Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming,Cell Expansion and Plant Development

Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.     

Light-Controlled Cytoplasmic Streaming in Vallisneria Mesophyll Cells

The effect of light irradiation on cytoplasmic streaming inVallisneria mesophyll cells was investigated. Red light (     

Studies on Independent Synthesis of Cytoplasmic Ribonucleic Acids in Acetabularia mediterranea

1. The RNA content of anucleate and nucleate fragments of Acetabularia has been measured. It was found that there is a net synthesis of RNA in nucleate fragments. On the other hand, the RNA content of anucleate fragments did not change significantly after enucleation. 2. Anucleate fragments, however, can readily incorporate ¹⁴C-labeled adenine, orotic acid, and carbon dioxide into their cytoplasmic RNA. 3. The results of experiments on ¹⁴CO₂ incorporation into the RNA of anucleate and nucleate fragments suggest that there is a mechanism for de novo synthesis of RNA in anucleate cytoplasm. 4. In Acetabularia, 81 per cent of the cytoplasmic RNA is bound to a large granule fraction, consisting mainly of chloroplasts. Even after removal of the nucleus, RNA is synthesized in this "chloroplast" fraction. The chloroplasts are thus a major site of RNA synthesis in the cytoplasm of these algae. Synthesis of "chloroplastic" RNA, in anucleate fragments, possibly occurs at the expense of the RNA present in other fractions (microsomes and supernatant). 5. 8-Azaguanine stimulates regeneration and cap formation in anucleate fragments and does not inhibit RNA synthesis in these fragments.     

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     

Motile Fibrils and Cytoplasmic Streaming in Germinated Pollen

The highly vigrous subprotoplasts were prepared from the germinated pollen of Lilium. As the protoplasm mass contracted, many cytoplasmic fibrils with free-ends which moved like animal sperm tails appeared at the surface of the mass. The winding movement of the fibril could tow the free-end's cytoplasm mass, but did not affect the particles moving along the fibril. Only when the fibril free-end adhered to the inner side of the cell membrane, could the, particle movement along the fibril occur, with the disappearance of the fibril’s winding movement. In vitro, the fibril contraction could make both cytoplasmic particles and subprotoplast move in unidirection, and the fibrils could specifically bind fluorescent beads coated with rabbit myosin. This indicates that the fibrils were composed of F-actin. We think that the cytoplasmic streaming may be based on the contraction of F-actin which must adhere to some points of the inner side of the cell membrane, and the contraction of F-actin drives the membrane-bound organells to move, at the same time, propels the sol cytoplasm thus forming the cytoplasmic streaming observed by light microscopy.     

Internal Water Distribution and Cytoplasmic Streaming in Physarum polycephalum

A fungal vacuolation factor causes vacuolation when appliedto the plasmodium ofP. polycephalum, and also results in negativechemotaxis of the plasmodium. This, and the relationship betweenthe normal distribution of vacuoles in the plasmodium and itsdirection of locomotion, suggests that a similar factor maybe operative in determining polarity. A vacuolation factor hasbeen extracted from P. polycephalum. A vacuolar-reticular system in the cytoplasm shows a cycle ofcontractions and expansions that, in the advancing lobe at least,is usually in phase with the oscillating streaming pattern ofthe plasmodium in that region. The possible significance ofthese findings for the mechanism of streaming is discussed.     

Temperature Dependency of Circadian Period in a Single Cell (Acetabularia)

The circadian rhythm in the oxygen production of 30 individual Acetabularia cells has been studied at different temperatures. The temperature induced period variation was continuously evaluated over the whole data record of each individual cell with an advanced spectral analysis technique. The observed circadian periods of O₂ production displayed a well established region of temperature compensation between 25 °C and 30 °C with a Q₁₀, value of 0.9, whereas between 15°C and 22°C a positive temperature coefficient was measured (Q₁₀ at 22 °C 0.9, Q₁₀ at 20°C 0.8, Q₁₀at 17°C 0.7).     

烟草培养细胞中钙离子,钙调素与细胞质流的关系

烟草愈伤组织的培养细胞中,当钙离子载体将Ca~(2 )导入细胞时,细胞质流停止.CaM拮抗剂试验表明,高钙使细胞质流停止的效应可能与CaM无关,除W7外的多种CaM拮抗剂都明显而且可逆地抑制细胞质流。酶联免疫吸附分析(ELISA)检出培养细胞中存在有CaM。间接酶标免疫组织化学分析进一步证明CaM存在于胞质条纹中。     

Lattice Structure of Cytoplasmic Microtubules in a Cultured Mammalian Cell

Tubulin can polymerize in two distinct arrangements: “B-lattices,” in which the α-tubulins of one protofilament lie next to α-tubulins in the neighboring protofilaments, or the “A” configuration, where α-tubulins lie beside β-tubulins. Microtubules (MTs) in flagellar axonemes and those assembled from pure tubulin in vitro display only B-lattices, but recent work shows that A-lattices are found when tubulin co-polymerizes in vitro with an allele of end-binding protein 1 that lacks C-terminal sequences. This observation suggests that cytoplasmic MTs, which form in the presence of this “tip-associating protein,” may have A-lattices. To test this hypothesis, we have decorated interphase MTs in 3T3 cells with monomeric motor domains from the kinesin-like protein Eg5. These MTs show only B-lattices, as confirmed by visual inspection of electron cryo-tomograms and power spectra of single projection views, imaged at higher electron dose. This result is significant because 13 protofilament MTs with B-lattices must include a “seam,” one lateral domain where adjacent dimers are in the A-configuration. It follows that cytoplasmic MTs are not cylindrically symmetric; they have two distinct faces, which may influence the binding patterns of functionally significant MT-interacting proteins.     

Cytoplasmic Inheritance of a Cell Surface Antigen in the Mouse

Mta is a cell surface antigen of the mouse and serves as a target for specific T killer lymphocytes. Using a killer cell assay, the antigen has been found in 72 strains of laboratory mice and, with one exception, in all tested samples of mice caught in the wild or bred from such, including Mus molossinus, Mus castaneus and Mus spretus. Five strains of rats, non-inbred NMRI mice, most substrains of NZB mice and the closely related strain NZO are negative for Mta. In reciprocal F₁ crosses between several Mta⁺ and two Mta^- strains, the antigen is maternally transmitted; that is, Mta⁺ females bear only positive offspring, whereas Mta^- females bear only negative offspring, regardless of the genotype of the male. Since 34 foster-nursed mice had the Mta type of their genetic mothers, the factor that determines expression of Mta must be transmitted before birth and not via the milk. The cytoplasmic genes of Mta⁺ strains have been combined with the chromosomal genes of Mta^- strains, and vice versa, by repeated backcrossing. All progeny retained the Mta type of their maternal lines. Thus, the Mta type is determined solely by maternal inheritance and is not influenced by chromosomal genes. We found no evidence of incompatibility between the cytoplasmic factors and nuclear genes of Mta^- and Mta ⁺ strains.     

The Presence of Contractile Proteins in Pollens ami Their Role in Cytoplasmic Streaming

Authors demonstrate the presence of actin and myosin in pollens from Luffa cylindricaand Zea mays in this report. The molecular weight of the heavy chain of pollen myosinis about 165000 daltons as analyzed by 4–30% SDS gradient polyacrylamide gel electrophoresis. The ATPase activity of pollen myosin is identical with the characteristics of rabbit ske-letal muscle myosin. In 0.5 mol/l KCl, the K+-EDTA activity is the highest and Mg2+ activitythe lowest. The Ca2+ activity is higher than Mg2+ activity and lower than K+-EDTA activity.Pollen actin from Zea mays was prepared by preparative SDS polyacrylamide gel electrophoresis Its molecular weight is 43,000 daltons which is the same as rabbit skeletal muscle actin. The effect of drugs on cytoplasmic streaming of pollen tubes were observed under opticalmicroscope Cytochalasin B (CB), chloropromazine (CPZ) and chlorotetracycline (CTC)inhibit cytoplasmic streaming obviously. But colchicine has no effect on the cytoplasmic streamrog. It is suggested that the motive force of cytoplasmic streaming may be the interaction ofmyosin and actin in the pollen tubes.     

Intracellular Ca2+ Concentration and Cytoplasmic Streaming in Vallisneria Mesophyll Cells

Intracellular Ca²⁺ concentration regulating the cytoplasmicstreaming in Vallisneria mesophyll cells was estimated. Theleaf segment was cut open at the middle of the mesophyll celllayers and the exposed mesophyll cells were treated with testsolutions of various Ca²⁺ concentrations in the dark. This allowedA23187 [GenBank] , a calcium ionophore, to exert its full effect on thecell membrane. The streaming was induced or maintained in solutions which containedCa²⁺ at lower than 10^–6M. However, Ca²⁺ at concentrationshigher than 10^–5M had a definite, inhibitory effect. Theinduction and cessation of streaming could be repeated by alternatelychanging the solutions. (Received March 14, 1986; Accepted May 15, 1986)     

Thioredoxin-Interacting Protein Regulates Glucose Metabolism and Affects Cytoplasmic Streaming in Mouse Oocytes

Thioredoxin-interacting protein (Txnip) regulates intracellular redox state and prompts oxidative stress by binding to and inhibiting Thioredoxin (Trx). In addition, via a Trx-independent mechanism, Txnip regulates glucose metabolism and thus maintains intracellular glucose levels. Previously, we found Txnip mRNA highly expressed in immature germinal vesicle (GV) oocytes, but currently there is no report describing the role of Txnip in oocytes. Therefore, we conducted the present study to determine the function of Txnip in mouse oocytes'' maturation and meiosis by using RNA interference (RNAi) method. Upon specific depletion of Txnip, 79.5% of oocytes were arrested at metaphase I (MI) stage. Time-lapse video microscopy analysis revealed that the formation of granules in the oocyte cytoplasm increased concurrent with retarded cytoplasmic streaming after Txnip RNAi treatment. Txnip RNAi-treated oocytes had upregulated glucose uptake and lactate production. To confirm the supposition that mechanism responsible for these observed phenomena involves increased lactate in oocytes, we cultured oocytes in high lactate medium and observed the same increased granule formation and retarded cytoplasmic streaming as found by Txnip RNAi. The MI-arrested oocytes exhibited scattered microtubules and aggregated chromosomes indicating that actin networking was disturbed by Txnip RNAi. Therefore, we conclude that Txnip is a critical regulator of glucose metabolism in oocytes and is involved in maintaining cytoplasmic streaming in mouse oocytes.     

Identification and Characterization of Higher Plant Myosins Responsible for Cytoplasmic Streaming

in vitro using these myosins and of localization studies using antiserum raised against each heavy chain, we suggested that both myosins are molecular motors for generating the motive force for cytoplasmic streaming in higher plant cells. The 170-kDa myosin is expressed not only in somatic cells but also in germinating pollen. In contrast, the 175-kDa myosin is distributed only in somatic cells. In the tip region of growing pollen tubes, it has been demonstrated that a tip-focused Ca²⁺ gradient is indispensable for growth and tube orientation. Cytoplasmic streaming in this region has been shown to be inactivated by high concentrations of Ca²⁺. The motile activity in vitro of 170-kDa myosin is suppressed by low (μM) levels of Ca²⁺ through its CaM light chain, suggesting that this suppression is one of the mechanisms for inactivating cytoplasmic streaming near the tip region of pollen tubes. The motile activity in vitro of 175-kDa myosin is also inhibited by Ca²⁺ at concentrations higher than 10⁻⁶M. It has been revealed that the elevation of cytosolic Ca²⁺ concentrations causes the cessation of cytoplasmic streaming even in somatic cells. Therefore, Ca²⁺-sensitivity of the motile activity of myosin appears to be a general molecular basis for Ca²⁺-induced cessation of cytoplasmic streaming. Received 6 September 2000/ Accepted in revised form 7 October 2000     

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole     

Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum

Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P(i)) were sufficient to simulate the data. Noticeably, both P(3) and P(4) contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (Glu204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.     

Mechanism of Inhibition of Cytoplasmic Streaming by Auxin in Root Hair Cells of Hydrocharis

It has been reported that auxin accelerates cytoplasmic streamingat low concentrations and inhibits it at high concentrationsin several plant cells. In the present study, the mechanismof inhibition of cytoplasmic streaming by naphthalene aceticacid (NAA) at high concentrations was analyzed in root haircells of Hydrocharis. Because the effective concentration ofNAA inhibiting cytoplasmic streaming decreased when the extracellularpH (pHo) was lowered, it was hypothesized that cytoplasmic streamingis inhibited by NAA via acidification of the cytoplasm. Thispossibility was supported by the fact that acetic acid, pro-pionicacid and decanoic acid also inhibited cytoplasmic streamingat low pHo. Acidification of the cytoplasm disturbed the orientationof actin filaments (AFs) and disrupted cortical microtubules(MTs). The effects of NAA were reversible; both cytoplasmicstreaming and organization of the cytoskeleton were recoveredupon removal of NAA. During the recovery, tracks of cytoplasmicstreaming in the subcortical region temporarily showed a helicalpattern along the longitudinal direction of the cell. Fluorescencestaining of cytoskeletons revealed that both AFs and MTs alignedobliquely to the longitudinal axis of the cell. The helicalstreaming returned to the original reverse fountain streamingafter several hours. The simultaneous changes in the organizationof both cytoskeletons supported our previous report that theorganization of AFs is regulated by MTs. ¹Author for correspondence. Fax, 81-7915-8-0175. e-mail: tomy-@sci.himeji-tech.ac.jp