Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

Myosin XI-B is involved in the transport of vesicles and organelles in pollen tubes of Arabidopsis thaliana

The movement of organelles and vesicles in pollen tubes depends on F-actin. However, the molecular mechanism through which plant myosin XI drives the movement of organelles is still controversial, and the relationship between myosin XI and vesicle movement in pollen tubes is also unclear. In this study, we found that the siliques of the myosin xi-b/e mutant were obviously shorter than those of the wild-type (WT) and that the seed set of the mutant was severely deficient. The pollen tube growth of myosin xi-b/e was significantly inhibited both in vitro and in vivo. Fluorescence recovery after photobleaching showed that the velocity of vesicle movement in the pollen tube tip of the myosin xi-b/e mutant was lower than that of the WT. It was also found that peroxisome movement was significantly inhibited in the pollen tubes of the myosin xi-b/e mutant, while the velocities of the Golgi stack and mitochondrial movement decreased relatively less in the pollen tubes of the mutant. The endoplasmic reticulum streaming in the pollen tube shanks was not significantly different between the WT and the myosin xi-b/e mutant. In addition, we found that myosin XI-B-GFP colocalized obviously with vesicles and peroxisomes in the pollen tubes of Arabidopsis. Taken together, these results indicate that myosin XI-B may bind mainly to vesicles and peroxisomes, and drive their movement in pollen tubes. These results also suggest that the mechanism by which myosin XI drives organelle movement in plant cells may be evolutionarily conserved compared with other eukaryotic cells.     

Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments

Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.     

Visualization of peroxisomes in living plant cells reveals acto-myosin-dependent cytoplasmic streaming and peroxisome budding

Here we examine peroxisomes in living plant cells using transgenic Arabidopsis thaliana plants expressing the green fluorescent protein (GFP) fused to the peroxisome targeting signal 1 (PTS1). Using time-lapse laser scanning confocal microscopy we find that plant peroxisomes exhibit fast directional movement with peak velocities approaching 10 microm s(-1). Unlike mammalian peroxisomes which move on microtubules, plant peroxisome movement is dependent on actin microfilaments and myosin motors, since it is blocked by treatment with latrunculin B and butanedione monoxime, respectively. In contrast, microtubule-disrupting drugs have no effect on peroxisome streaming. Peroxisomes were further shown to associate with the actin cytoskeleton by the simultaneous visualization of actin filaments and peroxisomes in living cells using GFP-talin and GFP-PTS1 fusion proteins, respectively. In addition, peroxisome budding was observed, suggesting a possible mechanism of plant peroxisome proliferation. The strong signal associated with the GFP-PTS1 marker also allowed us to survey cytoplasmic streaming in different cell types. Peroxisome movement is most intense in elongated cells and those involved in long distance transport, suggesting that higher plants use cytoplasmic streaming to help transport vesicles and organelles over long distances.     

Mitochondrial movement and morphology depend on an intact actin cytoskeleton in Aspergillus nidulans

Mitochondria are essential organelles for the oxidative energy metabolism in eukaryotic cells. Determinants of mitochondrial morphology as well as the machinery underlying their subcellular distribution are not well understood. In this study we constructed an Aspergillus nidulans strain, in which mitochondria are stained with the green-fluorescent protein (GFP) to visualize them and study their behavior in vivo (http://www.uni-marburg. de/mpi/movies/mitochondria/mitochondria.html). Mitochondria form a complex membranous system in the cytoplasm consisting of interconnected tubular structures. Mitochondrial tubes separate frequently or produce small organelles that migrate some distance with velocities of up to 15 microm/min before they fuse again with the reticulum. Experiments using cytochalasin A as an anti-cytoskeletal drug revealed that a functional actin cytoskeleton is crucial for mitochondrial morphology and the dynamic behavior of the mitochondrial network. Movement of organelles along actin filaments requires actin-dependent motor proteins, such as myosin. We found that MyoA, a class I myosin motor of A. nidulans involved in vesicle migration, is not responsible for mitochondrial movement.     

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells

Summary. Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.     

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.     

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     

The Contractile Proteins and Calcium Mediated Gelation and Contraction of Pollen Tube Extracts

The crude extracts of pollen tubes, like other nonmuscle ceils, showed gelation at Iow Ga2+ concentrations and ATP-dependent contraction at higher Ga2+ concentrations. The contracted cytoplasmic clots contained a lot of filaments which were mainly composed of actin, myosin, 105 kD, 67 kD, 48 kD, 38 kD, 34 kD and 28 kD proteins. It is likely that Ca2+ are able to mediate tranformation of acfin from a less ordered state to a more oriented filaments, which interact with actin-binding proteins to form the filamentous network, thus to induce the gel formation of cytoplasm, to regulate the interaction of actin and myosin which transform the chemical energy of ATP into mechanical work of contractile movement of cytoplasm.     

Characean actin bundles as a tool for stydying actomyosin-based motility

At the inner surface of the stagnant chloroplasts of Characeae cells, bundles of actin filaments having uniform polarity are anchored. These bundles are responsible for generating the motive force of cytoplasmic streaming. It is now possible to induce movement of either beads coated with foreign myosin or organelles associated with myosin along the characean actin bundles. The Ca²⁺ sensitivities of the reconstitued movements are consistent with those of the actin-activated myosin ATPases. The use of reconstituted systems is finding wide application in the detection of various myosins in materials from which myosin is not significantly purified. Furthermore, sliding velocities and the Ca²⁺ regulation of myosins bound to organelles are now being determined. Recipient of the Botanical Society Award for Young Scientists, 1987.     

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.     

Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro

Summary. Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5–15 μm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes. Correspondence: Shao-ling Zhang, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China.     

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.     

Characterization of in vitro motility assays using smooth muscle and cytoplasmic myosins

We have used two in vitro motility assays to study the relative movement of actin and myosin from turkey gizzards (smooth muscle) and human platelets. In the Nitella-based in vitro motility assay, myosin-coated polymer beads move over a fixed substratum of actin bundles derived from dissection of the alga, Nitella, whereas in the sliding actin filament assay fluorescently labeled actin filaments slide over myosin molecules adhered to a glass surface. Both assay systems yielded similar relative velocities using smooth muscle myosin and actin under our standard conditions. We have studied the effects of ATP, ionic strength, magnesium, and tropomyosin on the velocity and found that with the exception of the dependence on MgCl2, the two assays gave very similar results. Calcium over a concentration of pCa 8 to 4 had no effect on the velocity of actin filaments. Phosphorylated smooth muscle myosin propelled filaments of smooth muscle and skeletal muscle actin at the same rate. Phosphorylated smooth muscle and cytoplasmic myosin monomers also moved actin filaments, demonstrating that filament formation is not required for movement.     

Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of Lysosomes

An earlier report suggested that actin and myosin I alpha (MMIalpha), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIalpha were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIalpha. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIalpha impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIalpha contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.     

ATP-dependent movement of myosin in vitro: characterization of a quantitative assay

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP- dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP- dependent movement of myosin molecules on actin.     

Arabidopsis Formin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785–amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes'' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes.     

Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars

Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.     

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.