Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10-100 microm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, approximately 40 microm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization.     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。     

Effect of profilin on actin critical concentration: a theoretical analysis

To explain the effect of profilin on actin critical concentration in a manner consistent with thermodynamic constraints and available experimental data, we built a thermodynamically rigorous model of actin steady-state dynamics in the presence of profilin. We analyzed previously published mechanisms theoretically and experimentally and, based on our analysis, suggest a new explanation for the effect of profilin. It is based on a general principle of indirect energy coupling. The fluctuation-based process of exchange diffusion indirectly couples the energy of ATP hydrolysis to actin polymerization. Profilin modulates this coupling, producing two basic effects. The first is based on the acceleration of exchange diffusion by profilin, which indicates, paradoxically, that a faster rate of actin depolymerization promotes net polymerization. The second is an affinity-based mechanism similar to the one suggested in 1993 by Pantaloni and Carlier although based on indirect rather than direct energy coupling. In the model by Pantaloni and Carlier, transformation of chemical energy of ATP hydrolysis into polymerization energy is regulated by direct association of each step in the hydrolysis reaction with a corresponding step in polymerization. Thus, hydrolysis becomes a time-limiting step in actin polymerization. In contrast, indirect coupling allows ATP hydrolysis to lag behind actin polymerization, consistent with experimental results.     

Actin Monomers Activate Inverted Formin 2 by Competing with Its Autoinhibitory Interaction

INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.     

Maize profilin isoforms are functionally distinct

Profilin is an actin monomer binding protein that, depending on the conditions, causes either polymerization or depolymerization of actin filaments. In plants, profilins are encoded by multigene families. In this study, an analysis of native and recombinant proteins from maize demonstrates the existence of two classes of functionally distinct profilin isoforms. Class II profilins, including native endosperm profilin and a new recombinant protein, ZmPRO5, have biochemical properties that differ from those of class I profilins. Class II profilins had higher affinity for poly-l-proline and sequestered more monomeric actin than did class I profilins. Conversely, a class I profilin inhibited hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate by phospholipase C more strongly than did a class II profilin. These biochemical properties correlated with the ability of class II profilins to disrupt actin cytoplasmic architecture in live cells more rapidly than did class I profilins. The actin-sequestering activity of both maize profilin classes was found to be dependent on the concentration of free calcium. We propose a model in which profilin alters cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration. These results provide strong evidence that the maize profilin gene family consists of at least two classes, with distinct biochemical and live-cell properties, implying that the maize profilin isoforms perform distinct functions in the plant.     

The essential role of profilin in the assembly of actin for microspike formation.

Profilin was first identified as an actin monomer binding protein; however, recent reports indicate its involvement in actin polymerization. To date, there is no direct evidence of a functional role in vivo for profilin in actin cytoskeletal reorganization. Here, we prepared a profilin mutant (H119E) defective in actin binding, but retaining the ability to bind to other proteins. This mutant profilin I suppresses actin polymerization in microspike formation induced by N-WASP, the essential factor in microspike formation. Profilin associates both in vivo and in vitro with N-WASP at proline-rich sites different from those to which Ash/Grb2 binds. This association between profilin and N-WASP is required for N-WASP-induced efficient microspike elongation. Moreover, we succeeded in reconstituting microspike formation in permeabilized cells using profilin I combined with N-WASP and its regulator, Cdc42. These findings provide the first evidence that profilin is a key molecule linking a signaling network to rapid actin polymerization in microspike formation.     

How depolymerization can promote polymerization: the case of actin and profilin

Rapid polymerization and depolymerization of actin filaments in response to extracellular stimuli is required for normal cell motility and development. Profilin is one of the most important actin‐binding proteins; it regulates actin polymerization and interacts with many cytoskeletal proteins that link actin to extracellular membrane. The molecular mechanism of profilin has been extensively considered and debated in the literature for over two decades. Here we discuss several accepted hypotheses regarding the mechanism of profilin function as well as new recently emerged possibilities. Thermal noise is routine in molecular world and unsurprisingly, nature has found a way to utilize it. An increasing amount of theoretical and experimental research suggests that fluctuation‐based processes play important roles in many cell events. Here we show how a fluctuation‐based process of exchange diffusion is involved in the regulation of actin polymerization.     

The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin

Profilin inhibits the rate of nucleation of actin polymerization and the rate of filament elongation and also reduces the concentration of F-actin at steady state. Addition of profilin to solutions of F-actin causes depolymerization. The same steady state concentrations of polymerized and nonpolymerized actin are reached whether profilin is added before initiation of polymerization or after polymerization is complete. The KD for formation of the 1:1 complex between Acanthamoeba profilin and Acanthamoeba actin is in the range of 4 to 11 microM; the KD for the reaction between Acanthamoeba profilin and rabbit skeletal muscle actin is about 60 to 80 microM, irrespective of the concentrations of KCl or MgCl2. The critical concentration of actin for polymerization and the KD for the actin-profilin interaction are independent of each other; therefore, a change in the critical concentration of actin alters the amount of actin bound to profilin at steady state. As a consequence, the presence of profilin greatly amplifies the effects of small changes in the actin critical concentration on the concentration of F-actin. Profilin also inhibits the ATPase activity of monomeric actin, the profilin-actin complex being entirely inactive.     

Effects of profilin and profilactin on actin structure and function in living cells

Previous studies have yielded conflicting results concerning the physiological role of profilin, a 12-15-kD actin- and phosphoinositide-binding protein, as a regulator of actin polymerization. We have addressed this question by directly microinjecting mammalian profilins, prepared either from an E. coli expression system or from bovine brain, into living normal rat kidney (NRK) cells. The microinjection causes a dose-dependent decrease in F-actin content, as indicated by staining with fluorescent phalloidin, and a dramatic reduction of actin and alpha-actinin along stress fibers. In addition, it has a strong inhibitory effect toward the extension of lamellipodia. However, the injection of profilin causes no detectable perturbation to the cell-substrate focal contact and no apparent depolymerization of filaments in either the nonlamellipodial circumferential band or the contractile ring of dividing cells. Furthermore, cytokinesis of injected cells occurs normally as in control cells. In contrast to pure profilin, high-affinity profilin-actin complexes from brain induce an increase in total cellular F-actin content and an enhanced ruffling activity, suggesting that the complex may dissociate readily in the cell and that there may be multiple states of profilin that differ in their ability to bind or release actin molecules. Our results indicate that profilin and profilactin can function as effective regulators for at least a subset of actin filaments in living cells.     

The open nucleotide pocket of the profilin/actin x-ray structure is unstable and closes in the absence of profilin

The open nucleotide pocket conformation of actin in the profilin:actinCaATP x-ray structure has been hypothesized to be a crucial intermediate for nucleotide exchange in the actin depolymerization/polymerization cycle. The requirement for ancillary modification of actin for crystallization leads to ambiguities in this interpretation, however. We have used molecular dynamics simulations to model the thermodynamic properties of the actin x-ray structure, outside the crystal lattice, in an aqueous environment with profilin removed. Our simulations show that the open-nucleotide-pocket, profilin-free structure is actually unstable, and closes. The coordination of actin to the nucleotide in the molecular-dynamics-derived closed structure is virtually identical to that in the closed profilin:actinSrATP x-ray structure. Thus, there is currently no thermodynamically stable structure representing the open-nucleotide-pocket state of actin.     

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.     

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.     

Signal-mediated depolymerization of actin in pollen during the self-incompatibility response

Signal perception and the integration of signals into networks that effect cellular changes is essential for all cells. The self-incompatibility (SI) response in field poppy pollen triggers a Ca(2+)-dependent signaling cascade that results in the inhibition of incompatible pollen. SI also stimulates dramatic alterations in the actin cytoskeleton. By measuring the amount of filamentous (F-) actin in pollen before and during the SI response, we demonstrate that SI stimulates a rapid and large reduction in F-actin level that is sustained for at least 1 h. This represents quantitative evidence for stimulus-mediated depolymerization of F-actin in plant cells by a defined biological stimulus. Surprisingly, there are remarkably few examples of sustained reductions in F-actin levels stimulated by a biologically relevant ligand. Actin depolymerization also was achieved in pollen by treatments that increase cytosolic free Ca(2+) artificially, providing evidence that actin is a target for the Ca(2+) signals triggered by the SI response. By determining the cellular concentrations and binding constants for native profilin from poppy pollen, we show that profilin has Ca(2+)-dependent monomeric actin-sequestering activity. Although profilin is likely to contribute to stimulus-mediated actin depolymerization, our data suggest a role for additional actin binding proteins. We propose that Ca(2+)-mediated depolymerization of F-actin may be a mechanism whereby SI-induced tip growth inhibition is achieved.     

纤维蛋白影响花粉肌动蛋白体外聚合分析

The effects of different ratio of native profilin on maize (Zea mays L.) pollen actin polymerization in vitro were analyzed by using ultracentrifuging sedimentation and ultraviolet absorption spectrum measurement (the molar ratio of profilin to actin was 2∶1, 1.5∶1, 1∶1, 0.5∶1, 0.1∶1 respectively). Preliminary results showed that profilin bound to G-actin and inhibited its polymerization. The inhibition of actin polymerization by profilin increased with the increasing ratio of profilin to pollen actin. The dissociation constant （Kd） value of profilin for binding to actin was (1.30±0.33) μmol/L. No stimulation effect of profilin on actin polymerization was observed, suggesting that pollen profilin may affect actin organization by sequestering the G-actin.     

Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner

From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).     

Mechanism of the interaction of human platelet profilin with actin

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.     

Formin differentially utilizes profilin isoforms to rapidly assemble actin filaments

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.     

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.     

Mutant profilin suppresses mutant actin-dependent mitochondrial phenotype in Saccharomyces cerevisiae

In the Saccharomyces cerevisiae actin-profilin interface, Ala(167) of the actin barbed end W-loop and His(372) near the C terminus form a clamp around a profilin segment containing residue Arg(81) and Tyr(79). Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function.     

Individual actin filaments in a microfluidic flow reveal the mechanism of ATP hydrolysis and give insight into the properties of profilin

The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi) that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds) and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments.