A WASp homolog powers actin polymerization-dependent motility of endosomes in vivo

BACKGROUND: WASp/SCAR proteins activate the Arp2/3 complex to nucleate actin filament assembly and are thought to have important roles in endocytosis. WASp is required for efficient endocytosis of antigen receptors, N-WASp promotes actin polymerization-dependent movement of endomembrane vesicles, and Las17 (a yeast WASp homolog) is required for endocytic internalization. However, it is unknown whether movement of endosomes or other organelles requires activation of the Arp2/3 complex by members of the WASp/SCAR family. RESULTS: Fluorescence video microscopy of yeast cells expressing a GFP-tagged G protein-coupled receptor (Ste2-GFP) as an endocytic marker revealed that endosomes and the lysosome-like vacuole are highly motile. Endosome/vacuole motility required actin polymerization, as indicated by sensitivity to latrunculin A, whereas microtubules were uninvolved. Endosome/vacuole motility did not require actin cables or myosin V (a MYO2 gene product), which moves secretory vesicles and the Golgi apparatus and mediates vacuole segregation. However, endosome motility required Las17, a WASp homolog. In contrast to other processes involving Las17, endosome/vacuole motility required the WCA domain of Las17, which is necessary and sufficient to activate the Arp2/3 complex. CONCLUSIONS: Endosome/vacuole motility in vivo requires actin polymerization stimulated by the WASp homolog Las17. WASp/SCAR family members in mammalian cells may have similar functions. Defects in endosome/lysosome motility may contribute to deficits in lymphocyte or macrophage function observed in human patients lacking WASp or developmental defects in N-WASp-deficient mice.     

KATAMARI1/MURUS3 Is a novel golgi membrane protein that is required for endomembrane organization in Arabidopsis

In plant cells, unlike animal and yeast cells, endomembrane dynamics appear to depend more on actin filaments than on microtubules. However, the molecular mechanisms of endomembrane-actin filament interactions are unknown. In this study, we isolated and characterized an Arabidopsis thaliana mutant, katamari1 (kam1), which has a defect in the organization of endomembranes and actin filaments. The kam1 plants form abnormally large aggregates that consist of endoplasmic reticulum with actin filaments in the perinuclear region within the cells and are defective in normal cell elongation. Map-based cloning revealed that the KAM1 gene is allelic to the MUR3 gene. We demonstrate that the KAM1/MUR3 protein is a type II membrane protein composed of a short cytosolic N-terminal domain and a transmembrane domain followed by a large lumenal domain and is localized specifically on Golgi membranes. We further show that actin filaments interact with Golgi stacks via KAM1/MUR3 to maintain the proper organization of endomembranes. Our results provide functional evidence that KAM1/MUR3 is a novel component of the Golgi-mediated organization of actin functioning in proper endomembrane organization and cell elongation.     

Phospholipase D signaling regulates microtubule organization in the fucoid alga Silvetia compressa

Recent studies in higher plants or animals have shown that phospholipase D (PLD) signaling regulates many aspects of development, including organization of microtubules (MTs), actin and the endomembrane system. PLD hydrolyzes structural phospholipids to form the second messenger phosphatidic acid (PA). To begin to understand the signaling pathways and molecules that regulate cytoskeletal and endomembrane arrays during early development in the brown alga, Silvetia compressa, we altered PLD activity by applying butyl alcohols to zygotes. 1-Butanol activates PLD and is a preferred substrate, primarily forming phosphatidyl butanol (P-butanol), which is not a signaling molecule. Treatment with 1-butanol inhibited cell division and cytokinesis but not photopolarization or germination, suggesting an MT-based effect. Immunolabeling revealed that 1-butanol treatment rapidly disrupted MT arrays and caused zygotes to arrest in metaphase. MT arrays recovered rapidly following butanol washout, but subsequent development depended on the timing of the treatment regime. Additionally, treatment with 1-butanol early in development disrupted endomembrane organization, known to require functional MTs. Interestingly, treatment with higher concentrations of 2-butanol, which also activates PLD, mimicked the effects of 1-butanol. In contrast, the control t-butanol had no effect on MTs or development. These results indicate that S. compressa zygotes utilize PLD signaling to regulate MT arrays. In contrast, PLD signaling does not appear to regulate actin arrays or endomembrane trafficking directly. This is the first report describing the signaling pathways that regulate cytoskeletal organization in the stramenopile (heterokont) lineage.     

Cell-to-cell communication via plant endomembranes

Cell-to-cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC(6)). No cell-to-cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata.     

Breaking the WAVE complex: the point of Arabidopsis trichomes

Actin filaments comprise an essential cytoskeletal array that organizes the cytoplasm during growth and cell division. In growing cells, actin filaments carry out many functions. Actin filaments position the endomembrane system and act as a substrate on which organelle motility occurs. Other actin-filament arrays appear to be more dynamic and to reorganize in response to growth signals and external cues. The diverse cellular functions of the actin cytoskeleton are mediated by actin-binding proteins that nucleate, destabilize, and bundle actin filaments. The distorted trichome morphology mutants provide a simple genetic system in which to study mechanisms of actin-dependent morphogenesis. Recent results from several groups indicate that 'distorted group' genes encode subunits of the actin-related protein (Arp)2/3 and WAVE complexes, and function in a cell morphogenesis pathway.     

Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.     

Nuclear actin: ancient clue to evolution in eukaryotes?

Until recently it was widely accepted that the dynamic cytoskeletal matrix is exclusive to the cytoplasm of eukaryotes, evolving before the emergence of the cell nucleus to enable phagocytosis, cell motility and the sophisticated functioning of the endomembrane system within the cytosol. The discovery of the existence of a prokaryotic cytoskeleton has changed this picture significantly. As a result, the idea has taken shape that the appearance of actin occurred in the very first cell; therefore, the emergence of microfilaments precedes that of the eukaryotic cytoskeleton. The discovery of nuclear actin opened new perspective on the field, suggesting that the nuclear activities of actin reflect the functions of primordial actin-like proteins. In this paper, we review the recent literature to explore the evolutionary origin of nuclear actin. We conclude that both ancient and eukaryotic features of the actin world can be detected in the nucleus today, which supports the idea that the cytoskeleton attained significant eukaryotic innovations before the tandem evolution of the cytoskeleton and nucleus occurred.     

Apical F‐actin‐regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall

In tip‐confined growing pollen tubes, delivery of newly synthesized cell wall materials to the rapidly expanding apical surface requires spatial organization and temporal regulation of the apical F‐actin filament and exocytosis. In this study, we demonstrate that apical F‐actin is essential for the rigidity and construction of the pollen tube cell wall by regulating exocytosis of Nicotiana tabacum pectin methylesterase (NtPPME1). Wortmannin disrupts the spatial organization of apical F‐actin in the pollen tube tip and inhibits polar targeting of NtPPME1, which subsequently alters the rigidity and pectic composition of the pollen tube cell wall, finally causing growth arrest of the pollen tube. In addition to mechanistically linking cell wall construction and apical F‐actin, wortmannin can be used as a useful tool for studying endomembrane trafficking and cytoskeletal organization in pollen tubes.     

Rac1 function during fucoid development

Morphogenesis in fucoid algae begins with adhesive secretion and rhizoid germination, developmental events that secure the alga within the intertidal zone. The importance of the actin cytoskeleton during these processes has been well established; but in general, little is known about actin regulation within the stramenopile lineage. Based on conserved strategies for regulation of actin in other lineages, co-localization of the Arp2/3 complex with actin structures that are essential for rhizoid formation may implicate members of the Rho family of small GTPases in the signaling pathway(s) regulating actin polymerization during fucoid development. Our lab recently demonstrated Rac1 dependent regulation of endomembrane polarization, polarization of adhesive secretion, germination and tip growth in the fucoid brown alga Silvetia compressa. We also present new evidence revealing Rac1 localization during germination in S. compressa, and show that membrane localization is essential for proper Rac1 function.Key words: actin, Arp2/3 complex, manumycin A, NSC23766, Rac1, Rho GTPase, Scar/WAVE, Silvetia compressa     

THRUMIN1 is a light-regulated actin-bundling protein involved in chloroplast motility

Chloroplast movement in response to changing light conditions optimizes photosynthetic light absorption. This repositioning is stimulated by blue light perceived via the phototropin photoreceptors and is transduced to the actin cytoskeleton. Some actin-based motility systems use filament reorganizations rather than myosin-based translocations. Recent research favors the hypothesis that chloroplast movement is driven by actin reorganization at the plasma membrane, but no proteins affecting chloroplast movements have been shown to associate with both the plasma membrane and actin filaments in vivo. Here we identified THRUMIN1 as a critical link between phototropin photoreceptor activity at the plasma membrane and actin-dependent chloroplast movements. THRUMIN1 bundles filamentous actin in vitro, and it localizes to the plasma membrane and displays light- and phototropin-dependent localization to microfilaments in vivo. These results suggest that phototropin-induced actin bundling via THRUMIN1 is important for chloroplast movement. A mammalian homolog of THRUMIN1, GRXCR1, has been implicated in auditory responses and hair cell stereocilla development as a regulator of actin architecture. Studies of THRUMIN1 will help elucidate the function of this family of eukaryotic proteins.     

The role of the ER and cytoskeleton in plant viral trafficking

Plant viruses spread from cell to cell via plasmodesmata, which bridge the rigid cell wall and connect adjacent cells. The spread of infection is aided by interactions between the virus and the host components. These interactions have been intensively studied to understand the crosstalk between pathogen and host. The use of green fluorescent protein has shed new light on the close association between viral movement proteins and elements of the cytoskeleton and the endomembrane system.     

An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes

Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.     

Actin-based motility during endocytosis in budding yeast

Actin assembly nucleated by Arp2/3 complex has been implicated in the formation and movement of endocytic vesicles. The dendritic nucleation model has been proposed to account for Arp2/3-mediated actin assembly and movement. Here, we explored the model by examining the role of capping protein in vivo, with quantitative tracking analysis of fluorescence markers for different stages of endocytosis in yeast. Capping protein was most important for the initial movement of endocytic vesicles away from the plasma membrane, which presumably corresponds to vesicle scission and release. The next phase of endosome movement away from the plasma membrane was also affected, but less so. The results are consistent with the dendritic nucleation model's prediction of capping protein as important for efficient actin assembly and force production. In contrast, the movement of late-stage endocytic vesicles, traveling through the cytoplasm en route to the vacuole, did not depend on capping protein. The movement of these vesicles was found previously to depend on Lsb6, a WASp interactor, whereas Lsb6 was found here to be dispensable for early endosome movement. Thus, the molecular requirements for Arp2/3-based actin assembly differ in early versus later stages of endocytosis. Finally, acute loss of actin cables led to increased patch motility.     

Live cell imaging of the assembly, disassembly, and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae

Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as “conveyor belts” for retrograde movement and delivery of actin patches/endosomes to FM4-64–labeled internal compartments.     

Myosin cleft movement and its coupling to actomyosin dissociation

It has long been known that binding of actin and binding of nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, whereas actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin- and nucleotide-binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin head. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft.     

Interdependence of Endomembrane Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes

During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better understand such cross talk in the model plant Arabidopsis (Arabidopsis thaliana), we first established optimized concentrations of drugs that interfere with either endomembrane trafficking or the actin cytoskeleton, then examined pollen tube growth using fluorescent protein markers that label transport vesicles, endosomes, or the actin cytoskeleton. Both brefeldin A (BFA) and wortmannin disturbed the motility and structural integrity of ARA7- but not ARA6-labeled endosomes, suggesting heterogeneity of the endosomal populations. Disrupting endomembrane trafficking by BFA or wortmannin perturbed actin polymerization at the apical region but not in the longitudinal actin cables in the shank. The interference of BFA/wortmannin with actin polymerization was progressive rather than rapid, suggesting an indirect effect, possibly due to perturbed endomembrane trafficking of certain membrane-localized signaling proteins. Both the actin depolymerization drug latrunculin B and the actin stabilization drug jasplakinolide rapidly disrupted transport of secretory vesicles, but each drug caused distinct responses on different endosomal populations labeled by ARA6 or ARA7, indicating that a dynamic actin cytoskeleton was critical for some steps in endomembrane trafficking. Our results provide evidence of cross talk between endomembrane trafficking and the actin cytoskeleton in pollen tubes.Pollen tubes of flowering plants are specialized cells that deliver immotile sperm to the proximity of female gametes for successful reproduction (Johnson and Preuss, 2002). The growth of pollen tubes is both polar and directional (Hepler et al., 2001); many cellular activities contribute to such growth, the most important being the dynamics of the actin cytoskeleton system, targeted exocytosis, and endocytosis (Hepler et al., 2001).Pollen tubes contain longitudinal actin cables along the shank, which are important for providing structural support and acting as tracks for the movement of large organelles (Staiger et al., 1994). The apical area of pollen tubes instead contains dynamic filamentous actin (F-actin), as shown by fluorescently labeled actin-binding proteins (Kost et al., 1999; Fu et al., 2001; Chen et al., 2002; Wilsen et al., 2006). The dynamics of F-actin are critical for the polarized growth of pollen tubes. Genetically manipulating the activities of the small GTPases ROP (Kost et al., 1999; Fu et al., 2001; Cheung et al., 2008) and Rab (de Graaf et al., 2005), or of actin-binding proteins such as profilin and formin (Staiger et al., 1994; Chen et al., 2002; Cheung and Wu, 2004), disrupted F-actin dynamics and inhibited tube growth and caused apical bulges. Application of drugs such as latrunculin B (LatB) and jasplakinolide (Jas) showed similar effects (Gibbon et al., 1999; Vidali et al., 2001; Cardenas et al., 2005; Hörmanseder et al., 2005; Chen et al., 2007).Targeted exocytosis delivers building materials for cell membranes and cell walls and therefore is critical for maintaining growth polarity and directionality of growing pollen tubes (Hepler et al., 2001). Because targeted exocytosis brings more membrane and wall materials than needed to the apex of a pollen tube, an active endocytic system exists to retrieve excess secreted materials. In addition to this nonselective bulk membrane retrieval, pollen tubes may have selective and regulated endocytic trafficking pathways. For example, experiments using charged gold particles indicated the existence of two distinct endocytic pathways in tobacco (Nicotiana tabacum) pollen tubes (Moscatelli et al., 2007), and other studies showed that pollen tubes are able to take in materials from the extracellular matrix (Lind et al., 1996; Goldraij et al., 2006). The axis of targeted exocytosis correlated with the direction of tube growth and it asymmetrically changed toward the new apex during tube reorientation (Camacho and Malho, 2003; de Graaf et al., 2005). Disruption of membrane trafficking altered growth trajectories (de Graaf et al., 2005). Both suggest that membrane trafficking is a critical part of polarity maintenance and reorientation.As two important cellular processes in pollen tube growth, membrane trafficking and actin polymerization are conceivably dependent on each other. For example, several studies demonstrated that dynamic actin polymerization was essential for membrane trafficking (Hörmanseder et al., 2005; Wang et al., 2005; Chen et al., 2007; Lee et al., 2008), while others explored whether membrane trafficking affected actin polymerization (de Graaf et al., 2005; Hörmanseder et al., 2005). These studies, however, were mostly done with rapidly growing pollen tubes from tobacco or lily (Lilium longiflorum). For the model plant Arabidopsis (Arabidopsis thaliana), whose pollen tubes grow slower, little is known in this regard. Given a robust protocol for Arabidopsis pollen germination (Boavida and McCormick, 2007), it is now possible to investigate the interactions between these two cellular activities.In this study, we analyzed the effects of drug treatments on Arabidopsis pollen tubes expressing fluorescent protein probes for transport vesicles, endosomes, or the actin cytoskeleton. We show that perturbing actin dynamics by LatB or Jas treatments disrupted the V-shaped distribution of transport vesicles, caused aggregation, and finally dissipation of a subpopulation of endosomes, indicating that actin dynamics are critical at some steps of endomembrane trafficking. On the other hand, disturbing endomembrane trafficking with brefeldin A (BFA) or wortmannin abolished the F-actin structure at the apical region without affecting the longitudinal actin cables at the shank. These results provide evidence that endomembrane trafficking and actin dynamics interact at certain steps during polarized growth of Arabidopsis pollen tubes.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

Myosin IXb is a single-headed minus-end-directed processive motor

Myosin is an actin-based molecular motor that constitutes a diverse superfamily. In contrast to conventional myosin, which binds to actin for only a short time during cross-bridge cycling, recent studies have demonstrated that class V myosin moves along actin filaments for a long distance without dissociating. This would make it suitable for supporting cargo movement in cells. Because myosin V has a two-headed structure with an expanded neck domain, it has been postulated to 'walk' along the 36-nm helical repeat of the actin filament, with one head attached to the actin and leading the other head to the neighbouring helical pitch. Here, we report that myosin IXb, a single-headed myosin, moves processively on actin filaments. Furthermore, we found that myosin IXb is a minus-end-directed motor. In addition to class VI myosin, this is the first myosin superfamily member identified that moves in the reverse direction. The processive movement of the single-headed myosin IXb cannot be explained by a 'hand-over-hand' mechanism. This suggests that an alternative mechanism must be operating for the processive movement of single-headed myosin IXb.     

The Loop2 Insertion of Type IX Myosin Acts as an Electrostatic Actin Tether that Permits Processive Movement

Although class IX myosins are single-headed, they demonstrate characteristics of processive movement along actin filaments. Double-headed myosins that move processively along actin filaments achieve this by successive binding of the two heads in a hand-over-hand mechanism. This mechanism, obviously, cannot operate in single-headed myosins. However, it has been proposed that a long class IX specific insertion in the myosin head domain at loop2 acts as an F-actin tether, allowing for single-headed processive movement. Here, we tested this proposal directly by analysing the movement of deletion constructs of the class IX myosin from Caenorhabditis elegans (Myo IX). Deletion of the large basic loop2 insertion led to a loss of processive behaviour, while deletion of the N-terminal head extension, a second unique domain of class IX myosins, did not influence the motility of Myo IX. The processive behaviour of Myo IX is also abolished with increasing salt concentrations. These observations directly demonstrate that the insertion located in loop2 acts as an electrostatic actin tether during movement of Myo IX along the actin track.     

Change in the actin cytoskeleton during seismonastic movement of Mimosa pudica

The seismonastic movement of Mimosa pudica is triggered by a sudden loss of turgor pressure. In the present study, we compared the cell cytoskeleton by immunofluorescence analysis before and after movement, and the effects of actin- and microtubule-targeted drugs were examined by injecting them into the cut pulvinus. We found that fragmentation of actin filaments and microtubules occurs during bending, although the actin cytoskeleton, but not the microtubules, was involved in regulation of the movement. Transmission electron microscopy revealed that actin cables became loose after the bending. We injected phosphatase inhibitors into the severed pulvinus to examine the effects of such inhibitors on the actin cytoskeleton. We found that changes in actin isoforms, fragmentation of actin filaments and the bending movement were all inhibited after injection of a tyrosine phosphatase inhibitor. We thus propose that the phosphorylation status of actin at tyrosine residues affects the dynamic reorganization of actin filaments and causes seismonastic movement.