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H Ris

Publications and source records attributed to H Ris.

At least 19 recordsLinked to original sources
PubMed1989

A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum.

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.

Actins↗
PubMed1987

Structure of the cytoplasmic filament system in freeze-dried whole mounts viewed by HVEM.

Freeze-drying (FD) was used as an alternative to critical point-drying (CPD) for the preparation of whole mounts to study the cytoplasmic filament system in mammalian cultured cells by high voltage electron microscopy (HVEM). Rapid quenching methods such as plunging a grid into liquid propane cooled by LN2 or collision with a clean surface of a copper block cooled to LHe temperature were used to avoid ice crystal formation. For freeze-drying, a special apparatus was built that allowed the specimen to be kept at 145 K for 2-3 days at a vacuum of about 2 X 10(-7) Torr followed by gradual stepwise warming to room temperature. Purified skeletal muscle actin served as a test object for which the structure was known through independent techniques. PtK-1 cells, grown on gold grids and fixed in buffered glutaraldehyde, were used to study the structure of the cytoplasmic filament system. Under conditions that should avoid formation of ice crystals, the structure of actin fibres was as expected on the basis of previous studies, i.e. a tangle of independent uniform filaments about 7 nm thick, Using similar conditions, the cytoplasmic filaments of PtK-1 cells were equally distinct and evenly thick along their length and were usually associated with particles about 10 nm thick. On the other hand, when the sublimation time at the low temperature was cut short, both purified actin and the cytoplasmic filament system formed a network of tapering filaments devoid of particles, similar to that which has been described as a 'microtrabecular lattice'. We therefore conclude that when this structure is seen in FD preparations, it is probably the result of distortion produced by faulty FD procedure.

Actins↗
PubMed1986

A centriole-free Drosophila cell line. A high voltage EM study.

The problem of the absence of centrioles in cells of the 1182-4 line of Drosophila melanogaster has been reexamined with high voltage electron microscopy. A hypotonic treatment of the cells before fixation allowed a clear recognition of centrioles in 1 micron thick sections. Three different approaches were used to determine the presence of absence of centrioles in a Kc control cell line and in the 1182-4D cell line. 1) 1500 random 0.5 micron thick sections representing the equivalent of 60 whole cells of the 1182-4D line show no centrioles. In contrast, nearly all centrioles of the Kc cells were detected by this examination. 2) In a blind test 10 grids with either Kc or 1182-4D cells were correctly identified by the operator. In Kc cells, 4 to 6 diplosomes were observed by grid square on about 300 cell profiles, while no centrioles were seen in the sections of 1182-4D cells. 3) Complete serial sections 1 micron thick of whole 1182-4D cells were screened for presence or absence of centriole. No centriole was seen in any section. We conclude that these Drosophila 1182-4D cells, which have been maintained in culture for several years, are free of centrioles.

Animals↗
PubMed1985

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.

Actins↗
PubMed1985

Polarity of marginal-band microtubules in vertebrate erythrocytes.

Erythrocytes from three different vertebrate species (bullfrog, mudpuppy, and stinkpot) were isolated, and the polarity of the marginal-band microtubules was determined using a modification of the method described by Heidemann and McIntosh [12]. A considerable part of each marginal band was examined in serial sections to see whether any changes in the polarity pattern occurred along the length of the band. On the basis of analyses of over 45 marginal bands, the following observations were made: (i) different polarity patterns were present within a given species; (ii) the polarity pattern seen in one cross-section of the marginal band remained the same along its entire length; no evidence for a limited zone of overlap was obtained; (iii) centrioles were found in erythrocytes from all three species. The data on the polarity patterns and the presence of centrioles in erythrocytes are compatible with a model recently proposed for the organization of marginal-band microtubules in blood clams that invokes the activity of a microtubule organizing center [20]. We suggest that a modification of the process of marginal-band formation described in that model can account for our observations on the polarity of marginal-band microtubules.

Animals↗
PubMed1984

Electron microscopy of fine fibrin clots and fine and coarse fibrin films. Observations of fibers in cross-section and in deformed states.

Fine fibrin clots and coarse and fine fibrin films (both ligated and unligated), formed by shrinkage of clots in one dimension, were examined by electron microscopy. Specimens of clots were prepared by critical point drying and by embedding and sectioning; specimens of films were prepared by embedding and sectioning only. In the fine clots, network junctions appeared to be formed by fiber segments in which two or more protofibrils were gently twisted around each other for distances of the order of 200 nm and then diverged to give trifunctional branch points. This topology appeared to be preserved in the fine films. It is proposed that the strength of the junctions is primarily provided by the twisting topology, though reinforced by non-covalent bonding involving the B sites uncovered by thrombin. In coarse films, bundles of protofibrils, lying primarily in the film plane, had diameters of 40 to 200 nm and were gently twisted around each other to form thicker cables. Uniaxial stretching, up to 100%, of either fine or coarse film before fixing caused suprisingly extensive orientation of the protofibrils or bundles. However, random orientation was recovered if a stretched ligated film was allowed to retract to its original dimensions before fixing. In a stretched coarse film sectioned perpendicular to the stretch direction, fiber bundles could be seen in cross-section; these were roughly circular with scalloped edges. The changes with stretching and recovery are discussed in relation to possible mechanisms of deformation and elastic energy storage.

Animals↗
PubMed1984

Centriole distribution during tripolar mitosis in Chinese hamster ovary cells.

During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole.

Anaphase↗
PubMed1984

Novel organization of microtubules in cultured central nervous system neurons: formation of hairpin loops at ends of maturing neurites.

Using the high voltage electron microscope, we have examined cultured embryonic neurons in order to understand better the organization of microtubules in developing neurites. We found that, in embryonic chick retina neurons, microtubules were abundant in the ends of neurites and showed an unusual pattern of organization. Most striking was the presence of microtubule loops; after entering the flattened region of a growth cone, microtubules frequently made tight 180 degrees turns. Occasionally these looping microtubules re-entered the neurite and returned in the direction of the cell body. Positive identification of the loop structures as microtubules was made by specific immunocytochemical labeling. Quantitative analysis showed that more than half of the retina neurons that were dissociated on embryonic day 8 and kept in culture for 4 to 6 days (E8C4 and E8C6) contained at least one microtubule that made a 180 degrees turn at flat regions along or at the tips of neurites. The area within the loops typically contained larger membranous organelles, whereas only small vesicles were seen outside the loops. Fine filaments were seen to interconnect the loops at various places, suggesting the possibility that they played a role in maintaining the shape of microtubule loops. Examination of other neurons showed that tight microtubule loops were prominent in chick spinal cord neurons, but they were rarely seen in neurons of the sympathetic ganglia or dorsal root ganglia or in NG108-15 cloned cells. Developmentally, no loops were observed in E8C1 retina neurons, but retina neurons dissociated from older embryos (12 days) did show loops after 1 day in culture; these data suggest that microtubule loops may be abundant around embryonic day 12 to 13 in the chick retina. The possible significance of this unusual microtubule organization to the control of neurite growth and bidirectional transport is discussed.

Animals↗
PubMed1983

Ultrastructural networks in growth cones and neurites of cultured central nervous system neurons.

We have examined growth cones and neurites of cultured central nervous system neurons by high-voltage electron microscopy. Embryonic chicken retina cells were cultured on polylysine-treated and Formvar-coated gold grids for 2-6 days, fixed, and critical point dried. Growth cones and neurites were examined as unembedded whole mounts. Three-dimensional images from stereo-pair electron micrographs of these regions showed a high degree of ultrastructural articulation, with distinct, non-tapering filaments (5-9 nm in diameter) joining both cytoskeletal and membranous components. In the central regions of growth cones, interconnected structures included microtubules, large membranous sacs (up to 400 nm), and irregular vesicles (25-75 nm). A denser filamentous network was prevalent at the edges of growth cones. This network, which frequently adjoined the surface membrane, linked vesicles of uniform size (35-40 nm). Such vesicles often were seen densely packed in growth cone protrusions that were about the size of small synaptic boutons. Prevalent structural interconnections within growth cones conceivably could play a logistic role in specific membrane assembly, intracellular transport, endocytosis, and secretion. Because such processes are not unique to growth cones, the extensive linkages we have observed may have implications for cytoplasmic structure in general.

Animals↗
PubMed1983

Polarity of kinetochore microtubules in Chinese hamster ovary cells after recovery from a colcemid block.

The polarity of kinetochore microtubules was determined in a system for which kinetochore-initiated microtubule assembly has been demonstrated. Chinese hamster ovary cells were treated with 0.3 micrograms/ml colcemid for 8 h and then released from the block. Prior to recovery, microtubules were completely absent from the cells. The recovery was monitored using light and electron microscopy to establish that the cells progress through anaphase and that the kinetochore fibers are fully functional. Since early stages of recovery are characterized by short microtubule segments that terminate in the kinetochore fibrous corona rather than on the outer disk, microtubule polarity was determined at later stages of recovery when longer kinetochore bundles had formed, allowing us to establish unambiguously the spatial relationship between microtubules, kinetochores, and chromosomes. The cells were lysed in a detergent mixture containing bovine brain tubulin under conditions that allowed the formation of polarity-revealing hooks. 20 kinetochore bundles were assayed for microtubule polarity in either thick or thin serial sections. We found that 95% of the decorated kinetochore microtubules had the same polarity and that, according to the hook curvature, the plus ends of the microtubules were at the kinetochores. Hence, the polarity of kinetochore microtubules in Chinese hamster ovary cells recovering from a colcemid block is the same as in normal untreated cells. This result suggests that microtubule polarity is likely to be important for spindle function since kinetochore microtubules show the same polarity, regardless of the pattern of spindle formation.

Animals↗
PubMed1981

Structure of the mammalian kinetochore.

The structure of the mammalian trilaminar kinetochore was investigated using stereo electron microscopy of chromosomes in hypotonic solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D2O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed wihout pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore out layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observation of thick sections in stereo which made it possible to identify microtubules ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer.

Animals↗
PubMed1981

Structure of kinetochore fibers: microtubule continuity and inter-microtubule bridges.

To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 micron sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and in cells recovering from Colcemid arrest were similar in two respects: the number of microtubules per kinetochore (mean 14 and 12, respectively) and the nearest neighbor intermicrotubule distance (mean approximately 90 nm). In Colcemid recovered cells, over 90% of the microtubules in kinetochore fibers were attached to the kinetochore (i.e. kinetochore microtubules) are extended most or all of the distance to the pole. Few free microtubules were present in the kinetochore fibers; most non-kinetochore microtubules terminated in the pole. Since kinetochores in this Colcemid-recovered system have been demonstrated to nucleate microtubules (Witt et al., 1980), it seems likely that most if not all of these kinetochore microtubules originated at the kinetochore. Some of the reconstructed kinetochore fibers were attached to chromosomes with bipolar orientation, suggesting that kinetochore microtubules need not interact with many polar microtubules for orientation to occur. In Colcemid recovered cells lysed to reduce cytoplasmic background, microtubules in kinetochore fibers were preferentially preserved. The parallel and near-hexagonal order typical of microtubules in kinetochore fibers was maintained, as was the number of kinetochore microtubules (mean, 13). The intermicrotubule distance was slightly reduced in lysed cells (mean, 60 nm). Crossbridges about 5 nm wide and 30-40 nm long were visible in kinetochore fibers of lysed cells. Such crossbridges probably contribute to the stabilization and parallel order of microtubules in kinetochore fibers, any may have a functional role as well.

Animals↗
PubMed1980

Origin of kinetochore microtubules in Chinese hamster ovary cells.

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamsters ovary cells was reconstructed in three dimensions from 0.25 micrometer thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 microgram/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. -- In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubules were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.

Animals↗
PubMed1979

Electron microscopy of the spindle in locally heated cells.

Individual living cells in metaphase were exposed to a steep temperature gradient by placing a microheater near one spindle pole. The cells were then fixed and the spindle was examined by electron microscopy. The structure of the warmer half-spindle differed from the cooler half-spindle in several ways. Kinetochore microtubules were nearly parallel in the warmer half-spindle but were divergent in the cooler. The total length of microtubules in the warmer half-spindle was 52 per cent greater and the number of kinetochore microtubules per kinetochore averaged 16 per cent higher than in the cooler half-spindle. The warmer half-spindle was longer than the cooler. These observations clearly demonstrate a locally enhanced assembly of microtubules in the warmer half-spindle. The electron microscope study makes still clearer the unusual character of chromosome movement in the differentially heated cells: the structure of the warmer half-spindle is hard to distinguish from that in normal cells, yet chromosome movement there is far slower than normal (Nicklas, 1979).

Animals↗
PubMed1978

Comparison of active zones in spinal cord of anesthetized and unanesthetized rats: a high voltage electron microscope study.

High voltage electronmicroscopy was applied to the study of presynaptic nerve terminals and specifically the arrangement and staining properties of synaptic vesicles in and around the active zone. The zinc iodideosmium tetroxide technique was used for the impregnation of neuropil derived from the spinal cord of anesthetized (40 mg/kg pentobarbital sodium) and unanesthetized rats. Under optimal conditions of section thickness and tilt angles stereoscopic views of a paracrystalline lattice arrangement of synaptic vesicles could be ascertained. The ZIO affinity was significantly higher in synaptic vesicles of the anesthetized as compared with the unanesthetized preparations.

Animals↗