Dynamics of vacuoles and actin filaments in guard cells and their roles in stomatal movement

Vacuoles and actin filaments are important cytoarchitectures involved in guard cell function. The changes in the morphology and number of vacuoles and the regulation of ion channel activity in tonoplast of guard cells are essential for stomatal movement. A number of studies have investigated the regulation of ion channels in animal and plant cells; however, little is known about the regulating mechanism for vacuolar dynamics in stomatal movement. Actin filaments of guard cells are remodelling with the changes in the stomatal aperture; however, the dynamic functions of actin filaments in stomatal movement remain elusive. In this paper, we summarize the recent developments in the understanding of the dynamics of actin filaments and vacuoles of guard cells during stomatal movement. All relevant studies suggest that actin filaments might be involved in stomatal movement by regulating vacuolar dynamics and the ion channels in tonoplast. The future study could be focused on the linker protein mediating the interaction between actin filaments and tonoplast, which will provide insights into the interactive function of actin and vacuole in stomatal movement regulation.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.     

The DinI protein stabilizes RecA protein filaments

When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.     

The ARP2/3 complex mediates guard cell actin reorganization and stomatal movement in Arabidopsis

Guard cell actin reorganization has been observed in stomatal responses to a wide array of stimuli. However, how the guard cell signaling machinery regulates actin dynamics is poorly understood. Here, we report the identification of an allele of the Arabidopsis thaliana ACTIN-RELATED PROTEIN C2/DISTORTED TRICHOMES2 (ARPC2) locus (encoding the ARPC2 subunit of the ARP2/3 complex) designated high sugar response3 (hsr3). The hsr3 mutant showed increased transpirational water loss that was mainly due to a lesion in stomatal regulation. Stomatal bioassay analyses revealed that guard cell sensitivity to external stimuli, such as abscisic acid (ABA), CaCl(2), and light/dark transition, was reduced or abolished in hsr3. Analysis of a nonallelic mutant of the ARP2/3 complex suggested no pleiotropic effect of ARPC2 beyond its function in the complex in regard to stomatal regulation. When treated with ABA, guard cell actin filaments underwent fast disruption in wild-type plants, whereas those in hsr3 remained largely bundled. The ABA insensitivity phenotype of hsr3 was rescued by cytochalasin D treatment, suggesting that the aberrant stomatal response was a consequence of bundled actin filaments. Our work indicates that regulation of actin reassembly through ARP2/3 complex activity is crucial for stomatal regulation.     

Inorganic phosphate regulates the binding of cofilin to actin filaments

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers. These changes transform the nucleotide cleft of F-actin to G-actin-like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility.     

Glycine-rich region regulates cysteine-rich protein 1 binding to actin cytoskeleton

Cysteine-rich protein 1 (CRP1) has a unique structure with two well separated LIM domains, each followed by a glycine-rich region. Although CRP1 has been shown to interact with actin-binding proteins and actin filaments, the mechanism regulating localization to the actin cytoskeleton in cells is not clear. Experiments using truncated forms showed that the first LIM domain and glycine-rich region are necessary for CRP1 bundling of actin filaments and localization to the actin cytoskeleton. Furthermore, domain swapping experiments replacing the first glycine-rich region with the second resulted in the loss of CRP1 bundling activity and localization to the actin cytoskeleton, identifying seven critical amino acid residues. These results highlight the importance of the first glycine-rich region for CRP1 bundling activity and localization to the actin cytoskeleton. In addition, this work identifies the first LIM domain and glycine-rich region as a distinct actin filament bundling module.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

The plant-specific ssDNA binding protein OSB1 is involved in the stoichiometric transmission of mitochondrial DNA in Arabidopsis

Plant mitochondrial genomes exist in a natural state of heteroplasmy, in which substoichiometric levels of alternative mitochondrial DNA (mtDNA) molecules coexist with the main genome. These subgenomes either replicate autonomously or are created by infrequent recombination events. We found that Arabidopsis thaliana OSB1 (for Organellar Single-stranded DNA Binding protein1) is required for correct stoichiometric mtDNA transmission. OSB1 is part of a family of plant-specific DNA binding proteins that are characterized by a novel motif that is required for single-stranded DNA binding. The OSB1 protein is targeted to mitochondria, and promoter-beta-glucuronidase fusion showed that the gene is expressed in budding lateral roots, mature pollen, and the embryo sac of unfertilized ovules. OSB1 T-DNA insertion mutants accumulate mtDNA homologous recombination products and develop phenotypes of leaf variegation and distortion. The mtDNA rearrangements occur in two steps: first, homozygous mutants accumulate subgenomic levels of homologous recombination products; second, in subsequent generations, one of the recombination products becomes predominant. After the second step, the process is no longer reversible by backcrossing. Thus, OSB1 participates in controlling the stoichiometry of alternative mtDNA forms generated by recombination. This regulation could take place in gametophytic tissues to ensure the transmission of a functional mitochondrial genome.     

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.     

Arabidopsis AIP1-1 regulates the organization of apical actin filaments by promoting their turnover in pollen tubes

Apical actin filaments are highly dynamic structures that are crucial for rapid pollen tube growth, but the mechanisms regulating their dynamics and spatial organization remain incompletely understood. We here identify that AtAIP1-1 is important for regulating the turnover and organization of apical actin filaments in pollen tubes. AtAIP1-1 is distributed uniformly in the pollen tube and loss of function of AtAIP1-1 affects the organization of the actin cytoskeleton in the pollen tube. Specifically, actin filaments became disorganized within the apical region of aip1-1 pollen tubes. Consistent with the role of apical actin filaments in spatially restricting vesicles in pollen tubes, the apical region occupied by vesicles becomes enlarged in aip1-1 pollen tubes compared to WT. Using ADF1 as a representative actin-depolymerizing factor, we demonstrate that AtAIP1-1 enhances ADF1-mediated actin depolymerization and filament severing in vitro, although AtAIP1-1 alone does not have an obvious effect on actin assembly and disassembly. The dynamics of apical actin filaments are reduced in aip1-1 pollen tubes compared to WT. Our study suggests that AtAIP1-1 works together with ADF to act as a module in regulating the dynamics of apical actin filaments to facilitate the construction of the unique "apical actin structure" in the pollen tube.     

Myosin VI altered at threonine 406 stabilizes actin filaments in vivo

Myosin VI is a minus-end directed actin-based molecular motor implicated in uncoated endocytic vesicle transport. Recent kinetic studies have shown that myosin VI displays altered ADP release kinetics under different load conditions allowing myosin VI to serve alternately as a transporter or as an actin tether. We theorized that one potential regulatory event to modulate between these kinetic choices is phosphorylation at a conserved site, threonine 406 (T406) in the myosin VI motor domain. Alterations mimicking the phosphorylated (T406E) and dephosphorylated state (T406A) were introduced into a GFP-myosin VI fusion (GFP-M6). Live cell imaging revealed that GFP-M6(T406E) expression changed the path myosin VI took in its transport of uncoated endocytic vesicles. Rather than routing vesicles inwards as seen in GFP-M6 and GFP-M6(T406A) expressing cells, GFP-M6(T406E) moved vesicles into clusters at distinct peripheral sites. GFP-M6(T406E) expression also increased the density of the actin cytoskeleton. Filaments were enriched at the vesicle cluster sites. This was not due to a gross redistribution of the actin polymerization machinery. Instead the filament density correlated to the fixed positioning of GFP-M6(T406E)-associated vesicles on F-actin, leading to inhibition of actin depolymerization. Our study suggests that phosphorylation at T406 changes the nature of myosin VI's interaction with actin in vivo.     

Arabidopsis capping protein (AtCP) is a heterodimer that regulates assembly at the barbed ends of actin filaments

The precise regulation of actin filament polymerization and depolymerization is essential for many cellular processes and is choreographed by a multitude of actin-binding proteins (ABPs). In higher plants the number of well characterized ABPs is quite limited, and some evidence points to significant differences in the biochemical properties of apparently conserved proteins. Here we provide the first evidence for the existence and biochemical properties of a heterodimeric capping protein from Arabidopsis thaliana (AtCP). The purified recombinant protein binds to actin filament barbed ends with Kd values of 12-24 nM, as assayed both kinetically and at steady state. AtCP prevents the addition of profilin actin to barbed ends during a seeded elongation reaction and suppresses dilution-mediated depolymerization. It does not, however, sever actin filaments and does not have a preference for the source of actin. During assembly from Mg-ATP-actin monomers, AtCP eliminates the initial lag period for actin polymerization and increases the maximum rate of polymerization. Indeed, the efficiency of actin nucleation of 0.042 pointed ends created per AtCP polypeptide compares favorably with mouse CapZ, which has a maximal nucleation of 0.17 pointed ends per CapZ polypeptide. AtCP activity is not affected by calcium but is sensitive to phosphatidylinositol 4,5-bisphosphate. We propose that AtCP is a major regulator of actin dynamics in plant cells that, together with abundant profilin, is responsible for maintaining a large pool of actin subunits and a surprisingly small population of F-actin.     

Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.     

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis

The phragmoplast, a structure crucial for the completion of cytokinesis in plant cells, is composed of antiparallel microtubules (MTs) and actin filaments (AFs). However, how the parallel structure of phragmoplast MTs and AFs is maintained, especially during centrifugal phragmoplast expansion, remains elusive. Here, we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein (AtMAC). When AtMAC was deleted, the phragmoplast showed disintegrity during centrifugal expansion, and the resulting phragmoplast fragmentation led to incomplete cell plates. Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis. Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro, and the truncated AtMAC protein, N-CC1, was the key domain controlling the ability of AtMAC. Further analysis showed that N-CC1(51–154) is the key domain for binding MTs, and N-CC1(51–125) for binding AFs. In conclusion, AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion, which is required for complete cytokinesis.     

EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.