Biomedical subjects

Teruo Shimmen

Publications and source records attributed to Teruo Shimmen.

At least 19 recordsLinked to original sources

PubMed2007

Microtubule- and actin filament-dependent motors are distributed on pollen tube mitochondria and contribute differently to their movement.

The pollen tube exhibits cytoplasmic streaming of organelles, which is dependent on the actin-myosin system. Although microtubule-based motors have also been identified in the pollen tube, many uncertainties exist regarding their role in organelle transport. As part of our attempt to understand the role of microtubule-based movement in the pollen tube of tobacco, we investigated the cooperation between microtubules and actin filaments in the transport of mitochondria and Golgi vesicles, which are distributed differently in the growing pollen tube. The analysis was performed using in vitro motility assays in which organelles move along both microtubules and actin filaments. The results indicated that the movement of mitochondria and Golgi vesicles is slow and continuous along microtubules but fast and irregular along actin filaments. In addition, the presence of microtubules in the motility assays forces organelles to use lower velocities. Actin- and tubulin-binding tests, immunoblotting and immunogold labeling indicated that different organelles bind to identical myosins but associate with specific kinesins. We found that a 90 kDa kinesin (previously known as 90 kDa ATP-MAP) is associated with mitochondria but not with Golgi vesicles, whereas a 170 kDa myosin is distributed on mitochondria and other organelle classes. In vitro and in vivo motility assays indicate that microtubules and kinesins decrease the speed of mitochondria, thus contributing to their positioning in the pollen tube.

Actins↗

PubMed2006

Purification and characterization of plant dynamin from tobacco BY-2 cells.

We purified an 84 kDa polypeptide from the MAP (microtubule-associated protein) fraction of tobacco BY-2 cultured cells. LC/MS/MS (liquid chromatography-tandem mass spectrometry) analysis revealed that this polypeptide is a tobacco homolog of AtDRP3 (Arabidopsis thaliana dynamin-related protein 3). Electron microscopy revealed that NtDRP3 (Nicotiana tabacum dynamin-related protein 3) assembles to form a filamentous structure. When GDP was added to the NtDRP3 fraction, the filaments disappeared and many particles appeared. Biochemical analysis revealed that NtDRP3 could bind to and bundle both microtubules and actin filaments in vitro.

Arabidopsis Proteins↗

PubMed2006

n-Butanol induces depolymerization of microtubules in vivo and in vitro.

The effects of butanol on microtubules (MTs) were examined by immunofluorescence microscopy. Fragmentation of cortical MTs was induced by n-butanol, but not by s- and t-butanols, in cultured tobacco BY-2 cells. Taxol prevented n-butanol-induced MT fragmentation. Fragmented cortical MTs were still attached to the inner face of the plasma membrane when n-butanol-treated protoplasts were ruptured on the slide glass. Moreover, MTs were depolymerized in the presence of n-butanol in vitro. Therefore, n-butanol is not only an activator of phospholipase D but also an effective MT-depolymerizing agent.

1-Butanol↗

PubMed2006

Electrical perception of the 'death message' in Chara: characterization of K+ -induced depolarization.

When the nodal end of an internodal cell of Chara corallina was subjected to a pulse treatment with artificial cell sap, a depolarization lasted in artificial pond water. This depolarization could also be induced by pulse treatment with KCl solution, indicating that K+ in the artificial cell sap is responsible (K+ -induced depolarization). The depolarization was prolonged in the presence of 2 mM KCl and the prolonged depolarization was terminated by supplementing with either CaCl2, MgCl2 or NaCl. These results supported the hypothesis that K+ released from the killed cell is responsible for generation of the wound-induced membrane depolarization.

Calcium↗

PubMed2005

Electrical perception of the 'death message' in Chara: analysis of K+-sensitive depolarization.

Wounding electrical responses were studied in Chara corallina. Specimens comprising two adjoining internodal cells were prepared. When one cell (victim cell) was cut, the other cell (receptor cell) generated four kinds of depolarization: (i) rapid depolarization; (ii) long-lasting depolarization; (iii) action potentials; and (iv) small spikes. In the present study, attention was focused on the long-lasting depolarization. A decrease in the electrical resistance suggested activation of ion channel(s). The duration of the depolarization was sensitive to the external ions. K(+) significantly prolonged the depolarization. On the other hand, Ca(2+), Mg(2+) and Na(+) had a tendency to shorten the duration prolonged by K(+). When a nodal end was continuously flushed with a medium lacking K(+), the depolarization was significantly shortened. Treatment of the nodal end with artificial cell sap for 2 min induced a long-lasting depolarization similar to that induced by cutting the victim cell. These findings suggested the involvement of K(+) released from the victim cell in generating the long-lasting depolarization by the receptor cell.

Action Potentials↗

PubMed2005

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+).

Actins↗

PubMed2005

Peroxisomal localization of a myosin XI isoform in Arabidopsis thaliana.

The genome of Arabidopsis thaliana contains 13 myosin XI isoforms. Here we prepared a specific antibody against a peptide that mimics a unique C-terminal region from the myosin XI isoform, MYA2. The resulting antibody was used to demonstrate that MYA2 in Arabidopsis protein extracts co-sedimented with actin filaments and dissociated from the filaments with ATP treatment. Immunolocalization studies showed that MYA2 co-localized predominantly with actin filaments in clustered punctuate dots in leaf epidermal cells, root hair cells and suspension-cultured cells. In a transgenic plant in which peroxisomes are labeled with green fluorescent protein, some MYA2 signals were localized on peroxisomes in an actin-dependent manner. We propose that the peroxisome is one of the cargos translocated by MYA2 on actin filaments.

Actin Cytoskeleton↗

PubMed2005

Possible involvement of mechanosensitive Ca2+ channels of plasma membrane in mechanoperception in Chara.

When an internodal cell of Chara corallina was stimulated with a mechanical pulse of various amplitudes lasting for 0.1 s (mechanical stimulus), the cell generated a receptor potential, which was highly dependent not only on the strength of the stimulus but also on the extracellular Cl- concentration. Extracellular Ca2+ was indispensable for generating receptor potential, since removal of Ca2+ reversibly inhibited generation of the receptor potential. The cytoplasmic Ca2+ level transiently rose upon mechanical stimulation. The stronger the mechanical stimulus, the larger was the increase in the cytoplasmic level of Ca2+. It is proposed that the first step of receptor potential is an activation of mechanosensitive Ca2+ channels at the plasma membrane.

Calcium Channels↗

PubMed2005

Mechano-sensitive orientation of cortical microtubules during gravitropism in azuki bean epicotyls.

The orientation of cortical microtubules (cMT) during gravitropism was studied in epidermal cells of azuki epicotyls. The relative proportion of cells with longitudinal cMT increased in the upper epidermis, and those with transverse cMT increased in the lower epidermis. When epicotyls were kept straight during gravistimulation, no change in cMT orientation occurred in either the upper and lower epidermis. When epicotyls were forced to bend downward, cells with transverse cMT increased in the upper epidermis, and those with longitudinal cMT increased in the lower epidermis. When epicotyls were loaded with naphthylphthalamic acid, an inhibitor of auxin transport, both gravitropic bending and change in cMT orientation were inhibited. However, when a change in cMT orientation was induced by forced downward bending, cells with longitudinal cMT increased in the compressed (lower) side and those with transverse cMT increased in the extended (upper) side. It was suggested that cMT orientation was controlled by the bending of the epicotyl and not by a gravity signal per se. Loading with Gd3+, an inhibitor of the stretch-activated channel, did not inhibit gravitropic bending. However, it inhibited cMT reorientation induced by gravitropic bending and by forced bending. Involvement of the stretch-activated channel in mechano-sensitive orientation of cMT was suggested.

Fabaceae↗

PubMed2004

Saponin-induced release of single cells from filaments and rhizoid differentiation in Spirogyra.

Some species of Spirogyra living in streams can anchor to the substratum by differentiating a rhizoid from a terminal cell of a filament. Rhizoid differentiation occurs in the light but not in the dark. When a filament of Spirogyra sp. competent for rhizoid differentiation was incubated in a medium containing 0.1% saponin, terminal cells were released one by one, forming single cells. Single cells effectively differentiated to be rhizoids when saponin in the incubation medium was removed. The single-cell system developed in the present study seems suitable for analysis of gene expression during rhizoid differentiation of Spirogyra.

Cell Differentiation↗

PubMed2004

Studies on semipermeability and electrical characteristics in membranes of Chara cells fixed with glutaraldehyde.

When internodal cells of Chara corallina were incubated in a solution containing 1% glutaraldehyde, an increase in intracellular osmolality was observed, and this indicates that the plasma membrane maintained its semipermeability. The effect on the membrane potential was studied. Although the active component generated by the electrogenic proton pump was lost, the passive component (more negative than -100 mV) was maintained during glutaraldehyde treatment for more than 1-2 h. The membrane resistance increased significantly. It was found that the tonoplasts also maintained their semipermeability during glutaraldehyde treatment.

Cell Membrane Permeability↗

PubMed2004

21-kDa polypeptide, a low-molecular-weight cyclophilin, is released from pollen of higher plants into the extracellular medium in vitro.

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca2+. This phenomenon was also dependent on pH and on the concentrations of MgCl2 in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl2 concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis- trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.

Amino Acid Sequence↗

PubMed2004

Cytoplasmic streaming in plants.

Plant cells are surrounded by a cell wall composed of polysaccharides and hence can change neither their form nor their position. However, active movement of organelles (cytoplasmic streaming or protoplasmic streaming) is observed in plant cells, and involvement of the actin/myosin system in these processes has been suggested. Successful biochemical and biophysical approaches to studying myosins have extensively promoted the understanding of the molecular mechanism underlying these phenomena.

Actin Cytoskeleton↗

PubMed2004

Characterization of a 200 kDa microtubule-associated protein of tobacco BY-2 cells, a member of the XMAP215/MOR1 family.

A microtubule-associated protein composed of a 200 kDa polypeptide (MAP200) was isolated from tobacco-cultured BY-2 cells. Analysis of the partial amino acid sequence showed that MAP200 was identical to TMBP200, the tobacco MOR1/XMAP215 homolog. Although several homolog proteins in animal and yeast cells have been reported to promote MT dynamics in vitro, no such function has been reported for plant homologs. Turbidity measurements of tubulin solution suggested that MAP200 promoted tubulin polymerization, and analysis by dark-field microscopy revealed that this MAP increased both the number and length of microtubules (MTs). Electron microscopy and experiments using a chemical crosslinker demonstrated that MAP200 forms a complex with tubulin. Throughout the cell cycle, some MAP200 colocalized with MT structures, including cortical MTs, the preprophase band, spindle and phragmoplast, while some MAP200 was localized in areas lacking MTs. Based on our biochemical and immunofluorescence findings, the function of MAP200 in MT polymerization is discussed.

Biopolymers↗

PubMed2004

Functional characterization of contractile vacuole isolated from Amoeba proteus.

Contractile vacuoles (CVs) released from cells of Amoeba proteus were used to analyze its function in vitro. When CV was transferred to a hypertonic medium, its volume decreased within 10 sec. When it was subsequently returned to its original medium, it quickly started swelling. However, it ruptured before recovering its initial volume. These results suggested that the CV membrane is semi-permeable and that the fluid is collected by the osmotic gradient in vivo. The water permeability of membrane of isolated CV was calculated from the rate of osmotic volume change to be 0.94 microm/sec . OsM. This high value suggested that CV membrane is equipped with water channel. CV contracted (or burst) quickly upon addition of 1 mM ATP. Contraction was induced by ATP, but not by other nucleotides, GTP, ITP, ADP, or the analogues of ATP, AMP-PNP and ATPgammaS. It was suggested that the contraction of isolated CV was caused by increase in the tension of its membrane by ATP.

Adenosine Triphosphate↗

PubMed2003

Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity.

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.

Actins↗

PubMed2003

Studies on alkaline band formation in Chara corallina: ameliorating effect of Ca2+ on inhibition induced by osmotic shock.

Although the decrease in cell turgor by application of sorbitol to the external medium did not inhibit the alkaline band formation in Chara corallina, recovery of normal turgor severely inhibited it. Alkaline-loading analysis suggested that the inhibition of alkaline band formation was caused by inhibition of HCO(3)(-) influx but not that of OH(-) efflux. In the presence of 10 mM CaCl(2), the capacity of alkaline band formation was maintained during osmotic treatment. Cells could not form alkaline bands, when plasmolysis was induced by application of sorbitol at a higher concentration. Addition of 10 mM CaCl(2) could ameliorate the inhibition caused by plasmolyis.

Alkalies↗

PubMed2003

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.

Actin Cytoskeleton↗