Plant Cytoskeleton Research Articles

KinG Is a Plant-Specific Kinesin That Regulates Both Intra- and Intercellular Movement of SHORT-ROOT.

Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR.

Putative gravisensors among microtubule associated proteins.

Despite of long period of investigation (over 100 years), still a lot of questions remain unclear about molecular mechanisms of plant graviperception. This requires designing new experiments and new approaches to be applied in gravitational biology. Investigation of plant cell reactions under clinorotation (plant disorientation in respect to gravity vector) is of significant importance to such type of research. Clinorotation is known to cause changes of cell polarity and exert mechanical stress in plant cells. Microtubular cytoskeleton is highly dynamic structure and it responds to both of these stresses. Due to turgor pressure and cell elongation, endogenous mechanical forces influence microtubule orientation in order to coordinate cell growth. Rearrangements of microtubules are regulated by numerous associated proteins which functional activity is not fully clear. In this review, we discuss how MT associated proteins regulate cortical MT arrays under mechanical stress and consider how these proteins may act as plant cell gravisensors. Investigation of microtubule associated proteins under clinorotation might shed the light on molecular mechanism of plant cytoskeleton arrangement and its involvement in initial reactions of cell graviperception.

Cytoskeleton during aerenchyma formation in plants

Aerenchyma is a plant tissue characterized by prominent intercellular spaces facilitating gas diffusion between roots and the aerial environment. The classical formation of intercellular spaces is thought to be the result of schizogeny and lysogeny during development of wetland species and in some dry-land species in response to different abiotic stress, including drought, high temperature, and nutrient deficiency. The plant cytoskeleton is known to play a major role in cellular organization and signaling pathways. It is emphasized a lot of ambiguity as to the cytoskeleton function in the constitutive and induced aerenchyma development, especially at the earliest stages of this process. In the present review, we focus on some aspects of the cytoskeleton behavior during the formation of schizogenous and lysigenous aerenchyma in wetland and terrestrial plants growing both in the nature and experimental conditions.

Bioinformatic search for Ca2+- and calmodulin-dependent protein kinases potentially associated with the regulation of plant cytoskeleton

The paper presents the results of a bioinformatic search for Ca2+- and calmodulin-dependent protein kinases from Arabidopsis thaliana, which may potentially participate in cytoskeleton regulation. Homologues were chosen based on their similarity with the calmodulin-dependent protein kinases from Homo sapiens and Mus musculus, which modulate the structure and dynamic behavior of the cytoskeleton. In total, the sequences for the catalytic domains of 41 animal protein kinases and their known 42 plant homologues have been aligned. The closest animal and plant homologues have been determined using the methods of phylogenetic clusterization. According to the bioinformatic research results, the following plant protein kinases were selected as the most probable regulators of the plant cytoskeleton: CPK7, CPK14, CPK32, CPK17, CPK34, CPK20, CPK27, CPK16, CPK18, CPK28, CPK7, CRK2, CRK4, and CRK6.

Journey from the Center of the Cell - the intra- and intercellular transport of mRNA.

Transport and localized translation of mRNA is crucial for the proper spatiotemporal organization of proteins within cells. Distribution of RNAs to subcellular domains has recently emerged as a major mechanism for establishing functionally distinct compartments and structures in the cells. There is an emerging evidence that active transport of mRNA involves cytoskeleton and membrane trafficking pathways in fungi, plants and animals, suggesting that it is a common phenomenon among eukaryotes. The important highlights are that the RNA-binding proteins recognize the cargo mRNA and that RNPs are actively transported on the cytoskeletal tracks or co-transported with membranous compartments, such as the endoplasmic reticulum and endosomes. The interest of scientists has expanded over the past years in response to the discoveries that RNA can be exported from cells to play a role in the intercellular communication. In this review, we will focus on characterization of the RNA transport both, within a cell and between cells, and on the currently proposed mechanisms for RNA targeting.

The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection.

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.

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis.

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of ahlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.

Involvement of plant cytoskeleton in cellular mechanisms of metal toxicity

Literature data and results of the studies carried out us concerning the involvement of plant cell cytoskeleton in cellular mechanisms of metal toxicity are summarized. Characteristics of cytotoxic effect of metals on plant cytoskeleton and, in particular, on microtubules and actin filaments are reviewed. Particular attention is paid to cellular and molecular mechanisms of metal impact on cytoskeleton. The most probable binding sites of heavy metals, as well as alternative mechanisms of their impact on cytoskeleton, are discussed.

Microtubule Polymerization Functions in Hypersensitive Response and Accumulation of H2O2 in Wheat Induced by the Stripe Rust.

The plant cytoskeleton, including microtubules and microfilaments, is one of the important factors in determining the polarity of cell division and growth, as well as the interaction of plants with invading pathogens. In defense responses of wheat against the stripe rust (Puccinia striiformis f. sp. tritici) infection, hypersensitive response is the most crucial event to prevent the spread of pathogens. In order to reveal the effect of microtubules on the hypersensitive cell death and H2O2 accumulation in the interaction of wheat (Triticum aestivum) cv. Suwon 11 with an incompatible race, CYR23, wheat leaves were treated with microtubule inhibitor, oryzalin, before inoculation. The results showed that the frequency of infection sites with hypersensitive response occurrence was significantly reduced, and hypersensitive cell death in wheat leaves was suppressed compared to the control. In addition, the frequency and the incidence of infected cells with H2O2 accumulation were also reduced after the treatment with oryzalin. Those results indicated that microtubules are related to hypersensitive response and H2O2 accumulation in wheat induced by the stripe rust, and depolymerization of microtubules reduces the resistance of plants to pathogen infection in incompatible interaction, suggesting that microtubules play a potential role in the expression of resistance of wheat against the stripe rust fungus.

Formins: linking cytoskeleton and endomembranes in plant cells.

The cytoskeleton plays a central part in spatial organization of the plant cytoplasm, including the endomebrane system. However, the mechanisms involved are so far only partially understood. Formins (FH2 proteins), a family of evolutionarily conserved proteins sharing the FH2 domain whose dimer can nucleate actin, mediate the co-ordination between actin and microtubule cytoskeletons in multiple eukaryotic lineages including plants. Moreover, some plant formins contain transmembrane domains and participate in anchoring cytoskeletal structures to the plasmalemma, and possibly to other membranes. Direct or indirect membrane association is well documented even for some fungal and metazoan formins lacking membrane insertion motifs, and FH2 proteins have been shown to associate with endomembranes and modulate their dynamics in both fungi and metazoans. Here we summarize the available evidence suggesting that formins participate in membrane trafficking and endomembrane, especially ER, organization also in plants. We propose that, despite some methodological pitfalls inherent to in vivo studies based on (over)expression of truncated and/or tagged proteins, formins are beginning to emerge as candidates for the so far somewhat elusive link between the plant cytoskeleton and the endomembrane system.

Identification of Plant Homologues of Dual Specificity Yak1-Related Kinases

Currently, Dual Specificity YAK1-Related Kinases (MNB/DYRK) were found in slime molds, protista, fungi, and animals, but the existence of plant homologues is still unclear. In the present study, we have identified 14 potential plant homologues with the previously unknown functions, based on the strong sequence similarity. The results of bioinformatics analysis revealed their correspondence to DYRK1A, DYRK1B, DYRK3, and DYRK4. For two plant homologues of animal DYRK1A from Physcomitrella patens and Arabidopsis thaliana spatial structures of catalytic domains were predicted, as well as their complexes with ADP and selective inhibitor d15. Comparative analysis of 3D-structures of the human DYRK1A and plant homologues, their complexes with the specific inhibitors, and results of molecular dynamics confirm their structural and functional similarity with high probability. Preliminary data indicate the presence of potential MNB/DYRK specific phosphorylation sites in such proteins associated with plant cytoskeleton as plant microtubule-associated proteins WVD2 and WDL1, and FH5 and SCAR2 involved in the organization and polarity of the actin cytoskeleton and some kinesin-like microtubule motor proteins.

Quantitative analyses of the plant cytoskeleton reveal underlying organizational principles.

The actin and microtubule (MT) cytoskeletons are vital structures for cell growth and development across all species. While individual molecular mechanisms underpinning actin and MT dynamics have been intensively studied, principles that govern the cytoskeleton organization remain largely unexplored. Here, we captured biologically relevant characteristics of the plant cytoskeleton through a network-driven imaging-based approach allowing us to quantitatively assess dynamic features of the cytoskeleton. By introducing suitable null models, we demonstrate that the plant cytoskeletal networks exhibit properties required for efficient transport, namely, short average path lengths and high robustness. We further show that these advantageous features are maintained during temporal cytoskeletal rearrangements. Interestingly, man-made transportation networks exhibit similar properties, suggesting general laws of network organization supporting diverse transport processes. The proposed network-driven analysis can be readily used to identify organizational principles of cytoskeletons in other organisms.

The Plant Cytoskeleton, NET3C, and VAP27 Mediate the Link between the Plasma Membrane and Endoplasmic Reticulum

The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody.

BackgroundCertain members of the Camelidae family produce a special type of antibody with only one heavy chain. The antigen binding domains are the smallest functional fragments of these heavy-chain only antibodies and as a consequence have been termed nanobodies. Discovery of these nanobodies has allowed the development of a number of therapeutic proteins and tools.In this study a class of nanobodies fused to fluorescent proteins (chromobodies), and therefore allowing antigen-binding and visualisation by fluorescence, have been used. Such chromobodies can be expressed in living cells and used as genetically encoded immunocytochemical markers.ResultsHere a modified version of the commercially available Actin-Chromobody® as a novel tool for visualising actin dynamics in tobacco leaf cells was tested. The actin-chromobody binds to actin in a specific manner. Treatment with latrunculin B, a drug which disrupts the actin cytoskeleton through inhibition of polymerisation results in loss of fluorescence after less than 30 min but this can be rapidly restored by washing out latrunculin B and thereby allowing the actin filaments to repolymerise.To test the effect of the actin-chromobody on actin dynamics and compare it to one of the conventional labelling probes, Lifeact, the effect of both probes on Golgi movement was studied as the motility of Golgi bodies is largely dependent on the actin cytoskeleton. With the actin-chromobody expressed in cells, Golgi body movement was slowed down but the manner of movement rather than speed was affected less than with Lifeact.ConclusionsThe actin-chromobody technique presented in this study provides a novel option for in vivo labelling of the actin cytoskeleton in comparison to conventionally used probes that are based on actin binding proteins.The actin-chromobody is particularly beneficial to study actin dynamics in plant cells as it does label actin without impairing dynamic movement and polymerisation of the actin filaments.

The plant cytoskeleton controls regulatory volume increase

The ability to adjust cell volume is required for the adaptation to osmotic stress. Plant protoplasts can swell within seconds in response to hypoosmotic shock suggesting that membrane material is released from internal stores. Since the stability of plant membranes depends on submembraneous actin, we asked, whether this regulatory volume control depends on the cytoskeleton. As system we used two cell lines from grapevine which differ in their osmotic tolerance and observed that the cytoskeleton responded differently in these two cell lines. To quantify the ability for regulatory volume control, we used hydraulic conductivity (Lp) as readout and demonstrated a role of the cytoskeleton in protoplast swelling. Chelation of calcium, inhibition of calcium channels, or manipulation of membrane fluidity, did not significantly alter Lp, whereas direct manipulation of the cytoskeleton via specific chemical reagents, or indirectly, through the bacterial elicitor Harpin or activation of phospholipase D, was effective. By optochemical engineering of actin using a caged form of the phytohormone auxin we can break the symmetry of actin organisation resulting in a localised deformation of cell shape indicative of a locally increased Lp. We interpret our findings in terms of a model, where the submembraneous cytoskeleton controls the release of intracellular membrane stores during regulatory volume change.

Effects of phytohormones on the cytoskeleton of the plant cell

This review highlights the effects of “classic” phytohormones (auxins, cytokinins, gibberellins, abscisic acid, ethylene, and brassinosteroids) and also of important signaling molecules, such as jasmonic acid, strigolactones, and nitric oxide, on the main components of the plant cytoskeleton, microtubules and microfilaments. The effects of these growth regulators on orientation and organization of microtubules and actin filaments, realization of cytoskeleton-dependent processes, expression of tubulin and actin genes, and interaction of various phytohormones in their influence on the cytoskeleton are discussed.

ISOLATION, CLONING AND CHARACTERIZATION OF ACTIN-ENCODING cDNAs FROM Jatropha curcas L. IP-2P

Actin is a major component of the plant cytoskeleton, so all cells contain this protein. Actin is expressed constitutively and is involved in basic housekeeping functions required for cell maintenance. Because of this, it has been frequently used as an internal control to normalize changes in gene expressions analysis. Actually, the information of nucleotide sequence of actin gene of Jatropha curcas L. population IP-2P from Indonesia is not available yet. The objective of this research was to isolate, clone and characterize cDNA of actin genes of J. curcas IP-2P. Three partial actin gene sequences had been successfully isolated by PCR using total cDNA as template, and actin primer designed from conserved region of Arabidopsis thaliana. Nucleotide sequence analysis showed that the length of JcACT fragment is 610, 534, and 701 bp encoding 203, 177, and 234 amino acids respectively. Local alignment analysis based on mRNA sequences shows that JcACT fragment shares 98% similarity with actin mRNA of Hevea brasiliensis and 99% with actin mRNA of Ricinus communis. Based on deduced amino acid sequence, JcACT is 100% identical to actins from Prunus salicina, Gossypium hirsutum, and Betula luminifera. Even though these clones of cDNA are not completed yet, they can be used as reference in J. curcas L. gene expression analysis.

Computer simulation and mathematical models of the noncentrosomal plant cortical microtubule cytoskeleton

There is rising interest in modeling the noncentrosomal cortical microtubule cytoskeleton of plant cells, particularly its organization into ordered arrays and the mechanisms that facilitate this organization. In this review, we discuss quantitative models of this highly complex and dynamic structure both at a cellular and molecular level. We report differences in methodologies and assumptions of different models as well as their controversial results. Our review provides insights for future studies to resolve these controversies, in addition to underlining the common results between various models. We also highlight the need to compare the results from simulation and mathematical models with quantitative data from biological experiments in order to test the validity of the models and to further improve them. It is our hope that this review will serve to provide guidelines for how to combine quantitative and experimental techniques to develop higher-level models of the plant cytoskeleton in the future.

A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

Analysis of Differential Proteins Induced by Forchlorfenuron in Wheat

To identify differential proteins induced by N-(2-chloro-4-pyridy)-N′-phenylurea or forchlorfenuron (CPPU), two-dimensional electrophoresis was used to study the variations in protein expression of wheat seedlings induced with CPPU. The expressed protein spots were identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and protein data bank query. About 800 spots were reproducibly detected in three replicates of each sample. A total of 10 differential proteins were identified, including tRNA delta (2)-isopentenylpyrophosphate transferase (S1), probable peptide ABC transporter ATP-binding protein y4tS (S2), ribosomal RNA large subunit methyltransferase N (S3), microtubule-associated protein 6 (S4), actin-66 (Fragment) (S5), mitochondrial ATP synthase subunit alpha (S6), S-adenosylmethionine synthase 1 (S7), flavin-containing monooxygenase YUCCA4 (S8), GTPase obg (S9), and ribosomal RNA small subunit methyltransferase G (S10). These proteins were closely related to phytohormone synthesis, plant stress response, energy metabolism, protein synthesis, cytoskeleton, and transport. All of these processes may play important roles in wheat seedling development. These findings could provide valuable information for the further study of the functions and underlying mechanism of the action of CPPU. Thus, proteomic analysis revealed that CPPU treatment induces variations in many proteins involved in plant growth and development.