Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells

In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.     

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity

 A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421–1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes. Received: 23 February 1999 / Accepted: 22 April 1999     

The 135 kDa actin-bundling protein fromLilium longiflorum pollen is the plant homologue of villin

Summary Actin microfilaments, which are essential for cell growth and cytoplasmic streaming in pollen tubes, are closely dependent on actin-binding proteins for their organization and regulation. We have purified the plant 135 kDa actin-bundling protein (P-135-ABP) fromLilium longiflorum pollen and determined that its amino acid composition is highly similar to members of the villin-gelsolin family of proteins. We used antibodies against P-135-ABP to probe an expression cDNA library ofL. longiflorum pollen and isolated a full-length clone (ABP135) that corresponds to a 106 kDa polypeptide. The deduced amino acid sequence ofABP135 shows homology with members of the villin-gelsolin family of proteins and contains the characteristic six repeats of this family, as well as an extended carboxy-terminal domain that includes the villin headpiece preceded by a highly variable region. Using two-dimensional polyacrylamide gel electrophoresis we detected at least 5 isoforms of P-135-ABP, with isoelectric points (pI) ranging between 5.6 to 5.9. The most abundant P-135-ABP isoform has a pI of 5.8, closely approximating the pI predicted from the deducedABP135 amino acid sequence. These data, together with the partial amino acid sequence from a proteolytic peptide of the protein, indicate that P-135-ABP is a plant villin. Immuno-detection of Lilium villin in rapidly frozen pollen tubes localized it to actin bundles. Lilium villin is also ubiquitously expressed in all tissues tested. Since villins, like gelsolins, are also Ca²⁺-dependent severing, capping, and nucleating proteins, Lilium villin may participate in F-actin fragmentation and nucleation in the apex of the pollen tube where there is steep Ca²⁺ gradient.Abbreviations BMM butyl methyl-methacrylate - PPI polyphos-phoinositides - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

Possible involvement of protein phosphorylation in the regulation of cytoplasmic organization and streaming in root hair cells as revealed by a protein phosphatase inhibitor, calyculin A

Summary The effects of a protein phosphatase inhibitor, calyculin A (CA), on cytoplasmic streaming and cytoplasmic organization were examined in root hair cells ofLimnobium stoloniferum. CA at concentrations higher than 50 nM inhibited cytoplasmic streaming and also induced remarkable morphological changes in the cytoplasm. The transvacuolar strands, in which actin filament bundles were oriented parallel to the long axis, disappeared and spherical cytoplasmic bodies emerged in the CA-treated cells. In these spherical bodies, actin filaments were present and the spherical bodies were connected to each other by thin strands of actin filaments. Upon CA removal, transvacuolar strands, in which actin filament bundles were aligned, and cytoplasmic streaming reappeared. A nonselective inhibitor for protein kinases, K-252a, delayed the inhibitory effect of CA on cytoplasmic streaming and suppressed the CA-induced formation of the spherical bodies. From these results, it is suggested that phosphatases sensitive to CA regulate cytoplasmic streaming and are involved in the organization of the cytoplasm in root hair cells.Abbreviations APW artificial pond water - CA calyculin A     

Roles of actin filaments in cytoplasmic streaming and organization of transvacuolar strands in root hair cells ofHydrocharis

Summary Effects of cytochalasin B and mycalolide-B on cytoplasmic streaming, organizations of actin filaments and the transvacuolar strand were studied in root hair cells ofHydrocharis, which shows reverse fountain streaming. Both toxins inhibited cytoplasmic streaming and destroyed the organizations of actin filaments and transvacuolar strands. However, we found a great difference between these toxins with respect to reversibility. The effects of cytochalasin B were reversible but not those of mycalolide B. The present results suggest that actin filaments work as a track of cytoplasmic streaming and as a cytoskeleton to maintain the transvacuolar strand. The usefulness of root hair cells ofHydrocharis in studying the dynamic organization of actin filaments of plant is discussed.Abbreviations CB cytochalasin B - DMSO dimethylsulfoxide - ML-B mycalolide B     

Mechanism of inhibition of cytoplasmic streaming by a myosin inhibitor, 2,3-butanedione monoxime

Summary On the basis of the inhibition of myosin by 2,3-butanedione monoxime (BDM), the protein's involvement in various cell activities is discussed. However, it has not been established whether BDM inhibits plant myosin. In the present study, the effect of BDM on isolated plant myosin was analyzed in vitro. The sliding between myosin from lily (Lilium longiflorum) pollen tubes and actin filaments from skeletal muscle was inhibited to 25% at a concentration of 60 mM, indicating that BDM can be used as a myosin inhibitor for plant materials. Cytoplasmic streaming was completely inhibited by BDM at 30 mM in lily pollen tubes and at 70 mM in short root hair cells, and at 100 mM in long root hair cells ofHydrocharis dubia. However, BDM at high concentrations induced the disorganization of actin filament bundles in lily pollen tubes and short root hair cells. In addition, cortical microtubules were also fragmented in short root hair cells treated with BDM, suggesting a possible side effect of BDM.Abbreviations AF actin filament - BDM 2,3-butanedione monoxime - MT microtubule     

Actin filament organization and polarity in pollen tubes revealed by myosin II subfragment 1 decoration

The actin cytoskeleton plays a crucial role in pollen tube growth. In elongating pollen tubes the organization and arrangement of actin filaments (AFs) differs between the shank and apical region. However, the orientation of AFs in pollen tubes has not yet been successfully demonstrated. In the present work we have used myosin II subfragment 1 (S1) decoration to determine the polarity of AFs in pollen tubes. Electron microscopy studies revealed that in the shank of the tube bundles of AFs exhibit uniform polarity with those close to the cell cortex having their barbed ends oriented towards the tip of the pollen tube while those in the cell center have their barbed ends oriented toward the base of the tube. At the subapex, some AFs are organized in closely packed and longitudinally oriented bundles and some form curved bundles adjacent to the cell membrane. In contrast, few AFs are dispersed with random orientation in the extreme apex of the pollen tube. Our results confirm that the direction of cytoplasmic streaming within pollen tubes is determined by the polarity of AFs in the bundles.     

ACTIN BINDING PROTEIN 29 from Lilium pollen plays an important role in dynamic actin remodeling

Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.     

Fluorescence Microscopic Observations on Actin Filament Distribution in Corn Pollen and Gladiolus Pollen Protoplasts

Actin filament (AF) distribution in Zea mays pollen and Gladiolus gandavensis pollen protoplasts was localized by FITC conjugated phalloidin fluorescence microprobe. The pollen was incubated in Brewbaker and Kwack (BK) medium, and the pollen protoplasts were isolated enzymatically and cultured in K3 medium containing various supplements by a previously reported method. Samples were fixed for 30 min with 1.5% paraformaldehyde dissolved in 0.1 mol/1 phosphate buffer (pH 7), half strength of BK elements, 1 mol/1 EGTA and sucrose, stained for 30–60 min with 1 μg/ml FITC-phalloidin in the buffer solution, and observed by a fluorescence microscopy. In hydrated corn pollen grains, the AFs constituted an irregular network. Prior to germination a part of the pollen grains showed polarized pattern of Afs. At the opposite pole to the germ pore, there was a center from which AF bundles radiated and converged toward the pore, often making a spindle-shaped configuration. In just isolated gladiolus pollen protoplasts, the AFs appeared as irregular fine network. After 4–7h of culture, the AF distribution coincided in some cases with the unevenly regenerated new wall area as exhibited by FITC-phalloidin and Calcofluor White ST double staining, indicating a possible involvement of AF in wall synthesis. After 17–18 h of culture, a part of the pollen protoplasts went on germination. The AFs became polarized in such protoplasts and converged into the tubes produced, and ran longitudinally along the tubes just like in the tubes germinated from pollen grains. However, in ungerminated pollen protoplasts, the AFs behaved abnormalty, showing various irregular arrangements. When protoplasts bursted, the actin aggregates often located at the protrusion site from which the protoplasts would burst, and were discharged into the medium. In neither corn pollen nor gladiolus pollen protoplasts AFs were observed within the generative or sperm cells.     

Calcium-calmodulin suppresses the filamentous actin-binding activity of a 135-kilodalton actin-bundling protein isolated from lily pollen tubes

We have isolated a 135-kD actin-bundling protein (P-135-ABP) from lily (Lilium longiflorum) pollen tubes and have shown that this protein is responsible for bundling actin filaments in lily pollen tubes (E. Yokota, K. Takahara, T. Shimmen [1998] Plant Physiol 116: 1421-1429). However, only a few thin actin-filament bundles are present in random orientation in the tip region of pollen tubes, where high concentrations of Ca(2+) have also been found. To elucidate the molecular mechanism for the temporal and spatial regulation of actin-filament organization in the tip region of pollen tubes, we explored the possible presence of factors modulating the filamentous actin (F-actin)-binding activity of P-135-ABP. The F-actin-binding activity of P-135-ABP in vitro was appreciably reduced by Ca(2+) and calmodulin (CaM), although neither Ca(2+) alone nor CaM in the presence of low concentrations of Ca(2+) affects the activity of P-135-ABP. A micromolar order of Ca(2+) and CaM were needed to induce the inhibition of the binding activity of P-135-ABP to F-actin. An antagonist for CaM, W-7, cancelled this inhibition. W-5 also alleviated the inhibition effect of Ca(2+)-CaM, however, more weakly than W-7. These results suggest the specific interaction of P-135-ABP with Ca(2+)-CaM. In the presence of both Ca(2+) and CaM, P-135-ABP organized F-actin into thin bundles, instead of the thick bundles observed in the absence of CaM. These results suggest that the inhibition of the P-135-ABP activity by Ca(2+)-CaM is an important regulatory mechanism for organizing actin filaments in the tip region of lily pollen tubes.     

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     

Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner

From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).     

Fluorescence Microscopic Observations on the Distribution Patterns of Actin Filaments in the Cells of Developing Endosperm in Triticum aestivum

The presence and distribution patterns of actin filaments (AFs) in the cells of developing wheat (Triticum aestivum L. ) endosperm exhibiting intercellular protoplasmic movement were studied with fluorescence microscopy and video microscopy. By using TRITC-PhaIloidin as fluorescence probe and cytochalasin B (CB) treatment it was uncovered that there were a lot of AFs scattered throughout the cytoplasm and the patterns of AFs varied greatly with the actin localization. Four configurations of AFs could be recognized: an actin meshwork surrouding the nucleus; bundles of AFs radiating from nuclear “basket” and extending to the periplasm; numerous finer AFs densely and randomly distributed in the cortical cytoplasm and fusiform bodies composed of AFs appearing in the endosperm cells lying at the “cheek” of the caryopsis. Judging from the dynamic characters of intercellular movement of the cytoplasmic constituents and the reaction of cytoplasmic strands related to CB treatment, the authors have discussed and proposed that the exhibition of the two kinds of intercellular movement (extrusion of cytoplasmic strands and mass flow of ground substance) might also be in close relation to the different configurations of AF organization in the cytoplasm.     

Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii

The distribution of and relationship between F-actin and G-actin were investigated in pollen grains and pollen tubes of Lilium davidii Duch. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. Circular F-actin bundles were found to be the main form of microfilament cytoskeleton in pollen grains and pollen tubes. Consistent with cytoplasmic streaming in pollen tubes, there were no obvious F-actin bundles in the 10- to 20-microm tip region of long pollen tubes, only a few short F-actin fragments. Labeling with fluorescein isothiocyanate (FITC)-DNase I at first established the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of the tube, the concentration of G-actin being about twice as high in the 10- to 20-microm region of the tip as in other regions of the pollen tube. We also found that the distribution of G-actin was related negatively to that of the F-actin in pollen tubes of L. davidii. Caffeine treatment caused the G-actin tip-focused gradient to disappear, and F-actin to extend into the pollen tube tip. Based on these results, we speculate that the circular F-actin bundles may be the track for bidirectional cytoplasmic streaming in pollen tubes, and that in the pollen tube tip most of the F-actin is depolymerized into G-actin, leading to the absence of F-actin bundles in this region.     

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.     

Possible involvement of energy metabolism in the change of cytoplasm organization induced by a protein phosphatase inhibitor,calyculin A,in root hair cells of Limnobium stoloniferum

Summary.  In root hair cells of Limnobium stoloniferum, transvacuolar strands disperse and cytoplasmic spherical bodies (CSBs) emerge upon treatment with a protein phosphatase inhibitor, calyculin A (CA), whose effects were previously shown to be canceled by simultaneous treatment of the cells with a nonselective protein kinase inhibitor, K-252a. CSB formation is also suppressed by latrunculin B (LB) or cytochalasin D, actin filament depolymerization drugs, or 2,3-butanedione monoxime, an inhibitor of myosin activity. To confirm the involvement of myosin activity in CSB formation induced by CA, we examined the effect of an inhibitor of energy metabolism, NaN₃, on CSB formation in root hair cells pretreated simultaneously with CA and LB. In the presence of CA-LB, CSB formation was suppressed due to the depolymerization of actin filaments. When these drugs were removed, the actin filaments recovered and CSBs emerged even in the presence of K-252a. These results indicated that the phosphorylation level in the cells is elevated during the CA-LB treatment and that a phosphorylation level sufficient for the CSB formation was sustained even after CA removal. On the other hand, CSB formation after simultaneous treatment with CA and LB was significantly suppressed in the presence of NaN₃. In such cells, actin filament bundles recovered, although their organization was random. The present and previous results suggested that myosin activity is necessary for CSB formation induced by CA, and that myosin regulated by phosphorylation-dephosphorylation is implicated in the organization of the actin cytoskeleton in root hair cells. Received June 26, 2002; accepted October 18, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-1297, Japan.     

Disruption of actin filaments by latrunculin B affects cell wall construction in Picea meyeri pollen tube by disturbing vesicle trafficking

The involvement of actin filaments (AFs) in vesicle trafficking, cell wall construction and tip growth was investigated during pollen tube development of Picea meyeri. Pollen germination and tube elongation were inhibited in a dose-dependent manner by the latrunculin B (LatB) treatment. The fine AFs were broken down into disorganized fragments showing a tendency to aggregate. FM4-64 labeling revealed that the dynamic balance of vesicle trafficking was perturbed due to F-actin disruption and the fountain-like cytoplasmic pattern changed into disorganized Brownian movement. The configuration and/or distribution of cell wall components, such as pectins, callose and cellulose, as well as arabinogalactan proteins changed in obvious ways after the LatB application. Fourier transform infrared (FTIR) analysis further established significant changes in the chemical composition of the wall material. Our results indicate that depolymerization of AFs affects the distribution and configuration of cell wall components in Picea meyeri pollen tube by disturbing vesicle trafficking.     

Confocal Microscopic Observations on Actin Filament Distribution in Lily Pollen Protoplasts

Actin filaments (F-actin) were localized in the isolated pollen protoplasts of lily using TRITC-phalloidin probe and confocal microscopy. Two kinds of pollen protoplasts were examined: one from pollen grains of non-dehiscent anthers(referred to as ‘nearly mature’ pollen); and the other from pollen grains of just dehiscent anthers(referred to as ‘just mature’ pollen). In the cytoplasm of the pollen protoplasts of the ‘nearly mature’ pollen there was a very well organized actin network made up of thick actin bundles. Two types of bundle connections were seen in the network; namely ‘branch’ connections and 'junction' connections. The ‘branch’ connection (or branching points) was formed due to branching or merging of bundies. The ‘junction’ connection (or 'junction' point) had two or more bundles associated with it. Some of the ‘junction’ points might be actin filament organization: centres. The generative cell in iht pollen protoplasts of the ‘nearly mature’ pollen also contained an actin network. But this network was structurally quite loose and the pundles made up the network were short and thick. In the cytoplasm of the pollen protoplasts of the ‘just mature’ pollen the actin net work was more densely packed. The bundles made up the network were also thinner. The actin network in the generative cell was, however, less densely packed. If the pollen protoplasts from both the ‘nearly mature’ and the 'just mature' pollen grains were transferred from a B5 medium into a Brewbaker and Kwack medium supplemented with sucrose, protoplasts rapidly (i.e. within 2 to 3 hours) developed vacuoles and transvacuolar strand. In these va cuolated protoplasts the vegetative nucleus andthe generative cell became tightly surrounded by a new actin network. In the transvacuolar strands there were numerous actin bundles. The “ends” of some of these bundles appeared to be tightly attached to the protoplast membrane indicating that some kind of structures might be present in the protoplast membrane for actin filament attachment.     

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)