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Results for “Cytoplasmic Streaming”

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PubMed2003

The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP-Staufen.

The anteroposterior axis of Drosophila is defined during oogenesis, when the polarisation of the oocyte microtubule cytoskeleton directs the localisation of bicoid and oskar mRNAs to the anterior and posterior poles, respectively. Although maternal-effect lethal and female-sterile screens have identified many mutants that disrupt these processes, these screens could not recover mutations in essential genes. Here we describe a genetic screen in germline clones for mutants that disrupt the localisation of GFP-Staufen in living oocytes, which overcomes this limitation. As Staufen localises to the posterior with oskar mRNA and to the anterior with bicoid mRNA, it acts as a marker for both poles of the oocyte, allowing the identification of mutants that affect the localisation of either mRNA, as well as mutants that disrupt oocyte polarity. Using this approach, we have identified 23 novel complementation groups on chromosome 3R that disrupt anteroposterior axis formation. Analyses of new alleles of spn-E and orb show that both SPN-E and ORB proteins are required to organise the microtubule cytoskeleton at stage 9, and to prevent premature cytoplasmic streaming. Furthermore, yps mutants partially suppress the premature cytoplasmic streaming of orb mutants. As orb, yps and spn-E encode RNA-binding proteins, they may regulate the translation of unidentified RNAs necessary for the polarisation of the microtubule cytoskeleton.

Animals↗

PubMed1999

Biochemical and immunocytochemical characterization of two types of myosins in cultured tobacco bright yellow-2 cells

We have isolated a myosin (referred to as 170-kD myosin) from lily pollen tubes, which consists of 170-kD heavy chain and calmodulin (CaM) light chain and is responsible for cytoplasmic streaming. A 170-kD polypeptide that has similar antigenicity to the 170-kD myosin heavy chain of lily pollen tubes was also present in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, and possessed the ability to interact with F-actin in an ATP-dependent manner. In addition to this myosin, we identified biochemically another kind of myosin in BY-2 cells. This myosin consisted of a CaM light chain and a 175-kD heavy chain with antigenicity different from the 170-kD myosin heavy chain. In the present study, we referred to this myosin as 175-kD myosin. This myosin was able to translocate rhodamine-phalloidin (RP)-labeled F-actin at an average velocity of about 9 &mgr;m/s in the motility assay in vitro. In contrast, the sliding velocity of RP-labeled F-actin translocated by fractions containing the 170-kD myosin was 3 to 4 &mgr;m/s. The velocity of cytoplasmic streaming in living BY-2 cells ranged from 2 to 9 &mgr;m/s. The motile activity of 175-kD myosin in vitro was inhibited by Ca(2+) at concentrations higher than 10(-6) M. Immunoblot analyses using an antiserum against the heavy chain of 170- or 175-kD myosin revealed that in tobacco plants, the 175-kD myosin was expressed in leaf, stem, and root, but not in germinating pollen, while 170-kD myosin was present in all of these plant parts and in germinating pollen. These results suggest that the two types of myosins, 170 and 175 kD, presumably participate in cytoplasmic streaming in BY-2 cells and other somatic cells of tobacco plants.

Journal Article↗

PubMed1986

Growth and regrowth of actin bundles in Chara: bundle assembly by mechanisms differing in sensitivity to cytochalasin.

Cytochalasin is known to inhibit cytoplasmic streaming rapidly in characean cells without disassembling their actin bundles. Lower cytochalasin concentrations than those needed for streaming inhibition are now shown to disrupt bundle assembly and, over longer periods, assembled bundles. After local wounding, cytochalasin limited bundle regeneration to the production of polygons and straight, discontinuous bundles that rarely connected to bundles outside the wound. The regenerated bundles supported only scattered organelle movements, whereas long, oriented bundles of control cells were connected to those outside the wound and supported bulk endoplasmic streaming. Unwounded Chara plants cultured for up to 2 weeks in 1 microM-cytochalasin maintained normal bundle orientation and rapid cytoplasmic streaming, but the mean number of bundles per file of chloroplasts fell from 5.2 in controls to 2.0 in growing cells and 3.4 in nongrowing cells. These structural effects seem more likely than the streaming inhibition to reflect cytochalasin's in vitro effect of blocking extension at the barbed but not the pointed end of F-actin. In particular, cytochalasin inhibited the extension into the wound of bundles in which only the barbed ends of filaments would be exposed. However, short lengths of isolated bundles grew within the wound and bundle growth in the intact cell continued, albeit in modified form. It is suggested that these examples of continuing bundle growth involve cytochalasin-resistant mechanisms that are not wholly dependent on barbed-end filament growth.

Actins↗

PubMed2001

Aluminum toxicity studies in Vaucheria longicaulis var. macounii (Xanthophyta, Tribophyceae). I. Effects on cytoplasmic organization.

Using differential interference contrast (DIC) and epifluorescence microscopy, we tested the hypothesis that exposure to environmentally significant levels of aluminum (Al) would cause rapid changes in cytoplasmic organization in vegetative filaments of the coenocytic alga, Vaucheria longicaulis Hoppaugh var. macounii Blum resulting in the loss of cytoplasmic streaming. In untreated cells, DIC microscopy revealed the presence of cortical cytoplasmic strands that were oriented longitudinally to the cell axis as well as sub-cortical cytoplasmic strands that exhibited a reticulate morphology. Organelles such as chloroplasts and mitochondria translocated throughout the cell in close association with the cortical longitudinal cytoplasmic strands. Staining with the lipophilic dye, 3,3-dihexyloloxacarbocyanine, revealed structures that appeared to be endoplasmic reticulum (ER). This organelle closely resembled, in location and appearance, the cytoplasmic strands visualized using DIC microscopy. The addition of Al (80 µM) resulted in the inhibition of cytoplasmic streaming as well as the dissipation of the putative cortical longitudinal ER within one minute. Subsequently, the DIC-visible cortical cytoplasmic strands exhibited progressive degrees of disorganization. Throughout these changes, chloroplasts and mitochondria remained visibly associated with the cortical cytoplasmic strands.

Journal Article↗

PubMed1978

The influence of microinjected phalloidin on locomotion, protoplasmic streaming and cytoplasmic organization in Amoeba proteus and Physarum polycephalum.

Microinjected phalloidin induces both time and concentration-dependent changes in morphology and motility of amoebae and acellular slime moulds. In A. proteus injection of a 10(-3)M solution of the drug causes a separation of cortical hyaline plasma from central granular plasma. Simultaneously protoplasmic streaming and cellular locomotion are lost irreversibly. Lowering the concentration of phalloidin to 2 x 10(-4)M results in a reversible disturbance; amoebae recover after 30 to 60 minutes and show normal movement. In Ph. polycephalum the injection of a 10(-3)M solution of phalloidin into single veins induces a local gelation of the protoplasm followed by the separation of hyalo- and granuloplasm. In semi-thin and ultrathin sections the hyaline plasma regions contain a fine granular groundplasm rich in ribosomes but free of cellular organelles. The central granular plasma consists mainly of membrane-surrounded cellular compartments. The two morphologically distinct plasma regions are separated by a 0.5 to 1.0 micrometer layer of filamentous material. In A. proteus the filamentous layer is found shortly after phalloidin injection in close proximity to the plasma membrane, and consists of thin 5 to 6 nm filaments. With increasing time this layer contracts, separates from the inner plasma membrane and moves to the interior of the cell. During contraction thicker filaments with diameters of 10 to 30 nm and lengths of 300 to 500 nm are formed. The results indicate that the display and contraction of the phalloidin-induced filament layer can account for the changes observed in cellular movement and cytoplasmic organization. The resulting phenomena i.e. separation of hyaline plasma from granular plasma and changes in both the protoplasmic streaming pattern and locomotory activity of the cells, are discussed in terms of a general understanding of amoeboid movement.

Amoeba↗

PubMed2001

Role of Ca2+ in membrane excitation and cell motility in Characean cells as a model system.

The Characeae internodal cell is excitable and generates an action potential. The depolarizing phase of the action potential primarily reflects the activation of Cl- channels, which takes place in a Ca2+-dependent manner. Namely, an increase in the Ca2+ influx takes place at the very beginning of the action potential and heightens the Ca2+ concentration in the cytoplasm ([Ca2+]c). The high [Ca2+]c activates Cl- channels at the plasmalemma, resulting in a large depolarization. The high [Ca2+]c also acts as a signal to induce a tonoplast action potential and the instantaneous cessation of cytoplasmic streaming; the tonoplast action potential also is caused by Ca2+-induced activation of Cl- channels at the tonoplast, and the cessation is a result of inhibition of the actin-myosin interaction by Ca2+. When the cytoplasm of the Characeae cell, especially in Nitella flexilis, is hydrated rapidly, [Ca2+], also increases through Ca2+ release from an intracellular store(s). The release may be triggered by the stretching of endomembranes caused by osmotic swelling of the Ca2+ store lumens. Although the origin of Ca2+ is different from that in the case of an action potential, high [Ca2+]c not only induces membrane depolarization through activation of the Cl- channel in a Ca2+-dependent manner but also inhibits cytoplasmic streaming in Characeae.

Action Potentials↗

PubMed1979

Nitellopsis obtusa internodal cell birefringence change during action potential.

Birefringence change during excitation was studied by using Nitellopsis obtusa. The velocity change of cytoplasmic streaming during an action potential was measured simultaneously by fluctuation analysis of transmitted light intensity. The origin of the retardation change was discussed by comparing optical retardation change to the time course of the action potential, the cytoplasmic streaming velocity change and the cell contraction. By the time course analysis of retardation change, we concluded that the change of the birefringence might be the sum of the changes of cytoplasmic flow and that of the size of length and diameter of the cell. But it is still difficult to separate the change to its components.

Action Potentials↗

PubMed2003

Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity.

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.

Actins↗

PubMed2005

Mitochondrial behaviour in the early stages of ROS stress leading to cell death in Arabidopsis thaliana.

BACKGROUND AND AIMS: Reactive oxygen species (ROS) are involved in triggering cell death. To visualize mitochondrial behaviour under ROS stress, transgenic arabidopsis plants possessing mitochondrial-targeted GFP (S65T) were studied. METHODS: Arabidopsis leaves were treated with ROS and ROS-inducing chemicals such as hydrogen peroxide, paraquat and menadione. Microscopic observations were carried out using a confocal laser scanning microscope system, and electrolyte leakage was also monitored. KEY RESULTS: After treatment, mitochondria showed morphological changes from a bacillus-like to a round shape. The size of mitochondria treated with H(2)O(2) decreased by half compared with controls. Concurrently, cytoplasmic streaming was blocked and mitochondria eventually swelled. Treatment of leaves with butanedione monoxime, an inhibitor of myosin ATPase, resulted in similar behaviour of mitochondria to that under ROS stress. CONCLUSIONS: The results indicate that morphological changes of mitochondria and cessation of cytoplasmic streaming may interact, and this phenomenon is one of the features of ROS stress-induced cell death.

Arabidopsis↗

PubMed1991

The polar organization of the growing Chara rhizoid and the transport of statoliths are actin-dependent.

Horizontally positioned Chara rhizoids continue growth without gravitropic bending when the statoliths are removed from the apex by basipetal centrifugation. The transport of statoliths in centrifuged rhizoids is bidirectional: 50-60% of the statoliths are retransported on a straight course to the apex at velocities from 1 to 14 micrometers min-1, increasing towards the rhizoid tip The centrifuged statoliths which are located closest to the nucleus are basipetally transported and caught up in the cytoplasmic streaming of the cell. Those statoliths which are located near the apical side of the nucleus are transported either apically or basally. A de-novo-formation of statoliths was not observed. After retransport to the apex some statoliths transiently sediment, a process which can induce a local inhibition of cell wall growth. The rhizoid bends again gravitropically only if a few statoliths finally sediment in the apex; the more statoliths that sediment in the apex the shorter the radius of bending becomes. The transport of statoliths is mediated by actin filaments which form a network of thin filaments in the apical and subapical zone of the rhizoid, and thicker parallel bundles in the basal zone where cytoplasmic streaming occurs. Both subpopulations of actin filaments overlap in the nucleus zone.

Actin Cytoskeleton↗

PubMed1992

The contribution of the extracellular matrix to gravisensing in characean cells.

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

Amino Acid Sequence↗

PubMed1995

Myosin from alga Chara: unique structure revealed by electron microscopy.

Cytoplasmic streaming is a fundamental process for the transport of molecules and organelles in plant cells. A myosin-like protein responsible for cytoplasmic streaming in fresh water alga, Chara corallina, has been purified. Electron micrographs of this protein revealed that it has two heads with similar size and shape to those of muscle myosin (myosin II) but its tail was shorter than that of muscle myosin with a globular structure at its distal end like that of myosin V. The myosin cosedimented with vesicles composed of phosphatidyl serine like other non-muscle myosin species involved in intracellular transport.

Actins↗

PubMed1982

Hydrodynamic models of viscous coupling between motile myosin and endoplasm in characean algae.

Cytoplasmic streaming in characean algae is thought to be driven by interaction between stationary subcortical actin bundles and motile endoplasmic myosin. Implicit in this mechanism is a requirement for some form of coupling to transfer motive force from the moving myosin to the endoplasm. Three models of viscous coupling between myosin and endoplasm are presented here, and the hydrodynamic feasibility of each model is analyzed. The results show that individual myosinlike molecules moving along the actin bundles at reasonable velocities cannot exert enough viscous pull on the endoplasm to account for the observed streaming. Attachment of myosin to small spherical organelles improves viscous coupling to the endoplasm, but results for this model show that streaming can be generated only if the myosin-spheres move along the actin bundles in a virtual solid line at about twice the streaming velocity. In the third model, myosin is incorporated into a fibrous or membranous network or gel extending into the endoplasm. This network is pulled forward as the attached myosin slides along the actin bundles. Using network dimensions estimated from published micrographs of characean endoplasm, the results show that this system can easily generate the observed cytoplasmic streaming.

Actins↗

PubMed1986

Inhibitors of proton pumping: effect on passive proton transport.

Reported inhibitors of the Characean plasmalemma proton pump were tested for their ability to inhibit the passive H(+) conductance which develops in Chara corallina Klein ex Willd. at high pH. Diethylstilbestrol inhibits the proton pump and the passive H(+) conductance with about the same time course, at concentrations that have no effect on cytoplasmic streaming. N-Ethylmaleimide, a sulfhydryl reagent which is small and relatively nonpolar, also inhibits both pumping and passive conductance of H(+). However, it also inhibits cytoplasmic streaming with about the same time course, and therefore could not be considered a specific ATPase inhibitor. p-Chloromercuribenzene sulfonate (PCMBS), a sulfhydryl reagent which is large and charged and hence less able to penetrate the membrane, does not inhibit pumping or conductance at low concentration. At high concentration, PCMBS sometimes inhibits pumping without affecting H(+) conductance, but since streaming is also inhibited, the effect on the pump cannot be said to be specific. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water soluble carbodiimide, weakly inhibits both pump and conductance, apparently specifically.

Journal Article↗

PubMed1999

Calcium regulation of the actin-myosin interaction of Physarum polycephalum.

Plasmodia of Physarum polycephalum show vigorous cytoplasmic streaming, the motive force of which is supported by the actin-myosin interaction. Calcium is not required for the interaction but inhibits it. This calcium inhibition, a regulatory mode first discovered in Physarum, is the overwhelming mode of regulation of cytoplasmic streaming of plant cells and lower eukaryotes, and it is diametrically opposite to calcium activation of the interaction found in muscle and nonmuscle cells of the animal kingdom. Myosin, myosin II in myosin superfamily, is the most important protein for Ca2+ action. Its essential light chain, called calcium-binding light chain, is the sole protein that binds Ca2+. Although phosphorylation and dephosphorylation of myosin modify its properties, regulation of physiological significance is shown to be Ca-binding to myosin. The actin-binding protein of Physarum amplifies calcium inhibition when Ca2+ binds to calmodulin and other calcium-binding proteins. This review also includes characterization of this and other calcium-binding proteins of Physarum.

Actins↗

PubMed1997

Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains.

We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by 45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.

Allergens↗

PubMedDate not supplied

Comparative surface morphology of marine coelomic gregarines (Apicomplexa, Urosporidae): Pterospora floridiensis and Pterospora schizosoma.

Two species in the aseptate gregarine genus Pterospora from the Pacific and Gulf coasts were analyzed by scanning electron microscopy, which revealed characteristics not reported in other gregarines. The gamonts of these species had branching trunks that ended in terminal digits, and both species moved by cytoplasmic streaming and peristalsis. Pterospora floridiensis had surface pits and tracts of parallel ridges that bended and connected with one another. Pterospora schizosoma had irregular-shaped surface swellings that were usually arranged in rosette patterns. These unique surface features have not been reported for other gregarines, and are strikingly different from the surface features of many septate and aseptate gregarines that inhabit the intestinal lumena of their hosts and move by gliding. The correlation of Pterospora's unique pellicular features to the habitat and cytoplasmic streaming characteristic of the genus may be significant, and may reflect an adaptation for development in coelomic environments.

Animals↗

PubMed2004

Arabidopsis myosin XI mutant is defective in organelle movement and polar auxin transport.

Myosins are eukaryotic molecular motors moving along actin filaments. Only a small set of myosin classes is present in plants, in which myosins have been found to play a role in cytoplasmic streaming and chloroplast movement. Whereas most studies have been done on green algae, more recent data suggest a role of higher plant myosin at the postcytokinetic cell wall. Here we characterize a loss-of-function mutation for a myosin of plant-specific class XI and demonstrate myosin functions during plant development in Arabidopsis. T-DNA insertion in MYA2 caused pleiotropic effects, including flower sterility and dwarf growth. Elongation of epidermal cells, such as in hypocotyls and anther filaments, was reduced by up to 50% of normal length. This effect on anther filaments is responsible for flower sterility. In the meristems of root tips, it was evident that cell division was delayed and that cell plates were mislocated. Like zwichel, a kinesin-related mutation causing two-branched trichomes, the mya2 knockout causes branching defects, but here the trichomes remained unbranched. Growth was also impaired in pollen tubes and root hairs, cells that are highly dependent on vesicle transport. A failure in vesicle flow could be directly confirmed, because cytoplasmic streaming of vesicles and, more so, of large endoplasmic reticulum-based organelles was slowed. The defect in vesicle trafficking was accompanied by failures in basipetal auxin transport, measured in stem segments of inflorescences. This result strongly suggests a causal link between auxin-dependent processes and the distribution of vesicles and membrane-bound molecules by plant myosin.

Arabidopsis↗