Microtubule flux mediates poleward motion of acentric chromosome fragments during meiosis in insect spermatocytes.

We applied a combination of laser microsurgery and quantitative polarization microscopy to study kinetochore-independent forces that act on chromosome arms during meiosis in crane fly spermatocytes. When chromosome arms located within one of the half-spindles during prometa- or metaphase were cut with the laser, the acentric fragments (lacking kinetochores) that were generated moved poleward with velocities similar to those of anaphase chromosomes (approximately 0.5 microm/min). To determine the mechanism underlying this poleward motion of detached arms, we treated spermatocytes with the microtubule-stabilizing drug taxol. Spindles in taxol-treated cells were noticeably short, yet with polarized light, the distribution and densities of microtubules in domains where fragment movement occurred were not different from those in control cells. When acentric fragments were generated in taxol-treated spermatocytes, 22 of 24 fragments failed to exhibit poleward motion, and the two that did move had velocities attenuated by 80% (to approximately 0.1 microm/min). In these cells, taxol did not inhibit the disjunction of chromosomes nor prevent their poleward segregation during anaphase, but the velocity of anaphase was also decreased 80% (approximately 0.1 microm/min) relative to untreated controls. Together, these data reveal that microtubule flux exerts pole-directed forces on chromosome arms during meiosis in crane fly spermatocytes and strongly suggest that the mechanism underlying microtubule flux also is used in the anaphase motion of kinetochores in these cells.

Organelles are transported on sliding microtubules in Reticulomyxa.

Organelles and plasma membrane domains appear to be transported along Reticulomyxa's microtubule cytoskeleton. Previously we demonstrated that organelle and cell surface transport share the same enzymatic properties and suggested that both are powered by the same cytoplasmic dynein. Motility analysis in Reticulomyxa is complicated by the fact that the microtubules also are motile and appear to "slide" bidirectionally throughout the network. We have utilized laser ablation to address this frame-of-reference problem as to how each transport component (microtubule sliding vs. organelle translocations) contributes to reactivated bidirectional translocation of organelles along the microtubule cytoskeleton. Laser ablation was used to cut microtubule bundles from lysed networks into 4-15-microm segments. After examining these reactivated cut fragments, it appears that the majority of organelles did not move relative to microtubule fragments, but remained attached to microtubules and moved as the microtubules slid. Microtubule sliding stops after 1-2 min and cannot be reactivated even when perfused with fresh ATP. Furthermore, once sliding stops, organelle transport also stops. Our findings indicate that the majority of Reticulomyxa pseudopodial organelles do not move along the surface of the microtubules, rather it is the sliding of the microtubules to which they are attached that moves them.

Centrosome-independent mitotic spindle formation in vertebrates.

BACKGROUND: In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS: These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.

Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase.

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage.

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 microm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a "start anaphase" reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Charge Dependent Distribution of Endogenous Proteins within Vitellogenic Ovarian Follicles of Actias luna.

In vitellogenic ovarian follicles of Actis luna, internal Ca(2+) activity currents create an electrical gradient which influences the distribution of charged macromolecules between nurse cells and oocyte. We show that, between oocyte and nurse cells, there is an ionic gradient of 1-12 mV with the nurse cells being more electronegative than the oocyte by an average 3.5+/-0.2 mV(s.e.)(p<0.001). As previously reported for another saturniid, Hyalophora cecropia, the transbridge ionic gradient of luna: (1) is focused across the intercellular bridges, (2) is abolished by 200 &mgr;M vanadate and (3) includes a [Ca(2+)](i) gradient. Endogenous soluble proteins collected from control and from vanadate treated populations of nurse cells and oocytes were separated by two-dimensional (2-D) gel electrophoresis and visualized with sliver stain. Densitometric analysis showed that 14 out of the 19 acidic proteins and six of the eight basic proteins studied, changed their oocyte-to-nurse cell distribution in consort with change in the transbridge ionic gradient. This suggests that a transbridge ionic gradient may be, at least within the saturniidae, a method for maintaining different molecular concentrations in nurse cells compared to oocytes. Copyright 1997 Elsevier Science Ltd. All rights reserved

Chromosome fragments possessing only one kinetochore can congress to the spindle equator.

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.

A synergy of technologies: combining laser microsurgery with green fluorescent protein tagging.

When focused through an objective lens with a high numerical aperture, nanosecond pulses of high-intensity green (532-nm) laser light can be used to selectively destroy any cellular component whose boundaries can be defined by light microscopy. These components include, for example, chromosomes, spindle fibers, bundles of keratin, or actin filaments, mitochondria, vacuoles, and so forth. In addition, the definition of poorly resolved components can be enhanced for selective destruction by tagging one or more of their constituent proteins with green fluorescence protein (GFP). As a example we show that the centrosome in living PtK1 cells can be clearly defined, and then destroyed by green laser light, after transforming the cells with gamma-tubulin/GFP fusion protein. In some transformed cells it is even possible to target and selectively destroy just one of the centrioles.

Long crocidolite asbestos fibers cause polyploidy by sterically blocking cytokinesis.

Asbestos has been described as a physical carcinogen in that its carcinogenic effects appear to be related primarily to fiber dimensions. It has been hypothesized that long asbestos fibers may interfere with chromosome distribution during cell division, causing genomic changes that lead to cell transformation and neoplastic progression. Using high-resolution time-lapse light microscopy and serial-section electron microscopy, we have followed individual crocidolite asbestos fibers through the later stages of cell division in LLC-MK2 epithelial cells, and have detailed for the first time their effect on cytokinesis. We found that long fibers (15-55 microgram), trapped by the cleavage furrow, sterically blocked cytokinesis, sometimes resulting in the formation of a binucleated cell. The ends of blocking fibers were usually found within invaginations of the newly formed nuclei. Nuclear envelope-fiber attachment was evident when a chromatin strand ran with the fiber into the intercellular bridge. Such strands may break, causing chromosome structural rearrangements. Our data are the first to show that individual crocidolite fibers can cause genomic changes by sterically blocking cytokinesis and that fiber length and affinity for the nuclear envelope are important factors. Such genomic changes may be among the initial events leading to asbestos-induced cancers.

The force for poleward chromosome motion in Haemanthus cells acts along the length of the chromosome during metaphase but only at the kinetochore during anaphase.

The force for poleward chromosome motion during mitosis is thought to act, in all higher organisms, exclusively through the kinetochore. We have used time-lapse. video-enhanced, differential interference contrast light microscopy to determine the behavior of kinetochore-free "acentric" chromosome fragments and "monocentric" chromosomes containing one kinetochore, created at various stages of mitosis in living higher plant (Haemanthus) cells by laser microsurgery. Acentric fragments and monocentric chromosomes generated during spindle formation and metaphase both moved towards the closest spindle pole at a rate (approximately 1.0 microm/min) similar to the poleward motion of anaphase chromosomes. This poleward transport of chromosome fragments ceased near the onset of anaphase and was replaced. near midanaphase, by another force that now transported the fragments to the spindle equator at 1.5-2.0 microm/min. These fragments then remained near the spindle midzone until phragmoplast development, at which time they were again transported randomly poleward but now at approximately 3 microm/min. This behavior of acentric chromosome fragments on anastral plant spindles differs from that reported for the astral spindles of vertebrate cells, and demonstrates that in forming plant spindles, a force for poleward chromosome motion is generated independent of the kinetochore. The data further suggest that the three stages of non-kinetochore chromosome transport we observed are all mediated by the spindle microtubules. Finally, our findings reveal that there are fundamental differences between the transport properties of forming mitotic spindles in plants and vertebrates.

The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores.

During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.

Behavior of crocidolite asbestos during mitosis in living vertebrate lung epithelial cells.

Asbestos has been described as a physical carcinogen in that long thin fibers are generally more carcinogenic than shorter thicker ones. It has been hypothesized that long thin fibers disrupt chromosome behavior during mitosis, causing chromosome abnormalities which lead to cell transformation and neoplastic progression. Using high-resolution time lapse video-enhanced light microscopy and the uniquely suited lung epithelial cells of the newt Taricha granulosa, we have characterized for the first time the behavior of crocidolite asbestos fibers, and their interactions with chromosomes, during mitosis in living cells. We found that the keratin cage surrounding the mitotic spindle inhibited fiber migration, resulting in spindles with few fibers. As in interphase, fibers displayed microtubule-mediated saltatory movements. Fiber position was only slightly affected by the ejection forces of the spindle asters. Physical interactions between crocidolite fibers and chromosomes occurred randomly within the spindle and along its edge. Crocidolite fibers showed no affinity toward chromatin and most encounters ended with the fiber passively yielding to the chromosome. In a few encounters along the spindle edge the chromosome yielded to the fiber, which remained stationary as if anchored to the keratin cage. We suggest that fibers thin enough to be caught in the keratin cage and long enough to protrude into the spindle are those fibers with the ability to snag or block moving chromosomes.

Effects on individuals with mental retardation of changing Depakote to Depakene.

This study was conducted to determine whether Depakene could be substituted for Depakote, which would represent a significant financial savings, without sacrificing symptom control or drug tolerance. Over an 8-week period of intensive monitoring, we changed 77 patients from Depakote to Depakene. Results showed no change in seizure control, no adverse upper gastrointestinal side effect, no weight change, no sleep disturbance, no change in aberrant behavior, and no change in appetite. Patients were less less lethargic on Depakene than on Depakote. However, there was some increase in diarrhea, of uncertain cause. Some changes in psychiatric symptoms were also noted. Overall, this drug change was well-tolerated.

The force-producing mechanism for centrosome separation during spindle formation in vertebrates is intrinsic to each aster.

A popular hypothesis for centrosome separation during spindle formation and anaphase is that pushing forces are generated between interacting microtubules (MTs) of opposite polarity, derived from opposing centrosomes. However, this mechanism is not consistent with the observation that centrosomes in vertebrate cells continue to separate during prometaphase when their MT arrays no longer overlap (i.e., during anaphase-like prometaphase). To evaluate whether centrosome separation during prophase/prometaphase, anaphase-like prometaphase and anaphase is mediated by a common mechanism we compared their behavior in vivo at a high spatial and temporal resolution. We found that the two centrosomes possess a considerable degree of independence throughout all stages of separation, i.e., the direction and migration rate of one centrosome does not impart a predictable behavior to the other, and both exhibit frequent and rapid (4-6 microns/min) displacements toward random points within the cell including the other centrosome. The kinetic behavior of individual centrosomes as they separate to form the spindle is the same whether or not their MT arrays overlap. The characteristics examined include, e.g., total displacement per minute, the vectorial rate of motion toward and away from the other centrosome, the frequency of toward and away motion as well as motion not contributing to separation, and the rate contributed by each centrosome to the separation process. By contrast, when compared with prometaphase, anaphase centrosomes separated at significantly faster rates even though the average vectorial rate of motion away from the other centrosome was the same as in prophase/prometaphase. The difference in separation rates arises because anaphase centrosomes spend less time moving toward one another than in prophase/prometaphase, and at a significantly slower rate. From our data we conclude that the force for centrosome separation during vertebrate spindle formation is not produced by MT-MT interactions between opposing asters, i.e., that the mechanism is intrinsic to each aster. Our results also strongly support the contention that forces generated independently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by interactions between opposing astral MTs in the interzone.

Centriole duplication in lysates of Spisula solidissima oocytes.

A cell-free system has been developed that executes centriole duplication. Surf clam (Spisula solidissima) oocytes, arrested at late prophase of meiosis I, do not contain centrioles, centrosomes, or asters. Serial section high-voltage electron microscopy (HVEM) of asters and spindles isolated from potassium chloride-activated oocytes indicates that within 4 minutes oocytes assemble a single centriole that is duplicated by 15 minutes when assembly of the first meiotic spindle is complete. A mixture of lysates from unactivated oocytes and potassium chloride-activated oocytes induces centriole formation and duplication. Astral microtubule content in these lysate mixtures increases with time.

Crocidolite asbestos fibers undergo size-dependent microtubule-mediated transport after endocytosis in vertebrate lung epithelial cells.

The large respiratory epithelial cells within primary cultures of newt (Taricha granulosa) lung are uniquely suited for high resolution video-enhanced light-microscopic studies. We show here that these cells incorporate crocidolite asbestos fibers within 18 h by endocytosis. Once inside the cell, fibers less than 5 microns in length are seen by video light microscopy to undergo saltatory transport at a maximum velocity of 1.18 microns/s. By contrast, fibers over 5 microns long rarely exhibit saltatory motion. Over time, all of the fibers become preferentially located near the nucleus. This perinuclear accumulation is largely inhibited by disassembling the cytoplasmic microtubules with nocodazole. Same cell correlative light and electron microscopy reveal that fibers exhibiting saltatory behavior are enclosed within a membrane. From these observations we conclude that, upon incorporation into epithelial cells, asbestos fibers undergo size-dependent active transport along cytoplasmic microtubules. Our data are the first to link the dimension-dependent transforming ability of asbestos fibers to a basic cellular function, i.e., the microtubule-dependent transport of cellular components.

Shape, size, and distribution of cell structures by 3-D graphics reconstruction and stereology. I. The regulatory volume decrease of astroglial cells.

This report discusses fundamental limitations in attempting to derive cell size, shape, or distribution from the two-dimensional images provided by conventional electron microscopy. Morphometric or stereologic measurement of random thin sections is a convenient way to obtain some information of this type. However, it cannot provide complete, objective information about real size, shape, or connectivity of cells containing irregular or unevenly distributed structures or nonuniform populations of cells. Anisotropic structures require analysis of a complete set of serial sections. The analysis may utilize either stereo, mono, or tilted optical slices, and subsequent integration of this information into a single 3-D computer data set. In this study, we analyze stereo pairs of high-voltage electron micrographs of serial thick sections (0.5 micron) and critical-point-dried whole-cell mounts of rat brain astroglial cell cultures. The Z-axis resolution is increased by digitizing contours at discrete levels within each stereo view. This is accomplished with a new type of stereoscopic contouring device. We calculated area and volume changes accompanying hypo-osmolar swelling and spontaneous reversal of the swelling. (Regulatory Volume Decrease-RVD). An understanding of the mechanism of swelling of astroglial cells is important for improving the treatment of brain injury. The total cell-volume results are comparable with results previously obtained using nonmetabolized, radioactively tagged compounds that diffuse into various cell compartments. Our serial-section and whole-cell data also provide new information about the relative swelling of nucleus, cytoplasm, and individual organelles such as mitochondria. The basic biological problem being approached is whether homeostasis of cell function is accompanied by surface area and volume regulation of enzyme-rich membranes and organelles. Conversely, it is proposed to explore the possibility that abnormal organelle areas and volumes are indicators of perturbations of cell division, metabolism, or gene expression.

Resolution as a function of accelerating voltage in electron microscopy of semithick biological specimens.

In the past, biological sections ranging in thickness from 0.10- to 0.50-micron have usually been examined with high-voltage (greater than 500 kV) electron microscopes (HVEM). Now investigators are increasingly using intermediate voltage (200-500 kV) electron microscopes (IVEM), which are more readily available and demand less maintenance. In a study of "typical" plastic-embedded, stained sections of mouse liver ranging from 0.10 to 1.0 micron thick, we determined the resolution obtainable at 100, 200, and 1000 kV. At all three accelerating voltages the resolution (2.7 nm) for 0.10-micron sections was limited only by the sections stain granularity. For 0.25-micron thickness the resolutions were 5.8, 3.1, and 3.1 nm at 100, 200, and 1000 kV, respectively. The maximum usable thickness at 200 kV with resolution sufficient to resolve membranes clearly was between 0.75 and 1.0 micron, depending on the magnification. Resolution at 100 kV was adequate for screening sections up to 1.0-micron thick for preparation defects prior to examination with an IVEM or HVEM.