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C L Rieder

Publications and source records attributed to C L Rieder.

At least 55 records · Page 3Linked to original sources
PubMed1995

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.

Animals↗
PubMed1995

Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes.

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.

Animals↗
PubMed1995

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.

Anaphase↗
PubMed1995

Kinetochore motility after severing between sister centromeres using laser microsurgery: evidence that kinetochore directional instability and position is regulated by tension.

During mitosis in vertebrate somatic cells, the single attached kinetochore on a mono-oriented chromosome exhibits directional instability: abruptly and independently switching between constant velocity poleward and away from the pole motility states. When the non-attached sister becomes attached to the spindle (chromosome bi-orientation), the motility of the sister kinetochores becomes highly coordinated, one moving poleward while the other moves away from the pole, allowing chromosomes to congress to the spindle equator. In our kinetochore-tensiometer model, we hypothesized that this coordinated behavior is regulated by tension across the centromere produced by kinetochore movement relative to the sister kinetochore and bulk of the chromosome arms. To test this model, we severed or severely weakened the centromeric chromatin between sister kinetochores on bi-oriented newt lung cell chromosomes with a laser microbeam. This procedure converted a pair of tightly linked sister kinetochores into two mono-oriented single kinetochore-chromatin fragments that were tethered to their chromosome arms by thin compliant chromatin strands. These single kinetochore-chromatin fragments moved substantial distances off the metaphase plate, stretching their chromatin strands, before the durations of poleward and away from the pole movement again became similar. In contrast, the severed arms remained at or moved closer to the spindle equator. The poleward and away from the pole velocities of single kinetochore-chromatin fragments in prometaphase were typical of velocities exhibited by sister kinetochores on intact chromosomes from prometaphase through midanaphase A. However, severing the chromatin between sister kinetochores uncoupled the normally coordinated motility of sister kinetochores. Laser ablation also uncoupled the motilities of the single kinetochore fragments from the bulk of the arms. These results reveal that kinetochore directional instability is a fundamental property of the kinetochore and that the motilities of sister kinetochores are coordinated during congression by a stiff centromere linkage. We conclude that kinetochores act as tensiometers that sense centromere tension generated by differential movement of sister kinetochores and their chromosome arms to control switching between constant velocity P and AP motility states.

Animals↗
PubMed1994

Directed movement of chromosome arms and fragments in mitotic newt lung cells using optical scissors and optical tweezers.

A pulsed-laser microbeam at 532 nm wavelength (optical scissors) and a laser-induced optical trap at 1064 nm wavelength (optical tweezers) have been successively combined to dissect and manipulate chromosomes in live newt lung epithelial cells. These preliminary experimental results demonstrated that chromosome fragments dissected by laser microbeam surgery, regardless of their size, could be easily pulled or rotated by optical forces when positioned at the periphery of the mitotic spindle. In addition, chromosome arms which were not subjected to laser microsurgery also could be moved with the optical tweezers at the spindle periphery. In our previous study on rat kangaroo kidney cells (PTK2), this degree of facilty in manipulating chromosome movement was not possible, most likely due to the close proximity of the intermediate filament "cage" to the spindle. It is concluded herein that optical scissors and tweezers can be used in combination to study the interaction of chromosomes with the mitotic spindle in cells where the peripheral regions of the spindle are unobstructed by intermediate filaments. This can be performed on newt cells, where the diameter of the cage can be substantially larger than the diameter of the spindle.

Animals↗
PubMed1994

Meiosis in Drosophila males. I. The question of separate conjunctive mechanisms for the XY and autosomal bivalents.

The conjunctive mechanism of the XY bivalent is believed to differ from that of the autosomal bivalents in the achiasmate Drosophila melanogaster male. It has been proposed that hypothetical cohesive elements, termed collochores, hold the X and Y chromosomes together at or near their nucleolar organizing regions (NORs) and that collochores are not exhibited by autosomal bivalents. In electron micrographs, unique fibrillar material is observed between the X and Y chromosomes at the synaptic site. Recently, the 240 bp nontranscribed spacer associated with rRNA genes at the NOR has been implicated as the essential DNA sequence for XY pairing. To test whether this DNA sequence is always associated with XY pairing and to determine its relationship to the unique fibrillar material, we studied the XY bivalent in Drosophila simulans. The D. simulans Y chromosome has few, if any, rRNA genes, but does have a large block (3,000 kb or 12,500 copies) of the nontranscribed spacer repeat located at the distal end of its long arm. This is in contrast to the D. melanogaster Y, which has the repeat located among rRNA genes on its short arm. Using light and electron microscopy, we show that the X does indeed pair with the distal end of the long arm of the D. simulans Y. However, no fibrillar material is evident in serial thin sections of the D. simulans XY bivalent, suggesting that this material (in D. melanogaster) may be remnants of the NOR rather than a morphological manifestation of the hypothetical collochores.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Centrosome and kinetochore movement during mitosis.

During the past year important progress has been made in refining our understanding of how chromosomes become equally distributed to daughter cells during mitosis. Unlike the situation in diatoms and yeast, it now appears that spindle pole (centrosome) separation during spindle formation and anaphase B is mediated in vertebrates primarily by an astral pulling, and not a pushing, mechanism. Kinetochore motility is directionally unstable, which has important consequences for how chromosomes move to the equator of the forming spindle. Finally, the observation that sister chromatid disjunction occurs even in the presence of high levels of maturation promoting factor reveals that the series of biochemical events responsible for this phenomenon is not an obligatory part of the pathway by which the cell exits mitosis.

Anaphase↗
PubMed1994

Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle.

We argue that hypotheses for how chromosomes achieve a metaphase alignment, that are based solely on a tug-of-war between poleward pulling forces produced along the length of opposing kinetochore fibers, are no longer tenable for vertebrates. Instead, kinetochores move themselves and their attached chromosomes, poleward and away from the pole, on the ends of relatively stationary but shortening/elongating kinetochore fiber microtubules. Kinetochores are also "smart" in that they switch between persistent constant-velocity phases of poleward and away from the pole motion, both autonomously and in response to information within the spindle. Several molecular mechanisms may contribute to this directional instability including kinetochore-associated microtubule motors and kinetochore microtubule dynamic instability. The control of kinetochore directional instability, to allow for congression and anaphase, is likely mediated by a vectorial mechanism whose magnitude and orientation depend on the density and orientation or growth of polar microtubules. Polar microtubule arrays have been shown to resist chromosome poleward motion and to push chromosomes away from the pole. These "polar ejection forces" appear to play a key role in regulating kinetochore directional instability, and hence, positions achieved by chromosomes on the spindle.

Anaphase↗
PubMed1994

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

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

Anaphase↗
PubMed1994

Microtubule assembly and kinetochore directional instability in vertebrate monopolar spindles: implications for the mechanism of chromosome congression.

We have proposed previously a kinetochore motor-polar ejection model for chromosome congression to the metaphase plate where forces generated at the kinetochore are antagonized by away-from-the pole forces generated within each half-spindle on the chromosome arms. This model was based in large part on observations of the behavior of chromosomes on monopolar spindles. In these cells chromosomes typically become attached to the pole by only one kinetochore fiber. These mono-oriented chromosomes move to positions away from the pole even though they are pulled poleward at their kinetochores. Their arms are also ejected away from the pole when severed from the centromere. Here we have characterized further the properties of monopolar spindles in newt lung epithelial cells to determine the similarities between monopolar and bipolar spindles. We found no significant differences between monopolar and bipolar spindles over the parameters examined, which included: microtubule dynamics as measured by fluorescence redistribution after photobleaching; the ability of polar microtubule arrays to push chromosome arms away from the pole; the dependence of chromosome position relative to the pole on microtubule assembly; the number of kinetochore microtubules per kinetochore; and the directional instability of kinetochore motion during chromosome oscillations poleward and away-from-the-pole. As in bipolar spindles, kinetochore directional instability is characterized by abrupt switching between constant velocity phases of poleward and away-from-the-pole motion. From these data we conclude that the mechanism(s) responsible for chromosome positioning in monopolar spindles are fundamentally the same as those in bipolar spindles; only the geometry of the two spindle forms and the interplay between sister kinetochore directional instabilities are different. We also found no correlation in the kinetochore-to-pole distance with kinetochore microtubule number in monopolar spindles, but a strong qualitative correlation with microtubule density. This finding indicates that oscillations of mono-oriented chromosomes in both monopolar and bipolar spindles occur because chromosomes persist in poleward motion until they reach a density of polar microtubules sufficiently high to promote switching to away-from-the-pole motion. As the kinetochore and chromosome arms move away-from-the-pole, microtubule density decreases and the kinetochore switches to poleward motion, pulling the chromosome arms back into regions of higher microtubule density. The mechanism regulating kinetochore switching between poleward and away-from-the-pole motion is poorly understood, but may depend on tension at the kinetochore generated by pushing forces on the chromosome arms produced by the polar microtubule arrays.

Animals↗
PubMed1993

Structure of the colcemid-treated PtK1 kinetochore outer plate as determined by high voltage electron microscopic tomography.

High voltage electron microscopic tomography was used to determine the organization of the kinetochore plate and its attachment to the underlying chromosome. Six reconstructions were computed from thick sections of Colcemid-treated PtK1 cells and analyzed by a number of computer graphics methods including extensive thin slicing, three-dimensional masking, and volume rendering. When viewed en-face the kinetochore plate appeared to be constructed from a scaffold of numerous 10-20-nm thick fibers or rods. Although the fibers exhibited regions of parallel alignment and hints of a lattice, they were highly variable in length, orientation and spacing. When viewed in stereo, groups of these fibers were often seen oriented in different directions at different depths to give an overall matted appearance to the structure. When viewed "on edge," the plate was 35-40 nm thick, and in thin slices many regions were tripartite with electron-opaque domains, separated by a more translucent middle layer, forming the inner and outer plate boundaries. These domains were joined at irregular intervals. In some slices, each domain appeared as a linear array of 10-20-nm dots or rods embedded in a less electron-opaque matrix, and adjacent dots within or between domains often appeared fused to form larger blocks. The plate was connected to the underlying chromosome by less densely arrayed 10-20-nm thick fibers that contacted the chromosome-facing (i.e., inner) surface of the plate in numerous patches. These patches tended to be arrayed in parallel rows perpendicular to the long axis of the chromosome. In contrast to connecting fibers, corona fibers were more uniformly distributed over the cytoplasmic-facing (i.e., outer) surface of the plate. When large portions of the reconstructions were viewed, either en-face or in successive slices parallel to the long axis of the chromosome, the edges of the plate appeared splayed into multiple "fingers" that partly encircled the primary constriction. Together these observations reveal that regions of the kinetochore outer plate contain separate structural domains, which we hypothesize to serve separate functional roles. Our three-dimensional images of the kinetochore are largely consistent with the hypothesis that the outer plate is composed of multiple identical subunits (Zinkowski, R. P., J. Meyne, and B. R. Brinkley. 1991. J. Cell Biol. 113:1091-1110).

Animals↗
PubMed1993

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.

Anaphase↗
PubMed1992

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.

Animals↗
PubMed1992

Chromosome mal-orientation and reorientation during mitosis.

We argue that mal-orientation of mitotic chromosomes is not as rare as once believed. However, unlike bivalents during meiosis I, the reorientation of a mal-oriented mitotic chromosome has yet to be observed. This appears to be due, in part, to the difficulty in differentiating mal-oriented chromosomes from mono-oriented ones which are common during spindle formation in living mitotic cells. We assume that mitotic cells possess mechanisms for correcting chromosome mal-orientations that are similar to those operating during meiosis. However, unlike meiosis, where reorientation appears to be triggered when tension on a K-fiber is relieved or reduced, other factors related to the close proximity of sister kinetochores may also induce reorientation in mal-oriented mitotic chromosomes. We favor a model in which the reorientation of a mitotic kinetochore depends on, and is initiated by, the kinetochore capturing MTs from the pole to which it is reorienting.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Chromosome fiber dynamics and congression oscillations in metaphase PtK2 cells at 23 degrees C.

A bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2 cells. PtK2 cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 microns or greater. DTAF-tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF-tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti-beta tubulin antibody. At 2-5 minutes after injection, concentrated DTAF-tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF-tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 microns) of concentrated DTAF-tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF-containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF-containing segments: 1) a poleward flux of tubulin subunits (Mitchison, 1989) or 2) capture of DTAF-containing nonkinetochore microtubules.

Animals↗
PubMed1991

Mitosis: towards a molecular understanding of chromosome behavior.

The past year has seen important contributions made to resolving how chromosomes attach to and move on the mitotic spindle of animal cells. These include the findings that: kinetochore microtubules are derived from the asters (i.e. centrosomes); poleward chromosome motion need not be coupled to kinetochore microtubules disassembly; the motor for poleward chromosome motion is associated with the kinetochore; and immunological evidence that this motor is cytoplasmic dynein.

Anaphase↗