The attachment of chromosomes to the mitotic spindle and the production of aneuploidy in newt lung cells.
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Aneuploidy↗
Biomedical subjects
C L Rieder
Publications and source records attributed to C L Rieder.
Microtubules associate with actin-containing filaments at discrete sites along the ventral surface of Allogromia reticulopods.
We have investigated the distribution of actin and microtubules in pseudopodial networks (reticulopods) of the protozoan Allogromia sp., strain NF, in order to help elucidate the respective roles these components play in network organization and motility. Double-label fluorescence studies with tubulin antibodies and tetramethyl-rhodamine (TMR)-phalloidin reveal that microtubules and filamentous actin co-localize in regions where trunk pseudopods contact the substratum and splay to form the pseudopodial network; distal to these regions the network contains numerous microtubules but little or no F-actin. Similar results were obtained using various commercial actin antibodies. Correlative anti-actin immunofluorescence and high-voltage electron microscopy of serial 0.25 micron sections reveal that actin is contained within discrete electron-opaque, fan-shaped structures distributed along the cytoplasmic aspect of the ventral reticulopodial membrane. Electron microscopy of serial 100 nm sections from conventionally fixed specimens confirms that these actin-rich plaques are composed of a felt of roughly parallel, 5 nm diameter filaments. A subset of parallel and often bundled microtubules is enmeshed within, or contacts the periphery of, these filament plaques. Upon leaving a plaque, bundled microtubules frequently splay into smaller bundles. These observations are consistent with the hypothesis that interactions between microtubules and actin-containing microfilaments, particularly at substratum adhesion points, are involved in various aspects of reticulopodial motility, particularly network morphogenesis and cell body locomotion.
Actin Cytoskeleton↗
Flexible-substratum technique for viewing cells from the side: some in vivo properties of primary (9+0) cilia in cultured kidney epithelia.
Cells cultured on thin plastic (e.g. Formvar, Teflon, polycarbonate) membranes can be clearly imaged from the side in vivo by video microscopy. We have used this flexible-substratum technique to examine the behaviour and properties of primary cilia in confluent cultures of the kidney epithelial cell lines PtK1, PtK2, LLC-PK1, MDCK and BSC-40. In these cells primary cilia appear as rigid rods, up to 55 micron long, which project at various angles from the dorsal cell surface. The length distribution of primary cilia in confluent cultures is a distinct characteristic of each established kidney cell line examined, with LLC-PK1 exhibiting three distinct length populations. Primary cilia of kidney cell lines bend passively in response to flow but do not display propagated bending or vortical motions. Up to 26% of the cilia in the cell types examined possess one or more conspicuous swellings along the ciliary shaft. Treatment with 0.05% trypsin, which is sufficient to cause cell rounding, does not induce the resorption or shedding of the cilium. These direct observations demonstrate that kidney epithelial-cell primary cilia are non-motile and longer than previously thought, and suggest that their length represents a phenotypic marker for each cell line.
Animals↗
Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption.
The effects of colcemid (0.16-1.0 microM) and taxol (10 microM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high-voltage electron microscopy of serial 0.25-micron sections. Although these drugs induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubules, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5-7 h at 37 degrees C and then form 4N "micronucleated" restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration-dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16-0.25 microM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (greater than or equal to 1.0 microM) concentrations. However, even in the presence of 1.0 microM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell-cycle-dependent assembly and disassembly of axonemal microtubules in vivo.
Alkaloids↗
Naturally occurring tubulin-containing paracrystals in Allogromia: immunocytochemical identification and functional significance.
Bundles of microtubules (MTs) are readily visualized in vivo by videomicroscopy in highly flattened reticulopodia of the foraminiferan protozoan Allogromia sp. strain NF. In this report we use videomicroscopy, immunocytochemistry, and high-voltage electron microscopy to characterize the dynamic changes that occur in this extensive MT cytoskeleton, and in the associated cytoplasmic transport, during induced withdrawal and subsequent reextension of reticulopodia. Within seconds after application of the withdrawal stimulus (seawater substitute made hypertonic with MgCl2) intracellular bidirectional transport along linear MT-containing fibrils ceases and is replaced by an inward, constant-velocity flow of cytoplasm along the fibrils. As withdrawal continues, most fibrils become wavy and coalesce to form phase-dense pools. These wavy fibrils and phase-dense pools contain a paracrystalline material and few if any MTs. Same-section correlative immunofluorescence and high-voltage electron microscopy reveal that the paracrystalline material contains tubulin. During recovery linear fibrils (MTs) rapidly extend from the phase-dense pools (paracrystals), which concurrently shrink in size, thus reestablishing normal network morphology and motility. We conclude that the MT cytoskeleton in Allogromia reticulopodia is transformed during withdrawal into a tubulin-containing paracrystal, which serves as a temporary reservoir of MT protein and an initiation site for MT regrowth.
Animals↗
Novel extracellular matrix and microtubule cables associated with pseudopodia of Astrammina rara, a carnivorous Antarctic foraminifer.
Astrammina rara is a benthic foraminiferan protozoan which uses an extensive network of fine, branching, and anastomosing pseudopodia to capture and digest metazoans up to 12 mm long. During such predation the pseudopodia appear remarkably elastic and tensile. Electron microscopy has revealed a novel extracellular matrix of thin branching fibers associated with A. rara's pseudopodia, which are otherwise typical in appearance. These fibers are structurally associated with the pseudopodial glycocalyx. Cytoplasmic microtubules are often found in close juxtaposition to the plasma membrane and overlying extracellular fibers. In the main pseudopodial trunks bundles of 30-300 microtubules are surrounded by a lightly staining matrix and linked by occasional bridges. The microtubules follow straight trajectories in distal filopodia, but in pseudopodial trunks they appear to coil around one another to form cables. These microtubular cables and extracellular fibers are novel features of A. rara's pseudopodia and may provide the structural basis for their tensile strength.
Animals↗
Tomographic three-dimensional reconstruction of cilia ultrastructure from thick sections.
We have applied a computer-based tomographic technique to reconstruct the three-dimensional ultrastructure of newt lung cilia. Epon-embedded samples were cut into 0.25-micron-thick sections that were imaged at 1 MV with a high-voltage electron microscope. For the reconstruction shown, a tilt series of 53 micrographs was taken at tilt angles between -54 degrees and +50 degrees. The reconstruction was accomplished from these projections using a weighted back-projection algorithm. The 12-nm resolution of the reconstruction was sufficient to resolve the outer doublet and central pair microtubules, dynein arms, radial spokes, and central sheath structures. The reconstruction can be viewed from various angles and with appropriate parts cut away to reveal structural features of interest. The sense of depth in these views can be enhanced by stereo viewing of shaded surface images. From this reconstruction, we determined that newt lung cilia contain the more common triplet grouping of radial spokes.
Animals↗
Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle.
During mitosis a monooriented chromosome oscillates toward and away from its associated spindle pole and may be positioned many micrometers from the pole at the time of anaphase. We tested the hypothesis of Pickett-Heaps et al. (Pickett-Heaps, J. D., D. H. Tippit, and K. R. Porter, 1982, Cell, 29:729-744) that this behavior is generated by the sister kinetochores of a chromosome interacting with, and moving in opposite direction along, the same set of polar microtubules. When the sister chromatids of a monooriented chromosome split at the onset of anaphase in newt lung cells, the proximal chromatid remains stationary or moves closer to the pole, with the kinetochore leading. During this time the distal chromatid moves a variable distance radially away from the pole, with one or both chromatid arms leading. Subsequent electron microscopy of these cells revealed that the kinetochore on the distal chromatid is free of microtubules. These results suggest that the distal kinetochore is not involved in the positioning of a monooriented chromosome relative to the spindle pole or in its oscillatory movements. To test this conclusion we used laser microsurgery to create monooriented chromosomes containing one kinetochore. Correlative light and electron microscopy revealed that chromosomes containing one kinetochore continue to undergo normal oscillations. Additional observations on normal and laser-irradiated monooriented chromosomes indicated that the chromosome does not change shape, and that the kinetochore region is not deformed, during movement away from the pole. Thus movement away from the pole during an oscillation does not appear to arise from a push generated by the single pole-facing kinetochore fiber, as postulated (Bajer, A. S., 1982, J. Cell Biol., 93:33-48). When the chromatid arms of a monooriented chromosome are cut free of the kinetochore, they are immediately ejected radially outward from the spindle pole at a constant velocity of 2 micron/min. This ejection velocity is similar to that of the outward movement of an oscillating chromosome. We conclude that the oscillations of a monooriented chromosome and its position relative to the spindle pole result from an imbalance between poleward pulling forces acting at the proximal kinetochore and an ejection force acting along the chromosome, which is generated within the aster and half-spindle.
Anaphase↗
The reproduction of centrosomes: nuclear versus cytoplasmic controls.
The tight coordination normally found between nuclear events and the doubling of centrosomes at each cell cycle suggests that nuclear activities may be part of the mechanism that controls the reproduction of centrosomes. To determine if this is the case, we used a micropipette to completely remove the nucleus from eggs of the sea urchin Lytechinus variegatus at prophase of the first mitosis, leaving only one centrosome in the cell. The subsequent behavior of this centrosome was then followed in vivo with the polarization microscope. In all cases the centrosome reproduced in a precise 1:2:4:8 fashion with a periodicity that was slightly slower than the centrosome cycle of control eggs. The cell cycle-related changes in centrosome morphology were identical to those of control eggs in that: (a) the astral birefringence varied cyclically to a normal extent, (b) the astral focus enlarged and then flattened during the telophase equivalent, (c) cleavage furrows were initiated as the astral birefringence faded, and (d) daughter centrosomes separated before the increase in astral birefringence at the onset of each mitosis. To determine if centrioles also reproduced normally, enucleate eggs were followed in vivo until they contained eight centrosomes. They were then individually removed from the preparations, fixed, and embedded. Each egg was serially 0.25-micron sectioned for observation with the high voltage electron microscope. We completely reconstructed 23 centrosomes in four eggs; all centrosomes contained two centrioles apiece. These results demonstrate that the subunits for complete centrosome assembly can be stockpiled ahead of time and that the properly controlled use of these subunits for centrosome reproduction does not require nuclear transcription or nuclear DNA synthesis at each cell cycle.
Animals↗
Section thickness terminology: a source of confusion in microscopy.
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Histological Techniques↗
Centrosomes are required for the assembly of a bipolar spindle in animal cells.
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Animals↗
Digestion of prey in foraminifera is not anomalous: a correlation of light microscopic, cytochemical, and HVEM technics to study phagotrophy in two allogromiids.
Correlative light, high-voltage electron and conventional electron microscopic methods were used to investigate digestion in two allogromiid foraminiferans, Allogromia sp., strain NF, and A. laticollaris Arnold. Microscopic observations showed that bacterial prey are phagocytosed by reticulopodia and are transported to the allogromiid cell body within blister-like phagosomes. Larger prey (algae, diatoms) are transported along the reticulopodial surface and are either stored extrathalamously or phagocytosed at the oral opening (peduncle). Studies of allogromiids optimally fixed and labeled with an extracellular-space label (colloidal thorium) showed that phagocytosed prey are completely enclosed by a plasma membrane envelope; this finding was corroborated by a serial-section three-dimensional reconstruction of the oral zone of one allogromiid. Cytochemical staining for acid phosphatase showed that lysosomes are absent from reticulopods but abundant in the cell body, particularly in the oral zone cytoplasm. We conclude that digestion in allogromiid foraminiferans is accomplished by a vacuole-based digestive apparatus and not by extracellular digestion within a lacunary system, as has been suggested in earlier studies.
Acid Phosphatase↗
Centriole number and the reproductive capacity of spindle poles.
The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.
Animals↗
Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes.
We tested the ability of chromosomes in a mitotic cytoplasm to organize a bipolar spindle in the absence of centrosomes. Sea urchin eggs were treated with 5 X 10(-6) colcemid for 7-9 min before fertilization to block future microtubule assembly. Fertilization events were normal except that a sperm aster was not formed and the pronuclei remained up to 70 microns apart. After nuclear envelope breakdown, individual eggs were irradiated with 366-nm light to inactivate photochemically the colcemid. A functional haploid bipolar spindle was immediately assembled in association with the male chromosomes. In contrast to the male pronucleus, the female pronucleus in most of these eggs remained as a small nonbirefringent hyaline area throughout mitosis. High-voltage electron microscopy of serial semithick sections from individual eggs, previously followed in vivo, revealed that the female chromosomes were randomly distributed within the remnants of the nuclear envelope. No microtubules were found in these pronuclear areas even though the chromosomes were well-condensed and had prominent kinetochores with well-developed coronas. In the remaining eggs, a weakly birefringent monaster was assembled in the female pronuclear area. These observations demonstrate that chromosomes in a mitotic cytoplasm cannot organize a bipolar spindle in the absence of a spindle pole or even in the presence of a monaster. In fact, chromosomes do not even assemble kinetochore microtubules in the absence of a spindle pole, and kinetochore microtubules form only on kinetochores facing the pole when a monaster is present. This study also provides direct experimental proof for the longstanding paradigm that the sperm provides the centrosomes used in the development of the sea urchin zygote.
Animals↗
Evidence that cell surface motility in Allogromia is mediated by cytoplasmic microtubules.
We have previously shown that reticulopods of Allogromia sp. (strain NF) and Allogromia laticollaris display rapid, bidirectional saltatory transport of plasma membrane surface markers (i.e., polystyrene microspheres). Correlative video microscopic and electron microscopic methods were used to determine whether cytoskeletal components are involved in this surface transport. Such transport was observed only where the plasma membrane overlay cytoplasmic fibrils, which have been shown to be involved in organelle transport. Ultrastructural analysis indicated that these fibrils contain microtubules and an associated flocculent fibrillar material. In studies with nonionic detergents the surface marker particles remained bound to these microtubule-containing fibrils, even after the plasma membrane had been removed. Surface transport was inhibited when reticulopods were treated with agents that induce microtubule disassembly. Together these observations provide strong evidence that surface motility in Allogromia is mediated by labile cytoplasmic microtubules.
Animals↗
Correlative immunofluorescence and electron microscopy on the same section of epon-embedded material.
Semithick (0.25-0.50 micron) sections, cut from cells stained with fluorescein isothiocyanate (FITC)-conjugated antibodies prior to embedding in Epon, show high resolution patterns of immunofluorescence against a background void of autofluorescence. These same sections can then be viewed, after uranyl and lead staining, in the electron microscope. We clearly establish the specificity of this same-section correlative immunofluorescence-electron microscopy approach by showing that the immunofluorescent patterns observed in such sections of cells, stained prior to embedding for the indirect immunofluorescent localization of tubulin, follows the distribution of microtubules within the same sections as determined by electron microscopy. We then use this method to demonstrate for the first time that the 57 kD core protein of wound tumor virus is associated, at the ultrastructural level, with two distinct cellular inclusions in virally infected AC-20 cells. In some instances the fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section eliminates the need to employ more complex procedures for labeling antigens for ultrastructural detection. This technique, therefore, provides a rapid and simple first approach to many problems that require the ultrastructural localization of specific antigens.
Animals↗
Surface transport properties of reticulopodia: do intracellular and extracellular motility share a common mechanism?
The reticulopodial networks of the foraminiferan protozoans Allogromia sp., strain NF, and A. laticollaris display rapid (up to 11 microns/second) and bidirectional saltatory transport of membrane surface markers (polystyrene microspheres). Electron microscopy shows that microspheres adhere directly to the reticulopodial surface glycocalyx. A videomicroscopic analysis of this phenomenon reveals that microsphere movement is typically independent of pseudopod extension/withdrawal and that particles of different sizes and surface properties display similar motile characteristics. The motile properties of surface-associated microspheres appear identical to those of saltating intracellular organelles. Indeed, in some instances the surface-attached microspheres appear transiently linked in motion to these underlying organelles. Our observations suggest that, in reticulopodia, surface transport of microspheres and intracellular transport of organelles are driven by a common mechanism.
Animals↗
Multiple fission in Allogromia sp., strain NF (Foraminiferida): release, dispersal, and ultrastructure of offspring.
The release, dispersal, and ultrastructure of juveniles arising through multiple fission in the benthic foraminiferan Allogromia sp., strain NF (Lee & Pierce, 1963) has been examined by light and electron microscopy. An extensive reticulopodial network participates in the dispersal of fully differentiated young as they emerge from the fragmented parental test. During the earliest stages of release, offspring are of two classes--aroused and unaroused. Unaroused juveniles, which have not extended pseudopods, attach externally to the network and are transported bidirectionally along its surface. Aroused juveniles, which have extended pseudopods and are in protoplasmic continuity with the network, move quickly to the periphery of the network. Within 24 h, juveniles establish a communal "feeding reticulum" in which dispersed individuals are in protoplasmic continuity with neighbors via a common reticulopodial network. At the ultrastructural level, the cell body cytoplasm of unaroused juveniles contains numerous patches of a paracrystalline material, which disappears as their pseudopodia are extended to join the communal feeding reticulum. This paracrystalline material therefore appears to be a temporary reservoir of precursors required for pseudopod construction.
Animals↗