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Blebs are cell protrusions generated by local membrane–cortex detachments followed by expansion of the plasma membrane. Blebs are formed by some migrating cells, e.g. primordial germ cells of the zebrafish. While blebs occur randomly at each part of the membrane in unpolarized cells, a polarization process guarantees the occurrence of blebs at a preferential site and thereby facilitates migration toward a specified direction. Little is known about the factors involved in the controlled and directed bleb generation, yet recent studies revealed the influence of an intracellular flow and the stabilizing role of the membrane–cortex linker molecule Ezrin. Based on this information, we develop and analyse a coupled bulk-surface model describing a potential cellular mechanism by which a bleb could be induced at a controlled site. The model rests upon intracellular Darcy flow and a diffusion–advection–reaction system, describing the temporal evolution from a homogeneous to a strongly anisotropic Ezrin distribution. We prove the well-posedness of the mathematical model and show that simulations qualitatively correspond to experimental observations, suggesting that indeed the interaction of an intracellular flow with membrane proteins can be the cause of the Ezrin redistribution accompanying bleb formation.

Chemokine axes have been shown to mediate site-specific metastasis in breast cancer, but their relevance to different subtypes has been hardly addressed. Here, with the focus on the CCR7-CCL21 axis, patient datasets demonstrated that luminal-A tumors express relatively low CCR7 levels compared with more aggressive disease subtypes. Furthermore, lymph node metastasis was not associated with high CCR7 levels in luminal-A patients. The metastatic pattern of luminal-A breast tumors may be influenced by the way luminal-A tumor cells interpret signals provided by factors of the primary tumor microenvironment. Thus, CCR7-expressing human luminal-A cells were stimulated simultaneously by factors representing 3 tumor microenvironment arms typical of luminal-A tumors, hormonal, inflammatory, and growth stimulating: estrogen + TNF-α + epidermal growth factor. Such tumor microenvironment stimulation down-regulated the migration of CCR7-expressing tumor cells toward CCL21 and inhibited the formation of directional protrusions toward CCL21 in a novel 3-dimensional hydrogel system. CCL21-induced migration of CCR7-expressing tumor cells depended on PI3K and MAPK activation; however, when CCR7-expressing cancer cells were prestimulated by tumor microenvironment factors, CCL21 could not effectively activate these signaling pathways. In vivo, pre-exposure of the tumor cells to tumor microenvironment factors has put restraints on CCL21-mediated lymph node-homing cues and shifted the metastatic pattern of CCR7-expressing cells to the aggressive phenotype of dissemination to bones. Several of the aspects were also studied in the CXCR4-CXCL12 system, demonstrating similar patient and in vitro findings. Thus, we provide novel evidence to subtype-specific regulation of the CCR7-CCL21 axis, with more general implications to chemokine-dependent patterns of metastatic spread, revealing differential regulation in the luminal-A subtype.

Collective epithelial cell migration requires the maintenance of cell–cell junctions while enabling the generation of actin-rich protrusions at the leading edge of migrating cells. Ventral enclosure of Caenorhabditis elegans embryos depends on the collective migration of anterior-positioned leading hypodermal cells towards the ventral midline where they form new junctions with their contralateral neighbours. In this study, we characterized the zygotic function of RGA-7/SPV-1, a CDC-42/Cdc42 and RHO-1/RhoA-specific Rho GTPase-activating protein, which controls the formation of actin-rich protrusions at the leading edge of leading hypodermal cells and the formation of new junctions between contralateral cells. We show that RGA-7 controls these processes in an antagonistic manner with the CDC-42′s effector WSP-1/N-WASP and the CDC-42-binding proteins TOCA-1/2/TOCA1. RGA-7 is recruited to spatially distinct locations at junctions between adjacent leading cells, where it promotes the accumulation of clusters of activated CDC-42. It also inhibits the spreading of these clusters towards the leading edge of the junctions and regulates their accumulation and distribution at new junctions formed between contralateral leading cells. Our study suggests that RGA-7 controls collective migration and junction formation between epithelial cells by spatially restricting active CDC-42 within cell–cell junctions.

Collective motion occurs in many biological processes, such as wound healing, tumor invasion and embryogenesis. Experiments of cell monolayer migration have revealed the spontaneous formation of finger-like instabilities, with leader cells at their tips. We present a particle-based model for collective cell migration, based on several elements that have been found experimentally to influence cellular movement. Inside the bulk we include velocity alignment interactions between neighboring cells. At the border contour of the layer we introduce the following additional forces: surface-elasticity restoring force, curvature-dependent positive feedback, and contractile acto-myosin cables. We find that the curvature-driven instability at the layer edge is necessary and sufficient for the formation of cellular fingers, which are in good agreement with experimental observations.

Phosphate signaling and acquisition are critical for the bacterial response to phosphate limitation, and bacteria express multiple factors to scavenge phosphate. We previously found that multidrug-resistant strains of Pseudomonas aeruginosa from critically ill patients can form unusual outer-surface appendages harboring PstS proteins. Here, we have expanded our investigation to DING proteins that like PstS belong to the family of high-affinity phosphate-binding proteins but have strong similarity with eukaryotic DING proteins. We demonstrate the localization of DING on PstS-containing outer-surface appendages in both multidrug-resistant strain MDR25 and the PA14 strain of P. aeruginosa. However, the number of cells producing appendages and the amount of appendages on each cell in PA14 were found to be negligible, unless overexpression of either PstS or DING was achieved by transformation with constructed plasmids. We further noticed that DING expression under low phosphate conditions was significantly higher in MDR25 compared to PA14 which may explain the greater abundance of appendages in MDR25. Our finding that DING proteins are localized on extracellular appendages provides an opportunity to study the interaction of bacterial DING with host proteins by mimicking the action of host DINGs.

We previously reported that insulin-like growth factor-II mRNA-binding protein-3 (IMP-3) depletion (IMP-3(Δ)) was shown to inhibit invadopodia formation and extracellular matrix degradation capacity in oral squamous cell carcinoma (OSCC) cells. In this study, we found that IMP-3(Δ) cells significantly downregulated the podoplanin (PDPN) level, which resulted in a loss of extracellular matrix degradation activity, although invadopodia was still thriving. From RNA in situ hybridization using a digoxigenin-labeled 3'UTR recognition probe of PDPN and reporter assay with 3'UTR of the PDPN gene cloned downstream from the luciferase reporter gene, we revealed that IMP-3 depletion was shown to be downregulated, which most probably lowered PDPN gene expression by reducing mRNA stabilization. In a xenograft model, PDPN depletion was the cause of a decrease in tumor volume and regional infiltration into nearby stroma. Taken together, transforming growth factor beta 1 increased PDPN expression, which potentiated cancer invasion through increased invadopodia formation and extracellular matrix degradation in the low invasive OSCC cell line. Reciprocally, interleukin-1 beta secreted by OSCC cells, stimulated transforming growth factor beta 1 secretion from stromal fibroblasts to induce PDPN expression in OSCC cells. In addition, a retrospective investigation of OSCC patients found that IMP-3 and PDPN expression significantly correlated with lymph node metastasis of OSCC patients. Moreover, co-expression of IMP-3 and PDPN were frequently detected both in primary and lymph nodes metastatic OSCC cells using immunohistochemical dual staining. Thus, the IMP-3-PDPN axis may be a sensitive target molecule in anti-invadopodia therapy for the treatment of metastatic cancers.

The plasma membrane, the outermost surface of eukaryotic cells, contains various substructures, such as protrusions or invaginations, which are associated with diverse functions, including endocytosis and cell migration. These structures of the plasma membrane can be considered as tubules or inverted tubules (protrusions) of the membrane. There are six modes of membrane curvature at the plasma membrane, which are classified by the positive or negative curvature and the location of the curvature (tip, neck or shaft of the tubules). The BAR domain superfamily proteins have structurally determined positive and negative curvatures of membrane contact at their BAR, F-BAR and I-BAR domains, which generate and maintain such curved membranes by binding to the membrane. Importantly, the SH3 domains of the BAR domain superfamily proteins bind to the actin regulatory WASP/WAVE proteins, and the BAR/F-BAR/I-BAR domain-SH3 unit could orient the actin filaments towards the membrane for each subcellular structure. These membrane tubulations are also considered to function in membrane fusion and fission.

Pre-eclampsia is often associated with inadequate cytotrophoblast invasion and remodelling of the uterine spiral arteries. Examining a first trimester, 2D in vitro explant culture model which mimics in vivo placentation, including trophoblast column formation and extravillous cytotrophoblast (EVT) migration, we previously suggested that excessive maternal decidual natural killer cell interferon (IFN-gamma) limits EVT migration. Types-1 and -2 insulin-like growth factor (IGF-1, IGF-2) are trophic for EVT, act through their surface receptors, IGFR-1 and IGFR-2, and are regulated by the IGF-binding proteins (IGFBPs). Could the observed IFN-gamma-mediated inhibition of EVT outgrowth and migration be related to either expression changes of IGF-1 or IGF-2, their receptors, their binding proteins, or apoptosis? Using the 2D explant culture model, we examined the effect of IFN-gamma exposure on IGF-1 and -2, IGFR-1 and -2, IGFBPs and apoptosis. IFN-gamma relatively increased IGF-1 and -2 secretion. In EVT, IFN-gamma decreased IGFR-2, but not IGFR-1 expression. IGBP-2, -3 and -4 production were not influenced by IFN-gamma. IFN-gamma induced trophoblast apoptosis measured by the highly sensitive M30 neo-epitope, but not caspase 3 activity, in conditioned medium and EVT cell lysates. The observed IFN-gamma-mediated EVT migration inhibition may occur through the down-regulation of IGFR-2 and subtle induction of EVT apoptosis.

To investigate the hypothesis that caloric restriction alters mitochondrial function in situ, intact hepatocytes were isolated from fully fed and calorie-restricted (55% of control food intake, 4 months duration) male Brown-Norway rats at 6 months of age, and various parameters were determined. Overall, the production of reactive oxygen species was not affected by caloric restriction, neither were the mitochondrial membrane potential, oxygen consumption driving proton leak, or oxygen consumption driving ATP turnover. It is concluded that while isolated mitochondria from liver tissue of calorie-restricted animals display a reduction in the generation of reactive oxygen species, it was not possible to confirm this effect in isolated hepatocytes. Further work is required to establish what effect, if any, caloric restriction has on the rate of generation of reactive oxygen species in intact cells and tissues and importantly at the whole-animal level.

The pale aleurone color1 (pac1) locus, required for anthocyanin pigment in the aleurone and scutellum of the Zea mays (maize) seed, was cloned using Mutator transposon tagging. pac1 encodes a WD40 repeat protein closely related to anthocyanin regulatory proteins ANTHOCYANIN11 (AN11) (Petunia hybrida [petunia]) and TRANSPARENT TESTA GLABRA1 (TTG1) (Arabidopsis thaliana). Introduction of a 35S-Pac1 transgene into A. thaliana complemented multiple ttg1 mutant phenotypes, including ones nonexistent in Z. mays. Hybridization of Z. mays genomic BAC clones with the pac1 sequence identified an additional related gene, mp1. PAC1 and MP1 deduced protein sequences were used as queries to build a phylogenetic tree of homologous WD40 repeat proteins, revealing an ancestral gene duplication leading to two clades in plants, the PAC1 clade and the MP1 clade. Subsequent duplications within each clade have led to additional WD40 repeat proteins in particular species, with all mutants defective in anthocyanin expression contained in the PAC1 clade. Substantial differences in pac1, an11, and ttg1 mutant phenotypes suggest the evolutionary divergence of regulatory mechanisms for several traits that cannot be ascribed solely to divergence of the dicot and monocot protein sequences.

• Background and Aims There is strong support for the monophyly of the orchid subtribe Maxillariinae s.l., yet generic boundaries within it are unsatisfactory and need re‐evaluation. In an effort to assemble sets of morphological characters to distinguish major clades within this subtribe, the pollinarium morphology and floral rewards of representative Brazilian species of this subtribe were studied. • Methods The study was based on fresh material from 60 species and seven genera obtained from cultivated specimens. Variation of pollinarium structure and floral rewards was assessed using a stereomicroscope and by SEM analysis. • Key Results Four morphological types of pollinaria are described. Type 1 appears to be the most widespread and is characterized by a well‐developed tegula. Type 2 lacks a stipe and the pollinia are attached directly to the viscidium. Type 3 also lacks a stipe, and the viscidium is rigid and dark. In Type 4, the stipe consists of the whole median rostelar portion and, so far, is known only from Maxillaria uncata. Nectar, trichomes, wax‐like and resin‐like secretions are described as flower rewards for different groups of species within the genus Maxillaria. Data on the biomechanics and pollination biology are also discussed and illustrated. In Maxillariinae flowers with arcuate viscidia, the pollinaria are deposited on the scuttellum of their Hymenopteran pollinators. In contrast, some flowers with rounded to rectangular, pad‐like viscidia fix their pollinaria on the face of their pollinators. • Conclusions Pollinarium morphology and floral features related to pollination in Brazilian Maxillariinae are more diverse than previously suggested. It is hoped that the data presented herein, together with other data sources such as vegetative traits and molecular tools, will be helpful in redefining and diagnosing clades within the subtribe Maxillariinae.

• Background and aims Much recent study of plant trichomes has focused on various aspects of glandular secreting trichomes (GSTs) and differentiation of simple trichomes. This Botanical Briefing will highlight: ‐ research on various aspects of, and manipulation of glandular secreting trichomes; ‐ molecular aspects of the differentiation and development of simple trichomes of arabidopsis and cotton; ‐ how methods for manipulation of model systems used in the above work can be applied to expand our understanding of less studied surface structures of plants. • Scope The Briefing will cover: ‐ established and suggested roles of simple and glandular secreting trichomes; ‐ recent results regarding solute and ion movement in trichomes; ‐ methods for isolating trichomes; ‐ recent studies of trichome differentiation and development; ‐ attempts to modify metabolism in secreting trichomes; ‐ efforts to exploit trichomes for commercial and agronomic purposes.

Wiskott-Aldrich syndrome protein (WASP) is the product of the gene deficient in boys with X-linked Wiskott-Aldrich syndrome. We assessed the role of WASP in signaling through the high-affinity IgE receptor (FcepsilonRI) using WASP-deficient mice. IgE-dependent degranulation and cytokine secretion were markedly diminished in bone marrow-derived mast cells from WASP-deficient mice. Upstream signaling events that include FcepsilonRI-triggered total protein tyrosine phosphorylation, and protein tyrosine phosphorylation of FcepsilonRIbeta and Syk were not affected by WASP deficiency. However, tyrosine phosphorylation of phospholipase Cgamma and Ca(2+) mobilization were diminished. IgE-dependent activation of c-Jun N-terminal kinase, cell spreading and redistribution of cellular F-actin in mast cells were reduced in the absence of WASP. We conclude that WASP regulates FcepsilonRI-mediated granule exocytosis, cytokine production and cytoskeletal changes in mast cells.

Using live cell imaging, we demonstrate that immature dendritic cells (DC) derived from human peripheral blood monocytes undergo pronounced morphologic changes in vitro within minutes of exposure to unopsonized Escherichia coli, developing extensive membrane veils that efficiently capture additional bacteria. Internalization does not occur in the veils, but instead, bacteria are transported to the central region of the cell, where they sin directly into the plasma membrane. In contrast, exposure to polystyrene beads does not induce notable changes in cell morphology, and DC do not efficiently capture beads when introduced alone or mixed with bacteria. Long dendritic processes were also visualized in some cells that allowed capture of clumps of bacteria at a distance of more than 100 μm. These results demonstrate that immature DC can distinguish between inert particles and bacteria and alter their shape and phagocytic capacity in response to the latter.

Leaf trichomes protect plants from attack by insect herbivores and are often induced following damage. Hormonal regulation of this plant induction response has not been previously studied. In a series of experiments, we addressed the effects of artificial damage, jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Artificial damage and jasmonic acid caused significant increases in trichome production of leaves. The jar1-1 mutant exhibited normal trichome induction following treatment with jasmonic acid, suggesting that adenylation of jasmonic acid is not necessary. Salicylic acid had a negative effect on trichome production and consistently reduced the effect of jasmonic acid, suggesting negative cross-talk between the jasmonate and salicylate-dependent defense pathways. Interestingly, the effect of salicylic acid persisted in the nim1-1 mutant, suggesting that the Npr1/Nim1 gene is not downstream of salicylic acid in the negative regulation of trichome production. Last, we found that gibberellin and jasmonic acid had a synergistic effect on the induction of trichomes, suggesting important interactions between these two compounds.

Motivation: There is no consensus in the literature about how the deepest portions of protein structures are packed. Using an improved Voronoi procedure, we calculate reference packing densities for different regions in the protein interior. Furthermore, we want to clarify where cavities are located. Results: Sets of reference packing densities are provided for regions in proteins that differ in their distance to the surface and to internal cavities, supplementing previous data. Packing in the protein interior is tight but generally inhomogeneous. There are about 4.4 cavities per 100 amino acids in protein structures, they occur in all regions, most frequently in a depth of 2.5–3.6 Å underneath the Connolly surface. However, the deepest protein regions have a lower mean packing density than circumjacent regions, because more contacts to cavities occur in the core. Availability/Supplementary information: Calculation software and detailed packing data are available on request.

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.

The acquisition of a cell polarity is a crucial requirement for migration, activation, and apoptosis of leukocytes. The polarization of leukocytes involves the formation of two distinct poles: the leading edge—the attachment cell site to the substrate allowing directional movements of the cell—and on the opposite side, the uropod—mostly involved in cell-to-cell interaction and in a variety of leukocyte activities including activation and apoptosis. However, the uropod takes shape in neutrophils, monocytes, and natural killer cells, and the formation of this cell protrusion seems to exert an important role in immune interactions. In fact, the polarization sites of leukocytes are involved in a complex cross-talk between cells and extracellular matrix components, and a number of receptors and counter-receptors crowd in the contact sites to allow efficient cell-to-cell or cell–substrate interaction. The membrane/cytoskeleton interaction plays a crucial role in tuning these activities and in “predisposing” leukocytes to their function through the acquisition of a polarized phenotype. This review is focused on the mechanisms underlying the formation of the leukocyte uropod, the role of cytoskeleton in defining its structure and function, and the involvement of the uropod in the complex interplay between immune cells.

Polarized membrane trafficking during plant cytokinesis and cell expansion are critical for plant morphogenesis, yet very little is known about the molecular mechanisms that guide this process. Dynamin and dynamin-related proteins are large GTP binding proteins that are involved in membrane trafficking. Here, we show that two functionally redundant members of the Arabidopsis dynamin-related protein family, ADL1A and ADL1E, are essential for polar cell expansion and cell plate biogenesis. adl1A-2 adl1E-1 double mutants show defects in cell plate assembly, cell wall formation, and plasma membrane recycling. Using a functional green fluorescent protein fusion protein, we show that the distribution of ADL1A is dynamic and that the protein is localized asymmetrically to the plasma membrane of newly formed and mature root cells. We propose that ADL1-mediated membrane recycling is essential for plasma membrane formation and maintenance in plants.

SQUAMOSA PROMOTER BINDING PROTEIN–box genes (SBP-box genes) encode plant-specific proteins that share a highly conserved DNA binding domain, the SBP domain. Although likely to represent transcription factors, little is known about their role in development. In Arabidopsis, SBP-box genes constitute a structurally heterogeneous family of 16 members known as SPL genes. For one of these genes, SPL8, we isolated three independent transposon-tagged mutants, all of which exhibited a strong reduction in fertility. Microscopic analysis revealed that this reduced fertility is attributable primarily to abnormally developed microsporangia, which exhibit premeiotic abortion of the sporocytes. In addition to its role in microsporogenesis, the SPL8 knockout also seems to affect megasporogenesis, trichome formation on sepals, and stamen filament elongation. The SPL8 mutants described help to uncover the roles of SBP-box genes in plant development.

A positive correlation between cell size and DNA content has been recognized in many plant cell types. Conversely, misexpression of a dominant-negative cyclin-dependent kinase (CDK) or CDK inhibitor proteins (ICK/KRPs) in Arabidopsis and tobacco leaves has revealed that cell growth can be uncoupled from cell cycle progression and DNA content. However, cell growth also appears to be controlled in a non-cell-autonomous manner by organ size, making it difficult in a ubiquitous expression assay to judge the cell-autonomous function of putative cell growth regulators. Here, we investigated the function of the CDK inhibitor ICK1/KRP1 on cell growth and differentiation independent of any compensatory influence of an organ context using Arabidopsis trichomes as a model system. By analyzing cell size with respect to DNA content, we dissected cell growth in a DNA-dependent and a DNA-independent process. We further found that ICK1/KRP1 misexpression interfered with differentiation and induced cell death, linking cell cycle progression, differentiation, and cell death in plants. The function of ICK1/KRP1 in planta was found to be dependent on a C-terminal domain and regulated negatively by an N-terminal domain. Finally, we identified CDKA;1 and a D-type cyclin as possible targets of ICK1/KRP1 expression in vivo.

The chemical forms of zinc (Zn) in the Zn-tolerant and hyperaccumulator Arabidopsis halleri and in the non-tolerant and nonaccumulator Arabidopsis lyrata subsp. petraea were determined at the molecular level by combining chemical analyses, extended x-ray absorption spectroscopy (EXAFS), synchrotron-based x-ray microfluorescence, and μEXAFS. Plants were grown in hydroponics with various Zn concentrations, and A. halleri specimens growing naturally in a contaminated site were also collected. Zn speciation in A. halleri was independent of the origin of the plants (contaminated or non-contaminated) and Zn exposure. In aerial parts, Zn was predominantly octahedrally coordinated and complexed to malate. A secondary organic species was identified in the bases of the trichomes, which contained elevated Zn concentrations, and in which Zn was tetrahedrally coordinated and complexed to carboxyl and/or hydroxyl functional groups. This species was detected thanks to the good resolution and sensitivity of synchrotron-based x-ray microfluorescence and μEXAFS. In the roots of A. halleri grown in hydroponics, Zn phosphate was the only species detected, and is believed to result from chemical precipitation on the root surface. In the roots of A. halleri grown on the contaminated soil, Zn was distributed in Zn malate, Zn citrate, and Zn phosphate. Zn phosphate was present in both the roots and aerial part of A. lyrata subsp. petraea. This study illustrates the complementarity of bulk and spatially resolved techniques, allowing the identification of: (a) the predominant chemical forms of the metal, and (b) the minor forms present in particular cells, both types of information being essential for a better understanding of the bioaccumulation processes.

In Atropa belladonna hairy roots, exogeneously added salicylic acid (SA) is converted to methyl salicylate (MSA) through the reaction, which might be catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). Here we cloned a cDNA for A. belladonna SAMT (AbSAMT1), which consisted of 357 aa residues. It was expressed in E. coli, and the recombinant AbSAMT1 showed SAMT activity. When A. belladonna hairy roots were exposed to a high concentration of SA, AbSAMT1 mRNA begins to be expressed 12 h after the exposure, and steady expression continued over 144 h.

Plants of Ocimum basilicum L. grown under glass were exposed to short treatments with supplementary UV‐B. The effect of UV‐B on volatile essential oil content was analysed and compared with morphological effects on the peltate and capitate glandular trichomes. In the absence of UV‐B, both peltate and capitate glands were incompletely developed in both mature and developing leaves, the oil sacs being wrinkled and only partially filled. UV‐B was found to have two main effects on the glandular trichomes. During the first 4 d of treatment, both peltate and capitate glands filled and their morphology reflected their ‘normal’ mature development as reported in the literature. During the following days there was a large increase in the number of broken oil sacs among the peltate glands as the mature glands broke open, releasing volatiles. Neither the number of glands nor the qualitative or quantitative composition of the volatiles was affected by UV‐B. There seems to be a requirement for UV‐B for the filling of the glandular trichomes of basil.

Trichomes are specialized epidermal cells that produce secretions that are thought to provide a first line of defence against pests and pathogens. Many trichome-secreted compounds are used commercially as flavourings, medicines, etc. Described here is the cloning and characterization of the promoter of a tobacco trichome-specific P450 gene, CYP71D16. This promoter is shown to direct the specific expression of the reporter gene, beta-glucuronidase (GUS), in glandular trichomes of Nicotiana tabacum cv. T.I. 1068 at all developmental stages. With the full promoter, GUS activity was predominantly in the gland cell, with less in the stalk cell adjacent to the gland, and in lower stalk cells. GUS staining was also observed in the most distal trichome stalk cells of non-glandular trichomes found on variety T.I. 1112. Promoter deletion analysis revealed that the region from -223 to +111 bp is sufficient to direct trichome-specific expression, but not strong gland expression. Examination of the literature suggests that this is the first characterized trichome-specific-promoter shown to function at all stages of plant development. This promoter may provide efficient bioengineering to enhance pest and pathogen resistance, and for molecular farming based on the trichome gland system.

The control of the stoichiometric balance of alpha- and beta-tubulin is important during microtubule biogenesis. This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast. Here, we show that the TFC A gene is important in vivo in plants. The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene. Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A. kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched. Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants. The kis phenotype was rescued by overexpression of an alpha-tubulin, indicating that KIS is involved in the control of the correct balance of alpha- and beta-tubulin monomers.

Some species of Spirogyra anchor themselves to the substrate by differentiating rhizoids. A rhizoid is differentiated only from the terminal cell, suggesting that this cell can recognize its terminal position in a filament. In the present study, we have analyzed the mechanism for position sensing by the terminal cell. When a filament is cut, a new cell occupies the terminal position, and three phenomena are induced: (1) the cell wall of the cut cell detaches from the new terminal cell; (2) adhesive material is secreted by the terminal cell; and (3) the terminal cell begins to differentiate a rhizoid via tip growth. All of these phenomena were inhibited by adding sorbitol to the external medium, suggesting that turgor pressure is involved in position sensing by the terminal cell. The inhibition by sorbitol was reversible. Upon cutting a filament, the distal end of a new terminal cell became convex. However, when a filament was cut in the presence of sorbitol, the distal end of a new terminal cell became less convex. Either treatment with Gd(3+) or decrease in extracellular Ca(2+) resulted in inhibition of all these phenomena, suggesting possible involvement of stretch-activated ion channel in position sensing by terminal cells.

The structure of the essential oil secretory tissues of Prostanthera ovalifolia R.Br was investigated using bright- and dark-field optical microscopy, and scanning and transmission electron microscopy. The leaves of P. ovalifolia have glandular trichomes of the peltate type common to many Lamiaceae species. The trichomes consist of a basal cell embedded in the epidermis, a stalk cell with heavily cutinized walls and a 16-celled secretory head, but they differ from those of many previously reported Lamiaceae species in their morphological form defined by the elevated cuticle. The sub-cuticular space contains a mixture of lipid and aqueous phases. Secretory cells have dense cytoplasm with many leucoplasts present. Volatile terpenoids are eliminated from the cytoplasm into the sub-cuticular space, the site of essential oil accumulation, via granulocrine secretion.

The ability of neutrophils to sense and move to sites of infection is essential for our defense against pathogens. For motility, lamellipodium extension and stabilization are prerequisites, but how cells form such membrane protrusions is still obscure. Using contrast-enhanced video microscopy and Transwell® assays, we show that water-selective aquaporin channels regulate lamellipodium formation and neutrophil motility. Addition of anti-aquaporin-9 antibodies, HgCl2, or tetraethyl ammonium inhibited the function(s) of the channels and blocked motility-related shape changes. On human neutrophils, aquaporin-9 preferentially localized to the cell edges, where N-formyl peptide receptors also accumulated, as assessed with fluorescence microscopy. To directly visualize water fluxes at cell edges, cells were loaded with high dilution-sensitive, self-quenching concentrations of fluorophore. In these cells, motile regions always displayed increased fluorescence compared with perinuclear regions. Our observations provide the first experimental support for motility models where water fluxes play a pivotal role in cell-volume increases accompanying membrane extensions.

The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca²⁺ imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca²⁺ indicator. These AR and intracellular Ca²⁺ ([Ca²⁺]i) recordings revealed that the presence of [Ca²⁺]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca²⁺]i peak and ~40% of reacting spermatozoa also manifest a slow [Ca²⁺]i rise ~2 min before AR. Our findings uncover new AR features related to [Ca²⁺]i.

The rice plant (Oryza sativa L. cv Oochikara) is known to be a Si accumulator, but the mechanism responsible for the high uptake of Si by the roots is not well understood. We investigated the role of root hairs and lateral roots in the Si uptake using two mutants of rice, one defective in the formation of root hairs (RH2) and another in that of lateral roots (RM109). Uptake experiments with nutrient solution during both a short term (up to 12 h) and relatively long term (26 d) showed that there was no significant difference in Si uptake between RH2 and the wild type (WT), whereas the Si uptake of RM109 was much less than that of WT. The number of silica bodies formed on the third leaf in RH2 was similar to that in WT, but the number of silica bodies in RM109 was only 40% of that in WT, when grown in soil amended with Si under flooded conditions. There was also no difference in the shoot Si concentration between WT and RH2 when grown in soil under upland conditions. Using a multi-compartment transport box, the Si uptake at the root tip (0-1 cm, without lateral roots and root hairs) was found to be similar in WT, RH2, and RM109. However, the Si uptake in the mature zone (1-4 cm from root tip) was significantly lower in RM109 than in WT, whereas no difference was found in Si uptake between WT and RH2. All these results clearly indicate that lateral roots contribute to the Si uptake in rice plant, whereas root hairs do not. Analysis of F(2) populations between RM109 and WT showed that Si uptake was correlated with the presence of lateral roots and that the gene controlling formation of lateral roots and Si uptake is a dominant gene.