scholarly journals Electron Microscopic Analysis of Membrane Assemblies Formed by the Bacterial Chemotaxis Receptor Tsr

2003 ◽  
Vol 185 (12) ◽  
pp. 3636-3643 ◽  
Author(s):  
Robert M. Weis ◽  
Teruhisa Hirai ◽  
Anas Chalah ◽  
Martin Kessel ◽  
Peter J. Peters ◽  
...  
Keyword(s):  
Bacterial Chemotaxis ◽  
Microscopic Analysis ◽  
Large Family ◽  
Structural Constraints ◽  
Cytoplasmic Domains ◽  
Electron Microscopic ◽  
Chemotaxis Receptors ◽  
Receptor Dimer

ABSTRACT The serine receptor (Tsr) from Escherichia coli is representative of a large family of transmembrane receptor proteins that mediate bacterial chemotaxis by influencing cell motility through signal transduction pathways. Tsr and other chemotaxis receptors form patches in the inner membrane that are often localized at the poles of the bacteria. In an effort to understand the structural constraints that dictate the packing of receptors in the plane of the membrane, we have used electron microscopy to examine ordered assemblies of Tsr in membrane extracts isolated from cells engineered to overproduce the receptor. Three types of assemblies were observed: ring-like “micelles” with a radial arrangement of receptor subunits, two-dimensional crystalline arrays with approximate hexagonal symmetry, and “zippers,” which are receptor bilayers that result from the antiparallel interdigitation of cytoplasmic domains. The registration among Tsr molecules in the micelle and zipper assemblies was sufficient for identification of the receptor domains and for determination of their contributions to the total receptor length. The overall result of this analysis is compatible with an atomic model of the receptor dimer that was constructed primarily from the X-ray crystal structures of the periplasmic and cytoplasmic domains. Significantly, the micelle and zipper structures were also observed in fixed, cryosectioned cells expressing the Tsr receptor at high abundance, suggesting that the modes of Tsr assembly found in vitro are relevant to the situation in the cell.

scholarly journals Identification of Methylation Sites in Thermotoga maritima Chemotaxis Receptors

2006 ◽  
Vol 188 (11) ◽  
pp. 4093-4100 ◽  
Author(s):  
Eduardo Perez ◽  
Haiyan Zheng ◽  
Ann M. Stock
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Thermotoga Maritima ◽  
Consensus Sequence ◽  
Bacterial Chemotaxis ◽  
Cytoplasmic Domains ◽  
Consensus Sequences ◽  
Chemotaxis Proteins ◽  
Chemotaxis Receptors

ABSTRACT Adaptation in bacterial chemotaxis involves reversible methylation of specific glutamate residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins. The specific sites of methylation in Salmonella enterica and Escherichia coli chemoreceptors, identified 2 decades ago, established a consensus sequence for methylation by methyltransferase CheR. Here we report the in vitro methylation of chemoreceptors from Thermotoga maritima, a hyperthermophile that has served as a useful source of chemotaxis proteins for structural analysis. Sites of methylation have been identified by liquid chromatography-mass spectrometry/mass spectrometry. Fifteen sites of methylation were identified within the cytoplasmic domains of four different T. maritima chemoreceptors. The results establish a consensus sequence for chemoreceptor methylation sites in T. maritima that is distinct from the previously identified consensus sequence for E. coli and S. enterica. These findings suggest that consensus sequences for posttranslational modifications in one organism may not be directly extrapolated to analogous modifications in other bacteria.

scholarly journals DSS1 and ssDNA regulate oligomerization of BRCA2

2020 ◽  
Vol 48 (14) ◽  
pp. 7818-7833 ◽  
Author(s):  
Hang Phuong Le ◽  
Xiaoyan Ma ◽  
Jorge Vaquero ◽  
Megan Brinkmeyer ◽  
Fei Guo ◽  
...  
Keyword(s):  
Replication Fork ◽  
Microscopic Analysis ◽  
Filament Formation ◽  
Electron Microscopic ◽  
Microscopic Imaging ◽  
Cellular Events ◽  
Self Association ◽  
Oligomeric Forms

Abstract The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.

scholarly journals Silver Nanoparticles Embedded in Natural Rubber Films: Synthesis, Characterization, and Evaluation ofIn VitroToxicity

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Caroline S. Danna ◽  
Dalita G. S. M. Cavalcante ◽  
Andressa S. Gomes ◽  
Leandra E. Kerche-Silva ◽  
Eidi Yoshihara ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Natural Rubber ◽  
Chinese Hamster ◽  
Cell Lineage ◽  
Microscopic Analysis ◽  
Visible Spectrum ◽  
Electron Microscopic ◽  
Force Microscopy ◽  
Atomic Force

Natural rubber (NR) films can reduce silver metal ions forming embedded metal nanoparticles, a process that could be described as green synthesis. The NR films acting as a reactor generate and incorporate silver nanoparticles (AgNPs). Organic acids and amino acids play a crucial role in the formation of AgNPs. The plasmon extinction obtained in the UV-visible spectrum shows the presence of nanoparticles in the film after dipping the NR film into a solution of silver nitrate at 80°C. Electron microscopic analysis confirms the presence of AgNPs in the NR film and characterization by atomic force microscopy shows a change in the roughness of the NR film with AgNPs. In addition, our preliminary results fromin vitrotoxicity studies (MTT and comet assays) of the NR films and NR films with silver nanoparticles (NR/Ag) show that they are not toxic to cell lineage CHO-K1 (cells from the ovary of a Chinese hamster), an important result for potential medical applications.

scholarly journals Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice

Reproduction ◽  
2010 ◽  
Vol 139 (3) ◽  
pp. 587-598 ◽  
Author(s):  
Samu Myllymaa ◽  
Arja Pasternack ◽  
David G Mottershead ◽  
Matti Poutanen ◽  
Minna M Pulkki ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Microscopic Analysis ◽  
Corpora Lutea ◽  
Electron Microscopic ◽  
Oocyte Growth ◽  
Long Term Study ◽  
Ovarian Folliculogenesis

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain–Fc fusion protein (BMPR2ecd–Fc) is able to inhibit the actions of GDF9 and BMP15 in vitro. Here, we have produced bioactive BMPR2ecd–Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd–Fc during the postnatal days 4–12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd–Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4–28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd–Fc is a potent modulator of ovarian folliculogenesis in vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factors in vivo.

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