Association of Microtubules with the Plasma Membrane of Tobacco BY-2 Cells in Vitro

Membrane ghosts prepared from tobacco BY-2 cells bound microtubules(MTs) that had been assembled in an extract of evacuolated protoplastsof the same cells and, as a result, the number of MTs on theghosts increased. MTs that had formed on the ghosts after incubationwith the extract were more sensitive to cold-treatment thanthe preexisting MTs. Binding of MTs was also observed on ghoststhat had been treated with a cold solution of Ca²⁺ to removepreexisting MTs. This result suggests that a system that mediatedthe binding of MTs to the plasma membrane remained on the ghosts.No binding of MTs was observed to ghosts that had been treatedwith trypsin. Preexisting MTs were removed by treatment with0.6 M KCl or 0.1 M Na₂CO₃. Membrane ghosts treated with KClor Na₂CO₃ did not bind MTs that had been assembled in the extractin the absence of taxol. However, ghosts prepared in the sameway did bind MTs that had been assembled in the extract in thepresence of taxol. It seems that treatment with KCl or Na₂CO₃removes a component(s) from the system that mediates the bindingof MTs to the plasma membrane and that a factor(s), associatedwith MTs assembled in the extract in the presence of taxol,compensates for the loss of this component(s) from the system. (Received August 12, 1993; Accepted January 26, 1994)     

ATP-Sensitive Binding to Microtubules of Polypeptides Extracted from Isolated Phragmoplasts of Tobacco BY-2 Cells

Polypeptides of about 100 kDa, with the ability to bind to microtubulesassembled in vitro, were extracted from phragmoplasts isolatedfrom tobacco cultured cells by treatment with ATP. These 100-kDapolypeptides could also be extracted from isolated spindlesbut not from isolated interphase nuclei, an indication of theassociation in vivo of these polypeptides with the microtubulesof the spindle and of the phragmoplast. The association of thepolypeptides with microtubules was sensitive both to ATP andto AMPPNP, a nonhydrolyzable analogue of ATP, but not to GTP.Since the 100-kDa polypeptides showed little or no ATPase activity,it seems unlikely that they are species of microtubule-dependentmotility proteins. (Received August 28, 1991; Accepted May 6, 1992)     

Assembly of Microtubules in a Cytoplasmic Extract of Tobacco BY-2 Miniprotoplasts in the Absence of Microtubule-Stabilizing Agents

The assembly of microtubules occurred even in the absence ofmicrotubule-stabilizing agents in a cytoplasmic extract obtainedfrom evacuolated protoplasts (miniprotoplasts) of tobacco BY-2 cells. The assembled microtubules were arranged side by sideforming big bundles and two types of bridge-like structure wereobserved between adjacent microtubules. (Received October 31, 1991; Accepted March 20, 1992)     

CuCl2胁迫对烟草BY-2悬浮细胞死亡的诱导

本文以不含叶绿体的烟草BY-2悬浮细胞系为实验材料,研究了CuCl2胁迫对BY-2细胞呼吸速率、呼吸途径以及细胞内H2O2和ATP含量的影响。结果表明：0.5mmol·L-1CuCl2胁迫明显导致了烟草BY-2细胞的死亡,引起了胞内H2O2爆发和ATP含量下降,严重抑制了BY-2细胞的总呼吸和交替氧化酶途径（alternative oxidase pathway,AOX）。此外,CuCl2对BY-2细胞的线粒体电子传递具有即时的抑制作用。加入外源腺苷（ATP合成的底物）显著抑制了CuCl2胁迫引起的ATP损耗,并阻止了细胞死亡。上述结果表明CuCl2胁迫导致的ATP损耗在CuCl2诱导烟草BY-2细胞死亡过程中起重要的作用。     

Maturation of Cell-Substratum Focal Adhesions Induced by Depolymerization of Microtubules is Mediated by Increased Cortical Tension

Dynamics of alterations of focal adhesions (FA) induced by a microtubule-depolymerizing drug, colcemid, was examined in several types of fibroblastic cells. Evolution of individual FA in cultured cells was monitored by interference-reflection microscopy (IRM); at the end of the monitoring period (3 hours) the cells were fixed and immunofluorescence microscopy of the same FA was performed with an antibody against vinculin. Control and colcemid-treated cells remained non-motile and did not show lamellipodial activity at the edges. During the incubation, formation of new FA or disappearance of pre-existing FA did not occur in either colcemid-treated or control cultures. However, FA in colcemid-treated cells significantly increased in size in the course of a 3 hour incubation. The growth of FA was centripetal and sometimes was accompanied by the fusion of several adjacent FA.

Immunofluorescence examination showed that colcemid-induced growth of FA was accompanied by accumulation of several proteins specific for these structures including vinculin, talin, paxillin and pp125FAK kinase. Immunoblotting with anti-vinculin antibody showed that incubation with colcemid considerably increased the amount of vinculin associated with the ventral membranes due to its partial redistribution from a soluble pool into the growing adhesions. A substantial increase in tyrosine phosphorylation of pp125FAK was also observed in colcemid-treated cells. In cells plated on elastic silicone rubber films, colcemid induced formation of wrinkles in the films and these wrinkles relaxed after treatment with cytochalasin D. These results confirm that microtubule depolymerization increases traction transmitted to the substratum by the actin cortex and shows that an increase in cortical tension accompanies maturation of FA.

Taken together, these data show that short-term incubation with colcemid does not affect the formation of initial FA. In contrast, microtubule depolymerization considerably stimulates the maturation FA, manifested by their centripetal growth. Maturation is proposed to be mediated by increased cortical tension, which is caused by microtubule depolymerization.     

Synthesis of Polysaccharides in Phragmoplasts Isolated from Tobacco BY-2 Cells

Autoradiographic experiments using preparations of isolatedphragmoplast obtained from tobacco cultured cells revealed thatthe radioactivity incorporated into insoluble material fromUDP-[³H]glucose was exclusively present at the cell plate ofisolated phragmoplasts. Most of the radioactivity incorporatedinto isolated phragmoplasts from UDP-[¹⁴C]glucose was solubilizedby 1,3-ß-glucanase and the solubilized radioactivitywas associated only with glucose, indicating that most of theradioactivity was incorporated into 1,3-ß-glucan.In the presence of high concentrations of unlabeled UDP-glucose,isolated phragmoplasts incorporated radioactivity from UDP-[³H]xylose.Most of the radioactivity incorporated into insoluble materialwas present at several sites distributed around the nuclei,while only little was found at the cell plate. (Received October 2, 1991; Accepted February 24, 1992)     

CuCl2胁迫对烟草BY-2悬浮细胞死亡的诱导

本文以不含叶绿体的烟草BY-2悬浮细胞系为实验材料,研究了CuCl2胁迫对BY-2细胞呼吸速率、呼吸途径以及细胞内H2O2和ATP含量的影响。结果表明:0.5mmol·L-1CuCl2胁迫明显导致了烟草BY-2细胞的死亡,引起了胞内H2O2爆发和ATP含量下降,严重抑制了BY-2细胞的总呼吸和交替氧化酶途径(alternative oxidase pathway,AOX)。此外,CuCl2对BY-2细胞的线粒体电子传递具有即时的抑制作用。加入外源腺苷(ATP合成的底物)显著抑制了CuCl2胁迫引起的ATP损耗,并阻止了细胞死亡。上述结果表明CuCl2胁迫导致的ATP损耗在CuCl2诱导烟草BY-2细胞死亡过程中起重要的作用。     

Dynamic Organization of Plant Microtubules at the Three Distinct Transition Points During the Cell Cycle Progression of Synchronized Tobacco BY-2 Cells

Dynamic changes of microtubule (MT) configuration have been examined during the cell cycle progression in tobacco BY-2 cells, which have been highly synchronized by aphidicolin treatment. Although it has been shown previously that four cell cycle stages display characteristic features of MTs (Hasezawa et al., 1991), distinct changes of MT configuration were observed at the interfaces of G₂/M, M/G₁ and G₁/S, and the frequency of appearance of such distinct structures were quantitatively examined. Among others, it is the first observation that at M/G₁ disintegrating phragmoplasts coexisted with short MTs in the perinuclear envelopes, but the MTs disappeared in the later stage, when cortical MTs were organizing. Thus it is supposed that cortical MTs originate from the transiently observed short MTs in the perinuclear region. This observation offered also an experimental system to analyze the molecular changes of MTs at the three interfaces during cell cycle progression in plant cells, as the mass culture of tobacco BY-2 cells is readily available.     

Stabilization of Cortical Microtubules in Maize Mesocotyl Cells by Gibberellin A3

Effects of GA₃ on the stability of cortical microtubules (MTs)were studied in mesocotyl cells of etiolated maize seedlings.Propyzamide, an MT-disrupting agent specific for plant tubulin,disrupted cortical MTs in cells in the upper regions of mesocotylsof GA₃-untreated seedlings and caused swelling of the cells.GA₃ prevented propyzamide from disrupting MTs and from causingsuch swelling. Chilling of mesocotyls at 4°C for 60 minbrought about the disruption of cortical MTs in cells in theupper regions of mesocotyls of GA₃-untreated seedlings, butnot in cells in corresponding regions of GA₃-treated seedlings,suggesting that treatment with GA₃ increased the stability ofcortical MTs in maize mesocotyl cells. Cortical MTs in protoplastsisolated from mesocotyls of GA₃-untreated seedlings failed towithstand chilling at 0°C for 90 min, while those in protoplastsisolated from mesocotyls of GA₃-treated seedlings withstoodchilling successfully. It appears that the cell wall is notinvolved in the stabilization of cortical MTs by GA₃ in maizemesocotyl cells. (Received July 6, 1993; Accepted November 29, 1993)     

Acceleration of Vacuolar Regeneration and Cell Growth by Overexpression of an Aquaporin NtTIP1;1 in Tobacco BY-2 Cells

Aquaporin is a water channel that increases water permeabilitythrough membranous structures. In plants, vacuoles are essentialorganelles that undergo dynamic volume changes during cell growth.To understand the contribution of aquaporins to plant cell growth,we developed a transgenic tobacco BY-2 cell line overexpressingthe tonoplast intrinsic protein (TIP), TIP. Vacuolar membranesof isolated vacuoles from TIP-overexpressing cells showed higherwater permeation activities than those from wild-type cells.We then examined the role of TIP in vacuolar regeneration ofevacuolated tobacco BY-2 protoplasts (miniprotoplasts). Vacuolarregeneration from thin to thick tube-network vacuoles and subsequentdevelopment of large vacuoles was accelerated in miniprotoplastsof this cell line. A parallel increase in the rate of cell expansionindicated a tight relationship between vacuolar developmentand cellular volume increases. Interestingly, overexpressionof tobacco TIP also enhanced cell division. Thus, increasedvacuolar aquaporin activity may accelerate both cell expansionand cell division by increasing water permeability through thevacuolar membrane.     

Intrinsic Cell Polarity Coupled to Growth Axis Formation in Tobacco BY-2 Cells

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Actin Filaments Purified from Tobacco Cultured BY-2 Cells Can Be Translocated by Plant Myosin

Actin filaments (AFs) and microtubules (MTs) are essential constituentsof the cytoskeleton in plant cells. Sliding of motor proteinsalong these cytoskeletons is believed to be necessary in variouscellular functions. In our previous study [Yokota et al. (1995b)Plant Cell Physiol. 36: 1563], we succeeded in isolating tubulinfrom cultured tobacco BY-2 cells, which in its polymerized formcan be translocated by the MT-based motor protein, dynein, invitro. In the present study, the method was modified to purifyboth tubulin and actin. Purified actin could be polymerizedand decorated by subfragment-1 (S-1) of skeletal muscle myosin.In the motility assay in vitro, AFs, thus prepared, could betranslocated by plant myosin isolated from lily pollen tubes.The sliding velocity of those AFs was similar to that of animalAFs prepared from chicken breast muscle, and comparable withthe velocity of cytoplasmic streaming in living pollen tubesof lily. Using S-1, motility assay was carried out. The slidingvelocity of plant AFs and that of muscle AFs were also similar.As far as we know, this is the first report of the sliding ofisolated plant AFs with myosin. (Received April 30, 1999; Accepted September 7, 1999)     

Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform

A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.     

Nuclear-Cycle Dependence of the Development of the Preprophase Band in Tobacco BY-2 Cells

When tobacco BY-2 cells that had been treated with aphidicolinfor 24 h were cultured in the absence of aphidicolin, DNA synthesiswas initiated within 1 h. DNA synthesis was completed within4 h and then the preprophase band of microtubules (PPB) developed.However, when cells that had been treated with aphidicolin werecultured in the absence of aphidicolin for 1 h and then againin its presence, DNA synthesis, which was initiated during thehour in the absence of aphidicolin, was not completed withina further 10 h in the presence of aphidicolin. Moreover, thePPB did not develop even after the PPB had appeared and disappearedin cells that were cultured contemporaneously in the continuedabsence of aphidicolin. The development of the PPB seems to be causally associated withthe nuclear cycle of cell division in tobacco BY-2 cells. Thisprocess seems to require the completion of the replication ofall, or almost all, of the nuclear DNA. (Received July 25, 1992; Accepted November 24, 1992)     

Plasma Membrane Ghosts Form Differently When Produced from Microtubule-Free Tobacco BY-2 Cells

When lysed in an actin stabilizing buffer, protoplasts madefrom tobacco BY-2 suspension culture cells formed plasma membraneghosts that retained both cortical actin and microtubules. Distinctcytoskeletal arrays occurred: the most common ghost array (typeI) derived from protoplasts in interphase and had random actinand microtubules, although the alignment of the actin was dependent,at least partially, on microtubule organization. Type II ghostswere larger and more irregular in shape than type I ghosts,and were characterized by a lack of microtubules and the presenceof distinctive arrays of actin bundles in concentric arcs. Theseghosts derived from protoplasts lacking cortical microtubulesproduced when wall digestion occurred while the cells were incell division, or from protoplasts isolated in the presenceof 100 µM propyzamide. Because type II ghosts derivedfrom protoplasts of similar size to those that give rise totype I ghosts, and because type II ghosts retained ordered actinarrays while the parent protoplasts had random cortical actin,type II ghosts apparently form differently to type I ghosts.We speculate that instead of the protoplast being sheared offto produce a round ghost, the plasma membrane tears and collapsesonto the slide, ordering the actin bundles in the process. Oneimplication of this model would be that cortical microtubulesprovide structural support to the plasma membrane of the protoplastso that only in their absence do the type II ghosts form. (Received May 26, 1998; Accepted October 26, 1998)     

Effect of Ions on the Orientation of Cortical Microtubules in Spirogyra Cells

Effects of ions on the orientation of cortical micro-lubules(MTs) in Spirogyra cells were studied. After depo-lymerizalionwith amiprophos-methyl (APM), MTs were allowed to reorganizein NaCI solutions of various concentrations. As the concentrationof NaCI increased, the frequency of cells that had oblique MTsincreased. When cells in NaCI solution were transferred intoartificial pond water (APW) and incubated for 6 h, all the MTschanged to become transverse to the longitudinal axis of thecell. KC1 and MgCl₂ also had effects on the orientation of MTs.However, NH₄Cl, CaCl₂;, CoCl₂, and Co(NO₃)₂ did not show anyeffect. These results suggest that Na⁺, K⁺, and Mg²⁺have effectson MT orientation and that NH⁺₄, Ca²⁺, Co²⁺, Cl^–, andNO^–₃ have little effect. When MTs were reorganized ineither NaCl or KCl solutions, all the oblique MTs were organizedinto an S-helix. In contrast, some of the oblique MTs were foundas a Z-helix in the cells incubated in MgCl₂ or mannitol solutions.These results suggest that effects of Na⁺ and K⁺ on the orientationof MTs are not the same as those of Mg²⁺ and mannitol. Theseresults provide the first evidence that ions are involved inthe orientation of MTs in algae. (Received January 27, 1998; Accepted August 10, 1998)     

The Mode of Expression and Promoter Analysis of the arcA Gene, an Auxin-Regulated Gene in Tobacco BY-2 Cells

The arcA, a member of the G protein rß-subunit family,was isolated from tobacco BY-2 cells as an auxin-responsivegene. Characterization of arcA, which should help to elucidatethe function of the gene product in the plant cells, was performedwith emphasis on the mode of expression and the analysis ofits promoter. Accumulation of the arcA message was detectedonly after treatments with auxins and not after treatments withother phytohormones or CdCl₂, implying that responsiveness ofarcA was exclusive to auxin. The putative arcA promoter regionwas fused to a reporter gene for rß-glucuronidase(GUS), and transient expression was analyzed in tobacco BY-2cells. Two series of arcA promoter/GUS chimeric genes were constructed.One consisted of a set of 5' nested deletions of the arcA promoterconnected to the gene for GUS and the other consisted of a varietyof the arcA promoter fragments fused to a minimal promoter-GUSconstruct. The results indicated that the promoter sequencecovering four sets of direct repeats (– 562 to –167)was necessary for the sufficient response of arcA promoter toauxin in BY-2 cells. Moreover, irrespective of auxin treatment,elevated activity of GUS driven by this promoter fragment wasdetected, a result that implies that this region behaves anenhancer in BY-2 cells. (Received September 30, 1995; Accepted March 1, 1996)     

Auxin Deprivation Induces Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells

To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells.     

Pressure Induced Reorientation of Cortical Microtubules in Epidermal Cells of Lolium rigidum Leaves

In order to assess the effect on microtubule arrays of slowlypressurising cells over 50 s from 0.1 MPa (atmospheric pressure)to 55 MPa, microtubules in epidermal cells of Lolium rigidumleaves were visualised by immunofluorescent staining and fluorescencemicroscopy. In both control and pressure-treated leaves cellshape, measured as the ratio of cell length and width, can becorrelated to the arrangement of cortical microtubules. Microtubulearrays change from random to organised in cells whose lengthis greater than their width. In untreated leaves, elongatedcells have microtubules aligned predominantly transversely.In pressure-treated leaves, elongated cells have microtubulesaligned predominantly longitudinally. Thus, pressure treatmentresults in the rapid reorientation of organised cortical microtubulesfrom a transverse to a longitudinal orientation. (Received June 21, 1993; Accepted July 15, 1993)