Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.     

Cytokinin-induced root growth involves actin filament reorganization

Root architecture is developmentally plastic and affected by many intrinsic factors (e.g. plant hormones) and extrinsic factors (e.g. touch, gravity) in order to maximize nutrient and water acquisition. We have recently shown that asymmetrical exposure of cytokinin (CK) at the root tip causes root growth directional changes that is dependent on ethylene signaling and is potentiated by glucose signaling. Auxin homeostasis as maintained by auxin signaling and transport is also involved in CK-induced root cell elongation and differential growth. The signaling pathways eventually converge at actin filament organization since actin filament organization inhibitor latrunculin B (Lat B) can also induce similar growth. We, show that CK can actually alter actin filament organization as seen in actin binding protein 35S::GFP-ABD2-GFP transgenic lines as is also altered by auxin polar transport inhibitor 1-N-naphthylphthalamic acid (NPA) and Lat B in different manners.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings

In vivo visualization of filamentous actin in all cells of Arabidopsis thaliana seedlings is essential for understanding the numerous roles of the actin cytoskeleton in diverse processes of cell differentiation. A previously introduced reporter construct based on the actin-binding domain of mouse talin proved to be useful for unravelling some of these aspects in cell layers close to the organ surface. However, cells more deeply embedded, especially stelar cells active in polar transport of auxin, show either diffuse or no fluorescence at all due to the lack of expression of the fusion protein. The same problem is encountered in the root meristem. Recently introduced actin reporters based on fusions between A. thaliana fimbrin 1 and GFP gave brilliant results in organs from the root differentiation zone upwards to the leaves, however failed to depict the filamentous actin cytoskeleton in the transition zone of the root, in the apical meristem and the root cap. To overcome these problems, we have prepared new transgenic lines for the visualization of F-actin in vivo. We report here that a construct consisting of GFP fused to the C-terminal half of A. thaliana fimbrin 1 reveals dynamic arrays of F-actin in all cells of stably transformed A. thaliana seedlings.     

Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.     

甘蓝型油菜线粒体功能未知基因 ORF 117的载体构建与转化

本研究克隆了甘蓝型油菜线粒体功能未知基因 ORF 117,构建了带有线粒体转运信号肽序列（TP ）的植物过表达载体35S ∷TP-ORF117、35S ∷TP-EGFP 和35S ∷TP-ORF117-EGFP ,转化野生型拟南芥并进行抗除草剂筛选,共获得纯合的转基因拟南芥株系62个。qPCR 表明,ORF 117在转基因材料中得到高效过表达。用转35S ∷TP-EGFP 、35S ∷TP-ORF117-EGFP 拟南芥制备原生质体,经线粒体特异染料染色,在激光共聚焦显微镜下做线粒体、GFP 共定位检测,GFP 蛋白和 ORF117-EGFP 融合蛋白被准确定位到了线粒体。     

Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton.     

The actin-interacting protein AIP1 is essential for actin organization and plant development

Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions. In particular, actin interacting protein 1 (AIP1) is capable of capping F-actin and enhancing the activity of the small actin modulating protein, actin depolymerising factor (ADF) in vitro. Here, we analyze the effect of the inducible expression of AIP1 RNAi in Arabidopsis plants to assess AIP1s role in vivo. In intercalary growing cells, the normal actin organization is disrupted, and thick bundles of actin appear in the cytoplasm. Moreover, in root hairs, there is the unusual appearance of actin cables ramifying the root hair tip. We suggest that the reduction in AIP1 results in a decrease in F-actin turnover and the promotion of actin bundling. This distortion of the actin cytoskeleton causes severe plant developmental abnormalities. After induction of the Arabidopis RNAi lines, the cells in the leaves, roots, and shoots fail to expand normally, and in the severest phenotypes, the plants die. Our data suggest that AIP1 is essential for the normal functioning of the actin cytoskeleton in plant development.     

A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells

The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.     

Actin depolymerizing factor is essential for viability in plants, and its phosphoregulation is important for tip growth

Actin depolymerizing factor (ADF)/cofilin is important for regulating actin dynamics, and in plants is thought to be required for tip growth. However, the degree to which ADF is necessary has been elusive because of the presence of multiple ADF isoforms in many plant species. In the moss Physcomitrella patens , ADF is encoded by a single, intronless gene. We used RNA interference to demonstrate that ADF is essential for plant viability. Loss of ADF dramatically alters the organization of the F-actin cytoskeleton, and leads to an inhibition of tip growth. We show that ADF is subject to phosphorylation in vivo , and using complementation studies we show that mutations of the predicted phosphorylation site partially rescue plant viability, but with differential affects on tip growth. Specifically, the unphosphorylatable ADF S6A mutant generates small polarized plants with normal F-actin organization, whereas the phosphomimetic S6D mutant generates small, unpolarized plants with a disorganized F-actin cyotskeleton. These data indicate that phosphoregulation at serine 6 is required for full ADF function in vivo , and, in particular, that the interaction between ADF and actin is important for tip growth.     

Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.     

Actin cytoskeleton in Arabidopsis thaliana under blue and red light

BACKGROUND INFORMATION: Actin cytoskeleton is the basis of chloroplast-orientation movements. These movements are activated by blue light in the leaves of terrestrial angiosperms. Red light has been shown to affect the spatial reorganization of F-actin in water plants, where chloroplast movements are closely connected with cytoplasmic streaming. The aim of the present study was to determine whether blue light, which triggers characteristic responses of chloroplasts, i.e. avoidance and accumulation, also influences F-actin organization in the mesophyll cells of Arabidopsis thaliana. Actin filaments in fixed mesophyll tissue were labelled with Alexa Fluor 488-conjugated phalloidin. The configuration of actin filaments, expressed as a form factor (4 pi x area/perimeter(2)), was determined for all actin formations which were measured in fluorescence confocal images. RESULTS: In the present study, we compare form-factor distributions and the median form factors for strong and weak, blue- and red-irradiated tissues. Spatial organization of the F-actin network did not undergo any changes which could be attributed specifically to blue light. Actin patterns were similar in blue-irradiated wild-type plants and phot2 (phototropin 2) mutants which lack the avoidance response of chloroplasts. However, significant differences in the shape and distribution of F-actin formations were observed between mesophyll cells of phot2 mutants irradiated with strong and weak red light. These differences were absent in wild-type leaves. CONCLUSIONS: Actin does not appear to be the main target for the blue-light chloroplast-orientation signal. The modes of actin involvement in chloroplast translocations are different in water and terrestrial angiosperms. The results suggest that co-operation occurs between blue- and red-light photoreceptors in the control of the actin cytoskeleton architecture in Arabidopsis.     

Actin organization and root hair development are disrupted by ethanol-induced overexpression of Arabidopsis actin interacting protein 1 (AIP1)

* Actin organization and dynamics are essential for cell division, growth and cytoplasmic streaming. Here we analyse the effects of the overexpression of Actin Interacting Protein 1 (AIP1) on Arabidopsis development. * Arabidopsis plants were transformed with an ethanol-inducible AIP1 construct and the characteristics of these plants were analysed after induction. * When AIP1 was increased to approx. 90% above wild-type values, root hair development and actin organization in all cell types examined were disrupted. * Our data demonstrate that AIP1 is a key regulator of actin organization and that its regulation is essential for normal plant cell morphogenesis.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

Arabidopsis SMALL AUXIN UP RNA63 promotes hypocotyl and stamen filament elongation

Auxin regulates plant growth and development in part by activating gene expression. Arabidopsis thaliana SMALL AUXIN UP RNAs (SAURs) are a family of early auxin-responsive genes with unknown functionality. Here, we show that transgenic plant lines expressing artificial microRNA constructs (aMIR-SAUR-A or -B) that target a SAUR subfamily (SAUR61-SAUR68 and SAUR75) had slightly reduced hypocotyl and stamen filament elongation. In contrast, transgenic plants expressing SAUR63:GFP or SAUR63:GUS fusions had long hypocotyls, petals and stamen filaments, suggesting that these protein fusions caused a gain of function. SAUR63:GFP and SAUR63:GUS seedlings also accumulated a higher level of basipetally transported auxin in the hypocotyl than did wild-type seedlings, and had wavy hypocotyls and twisted inflorescence stems. Mutations in auxin efflux carriers could partially suppress some SAUR63:GUS phenotypes. In contrast, SAUR63:HA plants had wild-type elongation and auxin transport. SAUR63:GFP protein had a longer half-life than SAUR63:HA. Fluorescence imaging and microsomal fractionation studies revealed that SAUR63:GFP was localized mainly in the plasma membrane, whereas SAUR63:HA was present in both soluble and membrane fractions. Low light conditions increased SAUR63:HA protein turnover rate. These results indicate that membrane-associated Arabidopsis SAUR63 promotes auxin-stimulated organ elongation.     

Two domains of the epidermal growth factor receptor are involved in cytoskeletal interactions

Epidermal growth factor receptor can interact directly with F-actin through an actin-binding domain. In the present study, a mutant EGFR, lacking a previously identified actin-binding domain (ABD 1), was still able to bind elements of the cytoskeleton. A second EGFR actin-binding domain (ABD 2) was identified in the region of the receptor that includes Tyr-1148 by a yeast two-hybrid assay. GST fusion proteins comprising ABD 1 or ABD 2 bound actin in vitro and competed for actin-binding with the full-length EGFR. EGFR binding to actin was also studied in intact cells using fluorescence resonance energy transfer (FRET). The localization of the EGFR/actin-binding complex changed after EGF stimulation. Fusion proteins containing mutations in ABD1 or ABD2 did not display a FRET signal. The results lead to the conclusion that the interaction between ABD1 and ABD2 and actin during EGF-induced signal transduction, and thus between EGFR and actin, are important in cell activation.     

‘Fukusensor:’ a genetically engineered plant for reporting DNA damage in response to gamma radiation

Transgenic plants can be designed to be ‘phytosensors’ for detection of environmental contaminants and pathogens. In this study, we describe the design and testing of a radiation phytosensor in the form of green fluorescence protein (GFP)‐transgenic Arabidopsis plant utilizing a DNA repair deficiency mutant background as a host. Mutant lines of Arabidopsis AtATM (At3g48190), which are hypersensitive to gamma irradiation, were used to generate stable GFP transgenic plants in which a gfp gene was under the control of a strong constitutive CaMV 35S promoter. Mutant and nonmutant genetic background transgenic plants were treated with 0, 1, 5, 10 and 100 Gy radiation doses, respectively, using a Co‐60 source. After 1 week, the GFP expression levels were drastically reduced in young leaves of mutant background plants (treated by 10 and 100 Gy), whereas there were scant visible differences in the fluorescence of the nonmutant background plants. These early results indicate that transgenic plants could serve in a relevant sensor system to report radiation dose and the biological effects to organisms in response to radionuclide contamination.