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[This corrects the article DOI: 10.1371/journal.pbio.3000782.].
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As the stimulus-responsive mediator of actin dynamics, actin-depolymerizing factor (ADF)/cofilin is subject to tight regulation. It is well known that kinase-mediated phosphorylation inactivates ADF/cofilin. Here, however, we found that the activity of Arabidopsis ADF7 is enhanced by CDPK16-mediated phosphorylation. We found that CDPK16 interacts with ADF7 both in vitro and in vivo, and it enhances ADF7-mediated actin depolymerization and severing in vitro in a calcium-dependent manner. Accordingly, the rate of actin turnover is reduced in cdpk16 pollen and the amount of actin filaments increases significantly at the tip of cdpk16 pollen tubes. CDPK16 phosphorylates ADF7 at Serine128 both in vitro and in vivo, and the phospho-mimetic mutant ADF7S128D has enhanced actin-depolymerizing activity compared to ADF7. Strikingly, we found that failure in the phosphorylation of ADF7 at Ser128 impairs its function in promoting actin turnover in vivo, which suggests that this phospho-regulation mechanism is biologically significant. Thus, we reveal that CDPK16-mediated phosphorylation up-regulates ADF7 to promote actin turnover in pollen.
Assuntos
Actinas , Arabidopsis , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Destrina/metabolismo , Fosforilação , Tubo Polínico/metabolismoRESUMO
Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5-mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin-bundling activity. Notably, the absence of Val763-Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763-Ser823 sequence harbors a binding site for F-actin, suggesting that this linker-based F-actin binding site, in conjunction with the F-actin binding site localized in G1-G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments.
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Stomatal movement is critical for plant responses to environmental changes and is regulated by the important signaling molecule phosphatidylinositol 3-phosphate (PI3P). However, the molecular mechanism underlying this process is not well understood. In this study, we show that PI3P binds to stomatal closure-related actin-binding protein1 (SCAB1), a plant-specific F-actin-binding and -bundling protein, and inhibits the oligomerization of SCAB1 to regulate its activity on F-actin in guard cells during stomatal closure in Arabidopsis thaliana. SCAB1 binds specifically to PI3P, but not to other phosphoinositides. Treatment with wortmannin, an inhibitor of phosphoinositide kinase that generates PI3P, leads to an increase of the intermolecular interaction and oligomerization of SCAB1, stabilization of F-actin, and retardation of F-actin reorganization during abscisic acid (ABA)-induced stomatal closure. When the binding activity of SCAB1 to PI3P is abolished, the mutated proteins do not rescue the stability and realignment of F-actin regulated by SCAB1 and the stomatal closure in the scab1 mutant. The expression of PI3P biosynthesis genes is consistently induced when the plants are exposed to drought and ABA treatments. Furthermore, the binding of PI3P to SCAB1 is also required for vacuolar remodeling during stomatal closure. Our results illustrate a PI3P-regulated pathway during ABA-induced stomatal closure, which involves the mediation of SCAB1 activity in F-actin reorganization.
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Actinas/química , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas dos Microfilamentos/genética , Fosfatos de Fosfatidilinositol/metabolismo , Estômatos de Plantas/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas dos Microfilamentos/metabolismoRESUMO
Pollen tubes extend rapidly via tip growth. This process depends on a dynamic actin cytoskeleton, which has been implicated in controlling organelle movements, cytoplasmic streaming, vesicle trafficking, and cytoplasm organization in pollen tubes. In this update review, we describe the progress in understanding the organization and regulation of the actin cytoskeleton and the function of the actin cytoskeleton in controlling vesicle traffic and cytoplasmic organization in pollen tubes. We also discuss the interplay between ion gradients and the actin cytoskeleton that regulates the spatial arrangement and dynamics of actin filaments and the organization of the cytoplasm in pollen tubes. Finally, we describe several signaling components that regulate actin dynamics in pollen tubes.
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Proteínas de Arabidopsis , Arabidopsis , Tubo Polínico , Arabidopsis/fisiologia , Citoesqueleto de Actina , Actinas , CitoplasmaRESUMO
The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell-stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.
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Citoesqueleto de Actina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Polaridade Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Divisão Celular Assimétrica/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Movimento Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Células-Tronco Neurais/metabolismoRESUMO
ADF/cofilin is a central regulator of actin dynamics. We previously demonstrated that two closely related Arabidopsis class IIa ADF isovariants, ADF7 and ADF10, are involved in the enhancement of actin turnover in pollen, but whether they have distinct functions remains unknown. Here, we further demonstrate that they exhibit distinct functions in regulating actin turnover both in vitro and in vivo. We found that ADF7 binds to ADP-G-actin with lower affinity, and severs and depolymerizes actin filaments less efficiently in vitro than ADF10. Accordingly, in pollen grains, ADF7 more extensively decorates actin filaments and is less freely distributed in the cytoplasm compared to ADF10. We further demonstrate that ADF7 and ADF10 show distinct intracellular localizations during pollen germination, and they have non-equivalent functions in promoting actin turnover in pollen. We thus propose that cooperation and labor division of ADF7 and ADF10 enable pollen cells to achieve exquisite control of the turnover of different actin structures to meet different cellular needs.
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Proteínas de Arabidopsis , Arabidopsis , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pólen/metabolismo , Tubo Polínico/metabolismoRESUMO
Grain notching is a common deformation that decreases rice (Oryza sativa) quality; however, the underlying molecular basis causing grain notching remains unclear. We report mechanisms underlying grain notching in Small and notched grain (Sng) mutants, which contained an arginine to histidine substitution at amino acid position 422 (R422H) of the α-tubulin protein OsTUBA3. The R422H mutation decreased cell length and increased cell width/height of glumes and caryopses, but led to elongated caryopses compressed within shortened glumes, thus giving rise to notched and small grains. Glume and caryopsis cells had different dimensional orientations relative to the directions of organ elongation. Thus, the abnormal cell expansion induced in glumes and caryopses by the R422H mutation had different effects on elongation of these organs. The R422H mutation in OsTUBA3 compromised ß-tubulin binding and led to formation of defective heterodimers. This in turn affected tubulin incorporation and microtubule (MT) nucleation and regrowth, consequently leading to MT instability and reducing the transverse orientation. The defective MT dynamics affected cell expansion and shape, causing different alterations in glume and caryopsis dimensions and resulting in grain notching. These data indicate that Arg422 in OsTUBA3 is crucial for MT dynamics and that substitution with His causes grain notching, reducing grain quality and yield. These findings offer valuable insights into the molecular regulation underlying grain development in rice.
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Tight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/metabolismo , Desenvolvimento Vegetal/genética , Splicing de RNA/genética , Transcrição Gênica , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/metabolismo , Flores/fisiologia , Histonas/metabolismo , Mutação com Perda de Função , Lisina/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Fenótipo , Raízes de Plantas/citologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de AminoácidosRESUMO
Self-incompatibility (SI) in the poppy Papaver rhoeas triggers dramatic alterations in actin within pollen tubes. However, how these actin alterations are mechanistically achieved remains largely unexplored. Here, we used treatment with the Ca2+ ionophore A23187 to mimic the SI-induced elevation in cytosolic Ca2+ and trigger formation of the distinctive F-actin foci. Live-cell imaging revealed that this remodeling involves F-actin fragmentation and depolymerization, accompanied by the rapid formation of punctate actin foci and subsequent increase in their size. We established that actin foci are generated and enlarged from crosslinking of fragmented actin filament structures. Moreover, we show that villins associate with actin structures and are involved in this actin reorganization process. Notably, we demonstrate that Arabidopsis VILLIN5 promotes actin depolymerization and formation of actin foci by fragmenting actin filaments, and controlling the enlargement of actin foci via bundling of actin filaments. Our study thus uncovers important novel insights about the molecular players and mechanisms involved in forming the distinctive actin foci in pollen tubes.
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Actinas , Proteínas dos Microfilamentos , Tubo Polínico , Citoesqueleto de Actina , Actinas/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Tubo Polínico/genéticaRESUMO
Srv2p/CAP1 is an essential regulator of actin turnover, but its exact function in regulating actin polymerization, particularly the contribution of its actin nucleotide exchange activity, remains incompletely understood. We found that, although Arabidopsis CAP1 is distributed uniformly in the cytoplasm, its loss of function has differential effects on the actin cytoskeleton within different regions of the pollen tube. Specifically, the F-actin level increases in the shank but decreases in the apical region of cap1 pollen tubes. The reduction in apical F-actin results mainly from impaired polymerization of membrane-originated actin within cap1 pollen tubes. The actin nucleotide exchange activity of CAP1 is involved in apical actin polymerization. CAP1 acts synergistically with pollen ADF and profilin to promote actin turnover in vitro, and it can overcome the inhibitory effects of ADF and synergize with profilin to promote actin nucleotide exchange. Consistent with its role as a shuttle molecule between ADF and profilin, the cytosolic concentration of CAP1 is much lower than that of ADF and profilin in pollen. Thus, CAP1 synergizes with ADF and profilin to drive actin turnover in pollen and promote apical actin polymerization in pollen tubes in a manner that involves its actin nucleotide exchange activity.
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Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Oligopeptídeos/metabolismo , Tubo Polínico/metabolismo , Citoesqueleto de Actina/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas dos Microfilamentos/metabolismo , Pólen/metabolismo , Polimerização , Profilinas/metabolismoRESUMO
Stomatal movement is essential for plant growth. This process is precisely regulated by various cellular activities in guard cells. F-actin dynamics and vacuole morphology are both involved in stomatal movement. The sorting of cargoes by clathrin adaptor protein (AP) complexes from the Golgi to the vacuole is critical for establishing a normal vacuole morphology. In this study, we demonstrate that the medium subunit of the AP3 complex (AP3M) binds to and severs actin filaments in vitro and that it participates in the sorting of cargoes (such as the sucrose exporter SUC4) to the tonoplast, and thereby regulates stomatal closure in Arabidopsis thaliana Defects in AP3 or SUC4 led to more rapid water loss and delayed stomatal closure, as well as hypersensitivity to drought stress. In ap3m mutants, the F-actin status was altered compared to the wild type, and the sorted cargoes failed to localize to the tonoplast. AP3M contains a previously unidentified F-actin binding domain that is conserved in AP3M homologs in both plants and animals. Mutations in the F-actin binding domain of AP3M abolished its F-actin binding activity in vitro, leading to an aberrant vacuole morphology and reduced levels of SUC4 on the tonoplast in guard cells. Our findings indicate that the F-actin binding activity of AP3M is required for the precise localization of AP3-dependent cargoes to the tonoplast and for the regulation of vacuole morphology in guard cells during stomatal closure.
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Citoesqueleto de Actina/metabolismo , Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Estômatos de Plantas/metabolismo , Vacúolos/metabolismo , Citoesqueleto de Actina/genética , Complexo 3 de Proteínas Adaptadoras/genética , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Estômatos de Plantas/citologia , Estômatos de Plantas/genética , Vacúolos/genéticaRESUMO
Phosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PI-dependent modulation remain largely elusive, the effects of disturbed PI metabolism could be employed to identify regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice (Oryza sativa). Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related protein2 and -3 (Arp2/3) complex-nucleated actin-branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effects in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulting from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering cell development. These findings imply that the Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing insights into the relationship between PI homeostasis and plant growth and development.
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Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Oryza/genética , Fosfatases de Fosfoinositídeos/genética , Proteínas de Plantas/metabolismoRESUMO
Actin binding proteins and transcription factors are essential in regulating plant root hair growth in response to various environmental stresses; however, the interaction between these two factors in regulating root hair growth remains poorly understood. Apical and subapical thick actin bundles are necessary for terminating rapid elongation of root hair cells. Here, we show that Arabidopsis (Arabidopsis thaliana) actin-bundling protein Villin1 (VLN1) decorates filaments in shank, subapical, and apical hairs. vln1 mutants displayed significantly longer hairs with longer hair growing time and defects in the thick actin bundles and bundling activities in the subapical and apical regions, whereas seedlings overexpressing VLN1 showed different results. Genetic analysis showed that the transcription factor GLABRA2 (Gl2) played a regulatory role similar to that of VLN1 in hair growth and actin dynamics. Moreover, further analyses demonstrated that VLN1 overexpression suppresses the gl2 mutant phenotypes regarding hair growth and actin dynamics; GL2 directly recognizes the promoter of VLN1 and positively regulates VLN1 expression in root hairs; and the GL2-mediated VLN1 pathway is involved in the root hair growth response to osmotic stress. Our results demonstrate that the GL2-mediated VLN1 pathway plays an important role in the root hair growth response to osmotic stress, and they describe a transcriptional mechanism that regulates actin dynamics and thereby modulates cell tip growth in response to environmental signals.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodomínio/metabolismo , Raízes de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Pressão Osmótica , Raízes de Plantas/genéticaRESUMO
A population of dynamic apical actin filaments is required for rapid polarized pollen tube growth. However, the cellular mechanisms driving their assembly remain incompletely understood. It was postulated that formin is a major player in nucleating apical actin assembly, but direct genetic and cytological evidence remains to be firmly established. Here we found that both Arabidopsis formin 3 (AtFH3) and formin 5 (AtFH5) are involved in the regulation of apical actin polymerization and actin array construction in pollen tubes, with AtFH3 playing a more dominant role. We found that both formins have plasma membrane (PM) localization signals but exhibit distinct PM localization patterns in the pollen tube, and loss of their function reduces the amount of apical actin filaments. Live-cell imaging revealed that the reduction in filamentous actin is very likely due to the decrease in filament elongation. Furthermore, we found that the rate of tip-directed vesicle transport is reduced and the pattern of apical vesicle accumulation is altered in formin loss-of-function mutant pollen tubes, which explains to some extent the reduction in pollen tube elongation. Thus, we provide direct genetic and cytological evidence showing that formin is an important player in nucleating actin assembly from the PM at pollen tube tips.
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Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Genes de Plantas , Mutagênese Insercional , Plantas Geneticamente Modificadas , Tubo Polínico/genética , Polimerização , Multimerização ProteicaRESUMO
Here, we show that Arabidopsis ADF10 plays an important role in shaping the overall organization of apical actin filaments by promoting their turnover and ordering. ADF10 severs and depolymerizes actin filaments in vitro and is distributed throughout the entire pollen tube. In adf10 mutants, severing and monomer dissociation events for apical actin filaments are reduced, and the apical actin structure extends further toward the tube base than in wild-type tubes. In particular, the percentage of apical actin filaments that form large angles to the tube growth axis is much higher in adf10 pollen tubes, and the actin filaments are more randomly distributed, implying that ADF10 promotes their ordering. Consistent with the role of apical actin filaments in physically restricting the movement of vesicles, the region in which apical vesicles accumulate is enlarged at the tip of adf10 pollen tubes. Both tipward and backward movements of small vesicles are altered within the growth domain of adf10 pollen tubes. Thus, our study suggests that ADF10 shapes the organization of apical actin filaments to regulate vesicle trafficking and pollen tube growth.
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Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Tubo Polínico/metabolismo , Transporte Proteico/genética , Actinas/metabolismo , Arabidopsis/metabolismo , Pólen/genética , Pólen/metabolismoRESUMO
The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF.
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Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Estômatos de Plantas/metabolismo , Ácido Abscísico/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secas , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Estômatos de Plantas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus.
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Actinas/química , Núcleo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Trifosfato de Adenosina/química , Montagem e Desmontagem da Cromatina , Cristalização , Células HEK293 , Células HeLa , Humanos , Domínios ProteicosRESUMO
The actin cytoskeleton is involved in regulating stomatal movement, which forms distinct actin arrays within guard cells of stomata with different apertures. How those actin arrays are formed and maintained remains largely unexplored. Elucidation of the dynamic behavior of differently oriented actin filaments in guard cells will enhance our understanding in this regard. Here, we initially developed a program called 'guard cell microfilament analyzer' (GCMA) that enables the selection of individual actin filaments and analysis of their orientations semiautomatically in guard cells. We next traced the dynamics of individual actin filaments and performed careful quantification in open and closed stomata. We found that de novo nucleation of actin filaments occurs at both dorsal and ventral sides of guard cells from open and closed stomata. Interestingly, most of the nucleated actin filaments elongate radially and longitudinally in open and closed stomata, respectively. Strikingly, radial filaments tend to form bundles whereas longitudinal filaments tend to be removed by severing and depolymerization in open stomata. By contrast, longitudinal filaments tend to form bundles that are severed less frequently in closed stomata. These observations provide insights into the formation and maintenance of distinct actin arrays in guard cells in stomata of different apertures.
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Citoesqueleto de Actina/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Estômatos de Plantas/citologia , Estômatos de Plantas/metabolismo , Actinas/metabolismo , Ritmo Circadiano/fisiologia , PolimerizaçãoRESUMO
Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G-actin) and filamentous actin (F-actin) is crucial for actin assembly and array construction, and the local concentration of G-actin thus directly impacts actin assembly. The localization and dynamics of G-actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I-binding loop of ACT11 (GFPMet49 -ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFPMet49 -ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)-treated pollen tubes. Careful observations showed that G-actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G-actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.