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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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The actin cytoskeleton is one of the targets of the pH gradient in tip-growing cells, but how cytosolic pH regulates the actin cytoskeleton remains largely unknown. We here demonstrate that Arabidopsis ADF7 and ADF10 function optimally at different pH levels when disassembling actin filaments. This differential pH sensitivity allows ADF7 and ADF10 to respond to the cytosolic pH gradient to regulate actin dynamics in pollen tubes. ADF7 is an unusual actin-depolymerizing factor with a low optimum pH in in vitro actin depolymerization assays. ADF7 plays a dominant role in promoting actin turnover at the pollen tube apex. ADF10 has a typically high optimum pH in in vitro assays and plays a dominant role in regulating the turnover and organization of subapical actin filaments. Thus, functional specification and cooperation of ADF isovariants with different pH sensitivities enable the coordination of the actin cytoskeleton with the cytosolic pH gradient to support pollen tube growth.
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Factores Despolimerizantes de la Actina , Proteínas de Arabidopsis , Arabidopsis , Tubo Polínico , Actinas , Arabidopsis/genética , Diferenciación Celular , Proliferación Celular , Tubo Polínico/genética , Fuerza Protón-Motriz , Factores Despolimerizantes de la Actina/genética , Proteínas de Arabidopsis/genéticaRESUMEN
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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[This corrects the article DOI: 10.1371/journal.pbio.3000782.].
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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/fisiología , Citoesqueleto de Actina , Actinas , CitoplasmaRESUMEN
The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.
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Actinas , Dictyostelium , Proteínas Protozoarias , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Transducción de Señal , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
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.
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Actinas , Arabidopsis , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Destrina/metabolismo , Fosforilación , Tubo Polínico/metabolismoRESUMEN
The actin cytoskeleton plays an essential role in the regulation of polarized pollen tube growth, and its functions are dictated by its spatial organization and dynamics. Here we describe an assay to monitor the dynamics of actin filaments decorated with Lifeact-mEGFP in Arabidopsis pollen tubes using spinning disk confocal microscopy and measuring the parameters associated with their dynamics. The method allows us to assess the dynamics of actin filaments in growing Arabidopsis pollen tubes.
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Proteínas de Arabidopsis , Arabidopsis , Tubo Polínico , Actinas , Citoesqueleto de ActinaRESUMEN
Biomolecular condensation driven by liquid-liquid phase separation (LLPS) is key to assembly of membraneless organelles in numerous crucial pathways. It is largely unknown how cellular structures or components spatiotemporally regulate LLPS and condensate formation. Here we reveal that cytoskeletal dynamics can control the condensation of p62 bodies comprising the autophagic adaptor p62/SQSTM1 and poly-ubiquitinated cargos. Branched actin networks are associated with p62 bodies and are required for their condensation. Myosin 1D, a branched actin-associated motor protein, drives coalescence of small nanoscale p62 bodies into large micron-scale condensates along the branched actin network. Impairment of actin cytoskeletal networks compromises the condensation of p62 bodies and retards substrate degradation by autophagy in both cellular models and Myosin 1D knockout mice. Coupling of LLPS scaffold to cytoskeleton systems may represent a general mechanism by which cells exert spatiotemporal control over phase condensation processes.
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Actinas , Autofagia , Actinas/metabolismo , Animales , Autofagia/fisiología , Ratones , Miosinas/metabolismo , Proteína Sequestosoma-1/metabolismo , Ubiquitina/metabolismoRESUMEN
Plasmodesmata (PD) play an important role in plant growth and development and defense. The permeability of PD is strictly regulated. Here, we describe an assay for measuring the permeability of PD in Arabidopsis thaliana leaves, which relies on tracing intercellular movement of green fluorescent protein (GFP) upon transient expression of the protein-encoding plasmid delivered by particle bombardment. The method allows to assess GFP movement at single-cell resolution.
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Arabidopsis , Plasmodesmos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Permeabilidad , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plasmodesmos/metabolismo , Nicotiana/metabolismoRESUMEN
Asymmetric positioning of the mitotic spindle contributes to the generation of two daughter cells with distinct sizes and fates. Here, we investigated an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage. In this division, beginning with an asymmetrically positioned spindle, the daughter-cell size differences continuously increased during cytokinesis, and the smaller daughter cell in the posterior eventually underwent apoptosis. We found that Arp2/3-dependent F-actin assembled in the anterior but not posterior cortex during division, suggesting that asymmetric expansion forces generated by actin polymerization may enlarge the anterior daughter cell. Consistent with this, inhibition of cortical actin polymerization or artificially equalizing actin assembly led to symmetric cell division. Furthermore, disruption of the Wnt gradient or its downstream components impaired asymmetric cortical actin assembly and caused symmetric division. Our results show that Wnt signaling establishes daughter cell asymmetry by polarizing cortical actin polymerization in a dividing cell.
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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 , Polen/metabolismo , Tubo Polínico/metabolismoRESUMEN
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/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de Microfilamentos/genética , Fosfatos de Fosfatidilinositol/metabolismo , Estomas de Plantas/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Microfilamentos/metabolismoRESUMEN
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 , Polaridad Celular/fisiología , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , División Celular Asimétrica/fisiología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Movimiento Celular/fisiología , Proteínas de Microfilamentos/fisiología , Células-Madre Neurales/metabolismoRESUMEN
Cell-to-cell communication in plants is mediated by plasmodesmata (PD) whose permeability is tightly regulated during plant growth and development. The actin cytoskeleton has been implicated in regulating the permeability of PD, but the underlying mechanism remains largely unknown. Recent characterization of PD-localized formin proteins has shed light on the role and mechanism of action of actin in regulating PD-mediated intercellular trafficking. In this mini-review article, we will describe the progress in this area.
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In flowering plants, sexual reproduction involves a double fertilization event, which is facilitated by the delivery of two non-motile sperm cells to the ovule by the pollen tube. Pollen tube growth occurs exclusively at the tip and is extremely rapid. It strictly depends on an intact actin cytoskeleton, and is therefore an excellent model for uncovering the molecular mechanisms underlying dynamic actin cytoskeleton remodeling. There has been a long-term debate about the organization and dynamics of actin filaments within the apical and subapical regions of pollen tube tips. By combining state-of-the-art live-cell imaging with the usage of mutants which lack different actin-binding proteins, our understanding of the origin, spatial organization, dynamics and regulation of actin filaments within the pollen tube tip has greatly improved. In this review article, we will summarize the progress made in this area.
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PSGL-1 has recently been identified as an HIV restriction factor that inhibits HIV DNA synthesis and more potently, virion infectivity. But the underlying mechanisms of these inhibitions are unknown. Here we show that PSGL-1 directly binds to cellular actin filaments (F-actin) to restrict actin dynamics, which leads to inhibition of HIV DNA synthesis. PSGL-1 is incorporated into nascent virions and restricts actin dynamics in the virions, which partially accounts for the inhibition of virion infectivity. More potently, PSGL-1 inhibits incorporation of Env proteins into nascent virions, causing a loss of envelope spikes on the virions as shown by Cryo-electron microscopy and super-resolution imaging. This loss is associated with a profound defect in viral entry. Mechanistically, PSGL-1 binds gp41 and sequesters gp41 at the plasma membrane, explaining the inhibition of Env incorporation in nascent virions. PSGL-1's dual anti-HIV mechanisms represent novel strategies of human cells to defend against HIV infection.
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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 Homeodominio/metabolismo , Raíces de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Presión Osmótica , Raíces de Plantas/genéticaRESUMEN
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.