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1.
New Phytol ; 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31859367

RESUMO

The actin cytoskeleton is required for cell expansion and implicated in cellular responses to the phytohormone auxin. However, the mechanisms that coordinate auxin signaling, cytoskeletal remodeling and cell expansion are poorly understood. Previous studies examined long-term actin cytoskeleton responses to auxin, but plants respond to auxin within minutes. Before this work, an extracellular auxin receptor - rather than the auxin transporter AUXIN RESISTANT 1 (AUX1) - was considered to precede auxin-induced cytoskeleton reorganization. In order to correlate actin array organization and dynamics with degree of cell expansion, quantitative imaging tools established baseline actin organization and illuminated individual filament behaviors in root epidermal cells under control conditions and after indole-3-acetic acid (IAA) application. We evaluated aux1 mutant actin organization responses to IAA and the membrane-permeable auxin 1-naphthylacetic acid (NAA). Cell length predicted actin organization and dynamics in control roots; short-term IAA treatments stimulated denser and more parallel, longitudinal arrays by inducing filament unbundling within minutes. Although AUX1 is necessary for full actin rearrangements in response to auxin, cytoplasmic auxin (i.e. NAA) stimulated a lesser response. Actin filaments became more 'organized' after IAA stopped elongation, refuting the hypothesis that 'more organized' actin arrays universally correlate with rapid growth. Short-term actin cytoskeleton response to auxin requires AUX1 and/or cytoplasmic auxin.

2.
Plant Cell Physiol ; 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31738423

RESUMO

Recessively inherited mutant alleles of Mlo genes (mlo) confer broad-spectrum penetration resistance to powdery mildew pathogens in angiosperm plants. Although a few components are known to be required for mlo resistance, the detailed molecular mechanism underlying this type of immunity remains elusive. In the present study, we identified alloxan (5,5-dihydroxyl pyrimidine-2,4,6-trione) and some of its structural analogs as chemical suppressors of mlo-mediated resistance in monocotyledonous barley (Hordeum vulgare) and dicotyledonous Arabidopsis thaliana. Apart from mlo resistance, alloxan impairs non-host resistance in Arabidopsis. Histological analysis unraveled that the chemical reduces callose deposition and hydrogen peroxide accumulation at attempted fungal penetration sites. Fluorescence microscopy revealed that alloxan interferes with the motility of cellular organelles (peroxisomes, endosomes and the endoplasmic reticulum) and the pathogen-triggered redistribution of the PEN1/SYP121 t-SNARE protein. These cellular deficits are likely the consequence of disassembly of actin filaments and microtubules upon alloxan treatment. Similar to the situation in animal cells, alloxan elicited the temporary accumulation of reactive oxygen species (ROS) in cotyledons and rosette leaves of Arabidopsis plants. Our results suggest that alloxan may destabilize cytoskeletal architecture via induction of an early transient ROS burst, further leading to the failure of molecular and cellular processes that are critical for plant immunity.

3.
Plant Physiol ; 179(4): 1537-1555, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705068

RESUMO

In plants, cellulose is synthesized at the cell surface by plasma membrane (PM)-localized cellulose synthase (CESA) complexes (CSCs). The molecular and cellular mechanisms that underpin delivery of CSCs to the PM, however, are poorly understood. Cortical microtubules have been shown to interact with CESA-containing compartments and mark the site for CSC delivery, but are not required for the delivery itself. Here, we demonstrate that myosin XI and the actin cytoskeleton mediate CSC delivery to the PM by coordinating the exocytosis of CESA-containing compartments. Measurement of cellulose content indicated that cellulose biosynthesis was significantly reduced in a myosin xik xi1 xi2 triple-knockout mutant. By combining genetic and pharmacological disruption of myosin activity with quantitative live-cell imaging, we observed decreased abundance of PM-localized CSCs and reduced delivery rate of CSCs in myosin-deficient cells. These phenotypes correlated with a significant increase in failed vesicle secretion events at the PM as well as an abnormal accumulation of CESA-containing compartments at the cell cortex. Through high-resolution spatiotemporal assays of cortical vesicle behavior, we identified defects in CSC vesicle tethering and fusion at the PM. Furthermore, disruption of myosin activity reduced the delivery of several other secretory markers to the PM and reduced constitutive and receptor-mediated endocytosis. These findings reveal a previously undescribed role for myosin in vesicle secretion and cellulose production at the cytoskeleton-PM-cell wall nexus.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Exocitose , Glucosiltransferases/metabolismo , Miosinas/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular , Celulose/metabolismo , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Modelos Moleculares , Miosinas/genética , Miosinas/metabolismo
4.
Annu Rev Phytopathol ; 56: 513-533, 2018 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-29975609

RESUMO

The plant cytoskeleton is a dynamic framework of cytoplasmic filaments that rearranges as the needs of the cell change during growth and development. Incessant turnover mechanisms allow these networks to be rapidly redeployed in defense of host cytoplasm against microbial invaders. Both chemical and mechanical stimuli are recognized as danger signals to the plant, and these are perceived and transduced into cytoskeletal dynamics and architecture changes through a collection of well-recognized, previously characterized players. Recent advances in quantitative cell biology approaches, along with the powerful molecular genetics techniques associated with Arabidopsis, have uncovered two actin-binding proteins as key intermediaries in the immune response to phytopathogens and defense signaling. Certain bacterial phytopathogens have adapted to the cytoskeletal-based defense mechanism during the basal immune response and have evolved effector proteins that target actin filaments and microtubules to subvert transcriptional reprogramming, secretion of defense-related proteins, and cell wall-based defenses. In this review, we describe current knowledge about host cytoskeletal dynamics operating at the crossroads of the molecular and cellular arms race between microbes and plants.


Assuntos
Citoesqueleto/fisiologia , Doenças das Plantas/imunologia , Imunidade Vegetal/fisiologia , Transdução de Sinais/imunologia
5.
Dis Model Mech ; 11(3)2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29590639

RESUMO

Neutrophils are fast-moving cells essential for host immune functions. Although they primarily rely on glycolysis for ATP, isolated primary human neutrophils depend on mitochondrial membrane potential for chemotaxis. However, it is not known whether mitochondria regulate neutrophil motility in vivo, and the underlying molecular mechanisms remain obscure. Here, we visualized mitochondria in an interconnected network that localizes to the front and rear of migrating neutrophils using a novel transgenic zebrafish line. To disrupt mitochondrial function genetically, we established a gateway system harboring the CRISPR/Cas9 elements for tissue-specific knockout. In a transgenic line, neutrophil-specific disruption of mitochondrial DNA polymerase, polg, significantly reduced the velocity of neutrophil interstitial migration. In addition, inhibiting the mitochondrial electron transport chain or the enzymes that reduce mitochondrial reactive oxygen species also inhibited neutrophil motility. The reduced cell motility that resulted from neutrophil-specific knockout of sod1 was rescued with sod1 mRNA overexpression, or by treating with scavengers of reactive oxygen species. Together, our work has provided the first in vivo evidence that mitochondria regulate neutrophil motility, as well as tools for the functional characterization of mitochondria-related genes in neutrophils and insights into immune deficiency seen in patients with primary mitochondrial disorders.This article has an associated First Person interview with the first author of the paper.


Assuntos
Técnicas de Inativação de Genes , Mitocôndrias/metabolismo , Neutrófilos/metabolismo , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Movimento Celular , Clonagem Molecular , DNA Polimerase Dirigida por DNA/genética , Transporte de Elétrons , Dinâmica Mitocondrial , Especificidade de Órgãos , Oxirredução
6.
Sci Rep ; 8(1): 4381, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29531328

RESUMO

Flowering plants express multiple actin isoforms. Previous studies suggest that individual actin isoforms have specific functions; however, the subcellular localization of actin isoforms in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, as fluorescent-fusion proteins. By optimizing the linker sequence between fluorescent protein and actin, we succeeded in observing filaments that contained these expressed actin isoforms fused with green fluorescent protein (GFP) in Arabidopsis protoplasts. Different colored fluorescent proteins fused with AtACT2 and AtACT7 and co-expressed in Nicotiana benthamiana mesophyll cells co-polymerized in a segregated manner along filaments. In epidermal cells, surprisingly, AtACT2 and AtACT7 tended to polymerize into different types of filaments. AtACT2 was incorporated into thinner filaments, whereas AtACT7 was incorporated into thick bundles. We conclude that different actin isoforms are capable of constructing unique filament arrays, depending on the cell type or tissue. Interestingly, staining patterns induced by two indirect actin filament probes, Lifeact and mTalin1, were different between filaments containing AtACT2 and those containing AtACT7. We suggest that filaments containing different actin isoforms bind specific actin-binding proteins in vivo, since the two probes comprise actin-binding domains from different actin-binding proteins.


Assuntos
Citoesqueleto de Actina/química , Actinas/genética , Proteínas de Arabidopsis/química , Arabidopsis/química , Actinas/química , Actinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas dos Microfilamentos/metabolismo , Polimerização , Ligação Proteica , Isoformas de Proteínas
8.
PLoS Pathog ; 13(1): e1006186, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28129393

RESUMO

Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.


Assuntos
Citoesqueleto de Actina/patologia , Citoesqueleto/patologia , Interações Hospedeiro-Patógeno/fisiologia , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Células COS , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Doença dos Legionários/patologia , Espectrometria de Massas , Camundongos
9.
Plant Physiol ; 173(2): 1125-1136, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27909046

RESUMO

Plants perceive microbe-associated molecular patterns and damage-associated molecular patterns to activate innate immune signaling events, such as bursts of reactive oxygen species (ROS). The actin cytoskeleton remodels during the first 5 min of innate immune signaling in Arabidopsis (Arabidopsis thaliana) epidermal cells; however, the immune signals that impinge on actin cytoskeleton and its response regulators remain largely unknown. Here, we demonstrate that rapid actin remodeling upon elicitation with diverse microbe-associated molecular patterns and damage-associated molecular patterns represent a conserved plant immune response. Actin remodeling requires ROS generated by the defense-associated NADPH oxidase, RBOHD. Moreover, perception of flg22 by its cognate receptor complex triggers actin remodeling through the activation of RBOHD-dependent ROS production. Our genetic studies reveal that the ubiquitous heterodimeric capping protein transduces ROS signaling to the actin cytoskeleton during innate immunity. Additionally, we uncover a negative feedback loop between actin remodeling and flg22-induced ROS production.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Vegetal , Espécies Reativas de Oxigênio/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Alarminas , Arabidopsis/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Flagelina/metabolismo , Peróxido de Hidrogênio/farmacologia , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Padrões Moleculares Associados a Patógenos/metabolismo , Imunidade Vegetal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
10.
Plant Physiol ; 171(3): 2239-55, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27217495

RESUMO

The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type-III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high-resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling and a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms after pathogen infection, which can be complemented by actin-modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development.


Assuntos
Actinas/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas syringae/patogenicidade , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Bactérias/genética , Citoesqueleto/metabolismo , Teste de Complementação Genética , Interações Hospedeiro-Patógeno , Cinesina/metabolismo , Mutação , Tabaco/genética
11.
Mol Plant ; 9(6): 900-10, 2016 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-26996265

RESUMO

Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been implicated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively attaches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attachment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, competes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH14 moves processively on the elongating barbed end. At high concentration, AtFH14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Profilinas/metabolismo , Isoformas de Proteínas/metabolismo
12.
Cell Host Microbe ; 19(1): 7-9, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26764591

RESUMO

Pseudomonas syringae secretes effectors from its type III secretion system to infect plants. In this issue of Cell Host & Microbe, Guo et al. (2016) determine that the T3SS effector, HopE1, targets calmodulin and the microtubule-associated protein MAP65-1 to subvert plant immunity.


Assuntos
Proteínas de Bactérias/metabolismo , Pseudomonas syringae/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças das Plantas , Imunidade Vegetal
13.
Plant Physiol ; 170(1): 220-33, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26574597

RESUMO

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Células Vegetais/metabolismo , Profilinas/metabolismo , Citoesqueleto de Actina/ultraestrutura , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Mutação , Células Vegetais/efeitos dos fármacos , Células Vegetais/ultraestrutura , Epiderme Vegetal/citologia , Profilinas/genética , Tionas/farmacologia , Imagem com Lapso de Tempo , Uracila/análogos & derivados , Uracila/farmacologia
14.
Nat Commun ; 6: 7206, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-26018794

RESUMO

Plants and animals perceive diverse microbe-associated molecular patterns (MAMPs) via pattern recognition receptors and activate innate immune signalling. The actin cytoskeleton has been suggested as a target for innate immune signalling and a key transducer of cellular responses. However, the molecular mechanisms underlying actin remodelling and the precise functions of these rearrangements during innate immunity remain largely unknown. Here we demonstrate rapid actin remodelling in response to several distinct MAMP signalling pathways in plant epidermal cells. The regulation of actin dynamics is a convergence point for basal defence machinery, such as cell wall fortification and transcriptional reprogramming. Our quantitative analyses of actin dynamics and genetic studies reveal that MAMP-stimulated actin remodelling is due to the inhibition of capping protein (CP) by the signalling lipid, phosphatidic acid. In addition, CP promotes resistance against bacterial and fungal phytopathogens. These findings demonstrate that CP is a central target for the plant innate immune response.


Assuntos
Proteínas de Capeamento de Actina/imunologia , Citoesqueleto de Actina/imunologia , Alternariose/imunologia , Arabidopsis/imunologia , Imunidade Inata/imunologia , Epiderme Vegetal/imunologia , Alternaria/imunologia , Epiderme Vegetal/citologia , Reação em Cadeia da Polimerase em Tempo Real
15.
Annu Rev Plant Biol ; 66: 415-40, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25423079

RESUMO

Advances in microscopy techniques applied to living cells have dramatically transformed our view of the actin cytoskeleton as a framework for cellular processes. Conventional fluorescence imaging and static analyses are useful for quantifying cellular architecture and the network of filaments that support vesicle trafficking, organelle movement, and response to biotic stress. However, new imaging techniques have revealed remarkably dynamic features of individual actin filaments and the mechanisms that underpin their construction and turnover. In this review, we briefly summarize knowledge about actin and actin-binding proteins in plant systems. We focus on the quantitative properties of the turnover of individual actin filaments, highlight actin-binding proteins that participate in actin dynamics, and summarize the current genetic evidence that has been used to dissect specific aspects of the stochastic dynamics model. Finally, we describe some signaling pathways in which recent data implicate changes in actin filament dynamics and the associated cytoplasmic responses.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Transporte Proteico , Transdução de Sinais
16.
Plant Physiol ; 166(3): 1312-28, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25201878

RESUMO

The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Proteínas de Capeamento de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/genética , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Microssomos/metabolismo , Dados de Sequência Molecular , Plântula/genética , Plântula/metabolismo
17.
Plant Physiol ; 166(3): 1359-70, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25237128

RESUMO

Plant cell expansion relies on intracellular trafficking of vesicles and macromolecules, which requires myosin motors and a dynamic actin network. Arabidopsis (Arabidopsis thaliana) myosin XI powers the motility of diverse cellular organelles, including endoplasmic reticulum, Golgi, endomembrane vesicles, peroxisomes, and mitochondria. Several recent studies show that there are changes in actin organization and dynamics in myosin xi mutants, indicating that motors influence the molecular tracks they use for transport. However, the mechanism by which actin organization and dynamics are regulated by myosin XI awaits further detailed investigation. Here, using high spatiotemporal imaging of living cells, we quantitatively assessed the architecture and dynamic behavior of cortical actin arrays in a mutant with three Myosin XI (XI-1, XI-2, and XI-K) genes knocked out (xi3KO). In addition to apparent reduction of organ and cell size, the mutant showed less dense and more bundled actin filament arrays in epidermal cells. Furthermore, the overall actin dynamicity was significantly inhibited in the xi3KO mutant. Because cytoskeletal remodeling is contributed mainly by filament assembly/disassembly and translocation/buckling, we also examined the dynamic behavior of individual actin filaments. We found that the xi3KO mutant had significantly decreased actin turnover, with a 2-fold reduction in filament severing frequency. Moreover, quantitative analysis of filament shape change over time revealed that myosin XI generates the force for buckling and straightening of both single actin filaments and actin bundles. Thus, our data provide genetic evidence that three Arabidopsis class XI myosins contribute to actin remodeling by stimulating turnover and generating the force for filament shape change.


Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Miosinas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Técnicas de Silenciamento de Genes , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Mutação , Miosinas/genética , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Plântula/genética , Plântula/crescimento & desenvolvimento
19.
Mol Biol Cell ; 25(8): 1263-75, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24523291

RESUMO

A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament-filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls.


Assuntos
Proteínas de Capeamento de Actina/biossíntese , Citoesqueleto de Actina/metabolismo , Arabidopsis/metabolismo , Epiderme Vegetal/metabolismo , Raízes de Plantas/metabolismo , Proteínas de Capeamento de Actina/genética , Arabidopsis/crescimento & desenvolvimento , Hipocótilo/citologia , Processamento de Imagem Assistida por Computador , Proteínas dos Microfilamentos/metabolismo , Células Vegetais , Epiderme Vegetal/citologia , Raízes de Plantas/citologia , Processos Estocásticos
20.
Front Plant Sci ; 5: 5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24478785

RESUMO

The actin cytoskeleton plays a key role in the plant morphogenesis and is involved in polar cell growth, movement of subcellular organelles, cell division, and plant defense. Organization of actin cytoskeleton undergoes dynamic remodeling in response to internal developmental cues and diverse environmental signals. This dynamic behavior is regulated by numerous actin-binding proteins (ABPs) that integrate various signaling pathways. Production of the signaling lipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid affects the activity and subcellular distribution of several ABPs, and typically correlates with increased actin polymerization. Here we review current knowledge of the inter-regulatory dynamics between signaling phospholipids and the actin cytoskeleton in plant cells.

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