Inhibition of cell adhesion by phosphorylated Ezrin/Radixin/Moesin

Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated ERM, we modified phosphorylation status of ERM and investigated changes in cell adhesion and morphology. Treatment with Calyculin-A (Cal-A), a protein phosphatase inhibitor, dramatically augmented phosphorylated ERM (phospho-ERM). Cal-A-treatment or expression of phospho-mimetic Moesin mutant (Moesin-TD) induced cell rounding in adherent cells. Moreover, reattachment of detached cells to substrate was inhibited by either treatment. Phospho-ERM, Moesin-TD and actin cytoskeleton were observed at the plasma membrane of such round cells. Augmented cell surface rigidity was also observed in both cases.

Meanwhile, non-adherent KG-1 cells were rather rich in phospho-ERM. Treatment with Staurosporine, a protein kinase inhibitor that dephosphorylates phospho-ERM, up-regulated the integrin-dependent adhesion of KG-1 cells to substrate.

These findings strongly suggest the followings: (1) Phospho-ERM inhibit cell adhesion, and therefore, dephosphorylation of ERM proteins is essential for cell adhesion. (2) Phospho-ERM induce formation and/or maintenance of spherical cell shape. (3) ERM are constitutively both phosphorylated and dephosphorylated in cultured adherent and non-adherent cells.     

Inhibition of cell adhesion by phosphorylated Ezrin/Radixin/Moesin

Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated ERM, we modified phosphorylation status of ERM and investigated changes in cell adhesion and morphology. Treatment with Calyculin-A (Cal-A), a protein phosphatase inhibitor, dramatically augmented phosphorylated ERM (phospho-ERM). Cal-A-treatment or expression of phospho-mimetic Moesin mutant (Moesin-TD) induced cell rounding in adherent cells. Moreover, reattachment of detached cells to substrate was inhibited by either treatment. Phospho-ERM, Moesin-TD and actin cytoskeleton were observed at the plasma membrane of such round cells. Augmented cell surface rigidity was also observed in both cases.Meanwhile, non-adherent KG-1 cells were rather rich in phospho-ERM. Treatment with Staurosporine, a protein kinase inhibitor that dephosphorylates phospho-ERM, up-regulated the integrin-dependent adhesion of KG-1 cells to substrate.These findings strongly suggest the followings: (1) Phospho-ERM inhibit cell adhesion, and therefore, dephosphorylation of ERM proteins is essential for cell adhesion. (2) Phospho-ERM induce formation and/or maintenance of spherical cell shape. (3) ERM are constitutively both phosphorylated and dephosphorylated in cultured adherent and non-adherent cells.     

Formation of microvilli and phosphorylation of ERM family proteins by CD43, a potent inhibitor for cell adhesion: Cell detachment is a potential cue for ERM phosphorylation and organization of cell morphology

CD43/sialophorin/leukosialin, a common leukocyte antigen, is known as an inhibitor for cell adhesion. The ectodomain of CD43 is considered as a molecular barrier for cell adhesion, while the cytoplasmic domain has a binding site for Ezrin/Radixin/Moesin (ERM). We found expression of CD43 induced cell rounding, inhibition of cell re-attachment, augmentation of microvilli and phosphorylation of ERM in HE K293T cells. Mutant studies revealed the ectodomain of CD43, but not the intracellular domain, essential and sufficient for all these phenomena. We also found that forced cell detachment by itself induced phosphorylation of ERM in HE K293T cells. Taken together, these findings indicate that inhibition of cell adhesion by the ectodomain of CD43 induces phosphorylation of ERM, microvilli formation and eventual cell rounding. Furthermore, our study suggests a novel possibility that cell detachment itself induces activation of ERM and modification of cell shape.Key words: cell adhesion, CD43, microvilli, ERM, integrin, cell rounding, phosphorylation, mucin     

Formation of microvilli and phosphorylation of ERM family proteins by CD43, a potent inhibitor for cell adhesion

CD43/sialophorin/leukosialin, a common leukocyte antigen, is known as an inhibitor for cell adhesion. The ectodomain of CD43 is considered as a molecular barrier for cell adhesion, while the cytoplasmic domain has a binding site for Ezrin/Radixin/Moesin (ERM). We found expression of CD43 induced cell rounding, inhibition of cell re-attachment, augmentation of microvilli, and phosphorylation of ERM in HEK293T cells. Mutant studies revealed the ectodomain of CD43, but not the intracellular domain, essential and sufficient for all these phenomena. We also found that forced cell detachment by itself induced phosphorylation of ERM in HEK293T cells. Taken together, these findings indicate that inhibition of cell adhesion by the ectodomain of CD43 induces phosphorylation of ERM, microvilli formation, and eventual cell rounding. Furthermore, our study suggests a novel possibility that cell detachment itself induces activation of ERM and modification of cell shape.     

ERM proteins: from cellular architecture to cell signaling

ERM (ezrin/radixin/moesin) proteins, concentrated in actin rich cell-surface structures, cross-link actin filaments with the plasma membrane. They are involved in the formation of microvilli, cell-cell adhesion, maintenance of cell shape, cell motility and membrane trafficking. Recent analyses reveal that they are not only involved in cytoskeleton organization but also in signaling pathway. They play an important role in the activation of members of the Rho family by recruiting their regulators. The functions of ERM proteins are regulated by their conformational charges: the intramolecular interaction between the N- and C-terminal domains of ERM proteins charges masks several binding sites, leading to a dormant protein. Different activation signals regulate ERM proteins functions by modulating these intramolecular interactions. The involvement of ERM proteins in many signaling pathways has led to study their role during development of different species.     

Tetraspanins regulate the protrusive activities of cell membrane

Tetraspanins have gained increased attention due to their functional versatility. But the universal cellular mechanism that governs such versatility remains unknown. Herein we present the evidence that tetraspanins CD81 and CD82 regulate the formation and/or development of cell membrane protrusions. We analyzed the ultrastructure of the cells in which a tetraspanin is either overexpressed or ablated using transmission electron microscopy. The numbers of microvilli on the cell surface were counted, and the radii of microvillar tips and the lengths of microvilli were measured. We found that tetraspanin CD81 promotes the microvillus formation and/or extension while tetraspanin CD82 inhibits these events. In addition, CD81 enhances the outward bending of the plasma membrane while CD82 inhibits it. We also found that CD81 and CD82 proteins are localized at microvilli using immunofluorescence. CD82 regulates microvillus morphogenesis likely by altering the plasma membrane curvature and/or the cortical actin cytoskeletal organization. We predict that membrane protrusions embody a common morphological phenotype and cellular mechanism for, at least some if not all, tetraspanins. The differential effects of tetraspanins on microvilli likely lead to the functional diversification of tetraspanins and appear to correlate with their functional propensity.     

Characterization of a novel interaction between ELMO1 and ERM proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.     

A role for beta2 integrin (CD11/CD18)-mediated tyrosine signaling in extravasation of human polymorphonuclear neutrophils

During the recruitment of human polymorphonuclear neutrophils (PMN) to sites of inflammation, leukocyte adhesion molecules of the beta2 integrin (CD11/CD18) family mediate firm adhesion of these cells to the endothelial cell monolayer lining the vessel wall. This process is a prerequisite for shape change and spreading of PMN on the endothelium which eventually allows PMN emigration into the extravascular space. In order to elucidate the molecular mechanisms which mediate this sequence of events, intracellular protein tyrosine signaling was studied subsequent to beta2 integrin-mediated ligand binding. Using western blotting technique, beta2 integrin-mediated adhesion was found to induce tyrosine phosphorylation of different proteins. The effect was absent in PMN derived from CD18-deficient mice which lack any beta2 integrin expression on the cell surface demonstrating the specificity of the observed response. Inhibition of beta2 integrin-mediated tyrosine signaling by herbimycin A almost completely inhibited adhesion, shape change, and subsequent spreading of PMN. Herbimycin A also diminished chemotactic migration of these cells in response to the soluble mediator N-formyl-Met-Leu-Phe (fMLP). In contrast, treatment of PMN with cytochalasin D had no substantial effect on beta2 integrin-mediated signaling or adhesion but inhibited shape change, spreading, and chemotactic migration of PMN. This suggests that the signaling capacity exerted by beta2 integrins upon ligand binding was independent of an intact cytoskeleton. Moreover, the beta2 integrin-mediated activation of intracellular signal transduction pathways was critical for firm adhesion of PMN, the prerequisite subsequent shape change and spreading, which allows emigration of PMN into the extravascular space.     

Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases.

When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli (reviewed in [1] [2] [3] [4] [5]). Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) [6] [7] increased approximately 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho-family GTPases, Rac1 and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROKalpha or ROCK-II) [8] [9] [10]. The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27632, a specific inhibitor of ROCK kinases including Rho-kinase [11]. Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type Ialpha [12] [13] [14], but not a kinase-inactive mutant [15], increased approximately sixfold the level of CPERM, and induced microvilli. Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates ERM proteins in vitro [16], our data suggest that PIP(2), and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation.     

Age-related defects in moesin/ezrin cytoskeletal signals in mouse CD4 T cells

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.     

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo

The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell-cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell-cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.     

Phosphorylated ERM is responsible for increased T cell polarization, adhesion, and migration in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune/inflammatory disease characterized by autoantibody production and abnormal T cells that infiltrate tissues through not well-known mechanisms. We report that SLE T lymphocytes display increased levels of CD44, ezrin, radixin, and moesin (ERM) phosphorylation, stronger actin polymerization, higher polar cap formation, and enhanced adhesion and chemotactic migration compared with T cells from patients with rheumatoid arthritis and normal individuals. Silencing of CD44 by CD44 small interfering RNA in SLE T cells inhibited significantly their ability to adhere and migrate as did treatment with Rho kinase and actin polymerization inhibitors. Forced expression of T567D-ezrin, a phosphorylation-mimic form, enhanced remarkably the adhesion and migration rate of normal T cells. Anti-CD3/TCR autoantibodies present in SLE sera caused increased ERM phosphorylation, adhesion, and migration in normal T cells. pERM and CD44 are highly expressed in T cells infiltrating in the kidneys of patients with lupus nephritis. These data prove that increased ERM phosphorylation represents a key molecular abnormality that guides T cell adhesion and migration in SLE patients.     

Essential roles of integrin-mediated signaling for the enhancement of malignant properties of melanomas based on the expression of GD3

We reported that ganglioside GD3 enhances cell proliferation and invasion of melanomas causing stronger tyrosine-phosphorylation of p130Cas and paxillin after stimulation with fetal calf serum. Besides signals via growth factor/receptor, adhesion signals via integrin might be also enhanced by GD3. Here, roles of integrin-mediated signaling in the cell proliferation and invasion, and in the activation of adaptor molecules were examined, showing that integrin was also important for the cell growth and invasion. p130Cas and paxillin underwent stronger tyrosine-phosphorylation in GD3+ cells than in GD3− cells during the adhesion in the absence of serum. On the other hand, no proteins underwent tyrosine phosphorylation in GD3+ and GD3− cells in a suspension state when stimulated with fetal calf serum. These results suggested that integrin-mediated signaling is essential in the effects of GD3 on the malignant properties of melanomas. Co-localization of GD3 and integrin at the focal adhesion supported these results.     

Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins.

Ezrin/radixin/moesin (ERM) proteins have been thought to play a central role in the organization of cortical actin-based cytoskeletons including microvillar formation through cross-linking actin filaments and integral membrane proteins such as CD43, CD44, and ICAM-2. To examine the functions of these ERM-binding membrane proteins (ERMBMPs) in cortical morphogenesis, we overexpressed ERMBMPs (the extracellular domain of E-cadherin fused with the transmembrane/cytoplasmic domain of CD43, CD44, or ICAM-2) in various cultured cells. In cultured fibroblasts such as L and CV-1 cells, their overexpression significantly induced microvillar elongation, recruiting ERM proteins and actin filaments. When the ERM-binding domains were truncated from these molecules, their ability to induce microvillar elongation became undetectable. In contrast, in cultured epithelial cells such as MTD-1A and A431 cells, the overexpression of ERMBMPs did not elongate microvilli. However, in the presence of EGF, overexpression of ERMBMPs induced remarkable microvillar elongation in A431 cells. These results indicated that ERMBMPs function as organizing centers for cortical morphogenesis by organizing microvilli in collaboration with activated ERM proteins. Furthermore, immunodetection with a phosphorylated ERM-specific antibody and site-directed mutagenesis suggested that ERM proteins phosphorylated at their COOH-terminal threonine residue represent activated ERM proteins.     

Phosphorylation of ezrin enhances microvillus length via a p38 MAP-kinase pathway in an immortalized mouse hepatic cell line

The apical microvilli are closely related with the development and the maintenance of cell polarization, and the length of microvilli varies in a regular way among cell types. Ezrin, a member of the ezrin/radixin/moesin (ERM) family, seems to be involved in the formation and stabilization of the apical microvilli. We found that phosphorylation of ezrin caused elongation of microvilli via a p38 MAP-kinase signaling pathway in an immortalized mouse hepatic cell line. When, in the oncogenic Raf-1-transfected mouse hepatic cell line, epithelial to mesenchymal transition (EMT) indicated as down-regulation of E-cadherin and up-regulation of Snail occurred, loss of microvilli and down-regulation of ezrin but not radixin and moesin were also observed. In the Raf-1 transfectants treated with the MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580, the numbers of microvilli and the expression of ezrin, E-cadherin and Snail were recovered. More interestingly, treatment with SB203580 induced elongation of microvilli and increased phosphorylation of ezrin (at Thr-567 and Tyr-353). Phosphorylated ezrin-positive dots were colocalized with actin-positive dots on the surface of some Raf-1 transfectants treated with SB203580. These results suggested that phosphorylation of ezrin via the p38 MAP-kinase signaling pathway might be involved in the formation of microvilli during development of epithelial cell polarization.     

Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.     

Direct binding of neutral endopeptidase 24.11 to ezrin/radixin/moesin (ERM) proteins competes with the interaction of CD44 with ERM proteins

Neutral endopeptidase 24.11 (NEP) is a cell surface peptidase expressed by numerous tissues including prostatic epithelial cells. We reported that NEP inhibits prostate cancer cell proliferation and cell migration by enzymatic inactivation of neuropeptide substrates and through protein-protein interaction independent of catalytic function. The cytoplasmic domain of NEP contains a positively charged amino acid cluster, previously identified as a binding site for ezrin/radixin/moesin (ERM) proteins. We report here that NEP co-immunoprecipitates with ERM proteins in NEP-expressing LNCaP prostate cancer cells and MeWo melanoma cells. Co-immunoprecipitation showed that ERM proteins associate with wild-type NEP protein but not NEP protein containing a truncated cytoplasmic domain or point mutations replacing the positively charged amino acid cluster. In vitro binding assays showed that NEP binds directly to recombinant N terminus fragments of ERM proteins at the positively charged amino acid cluster within the NEP cytoplasmic domain. Binding of ERM proteins to NEP results in decreased binding of ERM proteins to the hyaluronan receptor CD44, a main binding partner of ERM proteins. Moreover, cells expressing wild-type NEP demonstrate decreased adhesion to hyaluronic acid and cell migration. These data suggest that NEP can affect cell adhesion and migration through direct binding to ERM proteins.     

CD43 interaction with ezrin-radixin-moesin (ERM) proteins regulates T-cell trafficking and CD43 phosphorylation

Cell polarization is a key feature of cell motility, driving cell migration to tissues. CD43 is an abundantly expressed molecule on the T-cell surface that shows distinct localization to the migrating T-cell uropod and the distal pole complex (DPC) opposite the immunological synapse via association with the ezrin-radixin-moesin (ERM) family of actin regulatory proteins. CD43 regulates multiple T-cell functions, including T-cell activation, proliferation, apoptosis, and migration. We recently demonstrated that CD43 regulates T-cell trafficking through a phosphorylation site at Ser-76 (S76) within its cytoplasmic tail. Using a phosphorylation-specific antibody, we now find that CD43 phosphorylation at S76 is enhanced by migration signals. We further show that CD43 phosphorylation and normal T-cell trafficking depend on CD43 association with ERM proteins. Interestingly, mutation of S76 to mimic phosphorylation enhances T-cell migration and CD43 movement to the DPC while blocking ERM association, showing that CD43 movement can occur in the absence of ERM binding. We also find that protein kinase CΘ can phosphorylate CD43. These results show that while CD43 binding to ERM proteins is crucial for S76 phosphorylation, CD43 movement and regulation of T-cell migration can occur through an ERM-independent, phosphorylation-dependent mechanism.     

Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.