Behavior of cytoplasmic streaming in Nitella during centrifugation as revealed by the television centrifuge-microscope.
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Embryonic axis specification in Drosophila melanogaster is achieved through the asymmetric subcellular localization of morphogenetic molecules within the oocyte. The cappuccino and spire loci are required for both posterior and dorsoventral patterning. Time-lapse confocal microscopic analyses of living egg chambers demonstrated that these mutations induce microtubule reorganization and the premature initiation of microtubule-dependent ooplasmic streaming. As a result, microtubule organization is altered and bulk ooplasm rapidly streams during the developmental stages in which morphogens are normally localized. These changes in oocyte cytoarchitecture and dynamics appear to disrupt axial patterning of the embryo.
After vacuolar perfusion of Chara internode cells, the cytoplasm remaining in situ can be reactivated by ATP to give full rates of streaming. Observations during both perfusion and reactivation indicated that the generation of the motive force was associated with fibres consisting of bundles of microfilaments. In the absence of ATP, the remaining endoplasmic organelles were immobilized along such fibres. When ATP was introduced, organelles moved along the fibres at speeds up to 50 mum S minus 1, but but were progressively released from contact to leave the fibres in a conspicuously clean state. Inorganic pyrophosphate freed the organelles from the fibres without supporting movements. Motility required millimolar Mg2nlevels, free Ca2nat 10 minus 7 M or less and was inhibited by high levels of Clminus and by pH's on either side of 7.0. The reactivated movements were rapidly and completely inhibited by 25mug ml minus 1 cytochalasin B. The results are interpreted in terms of actin filaments in the stationary cortex interacting with a myosin-like protein which is able to link to endoplasmic organelles. Movement results from an active shear type of mechanism.
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A method is described utilizing deuterium oxide stabilization of microtubules that allows the separation of stable axopodia from the cell body. In some instances the isolated axopodia are broken open and display two classes of cytoplasmic linear elements, microtubules and microfilaments, lying side by side. This morphological arrangement is consistent with the hypothesis that microtubules support a microfilament-based contractile apparatus in heliozoans and probably other cell types as well. Also included is a description of pseudopodial formation from axopodial cytoplasm in response to mechanical stimulation. The pseudopodial motility is discussed in relation to the particle saltations also observed in these cells and to different levels of organization or activation of the contractile machinery.
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Microtubules attached to the pellicle at the tips of tentacles pivot through about 140 degrees on these attachments, splay apart, and bend along their longitudinal axes when feeding occurs. The tubules could be bending in response to pellicular contractions; active bending, sliding, or contraction of the tubules may not be involved. Intertubule links apparently prevent tubules from splaying apart at certain levels. These links are probably under tension during feeding. They stretch; they sometimes become half as thick and eight times as long as they are before feeding. Often, tubules joined together by these links also change in shape; they become slightly flattened and elliptical in cross section. Cytoplasm from the ciliate Tetrahymena is drawn down a feeding tentacle inside an invagination of the Tokophrya cell membrane from the tentacle tip. The positions of arm-bearing microtubules around such invaginations indicate that arms are involved in moving invaginations along. The edges of the perforated Tetrahymena cell membrane are "sealed" to the cell membrane of Tokophrya around each feeding tentacle tip.
We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.
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Stromules are stroma-containing tubules which can grow from the surface of plastids, most commonly leucoplasts and chromoplasts, but also chloroplasts in some tissues. Their functions are obscure. Stills from video rate movies are presented here. They illustrate interaction of stromules with cytoskeletal strands and the anchoring of stromules to unidentified components at the cell surface. Anchoring leads to stretching and relaxation of stromules when forces arising from cytoplasmic streaming act on the attached, freely suspended plastid bodies. Data on stromule growth, retraction, and regrowth rates are provided. Formation and movement of stromular branches and bridges between plastids are described. The shedding of a tip region into the streaming cytoplasm is recorded in frame-by-frame detail, in accord with early observations.
In the streaming cytoplasm of the Characean algae cell, the movement of organelles along actin bundles occurs at a striking rate of up to 60 microns s-1. To further characterize the molecular mechanisms responsible for this phenomenon, we have reconstituted the movement of actin filaments in vitro using defined biochemical components. We report that only a soluble cytoplasmic fraction devoid of organelles and filamentous material supports the movement of fluorescent-labeled actin filaments on glass at a rate of up to 60 microns s-1. This fraction also contains the K(+)-EDTA ATPase and the actin-activated Mg2+ ATPase activities characteristic of myosin proteins. Therefore, on the basis of these observations, we conclude that Nitella cells have a soluble pool of non-filamentous myosin molecules with the mechanochemical properties expected for a motor responsible for cytoplasmic streaming in vivo. The preparation and conditions described here should be useful for the purification of this translocator.
Two small blobs and a channel excised from a slime mold plasmodium were allowed to fuse into a dumbbell-shaped mass in a thermally insulated Kamiya double chamber equipped with naked bead thermistors in contact with the blobs. Cyclic temperature differences of from 1 X 10(-4) to 5 X 10(-2) deg Celsius were recorded by a sensitive lock-in amplifier method with a basal noise level of less than 2 X 10(-2)deg Celsius and a time constant of 0.5 second. The temperature differences were caused by periodic bursts of heat production synchronized perfectly with the shuttle-streaming cycle and invariably localized at the source rather than the destination of the streaming cytoplasm. The results support the theory that the motive force for cytoplasmic streaming in the slime mold is pressure, probably generated by contraction of elements in the channel walls.
In mesophyll cells of the aquatic angiosperm Vallisneria gigantea, bundles of microfilaments (MFs) serve as tracks for the rotational streaming of the cytoplasm, which occurs along the two longer side walls and the two shorter end walls. The stationary organization of these bundles has been shown to depend on the association of the bundles with the plasma membrane at the end walls. To identify the sites of such association, the effects of cytochalasin B (CB) on the configuration of the bundles of MFs were examined. In the case of the side walls, MFs were completely disrupted after treatment with CB at 100 micrograms/ml for 24 hours. By contrast, in the case of the end walls, a number of partially disrupted MFs remained even after 48 hours of treatment. After removal of CB, a completely normal arrangement of bundles of MFs was once again evident within 24 hours after a rather complicated process of reassembly. When reassembly had been completed, the direction of cytoplasmic streaming was reversed only in a small fraction of the treated cells, suggesting that bundles of MFs are anchored and stabilized at the end walls of each cell and that the polarity of reorganized bundles and, therefore, the direction of the cytoplasmic streaming is determined in a manner that depends on the original polarity of MFs that remained in spite of the disruptive action of CB. By contrast, the direction of reinitiated cytoplasmic streaming was reversed in 50% of cells in which the bundles of MFs had been completely disrupted by exogenously applied trypsin prior treatment with CB.(ABSTRACT TRUNCATED AT 250 WORDS)