not fully compatible with those derived from IGS preparations. The results of indirect measurements are given in Figs. 10 and 11. Histograms (Fig. 10) showing the length distribution of half spindles (centrally located KFC) of individual cells at different stages of anaphase reveal an unexpected pattern. There is an increase in the length of the half-spindle between metaphase and early anaphase. This increase may be even larger than our measurements indicate due to inclusion in the measurements of metaphase and late prometaphase spindles, which cannot be distinguished in IGS preparations. The measurements in anaphase demonstrate that the length of the halfspindle remains unchanged until mid-anaphase, when the half-spindle rapidly shortens. The length distribution of KFs shows a continuous shortening, indicating that the half-spindle does not change in length or may even elongate at the time when the KF proper shortens.   Statistical analysis on a larger number of cells (Tables I-III) leads to the same conclusions. The difference between the means of groups for each set of measurements, the length of the KF at metaphase, early anaphase, mid-anaphase, and late anaphase, the length of the KFC at the same four stages, and the distance between the chromosome groups at early, mid-, and late anaphase, are all statistically highly significant (P ~< 0.001). The only exception is the two-tail probability of 0.46 between the measurements for early anaphase and mid-anaphase, since the length of the KFC at these two stages practically does not change.
The data are summarized in Fig. 11. The graphs show that the KF shortens at a constant rate from metaphase to the end of mid-anaphase, when chromosomes move with an approximately constant speed (Fig. 9). The KFC elongates at the metaphase/anaphase transition (see Discussion) and, while  K F shortens, the length of the KFC (which in our definition is the actual half-spindle length) remains unchanged during the first two-thirds of anaphase. Thus, during this period of anaphase the shortening of the K F is slower than chromosome movement during anaphase and is not concurrent with shortening of the half-spindle. The rapid shortening of the halfspindle occurs only in the last one-third of anaphase, when the half-spindle breaks down and the phragmoplast and astral (polar) MTs are formed abundantly.
To study the relationship between the change in length of polar MTs in mid-and late anaphase and chromosome movement, we plotted the length of the interzonal segments of polar MTs (measured from the kinetochores to their distal ends) against the distance between the chromosome groups in microns (Fig. 12). X-axis corresponds to the average time of anaphase in minutes (the average speed of anaphase chromosomes in Haemanthus is 1 #m/min). Therefore the mea-  configuration of polar MTs (e. g. Fig. 8, a-c), no statistical analysis was performed, and the numerical values are probably an underestimation of the MT elongation rate in the living cells (see Discussion). The distance between chromosome groups reflects the time and stage of anaphase and permits us to estimate the rate of polar MT elongation. This rate, in the range of 2 pm/min, is faster than chromosome movement at these stages. Anaphase-Telophase Transition. At the onset of tel•phase chromosomes stop their migration and trailing chromosome arms contract. A great variability exists in the appearance and growth of polar MTs that elongate toward the equator and may or may not form aster-like structures (7,8). In very flat and thin cells this process may start in mid-anaphase (Fig.  13). The development of a polar system always follows the same pattern. MTs grow away from the tel•phase chromosome group in a centrifugal direction until they reach the cell periphery. MTs, elongating toward the equator especially in the first stage of growth (mid-anaphase), are mostly straight and uniformly distributed (Fig. 3, a and b). The criss-crossing begins in this stage and is increasingly common in asters and phragmoplast where long, usually slightly curved MTs may cross the entire structure. This may indicate that these MTs elongated after the phragmoplast was already formed. In contrast to polar MTs growing toward the cell equator, the distal ends of those forming an aster do not show a tendency to a lateral association. Adjacent MTs have nearly identical lengths on the light microscopic level. This pattern of uniform distribution invariably reorganizes within 30-60 min.
The Development of Phragmoplast and Post-mitotic MTFTs. When the laterally growing post-mitotic or cyto-  plasmic phragmoplast reaches the cell periphery, MTFT(s) are often formed. Newly formed MTFTs grow along the cell periphery orthogonal to the cell plate. They resemble hairy tails and, within 60-90 min after preparation, may form gently twisting arched spirals up to 80 pm in length. These tail structures have been observed both in symmetrical (branches on two sides) and in asymmetrical (branches on one side) forms. Even though an entire tail structure may be arched and twisted, its constituent MT strands are usually straight. Similar formation of MTk--'rs is observed in phragmoplasts which form in the cytoplasm of interphase cells (Fig.  2 d). Therefore MTFTs, which basically are growing converging centers, appear at the distal (from the cell plate) regions of the phragmoplasts only in medium and larger cytoplasts and in intact cells. This demonstrates that processes in the living cell require a large tubulin pool and/or unknown associated components. Occasionally, MTFTs are also formed in the cytoplasm at the polar region of the spindle and may be arranged at an angle to the long axis of the spindle (33). They may be associated with very long single or few MTs detected with image enhancement system (Fig. 3c). Such long MTs are often found around the cell periphery and may form within the aster, where they are often arranged under the angle to the majority of astral MTs. Such MTFTs are of different size, with a dense MT cone as a trunk, and may not even be associated with the half-spindle (Fig. 6, d and f). Thus, the polar region of the spindle is often composed of multiple MTFTs: some are components of KF, and some are not associated with the mitotic spindle.

Discussion
The kinetochore/eentromere (41) and centriole-centrosome complex in vitro and probably in vivo modify some properties of MTs while facilitating the nucleation step (13). Activity of these structures yielded abundant information concerning MT nucleation centers in the living cell (11). MT (Tables I-III). Since metaphase in Haemanthus lasts up to 1 h, and there is an indistiguishable transition between prometaphase and metaphase, we do not know how long before the start of anaphase the metaphase measurements were taken. This and especially sudden jump apart of kinetochores at the onset ofanaphase may account partly for the abrupt change of length of the spindle between metaphase and the start of anaphase (cf, Discussion). endosperm occurs both in nucleated cells and in anucleate fragments (cytoplasts) in the absence of such discrete organizing organelles. Cytoplasts are the simplest known cytoplasmic unit where structural MT transformations can be followed and their time sequence deduced.

Nucleation and Self-Reorganization~Rearrangement of MTs
We do not know whether cytoplasts exist in intact ovules and, if present, whether they undergo similar reorganizations as after preparation. The final MT transformations (small phragmoplasts and spiral rings) were never seen a few minutes after preparation. This demonstrates that a rapid reorganization of MTs in cytoplasts is triggered in an unknown manner, perhaps due to specific properties of higher plant MTs. Animal MTs in cytoplasts with and without centrosomes do not reorganize in a similar manner (28). We believe, however, that our data may be related to formation of aster-like structures and tubulin gelation in vitro (44) and perhaps unusually rapid MT assembly-disassembly of some MTs (32). The first organized MT configuration in cytoplasts is a cone or converging center. The two final transformations are a closed bushy ring or a phragmoplast.
MT converging centers, the simplest ordered arrangement of MTs formed spontaneously, are not restricted to endosperm. They are common in plant and animal cells, both in controls and in experimental conditions when assembly is initiated. They appear in endosperm after a rapid recovery from the low temperature shock (7) or under the influence of taxol (Fig. 1 m in reference 34). They have been observed in the root of the fern Azolla (20) and in Haemanthus endosperm after various experimental treatments (7,35) that change the critical concentration of tubulin. A high temperature shock produces such converging centers in the streak stage of sea urchin eggs (cf. 19,39) and taxol in animal cytoplasts (28).
Spontaneous formation of MT converging centers, which in endosperm cytoplasts are unstable intermediates, must involve several steps. The irregular meshwork always present immediately after preparation in large anucleate fragments is composed of intermingled long MTs. The final structures (e.g., multiple phragmoplasts) are composed from shorter MTs. Therefore formation of an organized structure must involve either breaking of MTs or, more likely, their partial disassembly. The nucleation step in vitro is thermodynamically more difficult than the elongation. Only 8 subunits are necessary to form a seed (42) in vitro. However, in very small cytoplasmic fragments, the MT meshwork does not reorganize  Fig. 11. Explanation in text. 812 cells. E-, M-, and L-anaphase, early, mid-, and late anaphase. Stages of anaphase are defined on Fig. 11. Explanation in text. 339 cells. E-, M-, and L-anaphase, early, mid-,and late anaphase. Stages ofanaphase are defined on Fig. 11. Explanation m text. 576 cells. E-, M-, and L-anaphase, early, mid-, and late anaphase. even if it contains over 30,000 tubulin dimers (assuming ~ 1,700 dimers/1/~m of MT [33]). This suggests that a threshold concentration oftubulin pool, accessory proteins, number of free MT ends, etc., are needed in vivo for the MT reorganization to proceed and to form a specific structure. The second MT configuration, which is usually formed by large converging center(s), is the cytoplasmic spindle, which invariably transforms into a phragmoplast with a cell plate. Several MT configurations might be interpreted as intermediates between the converging center and the phragmoplast. Work in progress may determine whether phragmoplasts originate in two different ways, i.e., by fusion of two MT cones and/or internal reorganization. A phragmoplast, however, is always the final structure. This MT organization may therefore be related to intrinsic properties of MTs, either alone or with superimposed regulatory factors.
Our data do not exclude the existence of hypothetical polar organizers (diffuse centrosomes; 31). We believe, however, that the polar reorganization may also be determined without their existence by an increase of local concentration oftubulin in minute domains by a continuous assembly/disassembly of MT free ends, perhaps modified by free Ca 2÷ (29), calmodulin (41), and lateral association of polymerized MTs.
The transformation and rearrangement of MTs in endosperm, both in cytoplasts and mitotic spindles, may be related to the thermodynamically optimal polymer length in vivo and specific properties of (-) ends of MTs. Since the polymer size in vitro is limited and determined by specific conditions (37), the maximum length of cytoplasmic spindles/phragmoplasts may correspond to the optimal length of polymer (MTs) in vivo, similarly as suggested for cytoplasmic MTs (12). This assumption would also explain why in certain conditions existing MTs do not elongate and new MTs and/ or MTFTs are formed instead. Such configurations deserve special attention. Because they are found both in interphase and in mitosis, they are of general occurrence and may represent a basic unit in MT turnover in plant endosperm. The problem is more complex, however, because both MTFTs and single MTs form within the wide polar region and cytoplasm in anaphase/telophase and elongate in all directions. If converging ends of MTs are stabilized by capping, then the overall critical concentration decreases and assembly/disassembly is determined by the properties of (+) ends. Thus specific molecular conditions, especially critical concentration, in minute cell domains must be precisely regulated and determine formation of MTFTs vs. single MT and the direction of their growth. Thus our data are consistent with the model of MT nucleation proposed by De Brabander (13).
The Structural Polarity and Elongation of MTs. The structural polarity of cytoplasmic phragmoplasts and MTFTs is not known. The polarity of the mitotic spindle and phrag- Figure 13. Asters in mid-anaphase. Very flattened cell. There is asynchrony in aster development and they form between late mid-to late anaphase (cf. with Fig. 3). This particular cell has developed exceptionally large asters at mid-anaphase (cf. Fig. 6b), which is reproduced at the same magnification. The asters extend between the trailing chromosome arms into the interzone and converge toward a small defined polar region. Note criss-crossing of MTs within aster. MT arrangement and orientation strongly suggest that their proximal to the nucleus ends terminate over a wide area and not in well defined domain. Such arrangements are difficult to explain by discrete organelle type nucleating centers. Bar, 10 urn. Anaphase Assembly. The formation of polar MTs in the acentriolar Haemanthus spindle (9) involves MT assembly. Therefore unknown controls in the polar region must promote the nucleation and elongation of astral (polar) MTs concurrently with shortening of kinetochore MTs. The elongation may be the result of on/off rates at the (+) and (-) MT ends combined with local controls, which may turn out to be of primary importance. The few long or single MTs (our methods do not permit us to distinguish between a single and few MTs) detected often as extensions of the poles (Fig. 6a) may be MTs elongating at their (-) ends. Such polar organization is often pronounced when nucleation is inhibited but mitosis not arrested permanently (35).
Our technique does not permit a quantification of tubulin. However, the comparison of the sizes of asters, often seen by mid-anaphase (Fig. 13), and half-spindles (Fig. 6)

suggests that
Haemanthus asters and phragmoplasts together may contain more polymerized tubulin than the half-spindles at any stage. Thus, disassembly of the half-spindle is not sufficient to account for the formation of polar MTs, suggesting in turn that additional tubulin becomes competent to polymerize in anaphase-telophase. Asters in cytoplasmic phragmoplasts are always small or nonexistent, suggesting that chromosomes may be involved in regulation of the polymerizable tubulin pool, as suggested by Lambert (30).
Spindle Elongation. Centriole/centrosome complex and asters, if present, allow for little variation in the organization of the polar region. If present and detected in the light microscope, they also allow precise measurement of halfspindle length in vivo.
Acentriolar spindles of higher plants lack a known MT organizing organelle and their poles are usually not well defined. This prevents precise measurements of spindle length in vivo. The only organization that can be followed in vivo based on conventional (i.e., without image processing system), polarizing microscopy, and DIC, is that of the chromosomal fibers. It is impossible, however, to determine where they terminate. Thus, the only method to measure the entire halfspindle length in plants is an indirect method using immunolabeling of MTs. If chromosomal fibers are considered to be the measure of half-spindle length, then the acentriolar halfspindle shortens gradually during anaphase (22, 23). However, if the length of the KFC is measured, then the half-spindle length remains constant throughout a considerable part of anaphase and shortens only toward the end of chromosome migration (cf. schematic drawing, Fig. 14). A probable cause of the increase in length is the sudden jump apart of chromosomes at the onset of anaphase, which for Haemanthus is -4 um (30); it corresponds well to an increase of spindle length at metaphase/anaphase transition, reported here. These two sets of data obtained with different methodologies (polarizing microscope and IGS) are therefore complementary.
Previous observations (9,35) document that the assembly of new MTs occurs during anaphase. The present data demonstrate that during the first two-thirds of anaphase, when KF shorten, the length of the half-spindle remains constant. This indicates that the polar region, i.e., the region between poorly defined ends of KFs and the spindle periphery, or the ends of KFCs, elongate concurrently with shortening of KF. We also assume that the shape of the cell and the spindle may influence the direction of new MT growth. This partly explains the asynchrony of their formation and why they are pronounced in flattened cells and all syncytia. Moreover, a considerable variation of spindle elongation is expected in higher plant endosperm, as well as the existence of well developed polar MTs and small asters in long unflattened cells. Our observations are consistent with these assumptions, but the basic function of the half-spindle remains unknown (Fig. 14).
Our attempts to measure directly the elongation rate of polar MTs in living cells, using time-lapse films made with a high extinction polarizing microscope, but without image processing, were not conclusive due to the weak birefringence (see Fig. 8). Previous observations on living cells indicated, however, that chromosomes move faster than the spindle fibers decrease in length (2). Indirect measurements of polar MTs on IGS preparations suggest that their elongation is faster (2 um/min) than chromosome movement which in anaphase is 1-1.5 um/min. Although they are indirect, they are the only data available on the average rate of polar MT elongation. The actual rate of at least some MTs in vivo is probably higher. The rates are consistent, however, with our work on the transport of granules (in progress). These data and the analysis of particle movements suggest that some MTs in the living cells may elongate much faster than average, as implied by the work in vitro (3 !). The speed of kinetochore migration in anaphase is -1 um/min, while some acentric chromosome fragments move to the equator in anaphase 8 um/min (4), and the speed of 4 um/min is very common. These fragments are most likely pushed by the elongating MTs, as are backward-moving chromosomes in taxol-treated cells (8,34). This suggests that the elongation of polar MTs toward the equator may exert a considerable pushing force and may even break chromosomes (3). Such a force may contribute to chromosome movement and perhaps may be sufficient to push the half-spindles against the cytoplasm and account for spindle elongation (35).
MTFT and Spindle Organization. The basic question is whether MTFT spindle organization is the result of specific optical properties of immunogold as visualized by the light microscope (superimposing of optical images, etc.), increasing divergence of KFC during anaphase (18), preparatory methods (e.g., flattening of cells), etc. Extensive discussion of these problems is beyond the scope of this paper and will be presented elsewhere (10). These data demonstrate the existence of fir tree-like structure of numerous, but not all, individual KFC, both in flattened and unflattened spindles in controls. However, MTFT spindle organization of all KFC is very clear when the number of MTs is reduced, e.g., by low temperature (6"C) which does not arrest mitosis permanently, and chromosome segregation is completed (36). Similarly, when the assembly of branches of the MTFTs is inhibited by plant MT-specific drugs at physiological concentrations (micro-and nanomolar range), anaphase is instantly arrested (9,35). Thus, the whole functional spindle from prometaphase to late anaphase seems to be composed from associated MTFTs. Such organization may be essential for chromosome transport. It remained undetected until the introduction of IGS techniques and was demonstrated in the unflattened cells with the image enhancement system (10). The density of MT arrangement does not allow its easy detection with conventional light microscopy. The skew arrangement of MTs around the KFC in an umbrella-like fashion was practically impossible to detect in a polarizing microscope as the optical signal of the superimposed branches of the KFC cancel each other. The northern light phenomenon or flaming chromosomal fiber observed in Haemanthus (22) and in the meiotic spindle of grasshoppers (25) is most likely the result of continuous lateral rearrangements due to the formation of MTFTs. We conclude, therefore, that MTFT organization of KFC(s) may be necessary for the fiber(s) to function. We should caution, however, that the splaying of kinetochore fibers observed in vivo (18) in Haemanthus is difficult to distinguish from the formation of MT fir trees in unflattened cells. We hope that work in progress (10) may permit both better understanding of these structures and their role in mitosis. We believe also that such organization is probably of wide occurrence and is not restricted to the acentriolar spindle of the endosperm tissue. Spindles of other organisms contain large percentages of MTs that are skew to the long axis of the spindle. They have been demonstrated by electron microscopic analysis of crane fly spermatocytes (17), and the data of Schaap and Forer (38) are consistent with such organization. This suggests that the basic structure of the KFC is the same in both the acentriolar spindle of Haemanthus and the centriolar spindle of crane fly.
We conclude that the MTFT is a newly described structural and functional unit of MT organization in Haernanthus endosperm. It occurs in cell fragments and intact cells during interphase and mitosis. The reorganization of MTs in cell fragments reflects the intrinsic properties of MTs in vivo, which occur both in interphase and mitosis. Reorganization during anaphase leads to pronounced assembly of new polar MTs in front of disassembling KFs. Polar MTs elongating toward the equator may push half-spindles apart and contribute to polar separation.
Immunogold GAR-20 and GAR-15 and antibodies were a generous gift from Dr. Jan De Mey (Janssen Pharmaceutica, Beerse, Belgium). It is a pleasure to thank Dr. J. Wilczyfiski for help with statistical