Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven,auxin-dependent patterning

Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP₂) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.     

Leaf asymmetry as a developmental constraint imposed by auxin-dependent phyllotactic patterning

In a majority of species, leaf development is thought to proceed in a bilaterally symmetric fashion without systematic asymmetries. This is despite the left and right sides of an initiating primordium occupying niches that differ in their distance from sinks and sources of auxin. Here, we revisit an existing model of auxin transport sufficient to recreate spiral phyllotactic patterns and find previously overlooked asymmetries between auxin distribution and the centers of leaf primordia. We show that it is the direction of the phyllotactic spiral that determines the side of the leaf these asymmetries fall on. We empirically confirm the presence of an asymmetric auxin response using a DR5 reporter and observe morphological asymmetries in young leaf primordia. Notably, these morphological asymmetries persist in mature leaves, and we observe left-right asymmetries in the superficially bilaterally symmetric leaves of tomato (Solanum lycopersicum) and Arabidopsis thaliana that are consistent with modeled predictions. We further demonstrate that auxin application to a single side of a leaf primordium is sufficient to recapitulate the asymmetries we observe. Our results provide a framework to study a previously overlooked developmental axis and provide insights into the developmental constraints imposed upon leaf morphology by auxin-dependent phyllotactic patterning.     

The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

Switchgrass (Panicum virgatum L.; family Poaceae) is a warm-season C4 perennial grass. Tillering plays an important role in determining the morphology of aboveground parts and the final biomass yield of switchgrass. Auxin distribution in plants can affect a variety of important growth and developmental processes, including the regulation of shoot and root branching, plant resistance and biological yield. Auxin transport and gradients in plants are mediated by influx and efflux carriers. PvPIN1, a switchgrass PIN1-like gene that is involved in regulating polar transport, is a putative auxin efflux carrier. Neighbor-joining analysis using sequences deposited in NCBI databases showed that the PvPIN1gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Tiller emergence and development was significantly promoted in plants subjected toPvPIN1 RNA interference (RNAi), which yielded a phenotype similar to that of wild-type plants treated with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid). A transgenic approach that inducedPvPIN1 gene overexpression or suppression altered tiller number and the shoot/root ratio. These data suggest that PvPIN1plays an important role in auxin-dependent adventitious root emergence and tillering.     

Actin is involved in pollen tube tropism through redefining the spatial targeting of secretory vesicles

In order to accurately target the embryo sac and deliver the sperm cells, the pollen tube has to find an efficient path through the pistil and respond to precise directional cues produced by the female tissues. Although many chemical and proteic signals have been identified to guide pollen tube growth, the mechanism by which the tube changes direction in response to these signals is poorly understood. We designed an experimental setup using a microscope-mounted galvanotropic chamber that allowed us to induce the redirection of in vitro pollen tube growth through a precisely timed and calibrated external signal. Actin destabilization, reduced calcium concentration in the growth medium and inhibition of calcium channel activity decreased the responsiveness of the pollen tube to a tropic trigger. An increased calcium concentration in the medium enhanced this response and was able to rescue the effect of actin depolymerization. Time-lapse imaging revealed that the motion pattern of vesicles and the dynamics of the subapical actin array undergo spatial reorientation prior to the onset of a tropic response. Together these results suggest that the precise targeting of the delivery of new wall material represents a key component in the growth machinery that determines directional elongation in pollen tubes.     

The foot formation stimulating peptide pedibin is also involved in patterning of the head in hydra

Avian neural crest cells migrate on precise pathways to their target areas where they form a wide variety of cellular derivatives, including neurons, glia, pigment cells and skeletal components. In one portion of their pathway, trunk neural crest cells navigate in the somitic mesoderm in a segmental fashion, invading the rostral, while avoiding the caudal, half-sclerotome. This pattern of cell migration, imposed by the somitic mesoderm, contributes to the metameric organization of the peripheral nervous system, including the sensory and sympathetic ganglia. At hindbrain levels, neural crest cells also travel from the neural tube in a segmental manner via three migratory streams of cells that lie adjacent to even-numbered rhombomeres. In this case, the adjacent mesoderm does not possess an obvious segmental organization, compared to the somitic mesoderm at trunk levels. Thus, the mechanisms by which the embryo controls segmentally-organized cell migrations have been a fascinating topic over the past several years. Here, I discuss findings from classical and recent studies that have delineated several of the tissue, cellular and molecular elements that contribute to the segmental organization of neural crest migration, primarily in the avian embryo. One common theme is that neural crest cells are prohibited from entering particular territories in the embryo due to the expression of inhibitory factors. However, permissive, migration-promoting factors may also play a key role in coordinating neural crest migration.     

Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to the cell periphery

The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular approximately 70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.     

Bep4 protein is involved in patterning along the animal-vegetal axis in the Paracentrotus lividus embryo

In sea urchin embryos, the initial animal-vegetal (AV) axis is specified during oogenesis but the mechanism is largely unknown. By using chemical reagents such as lithium, it is possible to shift the principal embryonic territories toward a vegetal fate. We have investigated the possibility of obtaining the same morphological effect as with lithium by utilizing Fabs against the maternal Bep4 protein that is localized in the animal part of Paracentrotus lividus egg and embryos. Incubation of fertilized eggs with Fabs against Bep4 protein causes exogastrulation at 48 h of development of P. lividus embryos, similar to embryos treated with lithium. This vegetalizing effect was ascertained by utilizing territorial markers such as EctoV, EndoI, and Ig8. The effect of Fabs against Bep4 on gene expression was observed by monitoring spatial expression of the hatching enzyme gene. A decreased expression domain compared to its normal spatial distribution was detected and this effect was again comparable to those obtained with lithium treatment. Association of Bep4 with a cadherin was demonstrated by immunoprecipitation and immunostaining experiments, and an involvement in cell signaling is discussed. In addition, treatment of embryos with anti-Bep4 Fabs causes an enhancement in the level and an expansion in the pattern of nuclear beta-catenin. Moreover, this treatment also provokes a decrease of beta-catenin in adherens junctions. Together, these data indicate that anti-Bep4 Fabs provoke a shift of the animal-vegetal boundary toward the animal pole and suggest an active role of Bep4 protein in patterning along the AV axis.     

Drosophila TIEG is a modulator of different signalling pathways involved in wing patterning and cell proliferation

Acquisition of a final shape and size during organ development requires a regulated program of growth and patterning controlled by a complex genetic network of signalling molecules that must be coordinated to provide positional information to each cell within the corresponding organ or tissue. The mechanism by which all these signals are coordinated to yield a final response is not well understood. Here, I have characterized the Drosophila ortholog of the human TGF-β Inducible Early Gene 1 (dTIEG). TIEG are zinc-finger proteins that belong to the Krüppel-like factor (KLF) family and were initially identified in human osteoblasts and pancreatic tumor cells for the ability to enhance TGF-β response. Using the developing wing of Drosophila as "in vivo" model, the dTIEG function has been studied in the control of cell proliferation and patterning. These results show that dTIEG can modulate Dpp signalling. Furthermore, dTIEG also regulates the activity of JAK/STAT pathway suggesting a conserved role of TIEG proteins as positive regulators of TGF-β signalling and as mediators of the crosstalk between signalling pathways acting in a same cellular context.     

Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression

Plant development displays an exceptional plasticity and adaptability that involves the dynamic, asymmetric distribution of the phytohormone auxin. Polar auxin flow, which requires polarly localized transport facilitators of the PIN family, largely contributes to the establishment and maintenance of the auxin gradients. Functionally overlapping action of PIN proteins mediates multiple developmental processes, including embryo formation, organ development and tropisms. Here we show that PIN proteins exhibit synergistic interactions, which involve cross-regulation of PIN gene expression in pin mutants or plants with inhibited auxin transport. Auxin itself positively feeds back on PIN gene expression in a tissue-specific manner through an AUX/IAA-dependent signalling pathway. This regulatory switch is indicative of a mechanism by which the loss of a specific PIN protein is compensated for by auxin-dependent ectopic expression of its homologues. The compensatory properties of the PIN-dependent transport network might enable the stabilization of auxin gradients and potentially contribute to the robustness of plant adaptive development.     

A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice

Auxin transport affects a variety of important growth and developmental processes in plants, including the regulation of shoot and root branching. The asymmetrical localization of auxin influx and efflux carriers within the plasma membrane establishes the auxin gradient and facilitates its transport. REH1, a rice EIR1 (Arabidopsis ethylene insensitive root 1)-like gene, is a putative auxin efflux carrier. Phylogenetic analysis of 32 members of the PIN family, taken from across different species, showed that in terms of evolutionary relationship, OsPIN1 is closer to the PIN1 family than to the PIN2 family. It is, therefore, renamed as OsPIN1 in this study. OsPIN1 was expressed in the vascular tissues and root primordial in a manner similar to AtPIN1. Adventitious root emergence and development were significantly inhibited in the OsPIN1 RNA interference (RNAi) transgenic plants, which was similar to the phenotype of NPA (N-1-naphthylphalamic acid, an auxin-transport inhibitor)-treated wild-type plants. alpha-naphthylacetic acid (alpha-NAA) treatment was able to rescue the mutated phenotypes occurring in the RNAi plants. Overexpression or suppression of the OsPIN1 expression through a transgenic approach resulted in changes of tiller numbers and shoot/root ratio. Taken together, these data suggest that OsPIN1 plays an important role in auxin-dependent adventitious root emergence and tillering.     

Actin microfilaments are involved in scale formation of the chrysomonad cellSynura

Summary In the first step of scale formation several small vesicles originating from the Golgi fuse to form a large flattened primary vesicle associated with the surface of one plastid. In a second step this vesicle undergoes several morphogenetic events to form the mold of ornamented scales elaborated in the lumen of the scale forming vesicle (SFV). Thick filaments composed of a stack of actin microfilaments (MFs) settle on the membrane of the SFV turned toward the plastid. They are laterally cross-linked to each other and to the SFV membrane and assembled in a horseshoe figure at the place of the future base-plate of the scale. On the center of the vesicle free of actin MF, where the periplastidial endoplasmic reticulum (PER) and the SFV membranes are glued, a protrusion occurs to form a diverticulum which is to become the future hull or the spine of the finished scale. On the external side of the SFV a microfibrillar network covers the surface of the vesicle. Microtubules (MTs) originating near the kinetosomes change their rectilinear course to follow the two longitudinal margins and the diverticulum of the SFV. MTs are not directly attached to the membrane of the SFV but rather through the microfibrillar network. A set of observations suggest that actin MFs have a structural function in maintaining the shape of the vesicle rather than a role in the migration of the SFV on the surface of the plastid. MTs probably play a role in the migration and in the morphogenesis of the SFV in conjunction with microfibrils. Also, the formation of SFVs and silica deposition are compared to that in diatoms.     

NtGNL1 is involved in embryonic cell division patterning, root elongation, and pollen tube growth in tobacco

The function of the ARF-GEF family has drawn great attention recently, especially GNOM and GNL1, owing to their important role in plant development. A homolog of GBF was identified in Nicotiana tabacum, named NtGNL1, which is ubiquitously expressed throughout the tobacco life cycle. In NtGNL1 RNAi plants, irregular orientation of cell division and asynchronous cell development during early embryogenesis disrupted the symmetry of the developing embryo. In addition, root growth in transgenic lines was significantly slower than that in wild-type plants, although the structure of the root tip was largely intact. Pollen germination and pollen tube growth were also inhibited in the transgenic lines, and the tip of the pollen tube presented various aberrant morphologies in one of the transgenic lines. The phenotypes of different NtGNL1 RNAi transgenic lines suggest that the NtGNL1 is likely to be involved not only in embryogenesis and postembryonic development, but also in sexual reproduction; thus, NtGNL1 may play multiple and critical roles in plant development.     

Actin is oxidized during myocardial ischemia

Exposure of isolated rat hearts to 30 min global ischemia followed by 60 min reperfusion resulted in a significant 80% increase (p <.05) in actin content of carbonyl groups, which was associated with significant depression (p <.05) of postischemic contractile function. This result supports the hypothesis that one mechanism of postischemic contractile dysfunction may be oxidation of contractile proteins.     

Actin is unevenly distributed in the pituitary gland

Summary In view of the suggestion that actin-like proteins might be involved in the final steps leading to hormone secretion, the actin content of pituitary glands of adult rats was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (for total actin), by the DNAse method (which measures predominantly monomeric actin) and by immunocytochemistry. The amount of actin present in the neural lobe, expressed per mg total protein, was found to be comparable to that of other neural tissues. In contrast, in the anterior lobe, the ratio was significantly lower. The intensity of immunofluorescent staining with anti-actin antibodies was higher in the neural lobe than in either anterior or intermediate lobes. The intensity and distribution of tubulin immunofluorescent staining with anti-tubulin antibodies resembled that of anti-actin antibodies. Thus, three independent methods point to an uneven distribution of actin in the subdivisions of the pituitary gland, although all these subdivisions are believed to secrete their hormones by exocytosis. These data suggest that the bulk of actin present in pituitary cells is unlikely to be involved only in exocytosis, but may be implicated also in the intracellular translocation of secretory products.This paper is dedicated to Professor K. Akert, Zurich, on the occasion of his 60th birthday     

Mechanisms of auxin-dependent cell and tissue polarity

The establishment of cellular asymmetries and their coordination within the tissue layer are fundamental to the development of multicellular organisms. In plants, the induction and coordination of cell polarity have classically been attributed to involve the hormone auxin and its flow. However, the underlying mechanisms have only recently been addressed at the molecular level. We review progress on the characterisation of the auxin influx and efflux carrier properties of specific plasma membrane proteins, mechanisms underlying their delivery to and internalisation from the plasma membrane, their endocytic transport and degradation. We discuss mechanisms of auxin gradient, transport and response action during the coordination of polarity, along with the downstream involvement of Rho-of-plant small GTPases during the execution of cell polarity.     

Novel genetic screen for genes involved in posterior body patterning in Drosophila

Much of our current understanding of how cytoplasmic determinants are localized and activated stems from genetic analyses in Drosophila. Characterization of mutants defective in establishing the initial embryonic body pattern has revealed the importance of mRNA localization and translational control and identified several factors required for these processes. Nevertheless, many additional genes are likely to be involved. Here, we describe a novel genetic screen designed to identify genes that participate in posterior body patterning, an elaborate process involving the sequential use of two localized cytoplasmic determinants, the products of the oskar and nanos genes. From the screen, we recovered new alleles of genes known to be required for posterior body patterning, demonstrating the validity of the approach In addition, we isolated numerous other mutants. Further characterization of one mutant, P58, revealed that it is a novel allele of bullwinkle. We find that in bullwinkle mutants, oskar mRNA localization is not maintained in the embryo and oskar protein accumulates ectopically and to abnormally high levels. These defects are distinct from previously described perturbations in oskar activity and provide new insights into the regulation of oskar. © 1996 Wiley-Liss, Inc.