Involvement of an acrosinlike proteinase in the sulfhydryl-induced degradation of rabbit sperm nuclear protamine.

Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike.

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force.

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.

The effects of isopropyl N-phenyl carbamate on the green alga Oedogonium cardiacum. I. Cell division.

Cell division in vegetative filaments of the green alga Oedogonium cardiacum is presented as an experimental system. We report on how we have used this system to study the effects of isopropyl N-phenylcarbamate (IPC) on the mitotic apparatus and on the phycoplast, a planar array of cytokinetic microtubules. Polymerization of microtubules was prevented when filaments, synchronized by a light/dark regime and chilled (2 degrees C) while in metaphase or just before phycoplast formation, were exposed to 5.5 x 10(-4) M IPC and then returned to room temperature. Spindles reformed or phycoplasts formed when these filaments were transferred to growth medium free of IPC. However, the orientation of both microtubular systems was disturbed: the mitotic apparatus often contained three poles, frequently forming three daughter nuclei upon karyokinesis; the phycoplast was often stellate rather than planar, and it sometimes was displaced to the side of both daughter nuclei, resulting in a binucleate and an anucleate cell upon cytokinesis. Our results suggest that IPC (a) prevents the assembly of microtubules, (b) increases the number of functional polar bodies, and (c) affects the orientation of microtubules in O. cardiacum. High voltage (1,000 kV) electron microscopy of 0.5-microm thick sections allowed us to visualize the polar structures, which were not discernible in thin sections.

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.

Organization of spindle microtubules in Ochromonas danica.

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

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

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

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

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

On the mechanism of anaphase spindle elongation in Diatoma vulgare.

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

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.

An extended corona attached to metaphase kinetochores of the green alga Oedogonium.

Mitotic cells of the green alga Oedogonium were treated with the anti-microtubule agent oryzalin (1.0-0.1 microM) for 5 to 10 min. Within 5 min treatment of living cells, metaphase spindles became spherical with disorganized chromosomes, and anaphase spindles collapsed. At lower concentrations, the effects were slower, and partial recovery was observed about 10 to 20 min after the drug was washed out. Following breakdown of the spindle, considerable disorganized activity detected by time-lapse continued within the nucleus, isolated from the cytoplasm by its intact nuclear membrane. Under the electron microscope, spindle microtubules (MTs) were absent in oryzalin-treated cells. Paired metaphase kinetochores displayed an array of fine filamentous material extended, usually straight, about 3 microns into the nucleoplasm. In cells recovering from oryzalin treatment, MTs became associated with kinetochores in the usual manner. However, this filamentous array, the "extended corona" (EC), was almost undetectable, even when the MTs were short and poorly organized. The EC is appreciably larger by metaphase than the corona of prophase chromosomes and so it may assemble during early mitosis. Fine filaments interspersed with kinetochore MTs have been described in carefully fixed cells of this alga (M.J. Schibler, J.D. Pickett-Heaps, Eur. J. Cell Biol. 22, 687-698 (1980)). The EC apparently represents a less organized form of this material remaining after its scaffold of MTs has been removed. These fibers appear involved in MT capture upon spindle recovery from anti-MT drugs. They could function during prometaphase and even anaphase movement along spindle MTs.

Valve morphogenesis and the microtubule center of the diatom Hantzschia amphioxys.

The siliceous valve of Hantzschia is briefly described, preparatory to following its morphogenesis. While Hantzschia is morphologically very different to Pinnularia, there are several subtle but important similarities in detail. Bundles of microfilaments, significant in generating the gliding motion of these cells, line the cytoplasmic fissure of the raphe. Valve morphogenesis in Hantzschia has been followed in live cells and by using transmission electron microscopy. Major re-organization and translocation of individual organelles, and also the nascent wall and its associated system of structural cytoplasmic components, are described. The spindle always forms on the concave side of the cell, opposite the raphes in the keels. A "polar complex" near each spindle pole consolidates into the "microtubule center" (MC) after cytokinesis. Each MC moves around its daughter nucleus to the center of the completed cleavage furrow and comes to rest on the narrow silicalemma running along the center of the cell. Microfilaments line first one and then both sides of the silicalemma before it grows outwards and begins to secrete the wall. Microtubules (MTs) extend along the silicalemma from the MC and directly over the future raphe; a compact row of mitochondria is organized along these MTs, flanking each MC. After about one hour in this central position, the whole assemblage of MC, MTs, mitochondria and silicalemma with its forming wall, move laterally across to the convex side of the parent valves. Now, the valve thickens steadily. The fibulae that hold the wall together where it is perforated by the raphe, grow out as flanges fusing with the transverse ribs across the keel. As in Pinnularia, the raphe fissure is occluded precisely adjacent to the MC, which also suppresses formation of the fibulae in this restricted region. The nascent wall structure suggests that silica is being precipitated on to a fibrous base. After valve formation, each MC migrates back to its interphase position at the concave side of the daughter cell. The MC undergoes characteristic morphological changes during these morphogenetic events and remains throughout tightly associated with a pronounced evagination of the nucleus. Ultrastructural comparisons suggest that the Hantzschia symmetry could have originally been derived from that of Pinnularia.

Directionally controlled spindle disassembly after mitosis in the diatom Pinnularia.

Two aspects of late mitosis have been investigated further. First, the two interdigitated half spindles that form the central spindle of this organism appear to separate before their disassembly. This separation is not dependent upon the cleavage furrow breaking the spindle as appears to be the case of observing normal mitosis; it occurs even when cleavage is artificially prevented with cytochalasin D. After separation, the birefringence of each half spindle fades steadily from the overlap end (now, the "free" end) back to the pole. Disassembly commences about 1 1/2 min after the half spindles separate, synchronously in each daughter cell. This observation suggests that the microtubules in each half spindle are of uniform polarity and their disassembly, presumably influenced by this polarity, proceeds from only one end (the "free" end). The other end, however, may be "capped" by polar structures. Because there is this pause between separation and the initiation of disassembly, breakdown of microtubules is probably not initiated merely by their ends becoming free in the cytoplasm.

Mitosis in Oedogonium: spindle microfilaments and the origin of the kinetochore fiber.

New ultrastructural observations of mitosis in the closed spindle of Oedogonium cardiacum have been made using cells fixed with glutaraldehyde and tannic acid. Fine filaments 5 to 8 nm in diameter are attached to kinetochores from prophase through anaphase. Some are free in the early division nucleus while others emanate from forming kinetochores at prophase when few if any microtubules (MTs) are inside the nucleus. During prometaphase, MTs invade the nucleus from the poles and appear to interact with the microfilaments. Early in prometaphase, numerous MTs are laterally associated with kinetochores, and the kinetochore fiber is often formed first at one kinetochore of a pair. During metaphase and anaphase, the microfilaments are interspersed among the MTs of these kinetochore fibers. There also is an ill-defined matrix concentrated in the kinetochore fiber, and MTs are often coated irregularly with osmiophilic material. Live mitotic cells of Oedogonium were studied using time lapse cinematography, and we correlate these observations with the above results. We conclude that these microfilaments may constitute one structural component of the traction apparatus that moves chromosomes during metakinesis and anaphase, and that at least some (and possibly many) of the MTs of the kinetochore fiber are derived from those entering the nucleus at prometaphase.

The effects of colchicine and dinitrophenol on the in vivo rates of anaphase A and B in the diatom Surirella.

The rates of chromosome-to-pole movement (anaphase A) and pole-pole separation (anaphase B) in vivo were measured in the pennate diatom Surirella, using differential interference contrast (DIC) light microscopy. In control cells, the rate of anaphase A is 1.6 +/- 0.6 micron/min, the rate of anaphase B is 2.3 +/- 0.3 micron/min, and the extent of anaphase B is 26.7 +/- 9.7% of metaphase spindle length. Colchicine was added to metaphase cells in order to inhibit any further addition of microtubule (MT) subunits onto the spindle. Colchicine, which does not break down the well-ordered Surirella central spindle, caused no significant change in the rate of anaphase A (1.3 +/- 0.3 micron/min) while it significantly decreased both the rate of anaphase B (1.2 +/- 0.4 micron/min) and the extent of anaphase B (14.8 +/- 8.3% of metaphase spindle length). Surirella cells were also treated with the metabolic inhibitor 2-4-dinitrophenol (DNP) in order to test the effects of energy depletion on anaphase. When DNP was added early in anaphase A, prior to the completion of sister chromosome separation, anaphase A was inhibited. When DNP was added after initiation of sister chromosome separation, anaphase A continued to completion, although at a lower rate than control cells (0.5 +/- 0.2 micron/min). Anaphase B was completely inhibited by DNP, but upon recovery from DNP resumed at a normal rate (2.2 +/- 0.5 micron/min) and progressed to a slightly larger than normal extent (44.0 +/- 13.0% of metaphase length).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on kinetochore function in mitosis. I. The effects of colchicine and cytochalasin on mitosis in the diatom Hantzschia amphioxys.

Cytochalasin does not affect mitosis (including half spindle separation and disassembly) while cleavage is partially or totally inhibited. While colchicine stops central spindle growth, it resists breakdown even after prolonged exposure. Prometaphase chromosome movement soon ceases, and some attached chromosomes slowly detach; these phenomena are correlated with a loss of the numerous microtubules (MTs) that emanate from the poles, with which chromosomes interact. "C-anaphase", often seen, is marked in vivo by spindle elongation and unequal polar distribution of those chromosomes still attached to the central spindle; this stage is characterized ultrastructurally by the accumulation of dense matrix material, probably the "collar" previously described, at the poles. Kinetochores often remain tightly associated with this matrix. We believe this result is significant, since it clearly demonstrates that the kinetochores are attached to a spindle component other than microtubules. We suspect that this matrix is contractile and part of the mitotic machinery for moving chromosomes. These colchicine effects are not reversible.

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

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

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

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

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

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

UV-microbeam irradiations of the mitotic spindle: spindle forces and structural analysis of lesions.

Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region. Changes in organization of MTs in the irradiated region were analyzed by EM serial section analysis coupled with 3-D computer reconstruction. Metaphase cells irradiated 2 to 4 microns below the spindle pole (imaged by polarization optics) lost birefringence in the irradiated region. Peripheral spindle fibers, previously curved to focus on the pole, immediately splayed outwards when severed. We demonstrate via serial section analysis that following irradiation the lesion was devoid of MTs. Within 30 s to 1 min, recovery in live cells commenced as the severed spindle pole moved toward the metaphase plate closing the lesion. This movement was concomitant with the recovery of spindle birefringence and some of the severed fibers becoming refocused at the pole. Ultrastructurally we confirmed that this movement coincided with bridging of the lesion by MTs presumably growing from the pole. The non-irradiated half spindle also lost some birefringence and shortened until it resembled the recovered half spindle. Anaphase cells similarly irradiated did not show recovery of birefringence, and the pole remained disconnected from the remaining mitotic apparatus. Reconstructions of spindle structure confirmed that there were no MTs in the lesion which bridged the severed spindle pole with the remaining mitotic apparatus. These results suggest the existence of chromosome-to-pole spindle forces are dependent upon the existence of a MT continuum, and to a lesser extent to the loss of MT initiation capacity of the centrosome at the metaphase/anaphase transition.

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

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