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.     

Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.     

烟草丝束蛋白ABD2与微丝的体内体外相互作用

微丝骨架在细胞的生命活动中具有重要的功能,而其动态的解聚聚合特性是其实现功能的前提. 丝束蛋白（fimbrin/plastin）做为微丝结合蛋白质,是微丝骨架的重要调控因子之一,含有2个肌动蛋白结合结构域,目前对其结合微丝的机制并不清楚. 本文以烟草丝束蛋白的肌动蛋白结合结构域2(NtFAbd2) 为研究对象,通过原核细胞表达纯化NtFAbd2,利用体外沉淀法分析发现,NtFAbd2能够与微丝结合;利用激光共聚焦扫描显微镜分析发现,在烟草BY-2悬浮细胞内,NtAbd2-GFP与微丝共分布,这些结果为深入分析植物丝束蛋白的作用机制提供了新的数据.     

The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans

Filamentous fungi are ideal systems to study the process of polarized growth, as their life cycle is dominated by hyphal growth exclusively at the cell apex. The actin cytoskeleton plays an important role in this growth. Until now, there have been no tools to visualize actin or the actin-binding protein fimbrin in live cells of a filamentous fungus. We investigated the roles of actin (ActA) and fimbrin (FimA) in hyphal growth in Aspergillus nidulans . We examined the localization of ActA::GFP and FimA::GFP in live cells, and each displayed a similar localization pattern. In actively growing hyphae, cortical ActA::GFP and FimA::GFP patches were highly mobile throughout the hypha and were concentrated near hyphal apices. A patch-depleted zone occupied the apical 0.5 μm of growing hypha. Both FimA::GFP and Act::GFP also localize transiently to septa. Movement and later localization of both was compromised after cytochalasin treatment. Disruption of fimA resulted in delayed polarity establishment during conidium germination, abnormal hyphal growth and endocytosis defects in apolar cells. Endocytosis was severely impaired in apolar fimA disruption cells. Our data support a novel apical recycling model which indicates a critical role for actin patch-mediated endocytosis to maintain polarized growth at the apex.     

Lifeact: a versatile marker to visualize F-actin

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.     

Golgi body motility in the plant cell cortex correlates with actin cytoskeleton organization

The actin cytoskeleton is involved in the transport and positioning of Golgi bodies, but the actin-based processes that determine the positioning and motility behavior of Golgi bodies are not well understood. In this work, we have studied the relationship between Golgi body motility behavior and actin organization in intercalary growing root epidermal cells during different developmental stages. We show that in these cells two distinct actin configurations are present, depending on the developmental stage. In small cells of the early root elongation zone, fine filamentous actin (F-actin) occupies the whole cell, including the cortex. In larger cells in the late elongation zone that have almost completed cell elongation, actin filament bundles are interspersed with areas containing this fine F-actin and areas without F-actin. Golgi bodies in areas with the fine F-actin exhibit a non-directional, wiggling type of motility. Golgi bodies in areas containing actin filament bundles move up to 7 μm s?1. Since the motility of Golgi bodies changes when they enter an area with a different actin configuration, we conclude that the type of movement depends on the actin organization and not on the individual organelle. Our results show that the positioning of Golgi bodies depends on the local actin organization.     

AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament-crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin-binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament-crosslinking protein. AtFim1 binds pollen actin filaments (F-actin) in a calcium-independent manner, with an average dissociation constant (Kd) of 0.55+/-0.21 microM and with a stoichiometry at saturation of 1:4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F-actin by a calcium-independent mechanism, in contrast to crosslinking of plant actin by human T-plastin, a known calcium-sensitive actin-crosslinking protein. When micro-injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2-4 min. Using the 'nuclear displacement assay' as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)-induced depolymerization, in a dose-dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin-mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium-independent, actin filament-crosslinking protein that interacts with the actin cytoskeleton in living plant cells.     

Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton

Studies using drugs that cause the disassembly of filamentous actin (F-actin) have demonstrated the importance of an intact actin cytoskeleton for polarised secretion by yeast cells [1,2]. To address the level of dynamic turnover needed for such processes, however, drugs or mutants that confer stabilising properties on F-actin are needed. Jasplakinolide is the only readily available drug that stabilises F-actin structures both in vivo and in vitro [3-6]. Yeast strains have been generated in which two of the ABC multidrug resistance transporter genes have been deleted, rendering normally jasplakinolide-resistant yeast cells sensitive to its effects. Treatment of these cells with jasplakinolide caused rapid and dramatic effects on the actin cytoskeleton, resulting in the accumulation of single large actin structures in cells. These structures, however, still contained components that are normally associated with cortical actin patches. A dynamic actin cytoskeleton was found to be critical for the generation of cell polarity and endocytosis.     

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.     

Assembly of the brush border cytoskeleton: changes in the distribution of microvillar core proteins during enterocyte differentiation in adult chicken intestine

The assembly of the intestinal microvillus cytoskeleton was examined during the differentiation of enterocytes along the crypt-villus axis in adult chicken duodenum using light and electron microscopic immunolocalization techniques. Using antibodies reactive with villin, fimbrin, and the heavy chain (hc) of brush border (BB) myosin I (110K-calmodulin complex) and rhodamine-conjugated phalloidin as a probe for F-actin, we determined that while actin, villin, and fimbrin were all localized apically along the entire axis, BB myosin I (hc) did not assume this localization until the crypt-villus transition zone. In addition to their localization at the BB surface, all four proteins were present at significant levels along the lateral margins of enterocytes along the entire crypt-villus axis, suggesting that these proteins may be involved in the organization and function of the basolateral membrane cytoskeleton as well. The pattern of expression of the microvillar core proteins along the crypt-villus axis in the adult was comparable to that seen in the intestine of the late stage chicken embryo and suggests that a common program for brush border assembly may be used in both modes of enterocyte differentiation.     

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.     

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.     

Fluorescently-labeled fimbrin decorates a dynamic actin filament network in live plant cells

Recently it has been established, through a detailed biochemical analysis, that recombinant Arabidopsis thaliana fimbrin 1 (AtFim1) is a member of the fimbrin/plastin family of actin filament bundling or cross-linking proteins [D.R. Kovar et al. (2000) Plant J 24:625-636]. To determine whether AtFim1 can function as an F-actin-binding protein in the complex environment of the plant cell cytoplasm, we created a fluorescent protein analog and introduced it by microinjection into live Tradescantia virginiana L. stamen hair cells. AtFim1 derivatized with Oregon Green 488 had biochemical properties similar to unlabeled fimbrin, including the Kd value for binding to plant F-actin and the ability to cross-link filaments into higher-order structures. Fluorescent-fimbrin decorated an array of fine actin filaments in the cortical cytoplasm of stamen hair cells, which were shown with time-course studies to be highly dynamic. These data establish AtFim1 as a bona fide member of the fimbrin/plastin family, and represent the first use of a plant actin-binding protein as a powerful cytological tool for tracking the spatial and temporal redistribution of actin filaments in individual cells.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

多胺生物合成抑制剂D-精氨酸对拟南芥幼苗根系生长的影晌

为探讨多胺生物合成抑制剂D-精氨酸（D-arginine,D-Arg）对拟南芥根系生长的影响,首先用腐胺（0．1mmol‘L-1）和D—Arg（1．0mmol·L-1）处理种子萌发后生长2d的拟南芥幼苗。腐胺（0．1mmol·L-1）显著促进主根伸长,D-Arg（1．0mmol-L-1）显著抑制主根伸长,并对主根根尖的细胞形态有明显影响。为了进一步了解D—Arg影响拟南芥主根生长的机理,采用浓度梯度D．Arg处理幼苗根系。实验结果表明,随着D-Arg浓度增加（0．2～1．0mmol·L-1）,拟南芥幼苗主根生长受抑制的程度越严重。微分干涉观察主根根尖发现,外源施加D—Arg,引起拟南芥主根根尖分生区的细胞数目减少,使拟南芥幼苗表现出主根的伸长生长变缓。当分生区数目较少时,出现主根几乎不再仲长的现象。由此推测,多胺生物合成抑制剂D-Arg对拟南芥幼苗根生长的抑制作用机制,是D-Arg影响了其根尖分生区的细胞分裂活动,使分生区细胞数目减少,从而引起分生区长度减小,最终导致拟南芥主根仲长生长受到抑制。     

Localization of brush border cytoskeletal proteins in gastric oxynticopeptic cells from the bullfrog Rana catesbeiana

The contribution of brush border cytoskeletal proteins (actin, villin, fimbrin, and brush border myosin-1) to organization of the cytoskeletal network underlying apical plications of oxynticopeptic cells was examined by immunohistochemical techniques in frozen sections of gastric mucosa from the bullfrog, Rana catesbeiana. Apical localization of F-actin with phalloidin in oxynticopeptic cells inhibited with cimetidine revealed small, punctate domains within the apical cytoplasm that were consistent with the presence of short microvilli revealed by electron microscopy. Localization of F-actin in cells stimulated with forskolin was limited to a wide continuous band of cytoplasm corresponding to the location of numerous long surface folds. Inhibition of protein synthesis with cycloheximide did not prevent acid secretion or formation of actin filaments within surface folds in stimulated oxynticopeptic cells, suggesting that the formation of filaments does not require actin synthesis. Staining of gastric mucosae with fluorescent DNase-1 demonstrated that oxynticopeptic cells possess an unusually large pool of non-filamentous actin. Taken together, these results suggest that actin-filament formation in stimulated cells occurs by polymerization of an existing pool of non-filamentous actin. Localization of antibodies specific for villin and fimbrin revealed that these proteins were present within intestinal absorptive cells and gastric surface and neck cells but were not present within inhibited or stimulated oxynticopeptic cells. Brush border myosin-1, present in intestinal absorptive cells, was not present in gastric epithelium. Thus, we propose that actin-containing projections in oxynticopeptic cells are not organized like intestinal microvilli and that filament formation occurs after stimulation by modulating intracellular pools of filamentous and non-filamentous actin.     

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.     

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.