Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells.

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis.

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.

Cytoplasmic streaming velocity as a plant size determinant.

Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants.

Studies on vacuolar membrane microdomains isolated from Arabidopsis suspension-cultured cells: local distribution of vacuolar membrane proteins.

The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics.

Reconstruction of active regular motion in amoeba extract: dynamic cooperation between sol and gel states.

Amoeboid locomotion is one of the typical modes of biological cell migration. Cytoplasmic sol-gel conversion of an actomyosin system is thought to play an important role in locomotion. However, the mechanisms underlying sol-gel conversion, including trigger, signal, and regulating factors, remain unclear. We developed a novel model system in which an actomyosin fraction moves like an amoeba in a cytoplasmic extract. Rheological study of this model system revealed that the actomyosin fraction exhibits shear banding: the sol-gel state of actomyosin can be regulated by shear rate or mechanical force. Furthermore, study of the living cell indicated that the shear-banding property also causes sol-gel conversion with the same order of magnitude as that of shear rate. Our results suggest that the inherent sol-gel transition property plays an essential role in the self-regulation of autonomous translational motion in amoeba.

Ion gradients in xylem exudate and guttation fluid related to tissue ion levels along primary leaves of barley.

The concentration of ions in plant cells and tissues is an essential factor in determining physiological function. In the present study, we established that concentration gradients of mobile ions exist in both xylem exudates and tissues within a barley (Hordeum vulgare) primary leaf. For K(+) and NO3 (-) , ion concentrations generally decreased from the leaf base to the tip in both xylem exudates and tissues. Ion gradients were also found for Pi and Cl(-) in the xylem. The hydathode strongly absorbed Pi and re-translocated it to the rest of the plant, whereas Cl(-) was extruded. The ion concentration gradients developed early during leaf growth, increased as the tissue aged and remained under both high and low transpiration conditions. Measurement of the expression profiles of Pi, K(+) and NO3 (-) transporters along the longitudinal axis of the leaf revealed that some transporters are more expressed at the hydathode, but for most transporters, there was no significant variation along the leaf. The mechanisms by which longitudinal ion gradients develop in leaves and their physiological functions are discussed.

Transduction of pressure signal to electrical signal upon sudden increase in turgor pressure in Chara corallina.

By taking advantage of large cell size of Chara corallina, we analyzed the membrane depolarization induced by decreased turgor pressure (Shimmen in J Plant Res 124:639-644, 2011). In the present study, the response to increased turgor pressure was analyzed. When internodes were incubated in media containing 200 mM dimethyl sulfoxide, their intracellular osmolality gradually increased and reached a steady level after about 3 h. Upon removal of dimethyl sulfoxide, turgor pressure quickly increased. In response to the increase in turgor pressure, the internodes generated a transient membrane depolarization at its nodal end. The refractory period was very long and it took about 2 h for full recovery after the depolarizing response. Involvement of protein synthesis in recovery from refractoriness was suggested, based on experiments using inhibitors.

Calcium-induced mechanical change in the neck domain alters the activity of plant myosin XI.

Plant myosin XI functions as a motor that generates cytoplasmic streaming in plant cells. Although cytoplasmic streaming is known to be regulated by intracellular Ca(2+) concentration, the molecular mechanism underlying this control is not fully understood. Here, we investigated the mechanism of regulation of myosin XI by Ca(2+) at the molecular level. Actin filaments were easily detached from myosin XI in an in vitro motility assay at high Ca(2+) concentration (pCa 4) concomitant with the detachment of calmodulin light chains from the neck domains. Electron microscopic observations showed that myosin XI at pCa 4 shortened the neck domain by 30%. Single-molecule analysis revealed that the step size of myosin XI at pCa 4 was shortened to 27 nm under low load and to 22 nm under high load compared with 35 nm independent of the load for intact myosin XI. These results indicate that modulation of the mechanical properties of the neck domain is a key factor for achieving the Ca(2+)-induced regulation of cytoplasmic streaming.

Further electrophysiological studies on cellular effect of herbicide, bromoxynil, using characean cells.

In the previous paper, I reported that 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil) depolarizes the plasma membrane by inhibiting the electrogenic proton pump and discussed that the inhibition is caused by cytosol acidification due to influx of protonated bromoxynil and following release of proton (Shimmen in J Plant Res 123:715-722, 2010). However, a possibility of direct inhibition of the proton pump by bromoxynil flowed into the cell could not be excluded. In the present study, the direct effect of bromoxynil on the proton pump was unequivocally excluded.

Studies on conjugation of Spirogyra using monoclonal culture.

We succeeded in inducing conjugation of Spirogyra castanacea by incubating algal filaments on agar plate. Conjugation could be induced using clone culture. The scalariform conjugation was generally observed, while lateral conjugation was rarely. When two filaments formed scalariform conjugation, all cells of one filament behaved as male and those of other filament did as female. Very rarely, however, zygospores were formed in both of pair filaments. The surface of conjugation tube was stained with fluorescently labeled-lectins, such as Bandeiraea (Griffonia) simplicifolia lectin (BSL-I) and jacalin. BSL-I strongly stained the conjugation tubes, while weakly did the cell surface of female gamete first and then that of male gamete. Jacalin stained mainly the conjugation tubes. Addition of jacalin inhibited the formation of papilla, suggesting some important role of jacalin-binding material at the initial step of formation of the conjugation tubes.

Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells.

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

The myosin ATPase inhibitor, 2,3-butanedione 2-monoxime, prevents protein secretion by the basidiomycete Coprinopsis cinerea.

The plant-saprophytic basidiomycete, Coprinopsis cinerea, produces and secretes various cellulases during cellulose degradation as the main extracellular proteins. Although enzymatic characterization of such cellulases has been frequently reported, the mechanism of their secretion remains unclear. This study focused on myosins, actin-based motor proteins, involved in protein secretion in C. cinerea. During cultivation under cellulase-inducing condition, no cellulase activity was observed when the mycelia were treated with 2,3-butanedione 2-monoxime (BDM), a general inhibitor of myosin ATPase. Furthermore, BDM treatment disrupted the localization of the Golgi apparatus, but not that of the endoplasmic reticulum. Three genes encoding myosin-like proteins (CcMyo1, CcMyo2 and CcMyo5) were identified from the C. cinerea genome database. Transcription of these genes was promoted when the fungus was grown under cellulase-inducing condition.

Involvement of protein synthesis in recovery from refractory period of electrical depolarization induced by osmotic stimulation in Chara corallina.

Upon addition of sorbitol to the external medium of an internodal cell of Chara corallina, a transient depolarization is induced at its nodal end (Shimmen in Plant Cell Physiol 44:1215-1224, 2003). In the present study, refractory period was found to be very long, 2-4 h. Recovery from refractoriness was completely inhibited by inhibitors of eukaryote-type protein synthesis, cycloheximide or anisomysin, but not by inhibitors of prokaryote-type protein synthesis. This suggested that proteinous factor(s) responsible for generation of the depolarization is lost or inactivated upon depolarization and synthesized during the resting state. Low temperature, which is supposed to inhibit protein synthesis, also inhibited recovery from refractoriness. When unstimulated internodal cells were incubated in the medium containing an inhibitor of eukaryote-type protein synthesis, generation of the depolarization was almost completely inhibited. This result suggested that the factor is slowly turning over even in the absence of osmotic stimulation.

Proliferation of the hyperthermophilic archaeon Pyrobaculum islandicum by cell fission.

Pyrobaculum islandicum is a hyperthermophilic archaeon. P. islandicum cells have been suggested to multiply by constriction, budding and branching, as no septa were observed in cells by phase-contrast light microscopy. In this study, we observed the cells using transmission electron microscopy, scanning electron microscopy, and light microscopy with dark-field image analyses, and we report binary fission via septum formation to be the main mode of P. islandicum's proliferation. "Long cells" reported previously were found to comprise several cylindrical cells that align in tandem.

Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells.

Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to approximately 3.5 microm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.

Unique cellular effect of the herbicide bromoxynil revealed by electrophysiological studies using characean cells.

In a previous paper, we proposed that the primary action of the herbicide bromoxynil (BX; 3,5-dibromo-4-hydroxybenzonitrile) is cytosol acidification, based on the fact that bromoxynil induced the inhibition of cytoplasmic streaming and cell death of Chara corallina in acidic external medium (Morimoto and Shimmen in J Plant Res 121:227-233, 2008). In the present study, electrophysiological analysis of the BX effect was carried out in internodal cells of C. corallina. Upon addition of BX, a large and rapid pH-dependent depolarization was induced, supporting our hypothesis. Ioxynil, which belongs to the same group as bromoxynil, also induced a large and rapid membrane depolarization in a pH-dependent manner. On the other hand, four herbicides belonging to other groups of herbicides did not induce such a membrane depolarization. Thus, BX has a unique cellular effect. The decrease in the electro-chemical potential gradient for H(+) across the plasma membrane appears to result in inhibition of cell growth and disturbance of intracellular homeostasis in the presence of BX.

GTP is required for the microtubule catastrophe-inducing activity of MAP200, a tobacco homolog of XMAP215.

Widely conserved among eukaryotes, the microtubule-associated protein 215 (MAP215) family enhances microtubule dynamic instability. The family member studied most extensively, Xenopus laevis XMAP215, has been reported to enhance both assembly and disassembly parameters, although the mechanism whereby one protein can exert these apparently contradictory effects has not been clarified. Here, we analyze the activity of a plant MAP215 homolog, tobacco (Nicotiana tabacum) MAP200 on microtubule behavior in vitro. We show that, like XMAP215, MAP200 promotes both assembly and disassembly parameters, including microtubule growth rate and catastrophe frequency. When MAP200 is added to tubulin and taxol, strikingly long-coiled structures form. When GDP partially replaces GTP, the increase of catastrophe frequency by MAP200 is strongly diminished, even though this replacement stimulates catastrophe in the absence of MAP200. This implies that MAP200 induces catastrophes by a specific, GTP-requiring pathway. We hypothesize that, in the presence of MAP200, a catastrophe-prone microtubule lattice forms occasionally when elongated but nonadjacent protofilaments make lateral contacts.

The putative RNA-processing protein, THO2, is a microtubule-associated protein in tobacco.

THO2 is a component of the THO-TREX (transcription and export factor) complex that participates in mRNA metabolism and export from the nucleus in yeast and animal cells. Here we report that tobacco putative THO2-related protein (NtTHO2) is a microtubule-associated protein, which directly binds to microtubules in vitro and co-localizes with cortical microtubules in vivo. We purified endogenous NtTHO2 by cycles of microtubule polymerization-depolymerization from crude extracts of tobacco BY-2 miniprotoplasts. Purified NtTHO2 sedimented with microtubules in vitro. Immunofluorescence revealed that NtTHO2 was present in both the nucleus and cytoplasm. In interphase, cytoplasmic NtTHO2 was localized along cortical microtubules. In the mitotic phase, NtTHO2 was localized to the mitotic spindle but not to either the preprophase band or the phragmoplast. In mature cells of seedling roots, and in BY-2 cells in which proliferation was stopped by removing 2,4-D, NtTHO2 staining was confined mainly to the nucleolus. These results suggest that NtTHO2 is a multifunctional protein that participates in mRNA metabolism, and also functions within the cortical microtubules and mitotic spindle.

Involvement of actin filaments in rhizoid morphogenesis of Spirogyra.

The role of actin filaments in rhizoid morphogenesis was studied in Spirogyra. When the algal filaments were severed, new terminal cells started tip growth and finally formed rhizoids. Actin inhibitors, latrunculin B and cytochalasin D, reversibly inhibited the process. A mesh-like structure of actin filaments (AFs) was formed at the tip region. Gd(3+) inhibited tip growth and decreased AFs in the tip region. Either a decrease in turgor pressure or lowering of the external Ca(2+) concentration also induced similar results. It was suggested that the mesh-like AF structure is indispensable for the elongation of rhizoids. A possible organization mechanism of the mesh-like AF structure was discussed.

The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis.

The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.