Teruo Shimmen

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 ∼3.5 μm/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.

Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

SummaryThe mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 μm, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawaet al. 1976), we investigated the streaming rate with respect to the internal Ca2+ concentration. The rate was roughly identical to that of normal cells at a Ca2+ concentration of less than 10−7 M. It decreased with an increase in the internal Ca2+ concentration and was zero at 1 mM Ca2+.The above results, together with the two facts that Ca2+ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawaet al. 1976), lead us to conclude that a transient increase in the Ca2+ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca2+ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca2+ concentration in the cytoplasm causing streaming cessation were discussed.

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 μm/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was ∼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.

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis

Abstract. In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells.

Isolation and characterization of plant myosin from pollen tubes of lily

SummaryA plant myosin was isolated from pollen tubes of lily,Lilium longiflorum. Pollen tubes were homogenized in low ionic strength solution containing casein, and myosin from this crude extract was purified by co-precipitation with F-actin prepared from chicken breast muscle, followed by hydroxylapatite column and gel filtration column chromatography. Upon SDS-PAGE on 6% polyacrylamide gel, only 170 kDa polypeptide was detected in the purified myosin fraction. Furthermore, with immunoblotting using antiserum raised against 170 kDa polypeptide, only the 170 kDa component crossreacted in the crude sample of pollen tube proteins. This antiserum did not crossreact with the heavy chain of skeletal muscle myosin. The ATPase activity of pollen tube myosin was stimulated up to 60-fold by F-actin prepared from chicken breast muscle. The translocation velocity of rhodamine-phalloidin-labeled F-actin on a glass surface covered with pollen tube myosin ranged from 6.0 to 9.8 μm/s with an average of 7.7 μm/s. This velocity was similar to or a little faster than that of the cytoplasmic streaming that occurred in pollen tubes. These results suggested that myosin composed of a 170 kDa heavy chain produces the motive force for cytoplasmic streaming in pollen tube of lily.

Physiological and biochemical aspects of cytoplasmic streaming.

Publisher Summary This chapter discusses the physiological and biochemical aspects of cytoplasmic streaming. Cytoplasmic streaming has been reported in various plant species ranging from algae to higher plants and fungi. the motive force for cytoplasmic streaming in most plant cells is generated by the actin-myosin system, which is also responsible for generating the motive force in muscle contraction and ameboid movement, etc. Cytoplasmic streaming may play an important role not only in intracellular transport but also in other cell functions. In characean cells, it may affect photosynthesis by controlling the transport of ions or substrate through the plasma membrane. Also, cytoplasmic streaming is responsible for intercellular transport in Characeae and in the stem of some higher plants. The mechanism of cytoplasmic streaming is elucidated mostly by experiments using characean cells. It is reasonable to say that the motive force of cytoplasmic streaming in other plant cells is also generated by the same mechanism as that in characean cells, that is, the sliding of myosin associated with organelles along actin filaments using ATP energy.

Ca2+-induced fragmentation of actin filaments in pollen tubes

SummaryTo control the intracellular free Ca2+ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca2+ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca2+ concentration of the external medium ([Ca2+]) was raised to 5×10−6 M or higher. At [Ca2+] below 1×10−6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca2+] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca2+ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.

Control of cytoplasmic streaming by extracellular Ca2+ in permeabilizedNitella cells

SummaryCytoplasmic streaming in permeabilizedNitella cells was found to be controlled by Ca2+ of physiological concentration. The streaming driven by Mg · ATP was scarcely affected by 10−7 M Ca2+, but was inhibited significantly by 5 × 10−7 M Ca2+ and completely by 10−6 M Ca2+. The inhibition by Ca2+ was completely reversible even at 10−5 M.

Localization of a 170 kDa myosin heavy chain in plant cells

SummaryA polyclonal antibody directed against a 170 kDa myosin heavy chain from lily pollen tubes was employed to (a) assess the cellular distribution of the polypeptide using immunofluorescence methods, and (b) ascertain if similar polypeptides are present in pollen tubes and somatic cells of other species. Fluorescence is associated with particles of various size as well as an amorphous component, and is concentrated in the apical cytoplasm of lily and tobacco pollen tubes. Apical fluorescence is more extensive in lily than in tobacco, which may be related to different streaming patterns and apical zonation seen at the ultrastructural level. In suspension cells of tobacco andArabidopsis, fluorescence is concentrated around the nuclei. Dual localizations indicate that anti-myosin fluorescence may be associated with the presence of actin. Little or no staining was seen in controls consisting of either pre-immune serum or mono-specific IgG that had been preadsorbed with the 170 kDa polypeptide. Immunoblots show that a 170 kDa immunoreactive polypeptide is present in pollen tubes of tobacco andTradescantia virginiana in addition to lily, and in suspension culture cells of tobacco andArabidopsis and extracts of wholeArabidopsis seedlings. Our results show that a conserved 170 kDa myosin heavy chain is present in a variety of monocot and dicot cells. They are also consistent with the presence of multiple myosins in plants in general and pollen tubes in particular.

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

SummaryActin 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.