Cytoplasmic streaming in Chara corallina studied by laser light scattering.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46-7 mum s-1, S.D. +/- 4-8 at 20 degrees C) similar to those obtained for the same cells using the light microscope (44-3 mum s-1, S.D. +/- 5-3 at 20 degrees C). A narrow distribution of streaming velocities is indicated. The technique described provides a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.     

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.     

Cytoplasmic streaming in the cell model ofNitella

Summary The plasmalemma ofNitella internode was made freely permeable to solutes by treating the cell with detergent and EGTA under plasmolysis. After the treatment, the cytoplasmic streaming was stopped by bathing the cell in a medium lacking ATP. The streaming was reactivated by perfusing the exterior of the permeabilized cell with a medium containing both Mg²⁺ and ATP. The reactivated streaming could be reversibly stopped by depletion of ATP. However, depletion of Mg²⁺ irreversibly inhibited the streaming.Cytochalasin B at 5 g/ml irreversibly inhibited the reactivated streaming within a minute, showing that microfilaments are involved in the streaming.Abbreviations ATP adenosine-5-triphosphoric acid - CB cytochalasin B - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DMSO dimethylsulfooxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethylether)-N,N tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl-sulfonylfluoride     

Gliding mycoplasmas are inhibited by cytochalasin B and contain a polymerizable protein fraction

Studies are presented on the effect of cytochalasin B (CB) on the growth of five Mycoplasma species, three Acholeplasma species, and one Spiroplasma species. The three gliding mycoplasma species (M) gallisepticum, M pneumoniae and M pulmonis are the only mycoplasmas inhibited by CB. These are the only prolaryotes reported to be inhibited by CB. This suggested that these three mycoplasmas might have some sort of cytoskeletal structure. A protein fraction has been isolated from M gallisepticum which polymerizes in 0.6 M KC1 and depolymerizes when KC1 is removed. This fraction contains a major 58,000-dalton protein, a 46,000-dalton protein, and a minor 87,000-dalton protein.     

Cytoplasmic streaming in Amoeba proteus is inhibited by the actinspecific drug phalloidin

Phalloidin, applied by microinjection into Amoeba proteus inhibits specifically, in a concentration dependent manner, the process of cytoplasmic streaming. Preliminary ultrastructural analysis of phalloidin-injected amoebas shows that extensive arrays of microfilaments are formed. These results are discussed in relation to the specific interaction of phalloidin with actin known to occur in vitro, which involve promotion of actin polymerization and stabilization of F-actin structures.     

Pulsation of nuclei in insect oocytes is reversibly inhibited by cytochalasin B

Oocyte nuclei of the dipteran insect Heteropeza pygmaea display swift pulsating movements during in vitro follicle formation in the ovaries. Low doses of cytochalasin B (CB) completely inhibit the nuclear movements within a few minutes and cause the nuclei to assume spherical shapes. If the drug is removed, nuclear pulsation is resumed within 5–10 min. Phalloidin and colchicine do not affect the nuclear movements. Actin is shown by indirect immunofluorescence microscopy to be present in considerable amounts all over the cytoplasm of the oocytes. It is concluded that microfilaments are responsible for pulsation of the oocyte nuclei, whereas microtubules are not involved.     

The differential effects of cytochalasin B on microfilament populations and cytoplasmic streaming

Summary Two distinguishable populations of microfilaments (mfs) can be identified in the radish root hair. Bundles of mfs are found throughout the cytoplasm, excluding the tip region of the hair. Single mfs occur only as a cortical array, specifically associated with the microtubules. Both mf populations are oriented parallel to the direction of streaming. Hairs grown in 5 g/ml cytochalasin B (CB) exhibit site-specific differential responses to the drug in both their streaming pattern and sensitivity of their mfs. Cytochalasin B elicits the following responses: 1. cytoplasmic streaming is reduced in all regions of the hair; 2. small particles (<1 m in diameter) still stream, whereas large particles (>1 m in diameter) no longer stream but exhibit an oscillatory or rotational motion; 3. filament bundles show increasing sensitivity to CB along the length of the hair; 4. single mfs show decreasing sensitivity to CB along the hair length. The effects of CB on cytoplasmic streaming can be related to its effects on both mf populations, thus suggesting that although mf bundles are probably involved in streaming in the sub apical and basal regions of the hair, single mfs are most likely involved in generating the slower, more irregular streaming patterns exhibited in the hair tip and CB-treated hair base.     

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.     

Protoplasmic streaming, cytochalasin B, and growth of the pollen tube

Pollen tubes were grown in nutrient medium with increasing concentrations of cytochalasin B that inhibited cytoplasmic streaming to different degrees. The streaming of cytoplasmic particles and the growth rate of pollen tubes were measured by cinc photography and frame by frame analysis of the film. A direct correlation has been found between the concentration of cytochalasin B in the medium, the rate of cytoplasmic streaming, and the rate of growth of the pollen babe. The functioning of microfilaments is thus implicated in the control of pollen tube growth.     

Inhibition of mycoplasma cell division by cytochalasin B

Mycoplasma gallisepticum has subcellular organelles which may function as a primitive "mitotic-like" apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time "mitotic-like" structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle.     

Intercellular Transport and Cytoplasmic Streaming in Chara hispida

The correlation between the velocities of cytoplasmic streamingand of translocation of ¹⁴C-photosynthate and ³²P-phosphateassociated radioactivity has been investigated in whole plantsof the green freshwater alga Chara hispida L. Tracer was suppliedto the plant's rhizoid system in a split-chamber. The velocityof cytoplasmic streaming of 52±3.3 µm s^–1compares with 57±10 µm s^–1 found for ¹⁴C-transportand 32±20 µm s^–1 found for ³²P-transport.There was no indication of intercellular translocation at avelocity faster than visible streaming. Cytochalasin B inhibitedthe translocation of ³²P and cytoplasmic streaming. CytochalasinB becomes fully effective in inhibiting streaming and transportafter an incubation time of at least 5 h. Key words: Chara hispida, Cytoplasmic streaming, Intercellular transport     

Cytoplasmic alkalinization produced by the combination of anti-immunoglobulin antibody plus cytochalasin D in murine B lymphocytes

The intracellular pH of murine splenic B lymphocytes was measured using the pH-sensitive fluorescent dye, bis(carboxyethyl)carboxyfluorescein (BCECF). After stimulation of B lymphocytes with anti-immunoglobulin antibody plus cytochalasin D, two agents that act in synergy to promote S-phase entry, a late increase in pH was detected that occurred prior to the onset of DNA synthesis. The degree of alkalinization observed was comparable to that produced by two additional mitogenic regimens. Cytoplasmic alkalinization was not blocked by dimethylamiloride. Cytoplasmic alkalinization represents a sign of, and may play a role in, stimulation of B lymphocytes to enter S phase.     

The reconstituted ADP/ATP carrier from mitochondria is both inhibited and activated by anions

Anions were found to have a number of different effects on the reconstituted ADP/ATP carrier from mitochondria. (1) Binding of adenine nucleotides to the active site of the translocator is competitively inhibited by various anions. These anions can be arranged in a sequence of increasing competitive effect due to their order in a lyotropic series, and also due to increasing charge. (2) Apart from this competition effect, the presence of a sufficiently high concentration of anions turned out to be absolutely essential for functional ADP/ATP exchange in the reconstituted system. The activating anions too can be arranged in sequence, similar to that of the competition effect. The adenine nucleotide transport shows sigmoidal dependence on the stimulating anions with a Hill coefficient of n = 2. Addition of anions does not change the basic amount of functionally active translocator molecules. (3) The different effects of anions, i.e., inhibition and activation, were shown to take place at different sites and to be due to different mechanisms. Anions compete with substrates both at the outer (cytosolic) and at the inner (matrix) active site, whereas anion activation is observed solely by interaction with the cytosolic side of the translocator protein. (4) Activation of the reconstituted ADP/ATP exchange by anions could be discriminated from an activating influence of anionic phospholipids in the surroundings of the carrier protein.     

The Reconstituted ADP / ATP carrier from mitochondria is both inhibited and activated by anions

Anions were found to have a number of different effects on the reconstituted ADP / ATP carrier from mitochondria. (1) Binding of adenine nucleotides to the active site of the translocator is competitively inhibited by various anions. These anions can be arranged in a sequence of increasing competitive effect due to their order in a lyotropic series, and also due to increasing charge. (2) Apart from this competition effect, the presence of a sufficiently high concentration of anions turned out to be absolutely essential for functional ADP / ATP exchange in the reconstituted system. The activating anions too can be arranged in sequence, similar to that of the competition effect. The adenine nucleotide transport shows sigmoidal dependence on the stimulating anions with a Hill coefficient of n = 2. Addition of anions does not change the basic amount of functionally active translocator molecules. (3) The different effects of anions, i.e., inhibition and activation, were shown to take place at different sites and to be due to different mechanisms. Anions compete with substrates both at the outer (cytosolic) and at the inner (matrix) active site, whereas anion activation is observed solely by interaction with the cytosolic side of the translocator protein. (4) Activation of the reconstituted ADP / ATP exchange by anions could be discriminated from an activating influence of anionic phospholipids in the surroundings of the carrier protein.     

Action of cytochalasin B on cytoplasmic streaming systems and morphogenesis in Micrasterias torreyi (Conjugatophyceae)

The effects of cytochalasin B on the development and morphology of the Micrasterias cell vary according to different developmental stages. CB (especially 5–6 μm/ml) does not stop the growth of a developing semicell during early developmental stages and allows the formation of a three–lobed form, which is a kind of a basic form preceding the actual morphogenesis, further growth and development still being totally prevented by the same concentration. This is due to the occurrence of two separate cytoplasmic streaming systems in the developing semicell, one being cortical, more CB sensitive, the other being more central, not as sensitive to CB, both being microfilament based. The cortical streaming system supports tip growth, and many observations indicate that this system is also associated with the intermediation of the nuclear control regulating morphogenesis in Micrasterias. The effects of CB are specific: it has not been found to have distinct effects on the ultrastructure of the cell, on cell wall material production, or on the turgor pressure of the cell.