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At least 451 records · Page 25Linked to original sources
PubMed2006

Protein secretion in the Archaea: multiple paths towards a unique cell surface.

Archaea are similar to other prokaryotes in most aspects of cell structure but are unique with respect to the lipid composition of the cytoplasmic membrane and the structure of the cell surface. Membranes of archaea are composed of glycerol-ether lipids instead of glycerol-ester lipids and are based on isoprenoid side chains, whereas the cell walls are formed by surface-layer proteins. The unique cell surface of archaea requires distinct solutions to the problem of how proteins cross this barrier to be either secreted into the medium or assembled as appendages at the cell surface.

Archaea↗
PubMed2006

Interaction of non-adherent suspended neutrophils to complement opsonized pathogens: a new assay using optical traps.

Phagocytosis of opsonized pathogens by circulating non-adherent neutrophils is an essential step in host defense, which when overwhelmed contributes to sepsis. To investigate the role played by ligation of complement receptors CR3 and CR4 in non-adherent neutrophils, we designed a novel assay system utilizing dual optical traps, respectively, holding a suspended unactivated cell and presenting a specific ligand-coated bead to the cell surface. We chose anti-CD18 as an example ligand, mimicking the bacterial opsonizing complement fragment iC3b. Presentation of anti-CD18-coated beads elicited both pseudopodial protrusion and subsequent phagocytosis. This is in sharp contrast to previously reported responses of adherent neutrophils, which phagocytize opsonized particles without pseudopod formation. We used this same new assay to probe actomyosin pathways in the neutrophil's pseudopodial and phagocytic response. Disruption of actin or inhibition of myosin light-chain kinase dose-dependently reduced pseudopod formation and phagocytosis rates. In summary, i) the new dual trap assay can be used to study the responses of suspended neutrophils to a variety of ligands, and ii) in a first application of this technique, we found that local ligation of CR3/4 in unactivated neutrophils in suspension induces pseudopod formation and phagocytosis at that site, and that these events occur via an actomyosin-dependent pathway.

Adult↗
PubMed2005

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2.

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.

Animals↗
PubMed2005

Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP.

cAMP has been found to play a role in mediating the negative regulation of cell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese-hamster ovary) cells that express the EP2 subtype of PGE2 (prostaglandin E2) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE2 dose-dependently inhibited chemotaxis towards IGF-I (insulin-like growth factor-I), which is a Rac-dependent process, with the maximal 75% inhibition observed at 10(-8) M PGE2. EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositide 3-kinase or Akt in response to IGF-I, but potently and dose-dependently inhibited IGF-I-induced activation of cellular Rac activity and membrane ruffling. However, PGE2 failed to inhibit Val12-Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE2 inhibited PDGF (platelet-derived growth factor)-induced Rac activation and chemotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE2 on IGF-I-induced chemotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10(-8) M PGE2, and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3',5'-cyclic monophosphorothioate reduced PGE2 inhibition of Rac activation and chemotaxis. These results indicate that EP2 mediates Rac inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chemotaxis.

Actins↗
PubMed2002

Entopically additive expression of GLABRA2 alters the frequency and spacing of trichome initiation.

GLABRA2 (GL2)/ATHB-10 encodes a homeodomain protein that belongs to the homeodomain-leucine zipper family. Mutant studies have revealed that this gene is involved in trichome, root-hair and seed-coat development. We used reverse genetics to investigate the role of GL2 in trichome development. A transgene consisting of a GL2-coding fragment preceded by the cauliflower mosaic virus 35S promoter (35S::GL2) did not complement defects in the gl2-1 mutant. In the wild-type genetic background, 35S::GL2 caused gl2-mutant-like and scarcely viable phenotypes, suggesting that ectopic overexpression of GL2 interrupts endogenous GL2 function in trichome development and is toxic to plants. On the other hand, another GL2 transgene containing the GL2 promoter (pGL2::GL2) complemented the gl2-1 mutation. Entopically additive expression of GL2 by introduction of pGL2::GL2 in the wild-type genetic background noticably increased the number of trichomes and induced production of adjacent trichomes. Consistent with this result, gl2-1/+ heterozygous leaves, whose GL2 expression was expected to decrease, had fewer trichomes than +/+ leaves. These results indicate that GL2 quantitatively regulates the frequency of trichome initiation and is involved in determining trichome spacing.

Arabidopsis↗
PubMed2000

Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell.

Salmonella typhimurium translocates effector proteins into host cells via the SPI1 type III secretion system to induce responses such as membrane ruffling and internalization by non-phagocytic cells. Activation of the host cellular RhoGTPase Cdc42 is thought to be a key event during internalization. The translocated Salmonella protein SopE is an activator for Cdc42. Because SopE is absent from most S. typhimurium strains it remains unclear whether all S. typhimurium strains rely on activation of Cdc42 to invade host cells. We have identified SopE2, a translocated effector protein common to all S. typhimurium strains. SopE2 is a guanine nucleotide exchange factor for Cdc42 and shows 69% sequence similarity to SopE. Analysis of S. typhimurium mutants demonstrated that SopE2 plays a role in recruitment of the actin-nucleating Arp2/3 complex to the membrane ruffles and in efficient host cell invasion. Transfection experiments showed that SopE2 is sufficient to activate host cellular Cdc42, to recruit the actin-nucleating Arp2/3 complex and to induce actin cytoskeletal rearrangements and internalization. In conclusion, as a result of SopE2 all S. typhimurium strains tested have the capacity to activate Cdc42 signalling inside host cells which is important to ensure efficient entry.

Actin-Related Protein 2↗
PubMed2003

AtMYB103 regulates tapetum and trichome development in Arabidopsis thaliana.

The AtMYB103 gene is a member of the R2R3 MYB gene family in Arabidopsis thaliana. Using the GUS reporter gene, AtMYB103 expression was found to be restricted to the tapetum of developing anthers. Employing RT-PCR and in situ hybridisation, we now show that AtMYB103 is also expressed in trichomes. GUS expression in trichomes was obtained by incorporating the coding and 3'-untranslated regions of AtMYB103 into the promoter-GUS constructs. Sense and antisense technologies were used to downregulate AtMYB103 expression. In transgenic lines with reduced AtMYB103 transcript levels, pollen, tapetum and trichome development were altered. The majority of the pollen grains were distorted in shape and had reduced or no cytoplasmic content. Tapetal degeneration occurred early, and large opaque bodies appeared in the tapetal cytoplasm. In transgenic plants, trichomes on cauline and rosette leaves produced additional branches. These overbranched trichomes contained more nuclear DNA than the wild-type trichomes. The results indicate that AtMYB103 is required for tapetal development and microsporogenesis, and negatively regulates trichome endoreduplication linked to the trichome branching.

Antisense Elements (Genetics)↗
PubMed2003

The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis.

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.

Amino Acid Sequence↗
PubMed2004

The FUS3 transcription factor functions through the epidermal regulator TTG1 during embryogenesis in Arabidopsis.

Loss-of-function mutations in the FUSCA3 (FUS3) gene of Arabidopsis result in alterations in cotyledon identity, inability to complete late seed maturation processes, and the premature activation of apical and root embryonic meristems, which indicates that this transcription factor is an essential regulator of embryogenesis. Although FUS3 shows a complex pattern of expression in the embryo, this gene is only required in the protoderm to carry out its functions. Moreover, the epidermal morphogenesis regulator TRANSPARENT TESTA GLABRA1 (TTG1) is negatively regulated by FUS3 in the embryo. When a loss-of-function ttg1 mutation is introduced into a fus3 mutant, a number of fus3-related phenotypes are rescued, indicating a functional TTG1 gene is required to manifest the fus3 mutant phenotype. It therefore appears that one of the functions of FUS3 is to restrict the domain of expression of TTG1 during embryogenesis. The FUS3-TTG1 interaction is both maternal and zygotic, suggesting a complex relationship is required between these gene products to allow correct seed development.

Anthocyanins↗
PubMed2004

A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis.

The epidermis of higher plants generates the cuticle layer that covers the outer surface of each plant. The cuticle plays a crucial role in plant development, and some mutants with defective cuticle exhibit morphological abnormalities, such as the fusion of organs. The way in which the cuticle forms and its contribution to morphogenesis are poorly understood. Conventional detection of the cuticle by transmission electron microscopy (TEM) requires laborious procedures, which include fixation, staining with osmium, and preparation of ultra-thin sections. It is also difficult to survey entire surfaces of expanded leaves because of the limited size of specimens that can be examined. Thus, TEM is unsuitable for large-scale screening for mutants with defective cuticle. We describe here a rapid and inexpensive method, designated the toluidine-blue (TB) test, for detection of cuticular defects in whole leaves. We demonstrated the validity of the TB test using mutants of Arabidopsis thaliana, including abnormal leaf shape1 (ale1), fiddlehead (fdh), and five eceriferum (cer) mutants, in which the structure and/or function of the cuticle is abnormal. Genetic screening for mutants using the TB test allowed us to identify seven loci. The cuticle-defective regions of leaves of the mutants revealed five intrinsic patterns of surface defects (classes I through V), suggesting that formation of functional cuticle on leaves involves various spatially regulated factors.

Arabidopsis↗
PubMed2001

Protein kinase C activation by 12-0-tetradecanoylphorbol 13-acetate in CG-4 line oligodendrocytes stimulates turnover of choline and ethanolamine phospholipids by phospholipase D and induces rapid process contraction.

Treatment of [3H]-choline- or [14C]-ethanolamine-labelled undifferentiated bipolar and differentiated multipolar CG-4 line oligodendrocytes with 12-0-tetradecanoylphorbol 13-acetate (TPA) to activate protein kinase C stimulated the release of choline or ethanolamine metabolites to the medium over controls. Ro31-8220, a PKC inhibitor, reduced TPA-stimulated release of choline- and ethanolamine-metabolites to basal levels. TPA treatment of both bipolar and multipolar cells caused rapid contraction of processes leaving rounded up cells: this effect was blocked by Ro31-8220. After 12-15 h exposure to TPA, bipolar undifferentiated CG-4 line cells extended short processes again and the cells became multipolar. Nocodozole, an agent which disrupts microtubules and caused CG-4 line cells to round up, caused increased choline or ethanolamine-metabolite release to the medium over basal levels suggesting that some release during TPA-treatment might occur due to process fragmentation. However, the transphosphatidylation reaction confirmed that phospholipase D was active in these cells. Exposure of bipolar undifferentiated CG-4 line cells to TPA resulted in down-regulatation of PKC-alpha and PKC-beta which could not be detected by Western blotting after a few hours; PKC-epsilon was down-regulated much more slowly but PKCs delta, zeta and iota were not influenced by 48 h exposure of cells to TPA. Formation of phosphatidylethanol in the transphosphatidylation reaction was markedly reduced in TPA down-regulated cells indicating a role for PKCs alpha and beta in phospholipase D activation in CG-4 line oligodendrocytes.

Animals↗
PubMed2005

Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion.

Dynamic membrane protrusions such as lamellipodia and filopodia are driven by actin polymerization and often hijacked by intracellular microbes to enter host cells. The overall rate of membrane protrusion depends on the actin polymerization rate and the increase of localized cell volume. Although the signaling pathways involving actin polymerization are well characterized, the molecular mechanisms regulating local cell volume associated with membrane protrusion are unclear. Cryptosporidium parvum, an intracellular parasite, depends on host-cell membrane protrusion to accomplish cell entry and form the parasitophorous vacuole. Here, we report that C. parvum infection of cholangiocytes recruits host-cell SGLT1, a Na+/glucose cotransporter, and aquaporin 1 (AQP1), a water channel, to the attachment site. SGLT1-dependent glucose uptake occurs at the attachment site. Concordantly, the region of attachment displays localized water influx that is inhibited by either suppression of AQP1 by means of AQP1-small interfering RNA (siRNA) or inhibition of SGLT1 by a specific pharmacologic inhibitor, phlorizin. Inhibition of SGLT1 does not affect actin accumulation but decreases the membrane protrusion at the attachment site. Moreover, functional inhibition of host-cell AQP1 and SGLT1 hampers C. parvum invasion of cholangiocytes. Thus, glucose-driven, AQP-mediated localized water influx is involved in the membrane protrusion during C. parvum cellular invasion, phenomena that may also be relevant to the mechanisms of cell membrane protrusion in general.

Actins↗
PubMed2005

Nanoscale visualization and characterization of Myxococcus xanthus cells with atomic force microscopy.

Multicellular microbial communities are the predominant form of existence for microorganisms in nature. As one of the most primitive social organisms, Myxococcus xanthus has been an ideal model bacterium for studying intercellular interaction and multicellular organization. Through previous genetic and EM studies, various extracellular appendages and matrix components have been found to be involved in the social behavior of M. xanthus, but none of them was directly visualized and analyzed under native conditions. Here, we used atomic force microscopy (AFM) imaging and in vivo force spectroscopy to characterize these cellular structures under native conditions. AFM imaging revealed morphological details on the extracellular ultrastructures at an unprecedented resolution, and in vivo force spectroscopy of live cells in fluid allowed us to nanomechanically characterize extracellular polymeric substances. The findings provide the basis for AFM as a useful tool for investigating microbial-surface ultrastructures and nanomechanical properties under native conditions.

Bacterial Adhesion↗
PubMed2006

Molecular model of a type III secretion system needle: Implications for host-cell sensing.

Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are translocated, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex.

Amino Acid Sequence↗