RESUMO
Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and ß2-adrenergic receptor (ß2AR) optobodies suppressed endogenous gelsolin activity and ß2AR signaling, respectively.
Assuntos
Anticorpos/fisiologia , Gelsolina/fisiologia , Optogenética , Receptores Adrenérgicos beta 2/fisiologia , Animais , Células Cultivadas , HumanosRESUMO
Cancer-associated thrombosis is the second-leading cause of mortality in patients with cancer and presents a poor prognosis, with a lack of effective treatment strategies. NAD(P)H quinone oxidoreductase 1 (NQO1) increases the cellular nicotinamide adenine dinucleotide (NAD+) levels by accelerating the oxidation of NADH to NAD+, thus playing important roles in cellular homeostasis, energy metabolism, and inflammatory responses. Using a murine orthotopic 4T1 breast cancer model, in which multiple thrombi are generated in the lungs at the late stage of cancer development, we investigated the effects of regulating the cellular NAD+ levels on cancer-associated thrombosis. In this study, we show that dunnione (a strong substrate of NQO1) attenuates the prothrombotic state and lung thrombosis in tumor-bearing mice by inhibiting the expression of tissue factor and formation of neutrophil extracellular traps (NETs). Dunnione increases the cellular NAD+ levels in lung tissues of tumor-bearing mice to restore the declining sirtuin 1 (SIRT1) activity, thus deacetylating nuclear factor-kappa B (NF-κB) and preventing the overexpression of tissue factor in bronchial epithelial and vascular endothelial cells. In addition, we demonstrated that dunnione abolishes the ability of neutrophils to generate NETs by suppressing histone acetylation and NADPH oxidase (NOX) activity. Overall, our results reveal that the regulation of cellular NAD+ levels by pharmacological agents may inhibit pulmonary embolism in tumor-bearing mice, which may potentially be used as a viable therapeutic approach for the treatment of cancer-associated thrombosis.
Assuntos
Neoplasias da Mama/complicações , Armadilhas Extracelulares/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD/metabolismo , Naftoquinonas/farmacologia , Trombofilia/tratamento farmacológico , Tromboplastina/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Sirtuína 1/metabolismo , Trombofilia/etiologia , Trombofilia/prevenção & controle , Tromboplastina/antagonistas & inibidores , Trombose/tratamento farmacológico , Trombose/etiologia , Trombose/prevenção & controleRESUMO
Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Movimento Celular/genética , Fibroblastos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Retroalimentação Fisiológica , Fibroblastos/citologia , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Optogenética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Polimerização , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems.
Assuntos
Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Optogenética/métodos , Multimerização Proteica/efeitos da radiação , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cones de Crescimento/metabolismo , Cones de Crescimento/efeitos da radiação , Hipocampo/citologia , Transporte Proteico/efeitos da radiaçãoRESUMO
Despite large cell-to-cell variations in the concentrations of individual signaling proteins, cells transmit signals correctly. This phenomenon raises the question of what signaling systems do to prevent a predicted high failure rate. Here we combine quantitative modeling, RNA interference, and targeted selective reaction monitoring (SRM) mass spectrometry, and we show for the ubiquitous and fundamental calcium signaling system that cells monitor cytosolic and endoplasmic reticulum (ER) Ca(2+) levels and adjust in parallel the concentrations of the store-operated Ca(2+) influx mediator stromal interaction molecule (STIM), the plasma membrane Ca(2+) pump plasma membrane Ca-ATPase (PMCA), and the ER Ca(2+) pump sarco/ER Ca(2+)-ATPase (SERCA). Model calculations show that this combined parallel regulation in protein expression levels effectively stabilizes basal cytosolic and ER Ca(2+) levels and preserves receptor signaling. Our results demonstrate that, rather than directly controlling the relative level of signaling proteins in a forward regulation strategy, cells prevent transmission failure by sensing the state of the signaling pathway and using multiple parallel adaptive feedbacks.
Assuntos
Adaptação Fisiológica , Sinalização do Cálcio , Drosophila melanogaster/metabolismo , Animais , Cálcio/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retroalimentação Fisiológica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia , Transdução de SinaisRESUMO
Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.
Assuntos
Encéfalo/efeitos da radiação , DNA Nucleotidiltransferases/genética , Animais , Comportamento Animal/fisiologia , Encéfalo/enzimologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Nucleotidiltransferases/efeitos da radiação , Regulação da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Luz , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Recombinação GenéticaRESUMO
Pediatric brain tumors are highly associated with epileptic seizures1. However, their epileptogenic mechanisms remain unclear. Here, we show that the oncogenic BRAF somatic mutation p.Val600Glu (V600E) in developing neurons underlies intrinsic epileptogenicity in ganglioglioma, one of the leading causes of intractable epilepsy2. To do so, we developed a mouse model harboring the BRAFV600E somatic mutation during early brain development to reflect the most frequent mutation, as well as the origin and timing thereof. Therein, the BRAFV600E mutation arising in progenitor cells during brain development led to the acquisition of intrinsic epileptogenic properties in neuronal lineage cells, whereas tumorigenic properties were attributed to high proliferation of glial lineage cells. RNA sequencing analysis of patient brain tissues with the mutation revealed that BRAFV600E-induced epileptogenesis is mediated by RE1-silencing transcription factor (REST), which is a regulator of ion channels and neurotransmitter receptors associated with epilepsy. Moreover, we found that seizures in mice were significantly alleviated by an FDA-approved BRAFV600E inhibitor, vemurafenib, as well as various genetic inhibitions of Rest. Accordingly, this study provides direct evidence of a BRAF somatic mutation contributing to the intrinsic epileptogenicity in pediatric brain tumors and suggests that BRAF and REST could be treatment targets for intractable epilepsy.
Assuntos
Neoplasias Encefálicas/genética , Ganglioglioma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Repressoras/genética , Convulsões/genética , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/fisiopatologia , Criança , Modelos Animais de Doenças , Ganglioglioma/complicações , Ganglioglioma/diagnóstico por imagem , Ganglioglioma/fisiopatologia , Humanos , Camundongos , Mutação , Pediatria , Convulsões/complicações , Convulsões/diagnóstico por imagem , Convulsões/fisiopatologiaRESUMO
Pulsatile actomyosin contractility, important in tissue morphogenesis, has been studied mainly in apical but less in basal domains. Basal myosin oscillation underlying egg chamber elongation is regulated by both cell-matrix and cell-cell adhesions. However, the mechanism by which these two adhesions govern basal myosin oscillation and tissue elongation is unknown. Here we demonstrate that cell-matrix adhesion positively regulates basal junctional Rho1 activity and medio-basal ROCK and myosin activities, thus strongly controlling tissue elongation. Differently, cell-cell adhesion governs basal myosin oscillation through controlling medio-basal distributions of both ROCK and myosin signals, which are related to the spatial limitations of cell-matrix adhesion and stress fibres. Contrary to cell-matrix adhesion, cell-cell adhesion weakly affects tissue elongation. In vivo optogenetic protein inhibition spatiotemporally confirms the different effects of these two adhesions on basal myosin oscillation. This study highlights the activity and distribution controls of basal myosin contractility mediated by cell-matrix and cell-cell adhesions, respectively, during tissue morphogenesis.
Assuntos
Adesão Celular , Junções Célula-Matriz/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Miosina Tipo II/metabolismo , Óvulo/metabolismo , Actomiosina/metabolismo , Animais , Integrinas/metabolismo , Morfogênese , Optogenética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Mammalian tissue size is maintained by slow replacement of de-differentiating and dying cells. For adipocytes, key regulators of glucose and lipid metabolism, the renewal rate is only 10% per year. We used computational modeling, quantitative mass spectrometry, and single-cell microscopy to show that cell-to-cell variability, or noise, in protein abundance acts within a network of more than six positive feedbacks to permit pre-adipocytes to differentiate at very low rates. This reconciles two fundamental opposing requirements: High cell-to-cell signal variability is needed to generate very low differentiation rates, whereas low signal variability is needed to prevent differentiated cells from de-differentiating. Higher eukaryotes can thus control low rates of near irreversible cell fate decisions through a balancing act between noise and ultrahigh feedback connectivity.
Assuntos
Adipócitos/citologia , Adipogenia , Modelos Biológicos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Comunicação Celular , Diferenciação Celular , Linhagem Celular , Simulação por Computador , Retroalimentação Fisiológica , Espectrometria de Massas , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , RNA Interferente Pequeno/genética , Análise de Célula Única , Células-Tronco/citologiaRESUMO
Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs. Conversion begins by glucocorticoid and cAMP signals raising C/EBPß levels above a critical threshold, triggering three consecutive positive feedback loops: from PPARγ to C/EBPα, then to C/EBPß, and last to the insulin receptor. Experiments and modeling show that these feedbacks create a robust, irreversible transition to a terminally differentiated state by rejecting short- and low-amplitude stimuli. After the differentiation switch is triggered, insulin controls fat accumulation in a graded fashion. Altogether, our study introduces a regulatory motif that locks cells in a differentiated state by engaging a sequence of positive feedback loops.
Assuntos
Adipócitos/citologia , Células 3T3 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , AMP Cíclico/farmacologia , Glucocorticoides/farmacologia , Insulina/farmacologia , Lipídeos/biossíntese , Camundongos , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC). Using gel overlay assays, a Ca2+-dependent CaM-binding domain was identified in the C terminus of this protein. Specific binding of CaM to CaM-binding domain was corroborated by site-directed mutagenesis and a split-ubiquitin assay. Using a PCR-mediated random binding site selection method, we identified a DNA-binding sequence (CBNACBS) for CBNAC, which consisted of a GCTT core sequence flanked on both sides by other frequently repeating sequences (TTGCTTANNNNNNAAG). CBNAC was able to bind to CBNACBS, which resulted in the repression of transcription in Arabidopsis protoplasts. Interestingly, the transcriptional repression mediated by CBNAC was enhanced by CaM. These results suggest that CBNAC may be a CaM-regulated transcriptional repressor in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a DNA/genética , Proteínas Repressoras/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Ligação Proteica/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Repressoras/metabolismo , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.
Assuntos
Membrana Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , GTP Fosfo-Hidrolases/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Sistemas do Segundo Mensageiro , Transdução de Sinais , Eletricidade Estática , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.
Assuntos
Proteínas de Ligação a Calmodulina/isolamento & purificação , Oryza/química , Fatores de Transcrição/isolamento & purificação , Sequência de Aminoácidos , Arabidopsis/genética , Sequência de Bases , Sítios de Ligação , Calmodulina/genética , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Clonagem Molecular , DNA Complementar/genética , DNA de Plantas/química , DNA de Plantas/metabolismo , Escherichia coli/genética , Deleção de Genes , Expressão Gênica , Biblioteca Gênica , Glucuronidase/genética , Glutationa Transferase/genética , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oryza/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Sequências de Repetição em Tandem , Fatores de Transcrição/química , Fatores de Transcrição/genética , TransfecçãoRESUMO
Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus. The MAPK phosphorylates OsEREBP1 that binds to the GCC box element (AGCCGCC) of the several basic pathogenesis-related gene promoters, which in turn enhances DNA-binding activity of the factor to the cis element in vitro. Transient co-expression of the BWMK1 and OsEREBP1 in Arabidopsis protoplasts elevates the expression of the beta-glucuronidase reporter gene driven by the GCC box element. Furthermore, transgenic tobacco (Nicotiana tabacum) plants overexpressing BWMK1 expressed many pathogenesis-related genes at higher levels than wild-type plants with an enhanced resistance to pathogens. These findings suggest that MAPKs contribute to plant defense signal transduction by phosphorylating one or more transcription factors.
Assuntos
Núcleo Celular/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/enzimologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/genética , Sequência de Bases , Morte Celular , Ativação Enzimática , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Genes Reporter/genética , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Oryza/genética , Fosforilação , Filogenia , Proteínas de Plantas , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Protoplastos/metabolismo , Elementos de Resposta/genética , Nicotiana/citologia , Nicotiana/genética , Nicotiana/microbiologia , Fatores de Transcrição/químicaRESUMO
Hundreds of proteins involved in signaling pathways contain a Ca(2+)-dependent membrane-binding motif called the C2-domain. However, no small C2-domain proteins consisting of a single C2-domain have been reported in animal cells. We have isolated two cDNA clones, OsERG1a and OsERG1b, that encode two small C2-domain proteins of 156 and 159 amino acids, respectively, from a fungal elicitor-treated rice cDNA library. The clones are believed to have originated from a single gene by alternative splicing. Transcript levels of the OsERG1 gene are dramatically elevated by a fungal elicitor prepared from Magnaporthe grisea or by Ca(2+) ions. The OsERG1 protein produced in Escherichia coli binds to phospholipid vesicles in a Ca(2+)-dependent manner and is translocated to the plasma membrane of plant cells by treatment with either a fungal elicitor or a Ca(2+) ionophore. These results suggest that OsERG1 proteins containing a single C2-domain are involved in plant defense signaling systems.