Nukleation pflanzlicher Mikrotubuli: Expression relevanter Gene

Die wichtigsten Bestandteile des Cytoskeletts in pflanzlichen Zellen sind die Actinfilamente und die Mikrotubuli. Die Mikrotubuli spielen in der Organisation und der Morphogenese von pflanzlichen Zellen eine wichtige Rolle. Sie sind zusammen mit den Cellulosefibrillen an der Formgebung der Pflanzenzelle beteiligt. Sie bilden das Präprophaseband, das die Zellteilungsebene bestimmt und die Mitosespindel, die für die Trennung der Chromosomen sorgt, sowie den Phragmoplasten, der die Zellwand zwischen den Tochterzellen bildet. Weiterhin geben die Mikrotubuli durch Interaktion mit den Cellulose-Synthase-Komplexen die Richtung der Zellexpansion vor (GRANGER und CYR, 2001; LLOYD und CHAN, 2002; BASKIN, 2002). Die Mikrotubuli sind auch an der Stabilisierung der Zellform und an Transportprozessen beteiligt. Als Bestandteil der Mikrotubuli-organisierenden Zentren (MTOCs) wurde das γ-Tubulin identifiziert, das sehr wahrscheinlich an der Nukleation der Mikrotubuli beteiligt ist, indem es die Assemblierung der αβ-Tubulindimere zu Mikrotubuli einleitet. In tierischen Zellen ist durch intensive Forschung inzwischen relativ viel über die Funktion von γ-Tubulin, vor allem im Verlauf der Zellteilung bekannt, wie z. B. die Lokalisation in Centrosomen mit ihren paarweise angeordneten Centriolen, die die MTOCs darstellen. In pflanzlichen Zellen sind bisher nur wenige Funktionen des Proteins hinreichend geklärt. Die höheren Pflanzen besitzen keine Centriolen und keine Centrosomen. Über die Zellteilung hinaus gibt es kaum Anhaltspunkte über das Vorhandensein oder eventuelle Aufgaben von γ-Tubulin in expandierenden und voll expandierten Zellkulturen und Pflanzengeweben. In dieser Arbeit wurde die Expression über PCR und die Messung des Proteingehalts von cytoskelett-relevanten Proteinen in den Entwicklungsstadien der Zellsuspensionskultur (BY-2) und von Blattstadien der Tabakpflanze (SR1) von Nicotiana tabacum gemessen. Primäres Ziel war es eine Aussage zu erhalten, in welchem Ausmaß γ-Tubulin in expandierenden und voll expandierten Zellen noch exprimiert wird und ob bzw. wie eine Regulation (transkriptionell oder posttranskriptionell) des γ-Tubulins in der Pflanze stattfindet. Für den Nachweis des γ-Tubulins auf der Proteinebene wurde ein pflanzenspezifischer γ-Tubulin Antikörper zu entwickelt. Bei diesem Antikörper handelte es sich um einen polyklonalen Antikörper, der spezifisch gegen eine Sequenz in pflanzlichem γ-Tubulin gerichtet ist. Dabei zeigte der in der Arbeit entwickelte Antikörper gegen die pflanzliche JOSHI-Domäne spezifische Signale. Der erfolgte Nachweis von γ-Tubulin auf der Proteinebene und der Transkripte zeigte bis in die ältesten untersuchten Stadien der Zellsuspensionskultur (BY-2) und in Geweben der Blattstadien der Tabakpflanze (SR1) deutliche Signale für γ-Tubulin. Es war somit nicht nur in meristematisch aktiven Zellen und Geweben von Nicotiana tabacum, sondern auch in nichtmitotischen Zellen und Geweben vorhanden. Hierbei war über die Phasen der Zellteilung und der Zellformgebung hinweg auf beiden Ebenen eine parallele Entwicklung mit relativ konstanten starken Signalen zu beobachten. Nach dem Einstellen der Teilungsaktivität fiel der Gehalt an mRNA deutlich ab. Dabei nahm die Konzentration des Proteins im Vergleich zur mRNA zeitlich verzögert ab. Diese Ergebnisse bei der Zellsuspensionskultur (BY-2) und Tabakpflanze (SR1) gehen mit der möglichen Nukleationstätigkeit des Proteins konform. Es waren geringere aber doch deutlichen Signale bei Absterbenden Zellen der Zellkultur, bzw. bei expandierenden und voll expandierten und seneszenten Blättern der Tabakpflanze (SR1) nachzuweisen. Dies lässt die Folgerung zu, dass die nachgewiesene mRNA von γ-Tubulin nicht posttranskriptionell reguliert wird, sondern dass das γ-Tubulin auch eine wichtige Rolle außerhalb der Zellteilung in den postmitotischen Stadien, z. B. als organisierender Faktor bei der Umgestaltung oder Stabilisierung des Mikrotubuli-Cytoskeletts, spielt. Der γ-Tubulin-Gehalt in den Geweben der SR1-Pflanze zeigte über die Zellkultur hinaus, dass die Expression von α-Tubulin nach Einstellen der Teilungsaktivität kontinuierlich abnimmt. Dieses Ergebnis legt die Vermutung nahe, dass γ-Tubulin in älteren Blattgeweben zusätzliche Aufgaben übernehmen könnte, die nicht auf eine gleichzeitige Expression von α-Tubulin angewiesen sind. So kann beispielsweise eine Beteiligung von γ-Tubulin an der Stabilisierung der Mikrotubuli, und damit einhergehend eine Abnahme der dynamischen Instabilität dieser Filamente, eine denkbare Funktion des Proteins in expandierendem und voll expandiertem Gewebe sein. Die Aufgaben von γ-Tubulin in sehr altem Gewebe mit deutlichen Anzeichen der Seneszenz können allerdings nach dem derzeitigen Stand der Forschung nicht eindeutig beantwortet werden und bedürfen weitergehenden Untersuchungen, da dadurch ein die Komplexität und die Dynamik des pflanzlichen Cytoskeletts geklärt werden kann.

Chronic spontaneous activity generated in the somata of primary nociceptors is associated with pain-related behavior after spinal cord injury.

Mechanisms underlying chronic pain that develops after spinal cord injury (SCI) are incompletely understood. Most research on SCI pain mechanisms has focused on neuronal alterations within pain pathways at spinal and supraspinal levels associated with inflammation and glial activation. These events might also impact central processes of primary sensory neurons, triggering in nociceptors a hyperexcitable state and spontaneous activity (SA) that drive behavioral hypersensitivity and pain. SCI can sensitize peripheral fibers of nociceptors and promote peripheral SA, but whether these effects are driven by extrinsic alterations in surrounding tissue or are intrinsic to the nociceptor, and whether similar SA occurs in nociceptors in vivo are unknown. We show that small DRG neurons from rats (Rattus norvegicus) receiving thoracic spinal injury 3 d to 8 months earlier and recorded 1 d after dissociation exhibit an elevated incidence of SA coupled with soma hyperexcitability compared with untreated and sham-treated groups. SA incidence was greatest in lumbar DRG neurons (57%) and least in cervical neurons (28%), and failed to decline over 8 months. Many sampled SA neurons were capsaicin sensitive and/or bound the nociceptive marker, isolectin B4. This intrinsic SA state was correlated with increased behavioral responsiveness to mechanical and thermal stimulation of sites below and above the injury level. Recordings from C- and Aδ-fibers revealed SCI-induced SA generated in or near the somata of the neurons in vivo. SCI promotes the entry of primary nociceptors into a chronic hyperexcitable-SA state that may provide a useful therapeutic target in some forms of persistent pain.

Molecular mechanisms underlying a cellular analog of operant reward learning.

Operant conditioning is a ubiquitous but mechanistically poorly understood form of associative learning in which an animal learns the consequences of its behavior. Using a single-cell analog of operant conditioning in neuron B51 of Aplysia, we examined second-messenger pathways engaged by activity and reward and how they may provide a biochemical association underlying operant learning. Conditioning was blocked by Rp-cAMP, a peptide inhibitor of PKA, a PKC inhibitor, and by expressing a dominant-negative isoform of Ca2+-dependent PKC (apl-I). Thus, both PKA and PKC were necessary for operant conditioning. Injection of cAMP into B51 mimicked the effects of operant conditioning. Activation of PKC also mimicked conditioning but was dependent on both cAMP and PKA, suggesting that PKC acted at some point upstream of PKA activation. Our results demonstrate how these molecules can interact to mediate operant conditioning in an individual neuron important for the expression of the conditioned behavior.

Intracellular phosphorylation of ornithine decarboxylase: Regulation of enzyme activity

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis exists as two major and one minor ionic form in the macrophage cell line, RAW 264. The forms have the same molecular weight, 55,000, but differ in their isoelectric points, 5.2, 5.1, and 4.9-5.0. The hypothesis that phosphorylation accounts for the differences in the two major ionic forms and that phosphorylation is involved in the regulation of enzyme activity was investigated. Metabolic-radiolabeling of cells with $\sp{32}$P-orthophosphate indicated that only one of the major forms of the protein can be explained by phosphorylation: treatment of purified ODC with alkaline phosphatase resulted in the loss of the phosphorylated form of the protein, pl 5.1, with a concomitant increase in the unphosphorylated, pl 5.2, form of the protein. Characterization of the phosphorylation sites showed that serine was the present. Tryptic digests of $\sp{32}$P-labeled ODC, analyzed by either two dimensional tryptic peptide mapping or reverse-phase HPLC, contained only one major radiolabeled peptide.^ The role phosphorylation plays in the regulation of enzyme activity was also investigated. Treatment of purified ODC with alkaline phosphatase resulted in the loss of enzyme activity. A positive linear correlation exists between enzyme activity and the amount of phosphorylated form of the protein present.^ To ascertain if the two major forms of the protein were also found in animal cells, ODC was immunoprecipitated from various rat tissues, fractionated by isoelectric focusing, and detected by immunoblotting. ODC was present in rat tissues in a single major form, which comigrated with the pl 5.1, phosphorylated form of ODC present in RAW 264 cell.^ This study concludes that ODC exists as a phosphorylated form, pl 5.1, and an unphosphorylated form, pl 5.2 in RAW 264 cells. The amount of the phosphorylated form of ODC correlates well with the enzyme activity. ^

The Host Nonsense-Mediated mRNA Decay Pathway Restricts Mammalian RNA Virus Replication

In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we found that depletion of nonsense-mediated mRNA decay (NMD) pathway components Upf1, Smg5, and Smg7 led to increased levels of viral proteins and RNA and higher titers of released virus. The inhibitory effect of NMD was stronger when virus replication efficiency was impaired by mutations or deletions in the replicase proteins. Consequently, depletion of NMD components resulted in a more than 20-fold increase in production of these attenuated viruses. These findings indicate that a cellular mRNA quality control mechanism serves as an intrinsic barrier to the translation of early viral proteins and the amplification of +RNA viruses in animal cells.

Nonsense-mediated mRNA decay (NMD) restricts replication of mammalian RNA viruses

A genome-wide siRNA screen against host factors that affect the infection of Semliki Forest virus (SFV), a positive-strand (+)RNA virus, revealed that components of the nonsense-mediated mRNA decay (NMD) pathway restrict early, post-entry steps of the infection cycle. In HeLa cells and primary human fibroblasts, knockdown of UPF1, SMG5 and SMG7 leads to increased levels of viral proteins and RNA and to higher titers of released virus. The inhibitory effect of NMD was stronger when the efficiency of virus replication was impaired by mutations or deletions in the replicase proteins. Accordingly, impairing NMD resulted in a more than 20-fold increased production of these attenuated viruses. Our data suggest that intrinsic features of genomic and sub-genomic viral mRNAs, most likely the extended 3'-UTR length, make them susceptible to NMD. The fact that SFV replication is entirely cytoplasmic strongly suggests that degradation of the viral RNA occurs through the exon junction complex (EJC)-independent mode of NMD. Collectively, our findings uncover a new biological function for NMD as an intrinsic barrier to the translation of early viral proteins and the amplification of (+)RNA viruses in animal cells. Thus, in addition to its role in mRNA surveillance and post-transcriptional gene regulation, NMD also contributes to protect cells from RNA viruses.

Functional importance of conserved nucleotides at the histone RNA 3' processing site.

Histone pre-mRNA 3' processing is controlled by a hairpin element preceding the processing site that interacts with a hairpin-binding protein (HBP) and a downstream spacer element that serves as anchoring site for the U7 snRNP. In addition, the nucleotides following the hairpin and surrounding the processing site (ACCCA'CA) are conserved among vertebrate histone genes. Single to triple nucleotide mutations of this sequence were tested for their ability to be processed in nuclear extract from animal cells. Changing the first four nucleotides had no qualitative and little if any quantitative effects on histone RNA 3' processing in mouse K21 cell extract, where processing of this gene is virtually independent of the HBP. A gel mobility shift assay revealing HBP interactions and a processing assay in HeLa cell extract (where the contribution of HBP to efficient processing is more important) showed that only one of these mutations, predicted to extend the hairpin by one base pair, affected the interaction with HBP. Mutations in the next three nucleotides affected both the cleavage efficiency and the choice of processing sites. Analysis of these novel sites indicated a preference for the nucleotide 5' of the cleavage site in the order A > C > U > G. Moreover, a guanosine in the 3' position inhibited cleavage. The preference for an A is shared with the cleavage/polyadenylation reaction, but the preference order for the other nucleotides is different [Chen F, MacDonald CC, Wilusz J, 1995, Nucleic Acids Res 23:2614-2620].

Cycling in the nucleus: regulation of RNA 3' processing and nuclear organization of replication-dependent histone genes.

The histones which pack new DNA during the S phase of animal cells are made from mRNAs that are cleaved at their 3' end but not polyadenylated. Some of the factors used in this reaction are unique to it while others are shared with the polyadenylation process that generates all other mRNAs. Recent work has begun to shed light on how the cell manages the assignment of these common components to the two 3' processing systems, and how it achieves their cell cycle-regulation and recruitment to the histone pre-mRNA. Moreover, recent and older findings reveal multiple connections between the nuclear organization of histone genes, their transcription and 3' end processing as well as the control of cell proliferation.

Transcriptional regulation of the rat {\it mdr1b\/} gene

The expression of P-glycoproteins encoded by the mdr gene family is associated with the emergence of multidrug-resistance phenotype in animal cells. This gene family includes two members, MDR1 and MDR2, in humans, and three members, mdr1a, mdr1b, and mdr2, in rodents. Among them, the rat mdr1b is known to be highly activated during hepatocarcinogenesis, and its expression is sensitive to the treatment with growth factors, cytotoxic drugs, as well as other physical or chemical stresses. It is believed that the transcriptional regulation plays an important role in above events, however little has been known about mechanisms involved.^ To elucidate how mdr1b expression is regulated, we isolated the genomic sequence of the rat mdr1b and functionally dissected its 5$\prime$ promoter region. Our results demonstrated that: (1) the transcription start site of the rat mdr1b is identical to that of the murine mdr1b homologue; (2) a palindromic sequence from bp $-$189 to $-$180 bp is essential for the basal promoter function of the rat mdr1b, and binds to a specific protein that appears to be a novel transcription factor implicated in the regulation of the rat mdr1b expression; (3) a NF-$\kappa$B-binding site from bp $-$167 to $-$159 is also involved in the basal promoter function. The p65/p50 subunits of the NF-$\kappa$B and raf-1 kinase are implicated in the insulin-inducible promoter activity of the mdr1b, suggesting the important role of NF-$\kappa$B in the regulation of the mdr1b by growth factors; (4) a p53-binding site from bp $-$199 to $-$180 is not only essential for the basal promoter activity but also responsible for the induction of mdr1b by cytotoxic agents. In addition, we provided evidence showing that endogenous mdr1b expression can be modulated by wild-type p53. On the basis of these findings, a model of transcriptional regulation of the rat mdr1b was proposed. ^

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA–protein complexes into nuclei. Import of VirD2–ssDNA–VirE2 complexes was fast and efficient, and was shown to depended on importin α, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein–DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

Microtubule-based Endoplasmic Reticulum Motility in Xenopus laevis: Activation of Membrane-associated Kinesin during Development

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts of Xenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopus egg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development in Xenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute ∼19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower (∼0.4 μm/s) than minus end-directed motility (∼1.3 μm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system

Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.

Evidence for the existence of a sulfonylurea-receptor-like protein in plants: Modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium channel openers

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.