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C L Rieder

Publications and source records attributed to C L Rieder.

At least 19 recordsLinked to original sources
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↗
PubMed1991

Chromosome motion during attachment to the vertebrate spindle: initial saltatory-like behavior of chromosomes and quantitative analysis of force production by nascent kinetochore fibers.

Before forming a monopolar attachment to the closest spindle pole, chromosomes attaching in newt (Taricha granulosa) pneumocytes generally reside in an optically clear region of cytoplasm that is largely devoid of cytoskeletal components, organelles, and other chromosomes. We have previously demonstrated that chromosome attachment in these cells occurs when an astral microtubule contacts one of the kinetochores (Hayden, J., S. S. Bowser, and C. L. Rieder. 1990. J. Cell Biol. 111:1039-1045), and that once this association is established the chromosome can be transported poleward along the surface of the microtubule (Rieder, C. L., and S. P. Alexander. 1990. J. Cell Biol. 110:81-95). In the study reported here we used video enhanced differential interference contrast light microscopy and digital image processing to compare, at high spatial and temporal resolution (0.1 microns and 0.93 s, respectively), the microtubule-mediated poleward movement of attaching chromosomes and poleward moving particles on the spindle. The results of this analysis demonstrate obvious similarities between minus end-directed particle motion on the newt pneumocyte spindle and the motion of attaching chromosomes. This is consistent with the hypothesis that both are driven by a similar force-generating mechanism. We then used the Brownian displacements of particles in the vicinity of attaching chromosomes to calculate the apparent viscosity of cytoplasm through which the chromosomes were moving. From these data, and that from our kinetic analyses and previous work, we calculate the force-producing potential of nascent kinetochore fibers in newt pneumocytes to be approximately 0.1-7.4 x 10(-6) dyn/microtubule) This is essentially equivalent to that calculated by Nicklas (Nicklas, R.B. 1988. Annu. Rev. Biophys. Biophys. Chem. 17:431-449) for prometaphase (4 x 10(-6) dyn/microtubule) and anaphase (5 x 10(-6) dyn/microtubule) chromosomes in Melanoplus. Thus, within the limits of experimental error, there appears to be a remarkable consistency in force production per microtubule throughout the various stages of mitosis and between groups of diverse taxonomic affinities.

Animals↗
PubMed1991

Studies on the ejection properties of asters: astral microtubule turnover influences the oscillatory behavior and positioning of mono-oriented chromosomes.

The position of a mono-oriented chromosome changes as it oscillates to and from the pole to which it is attached. Such oscillatory behavior reveals that the net force on a mono-oriented chromosome is constantly changing. Fluctuations may occur in both the polewardly directed force acting at the kinetochore and the opposing outwardly directed force associated with the aster. We have examined the ejection properties of the aster--as well as the oscillatory behavior and positioning of mono-oriented chromosomes--in relation to astral microtubule turnover. We treated cells containing monopolar spindles with drugs that affect microtubule turnover, either by promoting the depletion of dynamically unstable astral microtubules (nocodazole and colcemid) or by augmenting their numbers and stability (taxol). Both types of drugs stopped the oscillatory behavior of mono-oriented chromosomes within seconds. The final position of the chromosomes depended on how microtubule turnover was affected. In the case of nocodazole and colcemid, non-kinetochore astral microtubules were depleted first and the kinetochore-to-pole distance shortened. In these cells chromosome fragments generated by laser microsurgery were no longer expelled from the center of the aster. By contrast, with taxol the number of non-kinetochore microtubules increased and the astral ejection force became stronger as shown by the finding that the chromosomes moved away from the pole to the periphery of the monaster. Moreover, arms severed from chromosomes at the periphery of the taxol monaster failed to move further away from the aster's center. From these observations we conclude that the oscillatory movements and changing position of a mono-oriented chromosome relative to the pole are mediated by changes in the number of astral microtubules. The dynamic instability of astral microtubules that leads to a rapid turnover may contribute to the astral ejection force by allowing the continual growth of microtubules out from the aster. Growing astral microtubules may exert a pushing force that their rigidity maintains until their depolymerization.

Alkaloids↗
PubMed1990

Keratin filaments restrict organelle migration into the forming spindle of newt pneumocytes.

When viewed by light microscopy the mitotic spindle of newt pneumocytes appears to assemble in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which is often much larger than the spindle. This clear area is also frequently larger than the region previously occupied by the nucleus. It forms even in prometaphase cells depleted of microtubules prior to nuclear envelope breakdown by colchicine treatment. Time-lapse video microscopy reveals that as prometaphase proceeds this clear area slowly and progressively collapses around the forming spindle so that it is greatly diminished or nonexistent by the onset of anaphase. The sharply defined nature of the boundary between the clear area and the remaining cytoplasm and the fact that organelles accumulate at its periphery suggest that a structural barrier is present at the boundary that limits organelle migration into the forming spindle. Immunofluorescence and electron microscopy, of cells previously followed in the living state, reveal that the periphery of the clear area contains prominent bundles of keratin filaments but lacks microtubules and actin. From our observations we conclude that keratin filaments form a loosely organized cage that surrounds the forming newt pneumocyte spindle. We propose that this cage functions, in part, to restrict the dispersion of chromosomes during nuclear envelope breakdown and to impede the bulk migration of organelles into the forming spindle.

Animals↗
PubMed1990

Diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20.

Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30-3.0-microns) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2-3-microns wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early-to-mild anaphase cells the separating sister plates are connected by chromatin-containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its pole-facing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1-0.3 micron dia.) rods varying considerably (0.15-2.3 microns) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.

Anaphase↗
PubMed1990

A simple pneumatic device for plunge-freezing cells grown on electron microscopy grids.

A detailed design for a simple and inexpensive variable-speed (1.0-5.8 m s-1) pneumatic plunge-freezing device is presented. Cultured cells, grown on Formvar-coated 75-mesh gold finder grids, are pneumatically driven into a stirring mixture of propane/isopentane (3:1) cooled by liquid nitrogen (LN2). Premature freezing of the sample in the cryogenic vapors above the cryogen is prevented by plunging through an entry tube into an insulating box, to which a partial vacuum is applied. The cryogenic vapors are drafted into the box at the level of the liquid cryogen by the vacuum, thereby preventing a layer of cold gas from collecting above the cryogen. To prevent the sample from thawing during transfer from the cryogen to the substitution medium, the box top is removed and compressed air is forced through a corrugated tube running the length of the box. The resulting boiling LN2 creates an atmosphere below -120 degrees C in which the transfer can be accomplished.

Animals↗
PubMed1990

Formation of the astral mitotic spindle: ultrastructural basis for the centrosome-kinetochore interaction.

The formation of the astral mitotic spindle is initiated at the time of nuclear envelope breakdown from an interaction between the replicated spindle poles (i.e. centrosomes) and the chromosomes. As a result of this interaction bundles of microtubules are generated which firmly attach the kinetochores on each chromosome to opposite spindle poles. Since these kinetochore fibers are also involved in moving the chromosomes, the mechanism by which they are formed is of paramount importance to understanding the etiology of force production within the spindle. As a prelude to outlining such a mechanism, the dynamics of spindle formation and chromosome behavior are examined in the living cell. Next, the properties of centrosomes and kinetochores are reviewed with particular emphasis on the structural and functional changes that occur within these organelles as the cell transits from interphase to mitosis. Finally, a number of recent observations relevant to the mechanism by which these organelles interact are detailed and discussed. From these diverse data it can be concluded that kinetochore fiber microtubules are derived from dynamically unstable astral microtubules that grow into, or grow by and then interact laterally with, the kinetochore. Moreover, the data clearly demonstrate that the interaction of a single astral microtubule with one of the kinetochores on an unattached chromosome is sufficient to attach the chromosome to the spindle, orient it towards a pole, and initiate poleward motion. As the chromosomes move into the region of the forming spindle more astral microtubules become incorporated into the nascent kinetochore fibers and chromosome velocity decreases dramatically. During this time the distribution of spindle microtubules changes from two overlapping radial arrays to the fusiform array characteristic of metaphase cells.

Animals↗
PubMed1990

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells.

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.

Animals↗
PubMed1990

Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control.

The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis. The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosome-specific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.

Animals↗
PubMed1990

Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells.

When viewed by light microscopy the mitotic spindle in newt pneumocytes assembles in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which can be much larger than the forming spindle. This unique optical property has allowed us to examine the behavior of individual microtubules, at the periphery of asters in highly flattened living prometaphase cells, by video-enhanced differential interference-contrast light microscopy and digital image processing. As in interphase newt pneumocytes (Cassimeris, L., N. K. Pryer, and E. D. Salmon. 1988. J. Cell Biol. 107:2223-2231), centrosomal (i.e., astral) microtubules in prometaphase cells appear to exhibit dynamic instability, elongating at a mean rate of 14.3 +/- 5.1 microns/min (N = 19) and shortening at approximately 16 microns/min. Under favorable conditions the initial interaction between a kinetochore and the forming spindle can be directly observed. During this process the unattached chromosome is repeatedly probed by microtubules projecting from one of the polar regions. When one of these microtubules contacts the primary constriction the chromosome rapidly undergoes poleward translocation. Our observations on living mitotic cells directly demonstrate, for the first time, that chromosome attachment results from an interaction between astral microtubules and the kinetochore.

Animals↗
PubMed1990

Stability of microtubule attachment to metaphase kinetochores in PtK1 cells.

Kinetochore microtubules are known to be differentially stable to a variety of microtubule depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubule attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtK1 spindles at 23 degrees C when microtubule assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34 microM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10-20 min interval. During this shortening process, the number of kinetochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24 +/- 5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubule attachment to the kinetochore was approximately 7.5 min. These results show that when microtubule assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubule surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.

Animals↗