A myxobacterial S-motility protein dances with poles

Coordinated movement of packs of S-motile Myxococcus xanthus cells relies on extrusion and retraction of pili that are located at one cell pole. At regular intervals the pili switch their polar location and cells reverse direction. Recently, the FrzS S-motility protein was observed to localize predominantly to the piliated pole. In time, FrzS was redeployed to the opposite pole and its sequestration at the new site coincided with cell reversal. The C-terminal region of FrzS, a response regulator homolog, is rich in coiled-coil motifs and is required for dynamic localization and proper motility. These results raise the possibility that proper spatial control of FrzS has an important role in the regulation of cell reversal and S-motility.     

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.     

A new diplectanid (Monogenea) genus and species from the gills of the black snook, Centropomus nigrescens (Perciformes: Centropomidae) of the Pacific coast of Mexico

Cornutohaptor nigrescensi n. sp. (Diplectanidae) is described from the gills of the black snook, Centropomus nigrescens (Perciformes: Centropomidae) from the Pacific coast of Mexico. Cornutohaptor n. gen. is proposed for this new species and is characterized by possessing 2 intestinal ceca terminating blindly; a germarium looping right intestinal cecum; bilobed testis; 2 seminal vesicles; 7 pairs of hooks, each with protruding thumb; a grooved ventral bar and coiled male copulatory organ (MCO); an accessory piece comprising a "baglike structure" with an appendage; dorsal bars associated parallelly to body midline; and no adhesive accessory organs on the haptor. Cornutohaptor differs from all confamilial genera by including species with anchors with straight and deep root longest, hook pair 1 reduced in size, MCO with counterclockwise rings, and by the morphology of the accessory piece. Cornutohaptor nigrescensi most closely resembles species of Murraytrema Price, 1937, Lobotrema Tripathi, 1937, and Murraytrematoides Yamaguti, 1958, because of the absence of squamodiscs or lamellodiscs on the haptor and tegumental scales on the posterior portion of the body. Cornutohaptor differs from these genera in the position and number of haptoral bars (2 bars in Lobotrema spp., dorsal bars transversally associated in Murraytrema and Murraytrematoides spp.) and in having a coiled MCO (copulatory organ is a comparatively straight, poorly sclerotized tube in Murraytrematoides spp.). This is the first diplectanid described from a centropomid along the Pacific coast of Mexico.     

Structure of Ceramide-1-Phosphate at the Air-Water Solution Interface in the Absence and Presence of Ca

Ceramide-1-phosphate, the phosphorylated form of ceramide, gained attention recently due to its diverse intracellular roles, in particular in inflammation mediated by cPLA₂α. However, surprisingly little is known about the physical chemical properties of this lipid and its potential impact on physiological function. For example, the presence of Ca²⁺ is indispensable for the interaction of Cer-1-P with the C2 domain of cPLA₂α. We report on the structure and morphology of Cer-1-P in monomolecular layers at the air/water solution interface in the absence and presence of Ca²⁺ using diverse biophysical techniques, including synchrotron x-ray reflectivity and grazing angle diffraction, to gain insight into the role and function of Cer-1-P in biomembranes. We show that relatively small changes in pH and the presence of monovalent cations dramatically affect the behavior of Cer-1-P. On pure water Cer-1-P forms a solid monolayer despite the negative charge of the phosphomonoester headgroup. In contrast, pH 7.2 buffer yields a considerably less solid-like monolayer, indicating that charge-charge repulsion becomes important at higher pH. Calcium was found to bind strongly to the headgroup of Cer-1-P even in the presence of a 100-fold larger Na⁺ concentration. Analysis of the x-ray reflectivity data allowed us to estimate how much Ca²⁺ is bound to the headgroup, ∼0.5 Ca²⁺ and ∼1.0 Ca²⁺ ions per Cer-1-P molecule for the water and buffer subphase respectively. These results can be qualitatively understood based on the molecular structure of Cer-1-P and the electrostatic/hydrogen-bond interactions of its phosphomonoester headgroup. Biological implications of our results are also discussed.     

Endocytosis and post-endocytic sorting of connexins

The connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells in solid tissues to directly exchange ions and small molecules. These channels assemble into distinct plasma membrane domains known as gap junctions. Gap junction intercellular communication plays critical roles in numerous cellular processes, including control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are dynamic plasma membrane domains, and there is increasing evidence that modulation of endocytosis and post-endocytic trafficking of connexins are important mechanisms for regulating the level of functional gap junctions at the plasma membrane. The emerging picture is that multiple pathways exist for endocytosis and sorting of connexins to lysosomes, and that these pathways are differentially regulated in response to physiological and pathophysiological stimuli. Recent studies suggest that endocytosis and lysosomal degradation of connexins is controlled by a complex interplay between phosphorylation and ubiquitination. This review summarizes recent progress in understanding the molecular mechanisms involved in endocytosis and post-endocytic sorting of connexins, and the relevance of these processes to the regulation of gap junction intercellular communication under normal and pathophysiological conditions. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Phosphatidylinositol-4,5-bisphosphate ionization and domain formation in the presence of lipids with hydrogen bond donor capabilities

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) is an important lipidic signaling molecule that is involved in a broad range of cellular processes. Its interaction with proteins and its lateral distribution are governed by the ionization state of the phosphomonoester groups and its ability to form intra- and intermolecular hydrogen bonds. In this study we have investigated the ionization state of PI(4,5)P(2) in ternary lipid vesicle systems that contain in addition to PI(4,5)P(2) and phosphatidylcholine (PC) either phosphatidylethanolamine (PE), phosphatidylserine (PS) or phosphatidylinositol (PI). In the presence of PE we find an increased ionization of PI(4,5)P(2), which can be attributed to increased deprotonation due to hydrogen bond formation between PE and the PI(4,5)P(2) phosphomonoester groups. However, the effect of PE on PI(4,5)P(2) ionization is significantly smaller than it had been found previously for phosphatidic acid in the presence of PE (Kooijman et al., 2005). The reduced impact of PE on PI(4,5)P(2) ionization can be attributed to competing intramolecular hydrogen bond formation between the phosphomonoester groups and neighboring hydroxyl groups. It is noteworthy that the presence of PE affects more strongly the ionization of the 5-phosphate group than that of the 4-phosphate, suggesting that the interaction of PE with the 5-phosphate is stronger. In PI(4,5)P(2)/PS/PC lipid vesicles, the presence of PS was expected to yield an increased protonation of the PI(4,5)P(2) phosphomonoester groups due to a decreased interfacial pH as a result of the increased negative interfacial charge. However, the effect of PS on PI(4,5)P(2) ionization is only minor, potentially suggesting that PS and PI(4,5)P(2) are demixed. The PI(4,5)P(2)/PI/PC vesicle system was characterized by a surprising mixing behavior that has potentially far reaching consequences: fluorescence microscopy measurements of giant unilammellar vesicles composed of PI(4,5)P(2)/PI/PC at physiological concentrations show that PI and PI(4,5)P(2) form macroscopic, fluid phase domains in contact with a fluid PC rich phase (fluid/fluid demixing). Despite the fact that PI and PI(4,5)P(2) co-localize, the effect of PI on PI(4,5)P(2) ionization behavior is only noticeable above pH 7. Apparently two opposing effects lead to the observed behavior: Due to the presence of the anionic PI, the interfacial pH drops, which is expected to lead to an enhanced protonation of the PI(4,5)P(2) phosphomonoester groups. In turn, hydrogen bond formation between PI and PI(4,5)P(2) would lead to the opposite, i.e. increased deprotonation of the phosphomonoester group. Apparently these two effects compensate each other for pH values smaller than about 7, while for higher pH values the increased interfacial pH in the presence of PI has a stronger impact than PI/PI(4,5)P(2) hydrogen bond formation. The cooperative formation of PI/PI(4,5)P(2) mixed domains has potentially important ramifications for the spatial organization of phosphoinositide mediated signaling events.     

Draft Genome Sequence of an Extremely Drug-Resistant KPC-Producing Klebsiella pneumoniae ST258 Epidemic Strain

Extremely drug-resistant (XDR) Klebsiella pneumoniae carbapenemase-producing clone ST258 has rapidly disseminated worldwide. We report here the draft genome sequence of the K. pneumoniae ST258 XDR clinical strain from Israel.     

The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.     

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIP^{S332A/−/−} cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP^+/−/− cells. Finally, CtIP^{S332A/−/−}BRCA1^−/− and CtIP^+/−/−BRCA1^−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.